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Sample records for cdk inhibitor p21

  1. AS160 controls eukaryotic cell cycle and proliferation by regulating the CDK inhibitor p21.

    Science.gov (United States)

    Gongpan, Pianchou; Lu, Yanting; Wang, Fang; Xu, Yuhui; Xiong, Wenyong

    2016-07-02

    AS160 (TBC1D4) has been implicated in multiple biological processes. However, the role and the mechanism of action of AS160 in the regulation of cell proliferation remain unclear. In this study, we demonstrated that AS160 knockdown led to blunted cell proliferation in multiple cell types, including fibroblasts and cancer cells. The results of cell cycle analysis showed that these cells were arrested in the G1 phase. Intriguingly, this inhibition of cell proliferation and the cell cycle arrest caused by AS160 depletion were glucose independent. Moreover, AS160 silencing led to a marked upregulation of the expression of the cyclin-dependent kinase inhibitor p21. Furthermore, whereas AS160 overexpression resulted in p21 downregulation and rescued the arrested cell cycle in AS160-depeleted cells, p21 silencing rescued the inhibited cell cycle and proliferation in the cells. Thus, our results demonstrated that AS160 regulates glucose-independent eukaryotic cell proliferation through p21-dependent control of the cell cycle, and thereby revealed a molecular mechanism of AS160 modulation of cell cycle and proliferation that is of general physiological significance.

  2. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    International Nuclear Information System (INIS)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid; Gjerloev, Simon; Birk, Jesper; Roepke, Carsten; Norrild, Bodil

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge, this is the first time a role for cathepsin B is reported in HPV-induced apoptotic signalling

  3. The functions of the cdk-cyclin kinase inhibitor p21{sup WAF1}; Les fonctions de l'inhibiteur de cdk-cycline kinase, p21{sup WAF1}

    Energy Technology Data Exchange (ETDEWEB)

    Boulaire, J. [Institut de Biologie Structurale J.P. Ebel, 38 - Grenoble (France); Fotedar, A.; Fotedar, R. [Sidney Kimmel Cancer Center, San Diego (United States)

    2000-04-01

    p21{sup WAF1} plays a critical role in regulating cell growth and the cell response to DNA damage. The primary targets of p21{sup WAF1} (hereafter referred to as p21) are the cdk-cyclins which regulate the progression of cukayotic cells through the cell cycle, and proliferating cell nuclear antigen (PCNA), an accessory protein of DNA polymerase {delta}. p21 forms complexes with a class of cdk-cyclins to inhibit their kinase activity and with PCNA to inhibit synthesis. These distinct properties map to the N- terminal and the C- terminal regions of p21, respectively. Cell cycle arrest in G-1 (G-1 checkpoint) following DNA damage is mediated by p53 and is deficient in p21 null cells. p53 thus up-regulates p21 expression in response to DNA damage, which in turn inhibits cdk2-associated kinase activity. Retinoblastoma protein is regulated by cdk-cyclin kinases, and acts as a downstream target of p21 in DNA damage-induced G-1 arrest. Furthermore, accumulating evidence indicates that p21 may play a role in maintaining G-2 arrest after DNA damage. Transcriptional control of p21 by factors other than p53 is critical for growth arrest and for cell differentiation in many instances. (authors)

  4. Development of mice without Cip/Kip CDK inhibitors

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    Tateishi, Yuki; Matsumoto, Akinobu; Kanie, Tomoharu [Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582 (Japan); CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan); Hara, Eiji [Cancer Institute, Japanese Foundation for Cancer Research, 3-8-31 Ariake, Koto-ku, Tokyo 135-8550 (Japan); Nakayama, Keiko [Department of Developmental Genetics, Center for Translational and Advanced Animal Research, Graduate School of Medicine, Tohoku University, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575 (Japan); Nakayama, Keiichi I., E-mail: nakayak1@bioreg.kyushu-u.ac.jp [Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, Fukuoka 812-8582 (Japan); CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 (Japan)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. Black-Right-Pointing-Pointer Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. Black-Right-Pointing-Pointer CDK2 kinase activity of the triple mutant MEFs is increased in G0 phase. -- Abstract: Timely exit of cells from the cell cycle is essential for proper cell differentiation during embryogenesis. Cyclin-dependent kinase (CDK) inhibitors (CKIs) of the Cip/Kip family (p21, p27, and p57) are negative regulators of cell cycle progression and are thought to be essential for development. However, the extent of functional redundancy among Cip/Kip family members has remained largely unknown. We have now generated mice that lack all three Cip/Kip CKIs (TKO mice) and compared them with those lacking each possible pair of these proteins (DKO mice). We found that the TKO embryos develop normally until midgestation but die around embryonic day (E) 13.5, slightly earlier than p27/p57 DKO embryos. The TKO embryos manifested morphological abnormalities as well as increased rates of cell proliferation and apoptosis in the placenta and lens that were essentially indistinguishable from those of p27/p57 DKO mice. Unexpectedly, the proliferation rate and cell cycle profile of mouse embryonic fibroblasts (MEFs) lacking all three Cip/Kip CKIs did not differ substantially from those of control MEFs. The abundance and kinase activity of CDK2 were markedly increased, whereas CDK4 activity and cyclin D1 abundance were decreased, in both p27/p57 DKO and TKO MEFs during progression from G{sub 0} to S phase compared with those in control MEFs. The extents of the increase in CDK2 activity and the decrease in CDK4 activity and cyclin D1 abundance were greater in TKO MEFs than in p27/p57 DKO MEFs. These results suggest that p27 and p57 play an essential role in mouse development after midgestation, and that p21 plays only an auxiliary role in

  5. Cyclin dependent kinase (CDK) inhibitors as anticancer drugs.

    Science.gov (United States)

    Sánchez-Martínez, Concepción; Gelbert, Lawrence M; Lallena, María José; de Dios, Alfonso

    2015-09-01

    Sustained proliferative capacity is a hallmark of cancer. In mammalian cells proliferation is controlled by the cell cycle, where cyclin-dependent kinases (CDKs) regulate critical checkpoints. CDK4 and CDK6 are considered highly validated anticancer drug targets due to their essential role regulating cell cycle progression at the G1 restriction point. This review provides an overview of recent advances on cyclin dependent kinase inhibitors in general with special emphasis on CDK4 and CDK6 inhibitors and compounds under clinical evaluation. Chemical structures, structure activity relationships, and relevant preclinical properties will be described. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Induction of p21(WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16(INK4a).

    Science.gov (United States)

    Mitra, J; Dai, C Y; Somasundaram, K; El-Deiry, W S; Satyamoorthy, K; Herlyn, M; Enders, G H

    1999-05-01

    The tumor suppressor p16(INK4a) inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27(KIP1), and p21(WAF1/CIP1). Concomitantly, the total cellular level of p21 increased severalfold via a posttranscriptional mechanism. Most cyclin E-cdk2 complexes associated with p21 and became inactive, expression of cyclin A was curtailed, and DNA synthesis was strongly inhibited. Induction of p21 alone, in a sibling clone, to the level observed during p16 induction substantially reproduced these effects. Overexpression of either cyclin E or A prevented p16 from mediating arrest. We then extended these studies to HCT 116 colorectal carcinoma cells and a p21-null clone derived by homologous recombination. In the parental cells, p16 expression also augmented total cellular and cdk2-bound p21. Moreover, p16 strongly inhibited DNA synthesis in the parental cells but not in the p21-null derivative. These findings indicate that p21-mediated inhibition of cdk2 contributes to the cell cycle arrest imposed by p16 and is a potential point of cooperation between the p16/pRB and p14(ARF)/p53 tumor suppressor pathways.

  7. Induction of p21WAF1/CIP1 and Inhibition of Cdk2 Mediated by the Tumor Suppressor p16INK4a

    OpenAIRE

    Mitra, Jayashree; Dai, Charlotte Y.; Somasundaram, Kumaravel; El-Deiry, Wafik S.; Satyamoorthy, Kapaettu; Herlyn, Meenhard; Enders, Greg H.

    1999-01-01

    The tumor suppressor p16INK4a inhibits cyclin-dependent kinases 4 and 6. This activates the retinoblastoma protein (pRB) and, through incompletely understood events, arrests the cell division cycle. To permit biochemical analysis of the arrest, we generated U2-OS osteogenic sarcoma cell clones in which p16 transcription could be induced. In these clones, binding of p16 to cdk4 and cdk6 abrogated binding of cyclin D1, p27KIP1, and p21WAF1/CIP1. Concomitantly, the total cellular level of p21 in...

  8. Identification and Characterization of an Irreversible Inhibitor of CDK2.

    Science.gov (United States)

    Anscombe, Elizabeth; Meschini, Elisa; Mora-Vidal, Regina; Martin, Mathew P; Staunton, David; Geitmann, Matthis; Danielson, U Helena; Stanley, Will A; Wang, Lan Z; Reuillon, Tristan; Golding, Bernard T; Cano, Celine; Newell, David R; Noble, Martin E M; Wedge, Stephen R; Endicott, Jane A; Griffin, Roger J

    2015-09-17

    Irreversible inhibitors that modify cysteine or lysine residues within a protein kinase ATP binding site offer, through their distinctive mode of action, an alternative to ATP-competitive agents. 4-((6-(Cyclohexylmethoxy)-9H-purin-2-yl)amino)benzenesulfonamide (NU6102) is a potent and selective ATP-competitive inhibitor of CDK2 in which the sulfonamide moiety is positioned close to a pair of lysine residues. Guided by the CDK2/NU6102 structure, we designed 6-(cyclohexylmethoxy)-N-(4-(vinylsulfonyl)phenyl)-9H-purin-2-amine (NU6300), which binds covalently to CDK2 as shown by a co-complex crystal structure. Acute incubation with NU6300 produced a durable inhibition of Rb phosphorylation in SKUT-1B cells, consistent with it acting as an irreversible CDK2 inhibitor. NU6300 is the first covalent CDK2 inhibitor to be described, and illustrates the potential of vinyl sulfones for the design of more potent and selective compounds. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  9. Antioxidants cause rapid expansion of human adipose-derived mesenchymal stem cells via CDK and CDK inhibitor regulation

    Science.gov (United States)

    2013-01-01

    Background Antioxidants have been shown to enhance the proliferation of adipose-derived mesenchymal stem cells (ADMSCs) in vitro, although the detailed mechanism(s) and potential side effects are not fully understood. In this study, human ADMSCs cultured in ImF-A medium supplemented with antioxidants (N-acetyl-l-cysteine and ascorbic acid-2-phosphate) and fibroblast growth factor 2 (FGF-2) were compared with ADMSCs cultured with FGF-2 alone (ImF) or with FGF-2 under 5% pO2 conditions (ImF-H). Results During log-phase growth, exposure to ImF-A resulted in a higher percentage of ADMSCs in the S phase of the cell cycle and a smaller percentage in G0/G1 phase. This resulted in a significantly reduced cell-doubling time and increased number of cells in the antioxidant-supplemented cultures compared with those supplemented with FGF-2 alone, an approximately 225% higher cell density after 7 days. Western blotting showed that the levels of the CDK inhibitors p21 and p27 decreased after ImF-A treatment, whereas CDK2, CDK4, and CDC2 levels clearly increased. In addition, ImF-A resulted in significant reduction in the expression of CD29, CD90, and CD105, whereas relative telomere length, osteogenesis, adipogenesis, and chondrogenesis were enhanced. The results were similar for ADMSCs treated with antioxidants and those under hypoxic conditions. Conclusion Antioxidant treatment promotes entry of ADMSCs into the S phase by suppressing cyclin-dependent kinase inhibitors and results in rapid cell proliferation similar to that observed under hypoxic conditions. PMID:23915242

  10. DACH1 regulates cell cycle progression of myeloid cells through the control of cyclin D, Cdk 4/6 and p21{sup Cip1}

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Jae-Woong; Kim, Hyeng-Soo; Kim, Seonggon; Hwang, Junmo; Kim, Young Hun; Lim, Ga Young [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Sohn, Wern-Joo [Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu 700-412 (Korea, Republic of); Yoon, Suk-Ran [Cell Therapy Research Center, Korea Research Institute of Bioscience and Biotechnology, Yuseong-gu, Daejeon 305-806 (Korea, Republic of); Kim, Jae-Young [Department of Biochemistry, School of Dentistry, IHBR, Kyungpook National University, Daegu 700-412 (Korea, Republic of); Park, Tae Sung [Department of Laboratory Medicine, Kyung Hee University School of Medicine, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-702 (Korea, Republic of); Park, Kwon Moo [Department of Anatomy, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of); Ryoo, Zae Young [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of); Lee, Sanggyu, E-mail: slee@knu.ac.kr [School of Life Science and Biotechnology, Kyungpook National University, Daegu 702-701 (Korea, Republic of)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer DACH1 increases cyclin D, F and Cdk 1, 4, 6 in mouse myeloid progenitor cells. Black-Right-Pointing-Pointer The knockdown of DACH1 blocked the cell cycle progression of HL-60 cells. Black-Right-Pointing-Pointer The novel effect of DACH1 related with cell cycle regulation and leukemogenesis. -- Abstract: The cell-fate determination factor Dachshund, a component of the Retinal Determination Gene Network (RDGN), has a role in breast tumor proliferation through the repression of cyclin D1 and several key regulators of embryonic stem cell function, such as Nanog and Sox2. However, little is known about the role of DACH1 in a myeloid lineage as a cell cycle regulator. Here, we identified the differential expression levels of extensive cell cycle regulators controlled by DACH1 in myeloid progenitor cells. The forced expression of DACH1 induced p27{sup Kip1} and repressed p21{sup Cip1}, which is a pivotal characteristic of the myeloid progenitor. Furthermore, DACH1 significantly increased the expression of cyclin D1, D3, F, and Cdk 1, 4, and 6 in myeloid progenitor cells. The knockdown of DACH1 blocked the cell cycle progression of HL-60 promyeloblastic cells through the decrease of cyclin D1, D3, F, and Cdk 1, 4, and 6 and increase in p21{sup Cip1}, which in turn decreased the phosphorylation of the Rb protein. The expression of Sox2, Oct4, and Klf4 was significantly up-regulated by the forced expression of DACH1 in mouse myeloid progenitor cells.

  11. Machine Learning Methods for Prediction of CDK-Inhibitors

    Science.gov (United States)

    Ramana, Jayashree; Gupta, Dinesh

    2010-01-01

    Progression through the cell cycle involves the coordinated activities of a suite of cyclin/cyclin-dependent kinase (CDK) complexes. The activities of the complexes are regulated by CDK inhibitors (CDKIs). Apart from its role as cell cycle regulators, CDKIs are involved in apoptosis, transcriptional regulation, cell fate determination, cell migration and cytoskeletal dynamics. As the complexes perform crucial and diverse functions, these are important drug targets for tumour and stem cell therapeutic interventions. However, CDKIs are represented by proteins with considerable sequence heterogeneity and may fail to be identified by simple similarity search methods. In this work we have evaluated and developed machine learning methods for identification of CDKIs. We used different compositional features and evolutionary information in the form of PSSMs, from CDKIs and non-CDKIs for generating SVM and ANN classifiers. In the first stage, both the ANN and SVM models were evaluated using Leave-One-Out Cross-Validation and in the second stage these were tested on independent data sets. The PSSM-based SVM model emerged as the best classifier in both the stages and is publicly available through a user-friendly web interface at http://bioinfo.icgeb.res.in/cdkipred. PMID:20967128

  12. Cyclin‑dependent kinase inhibitor p21 does not impact embryonic endochondral ossification in mice.

    Science.gov (United States)

    Chinzei, Nobuaki; Hayashi, Shinya; Hashimoto, Shingo; Kanzaki, Noriyuki; Iwasa, Kenjiro; Sakata, Shuhei; Kihara, Shinsuke; Fujishiro, Takaaki; Kuroda, Ryosuke; Kurosaka, Masahiro

    2015-03-01

    Endochondral ossification at the growth plate is regulated by a number of factors and hormones. The cyclin‑dependent kinase inhibitor p21 has been identified as a cell cycle regulator and its expression has been reported to be essential for endochondral ossification in vitro. However, to the best of our knowledge, the function of p21 in endochondral ossification has not been evaluated in vivo. Therefore, the aim of this study was to investigate the function of p21 in embryonic endochondral ossification in vivo. Wild‑type (WT) and p21 knockout (KO) pregnant heterozygous mice were sacrificed on embryonic days E13.5, E15.5 and E18.5. Sagittal histological sections of the forearms of the embryos were collected and stained with Safranin O and 5‑bromo‑2'‑deoxyuridine (BrdU). Additionally, the expression levels of cyclin D1, type II collagen, type X collagen, Sox9, and p16 were examined using immunohistochemistry, and the expression levels of p27 were examined using immunofluorescence. Safranin O staining revealed no structural change between the cartilage tissues of the WT and p21KO mice at any time point. Type II collagen was expressed ubiquitously, while type X collagen was only expressed in the hypertrophic zone of the cartilage tissues. No differences in the levels of Sox9 expression were observed between the two groups at any time point. The levels of cyclin D1 expression and BrdU uptake were higher in the E13.5 cartilage tissue compared with those observed in the embryonic cartilage tissue at subsequent time points. Expression of p16 and p27 was ubiquitous throughout the tissue sections. These results indicate that p21 may not be essential for embryonic endochondral ossification in articular cartilage of mice and that other signaling networks may compensate for p21 deletion.

  13. Frequent Genetic Aberrations in the CDK4 Pathway in Acral Melanoma Indicate the Potential for CDK4/6 Inhibitors in Targeted Therapy.

    Science.gov (United States)

    Kong, Yan; Sheng, Xinan; Wu, Xiaowen; Yan, Junya; Ma, Meng; Yu, Jiayi; Si, Lu; Chi, Zhihong; Cui, Chuanliang; Dai, Jie; Li, Yiqian; Yu, Huan; Xu, Tianxiao; Tang, Huan; Tang, Bixia; Mao, Lili; Lian, Bin; Wang, Xuan; Yan, Xieqiao; Li, Siming; Guo, Jun

    2017-11-15

    Purpose: Effective therapies for the majority of metastatic acral melanoma patients have not been established. Thus, we investigated genetic aberrations of CDK4 pathway in acral melanoma and evaluated the efficacy of CDK4/6 inhibitors in targeted therapy of acral melanoma. Experimental Design: A total of 514 primary acral melanoma samples were examined for the copy number variations (CNV) of CDK4 pathway-related genes, including Cdk4, Ccnd1 , and P16 INK4a , by QuantiGenePlex DNA Assay. The sensitivity of established acral melanoma cell lines and patient-derived xenograft (PDX) containing typical CDK4 aberrations to CDK4/6 inhibitors was evaluated. Results: Among the 514 samples, 203 cases, 137 cases, and 310 cases, respectively, showed Cdk4 gain (39.5%), Ccnd1 gain (26.7%), and P16 INK4a loss (60.3%). The overall frequency of acral melanomas that contain at least one aberration in Cdk4, Ccnd1 , and P16 INK4a was 82.7%. The median overall survival time for acral melanoma patients with concurrent Cdk4 gain with P16 INK4a loss was significantly shorter than that for patients without such aberrations ( P = 0.005). The pan-CDK inhibitor AT7519 and selective CDK4/6 inhibitor PD0332991 could inhibit the cell viability of acral melanoma cells and the tumor growth of PDX with Cdk4 gain plus Ccnd1 gain, Cdk4 gain plus P16 INK4a loss, and Ccnd1 gain plus P16 INK4a loss. Conclusions: Genetic aberration of CDK4 pathway is a frequent event in acral melanoma. Acral melanoma cell lines and PDX containing CDK4 pathway aberrations are sensitive to CDK4/6 inhibitors. Our study provides evidence for the testing of CDK4/6 inhibitors in acral melanoma patients. Clin Cancer Res; 23(22); 6946-57. ©2017 AACR . ©2017 American Association for Cancer Research.

  14. A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae [Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of); Lee, Janet [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Lee, Chang-Woo [Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Center for Molecular Medicine, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon, Gyeonggi 440-746 (Korea, Republic of); Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Suwon, Gyeonggi 440-746 (Korea, Republic of); Yang, Kwangmo [Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of); Department of Radiation Oncology, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of); Department of Radiation Oncology, Korea Institute of Radiological and Medical Sciences, Seoul 139-709 (Korea, Republic of); Lee, Chang Geun, E-mail: cglee@dirams.re.kr [Research Center, Dongnam Institute of Radiological and Medical Sciences, Busan 619-953 (Korea, Republic of)

    2013-01-25

    Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24{sup −}/CD44{sup +}) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer.

  15. A possible usage of a CDK4 inhibitor for breast cancer stem cell-targeted therapy

    International Nuclear Information System (INIS)

    Han, Yu Kyeong; Lee, Jae Ho; Park, Ga-Young; Chun, Sung Hak; Han, Jeong Yun; Kim, Sung Dae; Lee, Janet; Lee, Chang-Woo; Yang, Kwangmo; Lee, Chang Geun

    2013-01-01

    Highlights: ► A CDK4 inhibitor may be used for breast cancer stem cell-targeted therapy. ► The CDK4 inhibitor differentiated the cancer stem cell population (CD24 − /CD44 + ) of MDA-MB-231. ► The differentiation of the cancer stem cells by the CDK4 inhibitor radiosensitized MDA-MB-231. -- Abstract: Cancer stem cells (CSCs) are one of the main reasons behind cancer recurrence due to their resistance to conventional anti-cancer therapies. Thus, many efforts are being devoted to developing CSC-targeted therapies to overcome the resistance of CSCs to conventional anti-cancer therapies and decrease cancer recurrence. Differentiation therapy is one potential approach to achieve CSC-targeted therapies. This method involves inducing immature cancer cells with stem cell characteristics into more mature or differentiated cancer cells. In this study, we found that a CDK4 inhibitor sensitized MDA-MB-231 cells but not MCF7 cells to irradiation. This difference appeared to be associated with the relative percentage of CSC-population between the two breast cancer cells. The CDK4 inhibitor induced differentiation and reduced the cancer stem cell activity of MDA-MB-231 cells, which are shown by multiple marker or phenotypes of CSCs. Thus, these results suggest that radiosensitization effects may be caused by reducing the CSC-population of MDA-MB-231 through the use of the CDK4 inhibitor. Thus, further investigations into the possible application of the CDK4 inhibitor for CSC-targeted therapy should be performed to enhance the efficacy of radiotherapy for breast cancer

  16. Fragment-Based Discovery of 7-Azabenzimidazoles as Potent, Highly Selective, and Orally Active CDK4/6 Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Young Shin; Angove, Hayley; Brain, Christopher; Chen, Christine Hiu-Tung; Cheng, Hong; Cheng, Robert; Chopra, Rajiv; Chung, Kristy; Congreve, Miles; Dagostin, Claudio; Davis, Deborah J.; Feltell, Ruth; Giraldes, John; Hiscock, Steven D.; Kim, Sunkyu; Kovats, Steven; Lagu, Bharat; Lewry, Kim; Loo, Alice; Lu, Yipin; Luzzio, Michael; Maniara, Wiesia; McMenamin, Rachel; Mortenson, Paul N.; Benning, Rajdeep; O' Reilly, Marc; Rees, David C.; Shen, Junqing; Smith, Troy; Wang, Yaping; Williams, Glyn; Woolford, Alison J. -A.; Wrona, Wojciech; Xu, Mei; Yang, Fan; Howard, Steven

    2012-06-14

    Herein, we describe the discovery of potent and highly selective inhibitors of both CDK4 and CDK6 via structure-guided optimization of a fragment-based screening hit. CDK6 X-ray crystallography and pharmacokinetic data steered efforts in identifying compound 6, which showed >1000-fold selectivity for CDK4 over CDKs 1 and 2 in an enzymatic assay. Furthermore, 6 demonstrated in vivo inhibition of pRb-phosphorylation and oral efficacy in a Jeko-1 mouse xenograft model.

  17. In silico design of small molecule inhibitors of CDK9/cyclin T1 interaction.

    Science.gov (United States)

    Randjelovic, Jelena; Eric, Slavica; Savic, Vladimir

    2014-05-01

    In order to design a small molecule which potentially may interfere with CDK9/cyclin T1 complex formation and therefore influence its physiological role, a computational study of dynamics and druggability of CDK9 binding surface was conducted. Druggability estimates and pocket opening analyses indicated binding regions of cyclin T1 residues, Phe 146 and Lys 6, as starting points for the design of small molecules with the potential to inhibit the CDK9/cyclin T1 association. A pharmacophore model was created, based on these two residues and used to select potential inhibitor structures. Binding energies of the inhibitors were estimated with MM-GBSA. A good correlation of MM-GBSA energies and FTMap druggability predictions was observed. Amongst studied compounds a derivative of 2-amino-8-hydroxyquinoline was identified as the best potential candidate to inhibit CDK9/cyclin T1 interactions. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Circadian variation in expression of G1 phase cyclins D1 and E and cyclin-dependent kinase inhibitors p16 and p21 in human bowel mucosa

    Science.gov (United States)

    Griniatsos, John; Michail, Othon P; Theocharis, Stamatios; Arvelakis, Antonios; Papaconstantinou, Ioannis; Felekouras, Evangelos; Pikoulis, Emmanouel; Karavokyros, Ioannis; Bakoyiannis, Chris; Marinos, George; Bramis, John; Michail, Panayiotis O

    2006-01-01

    AIM: To evaluate whether the cellular proliferation rate in the large bowel epithelial cells is characterized by circadian rhythm. METHODS: Between January 2003 and December 2004, twenty patients who were diagnosed as suffering from primary, resectable, non-metastatic adenocarcinoma of the lower rectum, infiltrating the sphincter mechanism, underwent abdominoperineal resection, total mesorectal excision and permanent left iliac colostomy. In formalin-fixed and paraffin-embedded biopsy specimens obtained from the colostomy mucosa every six hours (00:00, 06:00, 12:00, 18:00 and 24:00), we studied the expression of G1 phase cyclins (D1 and E) as well as the expression of the G1 phase cyclin-dependent kinase (CDK) inhibitors p16 and p21 as indicators of cell cycle progression in colonic epithelial cells using immunohistochemical methods. RESULTS: The expression of both cyclins showed a similar circadian fashion obtaining their lowest and highest values at 00:00 and 18:00, respectively (P< 0.001). A circadian rhythm in the expression of CDK inhibitor proteins p16 and p21 was also observed, with the lowest levels obtained at 12:00 and 18:00 (P< 0.001), respectively. When the complexes cyclins D1 - p21 and E - p21 were examined, the expression of the cyclins was adversely correlated to the p21 expression throughout the day. When the complexes the cyclins D1 - p16 and E - p16 were examined, high levels of p16 expression were correlated to low levels of cyclin expression at 00:00, 06:00 and 24:00. Meanwhile, the highest expression levels of both cyclins were correlated to high levels of p16 expression at 18:00. CONCLUSION: Colonic epithelial cells seem to enter the G1 phase of the cell cycle during afternoon (between 12:00 and 18:00) with the highest rates obtained at 18:00. From a clinical point of view, the present results suggest that G1-phase specific anticancer therapies in afternoon might maximize their anti-tumor effect while minimizing toxicity

  19. Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models

    International Nuclear Information System (INIS)

    Natoni, Alessandro; Coyne, Mark R. E.; Jacobsen, Alan; Rainey, Michael D.; O’Brien, Gemma; Healy, Sandra; Montagnoli, Alessia; Moll, Jürgen; O’Dwyer, Michael; Santocanale, Corrado

    2013-01-01

    Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma

  20. Characterization of a Dual CDC7/CDK9 Inhibitor in Multiple Myeloma Cellular Models

    Energy Technology Data Exchange (ETDEWEB)

    Natoni, Alessandro [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland); Coyne, Mark R. E. [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland); Department of Medicine, National University of Ireland Galway, Galway (Ireland); Department of Haematology, Galway University Hospital, Galway (Ireland); Jacobsen, Alan; Rainey, Michael D.; O’Brien, Gemma; Healy, Sandra [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland); Montagnoli, Alessia; Moll, Jürgen [Nerviano Medical Sciences S.r.l., Via Pasteur 10, Nerviano 20014 (Italy); O’Dwyer, Michael, E-mail: michael.odwyer@nuigalway.ie [Department of Medicine, National University of Ireland Galway, Galway (Ireland); Department of Haematology, Galway University Hospital, Galway (Ireland); Santocanale, Corrado, E-mail: michael.odwyer@nuigalway.ie [Centre for Chromosome Biology, School of Natural Sciences National University of Ireland Galway, Galway (Ireland)

    2013-07-24

    Two key features of myeloma cells are the deregulation of the cell cycle and the dependency on the expression of the BCL2 family of anti-apoptotic proteins. The cell division cycle 7 (CDC7) is an essential S-phase kinase and emerging CDC7 inhibitors are effective in a variety of preclinical cancer models. These compounds also inhibit CDK9 which is relevant for MCL-1 expression. The activity and mechanism of action of the dual CDC7/CDK9 inhibitor PHA-767491 was assessed in a panel of multiple myeloma cell lines, in primary samples from patients, in the presence of stromal cells and in combination with drugs used in current chemotherapeutic regimens. We report that in all conditions myeloma cells undergo cell death upon PHA-767491 treatment and we report an overall additive effect with melphalan, bortezomib and doxorubicin, thus supporting further assessment of targeting CDC7 and CDK9 in multiple myeloma.

  1. Recent advances of highly selective CDK4/6 inhibitors in breast cancer

    Directory of Open Access Journals (Sweden)

    Hanxiao Xu

    2017-04-01

    Full Text Available Abstract Uncontrolled cell division is the hallmark of cancers. Full understanding of cell cycle regulation would contribute to promising cancer therapies. In particular, cyclin-dependent kinases 4/6 (CDK4/6, which are pivotal drivers of cell proliferation by combination with cyclin D, draw more and more attention. Subsequently, extensive studies were carried out to explore drugs inhibiting CDK4/6 and assess the efficacy and safety of these drugs in cancer, especially breast cancer. Due to the insuperable adverse events and the less activity observed in vivo, the drug development of the initial pan-CDK inhibitor flavopiridol was consequently discontinued, and then highly specific inhibitors were extensively researched and developed, including palbociclib (PD0332991, ribociclib (LEE011, and abemaciclib (LY2835219. Food and Drug Administration has approved palbociclib and ribociclib for the treatment of hormone receptor-positive, human epidermal growth factor receptor 2-negative advanced or metastatic breast cancer, and recent clinical trial data suggest that palbociclib significantly improved clinical outcome when combined with letrozole or fulvestrant. Besides, the favorable effects of abemaciclib on prolonging survival of breast cancer patients have also been observed in clinical trials both for single-agent and combination strategy. In this review, we outline the preclinical and clinical advancement of these three orally bioavailable and highly selective CDK4/6 inhibitors in breast cancer.

  2. Promotion of apoptosis in cancer cells by selective purine-derived pharmacological CDK inhibitors: one outcome, many mechanisms.

    Science.gov (United States)

    Węsierska-Gądek, Józefa; Maurer, Margarita

    2011-01-01

    The deregulation of apoptosis and the cell cycle are important steps in the onset of cancer, giving cells unlimited reproductive potential and increasing their likelihood of survival. The cell cycle is an essential and tightly regulated four-stage process that effects the accurate duplication and transmission of genetic content to cells' progeny. Cyclin-dependent kinases (CDKs) are key elements of the mammalian cell cycle machinery. Their activity is normally regulated via cyclin binding, phosphorylation events, and interactions with endogenous CDK inhibitors. Malfunctions in the control of the cell cycle can be specifically countered using pharmacological CDK inhibitors. Importantly, CDK inhibitors are very effective against both rapidly dividing and quiescent cancer cells; this is particularly relevant in the treatment of malignancies such as chronic lymphatic leukemia (CLL) and multiple myeloma (MM) that exhibit both a low mitotic index and apoptotic defects. The high efficacy of pharmacological CDK inhibitors against CLL and MM is attributable to their ability to eliminate leukemic cells by apoptosis. Indeed, not only do pharmacological CDK inhibitors block cell cycle progression; they also promote apoptosis and thereby destroy irrevocably malignant cells. This article focuses on the impact of inhibiting individual cellular CDKs on apoptosis. We discuss in detail the molecular mechanisms by which CDK inhibitors are able to bypass chemoresistance in tumor cells and trigger apoptosis. Remarkably, recent findings suggest that the pharmacological utility of CDK inhibitors may not be restricted to the treatment of cancer: some may be efficacious in the treatment of patients with neurodegenerative and cardiovascular diseases.

  3. The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression

    Directory of Open Access Journals (Sweden)

    Preeti Singh

    2017-03-01

    Full Text Available Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1, a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/− mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients.

  4. CDK9 inhibitors define elongation checkpoints at both ends of RNA polymerase II-transcribed genes.

    Science.gov (United States)

    Laitem, Clélia; Zaborowska, Justyna; Isa, Nur F; Kufs, Johann; Dienstbier, Martin; Murphy, Shona

    2015-05-01

    Transcription through early-elongation checkpoints requires phosphorylation of negative transcription elongation factors (NTEFs) by the cyclin-dependent kinase (CDK) 9. Using CDK9 inhibitors and global run-on sequencing (GRO-seq), we have mapped CDK9 inhibitor-sensitive checkpoints genome wide in human cells. Our data indicate that early-elongation checkpoints are a general feature of RNA polymerase (pol) II-transcribed human genes and occur independently of polymerase stalling. Pol II that has negotiated the early-elongation checkpoint can elongate in the presence of inhibitors but, remarkably, terminates transcription prematurely close to the terminal polyadenylation (poly(A)) site. Our analysis has revealed an unexpected poly(A)-associated elongation checkpoint, which has major implications for the regulation of gene expression. Interestingly, the pattern of modification of the C-terminal domain of pol II terminated at this new checkpoint largely mirrors the pattern normally found downstream of the poly(A) site, thus suggesting common mechanisms of termination.

  5. CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

    International Nuclear Information System (INIS)

    Kazantseva, Yuliya A.; Yarushkin, Andrei A.; Pustylnyak, Vladimir O.

    2014-01-01

    Highlights: • CAR activation decreased the level of Foxo1 in mouse livers. • CAR activation decreased the level of p21 in mouse livers. • CAR activation inhibited Foxo1 transcriptional activity in mouse livers. - Abstract: 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments

  6. Inhibitor of CDK interacting with cyclin A1 (INCA1) regulates proliferation and is repressed by oncogenic signaling

    DEFF Research Database (Denmark)

    Baumer, Nicole; Tickenbrock, Lara; Tschanter, Petra

    2011-01-01

    The cell cycle is driven by the kinase activity of cyclin/CDK complexes which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as interaction partner and substrate of cyclin A1 in complex with CDK2. On a functional level, we identified a novel cyclin binding site...... in the INCA1 protein. INCA1 inhibited CDK2 activity and cell proliferation. The inihibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic signals suppressed INCA1 expression, while it was induced by cell cycle arrest. We established a deletional mouse model that showed increased...... CDK2 activity in spleen with altered spleen architecture in Inca1-/- mice. Inca1-/- embryonic fibroblasts showed an increase in the fraction of S-phase cells. Furthermore, blasts from ALL and AML patients expressed significantly reduced INCA1 levels highlighting its relevance for growth control...

  7. Differential Regulation of G1 CDK Complexes by the Hsp90-Cdc37 Chaperone System

    Directory of Open Access Journals (Sweden)

    Stephen T. Hallett

    2017-10-01

    Full Text Available Summary: Selective recruitment of protein kinases to the Hsp90 system is mediated by the adaptor co-chaperone Cdc37. We show that assembly of CDK4 and CDK6 into protein complexes is differentially regulated by the Cdc37-Hsp90 system. Like other Hsp90 kinase clients, binding of CDK4/6 to Cdc37 is blocked by ATP-competitive inhibitors. Cdc37-Hsp90 relinquishes CDK6 to D3- and virus-type cyclins and to INK family CDK inhibitors, whereas CDK4 is relinquished to INKs but less readily to cyclins. p21CIP1 and p27KIP1 CDK inhibitors are less potent than the INKs at displacing CDK4 and CDK6 from Cdc37. However, they cooperate with the D-type cyclins to generate CDK4/6-containing ternary complexes that are resistant to cyclin D displacement by Cdc37, suggesting a molecular mechanism to explain the assembly factor activity ascribed to CIP/KIP family members. Overall, our data reveal multiple mechanisms whereby the Hsp90 system may control formation of CDK4- and CDK6-cyclin complexes under different cellular conditions. : Hallett et al. reconstitute CDK4/6 client kinase handover from Cdc37-Hsp90 to CDK regulatory partners and propose a model for the assembly factor activity of CIP/KIP CDK inhibitors. They find that CDK4/6 inhibitors in clinical use can displace G1 CDKs from the Cdc37-Hsp90 chaperone system at submicromolar concentrations. Keywords: Cdc37, CDK, chaperone, CIP/KIP, cyclin D, Hsp90, INK, kinase, palbociclib, ribociclib

  8. [Effect of CDK Inhibitor LS-007 on Acute Lymphoblastic Leukemia and Its Mechanism].

    Science.gov (United States)

    Lu, Jiang-Feng; Xu, Feng-Ling; Lu, Jun; Ren, Yu-Guo

    2017-04-01

    To study the effect of cyclin dependent kinase(CDK) inhibitor LS-007 on acute lymphoblastic leukemia and its mechanism. The acute lymphocytic leukemia cell line was cultured and treated by LS-007, flavopiridol and ABT-199, then the changes of apoptosis-related factor mRNA and protein levels were detected by using mRNA quantitative PCR and Werstern blot. quantitative PCR and Western blot detection showed that the levels of antiapoptotic protein decreased significantly in acute lymphoblastic leukemia cells after LS-007 treatment, and the pro-apoptotic effect of LS-007 combined with ABT-199 was much better. LS-007 can affect the phosphorylation of RNA polymerase sites and promote cell apoptosis through changing the activities of CDK, thus having some positive significance for relieving acute lymphoblastic leukemia.

  9. Structure-based drug design of a highly potent CDK1,2,4,6 inhibitor with novel macrocyclic quinoxalin-2-one structure.

    Science.gov (United States)

    Kawanishi, Nobuhiko; Sugimoto, Tetsuya; Shibata, Jun; Nakamura, Kaori; Masutani, Kouta; Ikuta, Mari; Hirai, Hiroshi

    2006-10-01

    The design of a novel series of cyclin-dependent kinase (CDK) inhibitors containing a macrocyclic quinoxaline-2-one is reported. Structure-based drug design and optimization from the starting point of diarylurea 2, which we previously reported as a moderate CDK1,2,4,6 inhibitor [J. Biol.Chem.2001, 276, 27548], led to the discovery of potent CDK1,2,4,6 inhibitor that were suitable for iv administration for in vivo study.

  10. Analysing the Effect of Mutation on Protein Function and Discovering Potential Inhibitors of CDK4: Molecular Modelling and Dynamics Studies.

    Directory of Open Access Journals (Sweden)

    Nagasundaram N

    Full Text Available The cyclin-dependent kinase 4 (CDK4-cyclin D1 complex plays a crucial role in the transition from the G1 phase to S phase of the cell cycle. Among the CDKs, CDK4 is one of the genes most frequently affected by somatic genetic variations that are associated with various forms of cancer. Thus, because the abnormal function of the CDK4-cyclin D1 protein complex might play a vital role in causing cancer, CDK4 can be considered a genetically validated therapeutic target. In this study, we used a systematic, integrated computational approach to identify deleterious nsSNPs and predict their effects on protein-protein (CDK4-cyclin D1 and protein-ligand (CDK4-flavopiridol interactions. This analysis resulted in the identification of possible inhibitors of mutant CDK4 proteins that bind the conformations induced by deleterious nsSNPs. Using computational prediction methods, we identified five nsSNPs as highly deleterious: R24C, Y180H, A205T, R210P, and R246C. From molecular docking and molecular dynamic studies, we observed that these deleterious nsSNPs affected CDK4-cyclin D1 and CDK4-flavopiridol interactions. Furthermore, in a virtual screening approach, the drug 5_7_DIHYDROXY_ 2_ (3_4_5_TRI HYDROXYPHENYL _4H_CHROMEN_ 4_ONE displayed good binding affinity for proteins with the mutations R24C or R246C, the drug diosmin displayed good binding affinity for the protein with the mutation Y180H, and the drug rutin displayed good binding affinity for proteins with the mutations A205T and R210P. Overall, this computational investigation of the CDK4 gene highlights the link between genetic variation and biological phenomena in human cancer and aids in the discovery of molecularly targeted therapies for personalized treatment.

  11. Natural aristolactams and aporphine alkaloids as inhibitors of CDK1/cyclin B and DYRK1A.

    Science.gov (United States)

    Marti, Guillaume; Eparvier, Véronique; Morleo, Barbara; Le Ven, Jessica; Apel, Cécile; Bodo, Bernard; Amand, Séverine; Dumontet, Vincent; Lozach, Olivier; Meijer, Laurent; Guéritte, Françoise; Litaudon, Marc

    2013-03-06

    In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

  12. Natural Aristolactams and Aporphine Alkaloids as Inhibitors of CDK1/Cyclin B and DYRK1A

    Directory of Open Access Journals (Sweden)

    Françoise Guéritte

    2013-03-01

    Full Text Available In an effort to find potent inhibitors of the protein kinases DYRK1A and CDK1/Cyclin B, a systematic in vitro evaluation of 2,500 plant extracts from New Caledonia and French Guyana was performed. Some extracts were found to strongly inhibit the activity of these kinases. Four aristolactams and one lignan were purified from the ethyl acetate extracts of Oxandra asbeckii and Goniothalamus dumontetii, and eleven aporphine alkaloids were isolated from the alkaloid extracts of Siparuna pachyantha, S. decipiens, S. guianensis and S. poeppigii. Among these compounds, velutinam, aristolactam AIIIA and medioresinol showed submicromolar IC50 values on DYRK1A.

  13. Abemaciclib: a CDK4/6 inhibitor for the treatment of HR+/HER2– advanced breast cancer

    Directory of Open Access Journals (Sweden)

    Corona SP

    2018-02-01

    Full Text Available Silvia Paola Corona,1 Daniele Generali2 1Radiation Oncology Department, Peter MacCallum Cancer Centre, Bentleigh East, VIC, Australia; 2Department of Medical, Surgery and Health Sciences, University of Trieste, Trieste, Italy Abstract: Although early breast cancer (BC is highly curable, advanced or metastatic disease poses numerous challenges in terms of medical management and treatment decisions and is associated with significantly worse prognosis. Among the new targeted agents, anticancer drugs exploiting the cell-cycle machinery have shown great potential in preclinical studies. CDK4/6 inhibitors target the cyclin D/CDK/retinoblastoma signaling pathway, inducing cell-cycle arrest, reduced cell viability and tumor shrinking. As the cyclin D/CDK complex is activated downstream of estrogen signaling, the combination of CDK4/6 inhibitors with standard endocrine therapies represents a rational approach to elicit synergic antitumor activity in hormone receptor-positive BC. The results of clinical trials have indeed confirmed the superiority of the combination of CDK4/6 inhibitors plus endocrine therapies over endocrine therapy alone. Currently approved are three compounds that exhibit similar structural characteristics as well as biological and clinical activities. Abemaciclib is the latest CDK4/6 inhibitor approved by the US Food and Drug Administration (FDA in view of the results of the MONARCH 1 and 2 trials. Further trials are ongoing as other important questions await response. In this review, we focus on abemaciclib to examine preclinical and clinical results, describing current therapeutic indications, open questions and ongoing clinical trials. Keywords: CDK4/6 inhibitor, abemaciclib, breast cancer, hormone receptor-positive BC, metastatic BC, mBC

  14. Efficacy and Safety of Abemaciclib, an Inhibitor of CDK4 and CDK6, for Patients with Breast Cancer, Non-Small Cell Lung Cancer, and Other Solid Tumors.

    Science.gov (United States)

    Patnaik, Amita; Rosen, Lee S; Tolaney, Sara M; Tolcher, Anthony W; Goldman, Jonathan W; Gandhi, Leena; Papadopoulos, Kyriakos P; Beeram, Muralidhar; Rasco, Drew W; Hilton, John F; Nasir, Aejaz; Beckmann, Richard P; Schade, Andrew E; Fulford, Angie D; Nguyen, Tuan S; Martinez, Ricardo; Kulanthaivel, Palaniappan; Li, Lily Q; Frenzel, Martin; Cronier, Damien M; Chan, Edward M; Flaherty, Keith T; Wen, Patrick Y; Shapiro, Geoffrey I

    2016-07-01

    We evaluated the safety, pharmacokinetic profile, pharmacodynamic effects, and antitumor activity of abemaciclib, an orally bioavailable inhibitor of cyclin-dependent kinases (CDK) 4 and 6, in a multicenter study including phase I dose escalation followed by tumor-specific cohorts for breast cancer, non-small cell lung cancer (NSCLC), glioblastoma, melanoma, and colorectal cancer. A total of 225 patients were enrolled: 33 in dose escalation and 192 in tumor-specific cohorts. Dose-limiting toxicity was grade 3 fatigue. The maximum tolerated dose was 200 mg every 12 hours. The most common possibly related treatment-emergent adverse events involved fatigue and the gastrointestinal, renal, or hematopoietic systems. Plasma concentrations increased with dose, and pharmacodynamic effects were observed in proliferating keratinocytes and tumors. Radiographic responses were achieved in previously treated patients with breast cancer, NSCLC, and melanoma. For hormone receptor-positive breast cancer, the overall response rate was 31%; moreover, 61% of patients achieved either response or stable disease lasting ≥6 months. Abemaciclib represents the first selective inhibitor of CDK4 and CDK6 with a safety profile allowing continuous dosing to achieve sustained target inhibition. This first-in-human experience demonstrates single-agent activity for patients with advanced breast cancer, NSCLC, and other solid tumors. Cancer Discov; 6(7); 740-53. ©2016 AACR.See related commentary by Lim et al., p. 697This article is highlighted in the In This Issue feature, p. 681. ©2016 American Association for Cancer Research.

  15. Design, synthesis and biological evaluation of tetrahydronaphthyridine derivatives as bioavailable CDK4/6 inhibitors for cancer therapy.

    Science.gov (United States)

    Zha, Chuantao; Deng, Wenjia; Fu, Yan; Tang, Shuai; Lan, Xiaojing; Ye, Yan; Su, Yi; Jiang, Lei; Chen, Yi; Huang, Ying; Ding, Jian; Geng, Meiyu; Huang, Min; Wan, Huixin

    2018-03-25

    CDK4/6 pathway is an attractive chemotherapeutic target for antitumor drug discovery and development. Herein, we reported the structure-based design and synthesis of a series of novel tetrahydronaphthyridine analogues as selective CDK4/6 inhibitors. Compound 5 was identified as a hit and then systematically structure optimization study was conducted. These efforts led to compound 28, which exhibited excellent in vitro potencies against CDK4/6 enzymatic activity with high selectivity over CDK1, and against Colo-205 cell growth. The compound demonstrated favorable in vitro metabolic and robust mice pharmacokinetic properties. In Colo-205 xenograft models, compound 28 showed potent tumor growth inhibition with acceptable toxic effects, which could serve as a novel anticancer agent for further preclinical study. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  16. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation...

  17. Palbociclib: First CDK4/6 Inhibitor in Clinical Practice for the Treatment of Advanced HR-Positive Breast Cancer.

    Science.gov (United States)

    Ettl, Johannes

    2016-06-01

    Palbociclib is the first inhibitor of the cyclin-dependent kinases (CDK) 4 and 6 to be introduced into clinical practice. Preclinical investigations led to its clinical development in advanced hormone receptor (HR)-positive breast cancer. To date, 2 significant clinical trials have been fully published. In this article, the results of these trials and their clinical relevance for the management of HR-positive advanced breast cancer are discussed.

  18. Maintenance of leukemia-initiating cells is regulated by the CDK inhibitor Inca1.

    Directory of Open Access Journals (Sweden)

    Nicole Bäumer

    Full Text Available Functional differences between healthy progenitor and cancer initiating cells may provide unique opportunities for targeted therapy approaches. Hematopoietic stem cells are tightly controlled by a network of CDK inhibitors that govern proliferation and prevent stem cell exhaustion. Loss of Inca1 led to an increased number of short-term hematopoietic stem cells in older mice, but Inca1 seems largely dispensable for normal hematopoiesis. On the other hand, Inca1-deficiency enhanced cell cycling upon cytotoxic stress and accelerated bone marrow exhaustion. Moreover, AML1-ETO9a-induced proliferation was not sustained in Inca1-deficient cells in vivo. As a consequence, leukemia induction and leukemia maintenance were severely impaired in Inca1-/- bone marrow cells. The re-initiation of leukemia was also significantly inhibited in absence of Inca1-/- in MLL-AF9- and c-myc/BCL2-positive leukemia mouse models. These findings indicate distinct functional properties of Inca1 in normal hematopoietic cells compared to leukemia initiating cells. Such functional differences might be used to design specific therapy approaches in leukemia.

  19. Azolium analogues as CDK4 inhibitors: Pharmacophore modeling, 3D QSAR study and new lead drug discovery

    Science.gov (United States)

    Rondla, Rohini; Padma Rao, Lavanya Souda; Ramatenki, Vishwanath; Vadija, Rajender; Mukkera, Thirupathi; Potlapally, Sarita Rajender; Vuruputuri, Uma

    2017-04-01

    The cyclin-dependent kinase 4 (CDK4) enzyme is a key regulator in cell cycle G1 phase progression. It is often overexpressed in variety of cancer cells, which makes it an attractive therapeutic target for cancer treatment. A number of chemical scaffolds have been reported as CDK4 inhibitors in the literature, and in particular azolium scaffolds as potential inhibitors. Here, a ligand based pharmacophore modeling and an atom based 3D-QSAR analyses for a series of azolium based CDK4 inhibitors are presented. A five point pharmacophore hypothesis, i.e. APRRR with one H-bond acceptor (A), one positive cationic feature (P) and three ring aromatic sites (R) is developed, which yielded an atom based 3D-QSAR model that shows an excellent correlation coefficient value- R2 = 0.93, fisher ratio- F = 207, along with good predictive ability- Q2 = 0.79, and Pearson R value = 0.89. The visual inspection of the 3D-QSAR model, with the most active and the least active ligands, demonstrates the favorable and unfavorable structural regions for the activity towards CDK4. The roles of positively charged nitrogen, the steric effect, ligand flexibility, and the substituents on the activity are in good agreement with the previously reported experimental results. The generated 3D QSAR model is further applied as query for a 3D database screening, which identifies 23 lead drug candidates with good predicted activities and diverse scaffolds. The ADME analysis reveals that, the pharmacokinetic parameters of all the identified new leads are within the acceptable range.

  20. PD-0332991, a CDK4/6 Inhibitor, Significantly Prolongs Survival in a Genetically Engineered Mouse Model of Brainstem Glioma

    Science.gov (United States)

    Barton, Kelly L.; Misuraca, Katherine; Cordero, Francisco; Dobrikova, Elena; Min, Hooney D.; Gromeier, Matthias; Kirsch, David G.; Becher, Oren J.

    2013-01-01

    Diffuse intrinsic pontine glioma (DIPG) is an incurable tumor that arises in the brainstem of children. To date there is not a single approved drug to effectively treat these tumors and thus novel therapies are desperately needed. Recent studies suggest that a significant fraction of these tumors contain alterations in cell cycle regulatory genes including amplification of the D-type cyclins and CDK4/6, and less commonly, loss of Ink4a-ARF leading to aberrant cell proliferation. In this study, we evaluated the therapeutic approach of targeting the cyclin-CDK-Retinoblastoma (Rb) pathway in a genetically engineered PDGF-B-driven brainstem glioma (BSG) mouse model. We found that PD-0332991 (PD), a CDK4/6 inhibitor, induces cell-cycle arrest in our PDGF-B; Ink4a-ARF deficient model both in vitro and in vivo. By contrast, the PDGF-B; p53 deficient model was mostly resistant to treatment with PD. We noted that a 7-day treatment course with PD significantly prolonged survival by 12% in the PDGF-B; Ink4a-ARF deficient BSG model. Furthermore, a single dose of 10 Gy radiation therapy (RT) followed by 7 days of treatment with PD increased the survival by 19% in comparison to RT alone. These findings provide the rationale for evaluating PD in children with Ink4a-ARF deficient gliomas. PMID:24098593

  1. PD-0332991, a CDK4/6 inhibitor, significantly prolongs survival in a genetically engineered mouse model of brainstem glioma.

    Directory of Open Access Journals (Sweden)

    Kelly L Barton

    Full Text Available Diffuse intrinsic pontine glioma (DIPG is an incurable tumor that arises in the brainstem of children. To date there is not a single approved drug to effectively treat these tumors and thus novel therapies are desperately needed. Recent studies suggest that a significant fraction of these tumors contain alterations in cell cycle regulatory genes including amplification of the D-type cyclins and CDK4/6, and less commonly, loss of Ink4a-ARF leading to aberrant cell proliferation. In this study, we evaluated the therapeutic approach of targeting the cyclin-CDK-Retinoblastoma (Rb pathway in a genetically engineered PDGF-B-driven brainstem glioma (BSG mouse model. We found that PD-0332991 (PD, a CDK4/6 inhibitor, induces cell-cycle arrest in our PDGF-B; Ink4a-ARF deficient model both in vitro and in vivo. By contrast, the PDGF-B; p53 deficient model was mostly resistant to treatment with PD. We noted that a 7-day treatment course with PD significantly prolonged survival by 12% in the PDGF-B; Ink4a-ARF deficient BSG model. Furthermore, a single dose of 10 Gy radiation therapy (RT followed by 7 days of treatment with PD increased the survival by 19% in comparison to RT alone. These findings provide the rationale for evaluating PD in children with Ink4a-ARF deficient gliomas.

  2. A specific inhibitor of CDK1, RO-3306, reversibly arrests meiosis during in vitro maturation of porcine oocytes.

    Science.gov (United States)

    Jang, Woo-In; Lin, Zi-Li; Lee, Sung Hyun; Namgoong, Suk; Kim, Nam-Hyung

    2014-01-30

    CDK1 plays pivotal role in meiotic progression of oocytes from G2 to metaphase II (MII) stage. In this study, we investigated the possibility of utilizing a selective inhibitor of CDK1, RO-3306, as a novel agent for the synchronization of oocyte maturation. Two groups of cumulus-oocyte complexes (COCs) were treated with 10 μM RO-3306. The first group was treated for 44 h, whereas the second group was transferred to drug-free medium after a 20 h treatment. MII-stage oocytes from each group were confirmed by cytoplasmic maturation and embryonic development assays. Treatment of immature porcine oocytes with RO-3306 for 20 h arrested them at the germinal vesicle (GV) stage. The GV-arrest effect of RO-3306 was reversible: when RO-3306-arrested COCs were subsequently cultured for 24h in the absence of RO-3306, 76.19 ± 2.68% of these oocytes reached the MII stage after 44 h of in vitro maturation, a rate similar to that of non-treated control oocytes (79.08 ± 3.23%). Furthermore, RO-3306-treated oocytes transferred to drug-free media did not differ significantly from controls (P>0.05) with respect to cleavage and blastocyst formation upon parthenogenetic activation. To explore the underlying molecular mechanisms, we examined the expression patterns of four representative maternal transcripts, CDK1, Cyclin B1, GDF9, and BMP15, by real-time polymerase chain reaction (PCR) and poly(A)-test PCR (PAT assay). RO-3306 treatment increased expression of CDK1 but had no effect on the expression of the other genes. These data suggest that RO-3306 efficiently blocks and synchronizes the meiotic progression of porcine oocytes at the GV stage without affecting their meiotic and cytoplasmic maturation. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Prevention of radiation-induced salivary gland dysfunction utilizing a CDK inhibitor in a mouse model.

    Directory of Open Access Journals (Sweden)

    Katie L Martin

    Full Text Available Treatment of head and neck cancer with radiation often results in damage to surrounding normal tissues such as salivary glands. Permanent loss of function in the salivary glands often leads patients to discontinue treatment due to incapacitating side effects. It has previously been shown that IGF-1 suppresses radiation-induced apoptosis and enhances G2/M arrest leading to preservation of salivary gland function. In an effort to recapitulate the effects of IGF-1, as well as increase the likelihood of translating these findings to the clinic, the small molecule therapeutic Roscovitine, is being tested. Roscovitine is a cyclin-dependent kinase inhibitor that acts to transiently inhibit cell cycle progression and allow for DNA repair in damaged tissues.Treatment with Roscovitine prior to irradiation induced a significant increase in the percentage of cells in the G(2/M phase, as demonstrated by flow cytometry. In contrast, mice treated with radiation exhibit no differences in the percentage of cells in G(2/M when compared to unirradiated controls. Similar to previous studies utilizing IGF-1, pretreatment with Roscovitine leads to a significant up-regulation of p21 expression and a significant decrease in the number of PCNA positive cells. Radiation treatment leads to a significant increase in activated caspase-3 positive salivary acinar cells, which is suppressed by pretreatment with Roscovitine. Administration of Roscovitine prior to targeted head and neck irradiation preserves normal tissue function in mouse parotid salivary glands, both acutely and chronically, as measured by salivary output.These studies suggest that induction of transient G(2/M cell cycle arrest by Roscovitine allows for suppression of apoptosis, thus preserving normal salivary function following targeted head and neck irradiation. This could have an important clinical impact by preventing the negative side effects of radiation therapy in surrounding normal tissues.

  4. Understanding the potency of competitive CDK2 inhibitors using quantum chemical scoring

    Czech Academy of Sciences Publication Activity Database

    Deepa, Palanisamy; Fanfrlík, Jindřich; Brahmkshatriya, Pathik; Brynda, Jiří; Cankař, P.; Kryštof, V.; Řezáč, Jan; Lepšík, Martin; Hobza, Pavel

    2012-01-01

    Roč. 156, Suppl. 1 (2012), S75-S75 ISSN 1213-8118. [International Congress Natural Anticancer Drugs. 30.06.2012-04.07.2012, Olomouc] R&D Projects: GA ČR GBP208/12/G016 Grant - others:European Science Foundation(XE) CZ.1.05/2.1.00/03.0058 Institutional support: RVO:61388963 Keywords : cyclin -dependent kinases * CDK2 * quantum chemical scoring Subject RIV: CF - Physical ; Theoretical Chemistry

  5. Fluorine Substituted 1,2,4-Triazinones as Potential Anti-HIV-1 and CDK2 Inhibitors

    Directory of Open Access Journals (Sweden)

    Mohammed S. I. Makki

    2014-01-01

    Full Text Available Fluorine substituted 1,2,4-triazinones have been synthesized via alkylation, amination, and/or oxidation of 6-(2-amino-5-fluorophenyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H-one 1 and 4-fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro-1,2,4-triazin-6-ylphenylbenzamide 5 as possible anti-HIV-1 and CDK2 inhibitors. Alkylation on positions 2 and 4 in 1,2,4-triazinone gave compounds 6–8. Further modification was performed by selective alkylation and amination on position 3 to form compounds 9–15. However oxidation of 5 yielded compounds 16–18. Structures of the target compounds have been established by spectral analysis data. Five compounds (5, 11, 14, 16, and 17 have shown very good anti-HIV activity in MT-4 cells. Similarly, five compounds (1, 3, and 14–16 have exhibited very significant CDK2 inhibition activity. Compounds 14 and 16 were found to have dual anti-HIV and anticancer activities.

  6. TGFbeta influences Myc, Miz-1 and Smad to control the CDK inhibitor p15INK4b

    DEFF Research Database (Denmark)

    Seoane, J; Pouponnot, C; Staller, P

    2001-01-01

    Transforming growth factor-beta (TGFbeta) is a cytokine that arrests epithelial cell division by switching off the proto-oncogene c-myc and rapidly switching on cyclin-dependent kinase (CDK) inhibitors such as p15INK4b. Gene responses to TGFbeta involve Smad transcription factors that are directly...... activated by the TGFbeta receptor. Why downregulation of c-myc expression by TGFbeta is required for rapid activation of p15INK4b has remained unknown. Here we provide evidence that TGFbeta signalling prevents recruitment of Myc to the p15INK4b transcriptional initiator by Myc-interacting zinc......-finger protein 1 (Miz-1). This relieves repression and enables transcriptional activation by a TGFbeta-induced Smad protein complex that recognizes an upstream p15INK4b promoter region and contacts Miz-1. Thus, two separate TGFbeta-dependent inputs - Smad-mediated transactivation and relief of repression by Myc...

  7. Lidocaine inhibits NIH-3T3 cell multiplication by increasing the expression of cyclin-dependent kinase inhibitor 1A (p21).

    Science.gov (United States)

    Desai, Sukumar P; Kojima, Koji; Vacanti, Charles A; Kodama, Shohta

    2008-11-01

    We explored molecular mechanisms by which lidocaine inhibits growth in the murine embryonic fibroblast cell line NIH-3T3. Local anesthetics can adversely affect cell growth in vitro. Their effects on wound healing are controversial. We examined the effects and novel mechanisms by which lidocaine affects in vitro multiplication of the murine fibroblast cell line NIH-3T3. NIH-3T3 cells were grown in culture with lidocaine [0, 0.05, 0.5, 1, 2, and 5 mM]. Cell multiplication was assessed by determining cell counts on subsequent days, while mechanisms by which inhibition occurred were evaluated by bromodeoxyuridine uptake, gene expression using polymerase chain reaction array, and Western blot analysis to verify increased levels of affected proteins. Lidocaine caused dose-dependent inhibition of multiplication of NIH-3T3 cells. Effects ranged from no inhibition [0.05 and 0.5 mM] and mild inhibition [1 mM], to severe inhibition [2 and 5 mM] [P = 0.006]. Lidocaine 2 mM inhibited bromodeoxyuridine uptake at day 3.5 [P = 0.02 versus control, and P = 0.0495 vs 1 mM lidocaine]. On day 1.5, lidocaine upregulated expression of cyclin-D1 and cyclin-dependent kinase inhibitor 1A [p21]. On day 2.5, lidocaine increased the levels of p21 protein. Low concentrations of lidocaine, as would be seen in plasma after spinal, epidural, or plexus anesthesia, do not significantly affect multiplication of fibroblasts. Higher doses of lidocaine arrest cell multiplication at the S-phase of the growth cycle by upregulation of p21, an extremely potent inhibitor of cell multiplication. Higher concentrations, as would be seen after tissue infiltration, severely inhibit fibroblast multiplication and thus may impair wound healing.

  8. Discovery of a highly potent, selective and novel CDK9 inhibitor as an anticancer drug candidate.

    Science.gov (United States)

    Li, Yongtao; Guo, Qingxiang; Zhang, Chao; Huang, Zhi; Wang, Tianqi; Wang, Xin; Wang, Xiang; Xu, Guangwei; Liu, Yanhua; Yang, Shengyong; Fan, Yan; Xiang, Rong

    2017-08-01

    A series of novel hybrid structure derivatives, containing both LEE011 and Cabozantinib pharmacophore, were designed, synthesized and evaluated. Surprisingly, a compound 4d was discovered that highly exhibited effective and selective activity of CDK9 inhibition with IC 50 =12nM. It effectively induced apoptosis in breast and lung cancer cell lines at nanomolar level. Molecular docking of 4d to ATP binding site of CDK9 kinase demonstrated a new hydrogen bonding between F atom of 4-(3-fluorobenzyloxy) group and ASN116 residue, compared with the positive control, LEE011. The compound 4d could block the cell cycle both in G0/G1 and G2/M phase to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with compound 4d showed significant suppression of cancer with low toxicity. Taken together, this novel compound 4d could be a promising drug candidate for clinical application. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Antitumor action of CDK inhibitor LS-007 as a single agent and in combination with ABT-199 against human acute leukemia cells.

    Science.gov (United States)

    Xie, Shao; Jiang, Hui; Zhai, Xiao-Wen; Wei, Fan; Wang, Shu-Dong; Ding, Jian; Chen, Yi

    2016-11-01

    LS-007 is a CDK inhibitor, which exhibits potent antitumor activity against chronic lymphocytic leukemia and ovarian cancer cells. In this study, we further evaluated the antitumor activity of LS-007 alone and in combination with a Bcl-2 inhibitor ABT-199 in acute leukemia (AL) cells. Cell viability was detected using resazurin assay, and cell apoptosis was examined using Annexin V/PI double staining and flow cytometry. The inhibition of LS-007 on kinases was evaluated with the mobility shift assay or ELISA. The expression of relevant signaling molecules was assessed using Western blotting and RT-PCR. Primary lymphocytes from patients with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) were separated using Ficoll-Paque PLUS. LS-007 inhibited the proliferation of 6 AL cell lines with IC 50 values of 100-200 nmol/L, and decreased the survival of ALL and AML patient-derived lymphocytes with mean LD 50 value of 67 and 102 nmol/L, respectively. In kinase assays in vitro, LS-007 was more selective for the CDK family, inhibiting CDK2, CDK9, CDK1 and CDK4 at low nanomolar concentrations. In HL-60 and CCRF-CEM cells, LS-007 (0.1-0.4 μmol/L) dose-dependently induced cell apoptosis predominantly through CDK9 inhibition-related dephosphorylation at the ser2 residue of RNA pol II and the corresponding depletion of anti-apoptotic proteins, especially Mcl-1 and XIAP. LS-007 (0.2 and 0.4 μmol/L) also induced cell apoptosis in the patient-derived lymphocytes. In HL-60, CCRF-CEM and Molt-4 cells, combined application of LS-007 with ABT-199 (1 or 2 μmol/L) markedly increased cell apoptosis with a maximal decrease in the XIAP levels as compared with either drug used alone. CDK inhibitor LS-007 potently inhibits the established human AL cell lines and primary AL blasts, and it also shows remarkable synergy with Bcl-2 inhibitor ABT-199.

  10. Overcoming Endocrine Resistance in Hormone-Receptor Positive Advanced Breast Cancer-The Emerging Role of CDK4/6 Inhibitors.

    Science.gov (United States)

    O'Sullivan, Ciara C

    Dysregulation of the cyclin D and cyclin-dependent kinase (CDK) pathway in cancer cells may inhibit senescence and promote cellular proliferation. By using various different mechanisms, malignant cells may increase cyclin D-dependent activity. The cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6)-retinoblastoma (Rb) pathway controls the cell cycle restriction point, and is commonly dysregulated in breast cancer; making it a rational target for anticancer therapy. To date, three oral highly selective cyclin-dependent kinase 4/6 inhibitors (CDK4/6i) are in various stages of clinical development: PD0332991 (palbociclib), LEE011 (ribociclib) and LY2835219 (abemaciclib). Results from phase I, II and III trials in hormone-receptor (HR)-positive breast cancer have been encouraging, demonstrating convincing efficacy and a tolerable side-effect profile (mainly uncomplicated neutropenia). This article will review the preclinical and clinical development of the CDK4/6i, as well as reviewing the existing preclinical evidence regarding combination of these agents with chemotherapy and other targeted therapies. Future and ongoing clinical trials, which may expand the potential application of these agents, will also be discussed. In summary, CDK4/6i are exciting compounds which may change the therapeutic landscape of HR-positive breast cancer.

  11. The expression of cyclin-dependent kinase inhibitors p15, p16, p21, and p27 during ovarian follicle growth initiation in the mouse

    Directory of Open Access Journals (Sweden)

    Bayrak Aykut

    2003-05-01

    Full Text Available Abstract Background Cyclins regulate the cell cycle in association with cyclin dependent kinases (CDKs. CDKs are under inhibitory control of cyclin dependent kinase inhibitors (CDKIs. Method In this study we tested the expression of CDKIs p15, p16, p21 and p27 by immunohistochemistry to determine the role of CDKIs in the initiation of primordial follicle growth. Ovaries were collected from 60-day-old cycling B6D2F1/J mice (n = 16. Results Expression of p15, p16, p21 and p27 did not vary in granulosa and theca cells by the follicle stage. However, p16 staining was stronger (++ in the oocytes of all primordial, and 57.4 ± 3.1% of primary follicles compared to the remaining primary and more advanced follicles (+. Interestingly, primary follicles with weaker (+ oocyte staining for p16 had significantly larger mean follicle diameter compared to the primary and primordial follicles with stronger (++ oocyte staining (55.6 ± 2.1 vs. 32.0 ± 1.0 and 26.5 ± 0.7 μm, respectively, p Conclusions These preliminary findings suggest that the initiation of oocyte growth, which seems to lead follicle growth, is associated with diminished p16 expression in the mouse ovary. Further studies are needed to investigate the factors that regulate the expression of p16 in the oocyte, which might also govern the initiation of primordial follicle growth.

  12. Jumping the nuclear envelop barrier: Improving polyplex-mediated gene transfection efficiency by a selective CDK1 inhibitor RO-3306.

    Science.gov (United States)

    Zhou, Xuefei; Liu, Xiangrui; Zhao, Bingxiang; Liu, Xin; Zhu, Dingcheng; Qiu, Nasha; Zhou, Quan; Piao, Ying; Zhou, Zhuxian; Tang, Jianbin; Shen, Youqing

    2016-07-28

    Successful transfection of plasmid DNA (pDNA) requires intranuclear internalization of pDNA effectively and the nuclear envelope appears to be one of the critical intracellular barriers for polymer mediated pDNA delivery. Polyethylenimine (PEI), as the classic cationic polymer, compact the negatively charged pDNA tightly and make up stable polyplexes. The polyplexes are too large to enter the nuclear through nuclear pores and it is believed that the nuclear envelope breakdown in mitosis could facilitate the nuclear entry of polyplexes. To jump the nuclear envelope barrier, we used a selective and reversible CDK1 inhibitor RO-3306 to control the G2/M transition of the cell cycle and increased the proportion of mitotic cells which have disappeared nuclear envelope during transfection. Herein, we show that RO-3306 remarkably increases the transfection efficiency of PEI polyplexes through enhanced nuclear localization of PEI and pDNA. However, RO-3306 is less effective to the charge-reversal polymer poly[(2-acryloyl)ethyl(p-boronic acid benzyl)diethylammonium bromide] (B-PDEAEA) which responses to cellular stimuli and releases free pDNA in cytoplasm. Our findings not only offer new opportunities for improving non-viral based gene delivery but also provide theoretical support for the rational design of novel functional polymers for gene delivery. We also report current data showing that RO-3306 synergizes TRAIL gene induced apoptosis in cancer cells. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Potential Clinical Uses of CDK Inhibitors: Lessons from Synthetic Lethality Screens

    Czech Academy of Sciences Publication Activity Database

    Vymětalová, Ladislava; Kryštof, Vladimír

    2015-01-01

    Roč. 35, č. 6 (2015), s. 1156-1174 ISSN 0198-6325 R&D Projects: GA MŠk(CZ) LO1204; GA ČR(CZ) GA15-15264S Institutional support: RVO:61389030 Keywords : cyclin-dependent kinase * inhibitor * cancer Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 9.135, year: 2015

  14. AZD5438, an Inhibitor of Cdk1, 2, and 9, Enhances the Radiosensitivity of Non-Small Cell Lung Carcinoma Cells

    Energy Technology Data Exchange (ETDEWEB)

    Raghavan, Pavithra; Tumati, Vasu; Yu Lan [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Chan, Norman [Departments of Medical Biophysics and Radiation Oncology, Princess Margaret Hospital, University Health Network, University of Toronto, Ontario (Canada); Tomimatsu, Nozomi [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Burma, Sandeep [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Simmons Comprehensive Cancer Center, Dallas, Texas (United States); Bristow, Robert G. [Departments of Medical Biophysics and Radiation Oncology, Princess Margaret Hospital, University Health Network, University of Toronto, Ontario (Canada); Saha, Debabrata, E-mail: debabrata.saha@utsouthwestern.edu [Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas (United States); Simmons Comprehensive Cancer Center, Dallas, Texas (United States)

    2012-11-15

    Purpose: Radiation therapy (RT) is one of the primary modalities for treatment of non-small cell lung cancer (NSCLC). However, due to the intrinsic radiation resistance of these tumors, many patients experience RT failure, which leads to considerable tumor progression including regional lymph node and distant metastasis. This preclinical study evaluated the efficacy of a new-generation cyclin-dependent kinase (Cdk) inhibitor, AZD5438, as a radiosensitizer in several NSCLC models that are specifically resistant to conventional fractionated RT. Methods and Materials: The combined effect of ionizing radiation and AZD5438, a highly specific inhibitor of Cdk1, 2, and 9, was determined in vitro by surviving fraction, cell cycle distribution, apoptosis, DNA double-strand break (DSB) repair, and homologous recombination (HR) assays in 3 NSCLC cell lines (A549, H1299, and H460). For in vivo studies, human xenograft animal models in athymic nude mice were used. Results: Treatment of NSCLC cells with AZD5438 significantly augmented cellular radiosensitivity (dose enhancement ratio rangeing from 1.4 to 1.75). The degree of radiosensitization by AZD5438 was greater in radioresistant cell lines (A549 and H1299). Radiosensitivity was enhanced specifically through inhibition of Cdk1, prolonged G{sub 2}-M arrest, inhibition of HR, delayed DNA DSB repair, and increased apoptosis. Combined treatment with AZD5438 and irradiation also enhanced tumor growth delay, with an enhancement factor ranging from 1.2-1.7. Conclusions: This study supports the evaluation of newer generation Cdk inhibitors, such as AZD5438, as potent radiosensitizers in NSCLC models, especially in tumors that demonstrate variable intrinsic radiation responses.

  15. Targeting transcriptional addictions in small cell lung cancer with a covalent CDK7 inhibitor.

    Science.gov (United States)

    Christensen, Camilla L; Kwiatkowski, Nicholas; Abraham, Brian J; Carretero, Julian; Al-Shahrour, Fatima; Zhang, Tinghu; Chipumuro, Edmond; Herter-Sprie, Grit S; Akbay, Esra A; Altabef, Abigail; Zhang, Jianming; Shimamura, Takeshi; Capelletti, Marzia; Reibel, Jakob B; Cavanaugh, Jillian D; Gao, Peng; Liu, Yan; Michaelsen, Signe R; Poulsen, Hans S; Aref, Amir R; Barbie, David A; Bradner, James E; George, Rani E; Gray, Nathanael S; Young, Richard A; Wong, Kwok-Kin

    2014-12-08

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive to transcription-targeting drugs, in particular to THZ1, a recently identified covalent inhibitor of cyclin-dependent kinase 7. We find that expression of super-enhancer-associated transcription factor genes, including MYC family proto-oncogenes and neuroendocrine lineage-specific factors, is highly vulnerability to THZ1 treatment. We propose that downregulation of these transcription factors contributes, in part, to SCLC sensitivity to transcriptional inhibitors and that THZ1 represents a prototype drug for tailored SCLC therapy. Copyright © 2014 Elsevier Inc. All rights reserved.

  16. A short motif in Arabidopsis CDK inhibitor ICK1 decreases the protein level, probably through a ubiquitin-independent mechanism.

    Science.gov (United States)

    Li, Qin; Shi, Xianzong; Ye, Shengjian; Wang, Sheng; Chan, Ron; Harkness, Troy; Wang, Hong

    2016-09-01

    The ICK/KRP family of cyclin-dependent kinase (CDK) inhibitors modulates the activity of plant CDKs through protein binding. Previous work has shown that changing the levels of ICK/KRP proteins by overexpression or downregulation affects cell proliferation and plant growth, and also that the ubiquitin proteasome system is involved in degradation of ICK/KRPs. We show in this study that the region encompassing amino acids 21 to 40 is critical for ICK1 levels in both Arabidopsis and yeast. To determine how degradation of ICK1 is controlled, we analyzed the accumulation of hemagglutinin (HA) epitope-tagged ICK1 proteins in yeast mutants defective for two ubiquitin E3 ligases. The highest level of HA-ICK1 protein was observed when both the N-terminal 1-40 sequence was removed and the SCF (SKP1-Cullin1-F-box complex) function disrupted, suggesting the involvement of both SCF-dependent and SCF-independent mechanisms in the degradation of ICK1 in yeast. A short motif consisting of residues 21-30 is sufficient to render green fluorescent protein (GFP) unstable in plants and had a similar effect in plants regardless of whether it was fused to the N-terminus or C-terminus of GFP. Furthermore, results from a yeast ubiquitin receptor mutant rpn10Δ indicate that protein ubiquitination is not critical in the degradation of GFP-ICK1(1-40) in yeast. These results thus identify a protein-destabilizing sequence motif that does not contain a typical ubiquitination residue, suggesting that it probably functions through an SCF-independent mechanism. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  17. Internal Tandem Duplication in FLT3 Attenuates Proliferation and Regulates Resistance to the FLT3 Inhibitor AC220 by Modulating p21Cdkn1a and Pbx1 in Hematopoietic Cells

    Science.gov (United States)

    Abe, Mariko; Pelus, Louis M.; Singh, Pratibha; Hirade, Tomohiro; Onishi, Chie; Purevsuren, Jamiyan; Taketani, Takeshi; Yamaguchi, Seiji; Fukuda, Seiji

    2016-01-01

    Internal tandem duplication (ITD) mutations in the Fms-related tyrosine kinase 3 (FLT3) gene (FLT3-ITD) are associated with poor prognosis in patients with acute myeloid leukemia (AML). Due to the development of drug resistance, few FLT3-ITD inhibitors are effective against FLT3-ITD+ AML. In this study, we show that FLT3-ITD activates a novel pathway involving p21Cdkn1a (p21) and pre-B cell leukemia transcription factor 1 (Pbx1) that attenuates FLT3-ITD cell proliferation and is involved in the development of drug resistance. FLT3-ITD up-regulated p21 expression in both mouse bone marrow c-kit+-Sca-1+-Lin- (KSL) cells and Ba/F3 cells. The loss of p21 expression enhanced growth factor-independent proliferation and sensitivity to cytarabine as a consequence of concomitantly enriching the S+G2/M phase population and significantly increasing the expression of Pbx1, but not Evi-1, in FLT3-ITD+ cells. This enhanced cell proliferation following the loss of p21 was partially abrogated when Pbx1 expression was silenced in FLT3-ITD+ primary bone marrow colony-forming cells and Ba/F3 cells. When FLT3-ITD was antagonized with AC220, a selective inhibitor of FLT3-ITD, p21 expression was decreased coincident with Pbx1 mRNA up-regulation and a rapid decline in the number of viable FLT3-ITD+ Ba/F3 cells; however, the cells eventually became refractory to AC220. Overexpressing p21 in FLT3-ITD+ Ba/F3 cells delayed the emergence of cells that were refractory to AC220, whereas p21 silencing accelerated their development. These data indicate that FLT3-ITD is capable of inhibiting FLT3-ITD+ cell proliferation through the p21/Pbx1 axis and that treatments that antagonize FLT3-ITD contribute to the subsequent development of cells that are refractory to a FLT3-ITD inhibitor by disrupting p21 expression. PMID:27387666

  18. Hair Growth Promoting and Anticancer Effects of p21-activated kinase 1 (PAK1 Inhibitors Isolated from Different Parts of Alpinia zerumbet

    Directory of Open Access Journals (Sweden)

    Nozomi Taira

    2017-01-01

    Full Text Available PAK1 (p21-activated kinase 1 is an emerging target for the treatment of hair loss (alopecia and cancer; therefore, the search for PAK1 blockers to treat these PAK1-dependent disorders has received much attention. In this study, we evaluated the anti-alopecia and anticancer effects of PAK1 inhibitors isolated from Alpinia zerumbet (alpinia in cell culture. The bioactive compounds isolated from alpinia were found to markedly promote hair cell growth. Kaempferol-3-O-β-d-glucuronide (KOG and labdadiene, two of the isolated compounds, increased the proliferation of human follicle dermal papilla cells by approximately 117%–180% and 132%–226%, respectively, at 10–100 μM. MTD (2,5-bis(1E,3E,5E-6-methoxyhexa-1,3,5-trien-1-yl-2,5-dihydrofuran and TMOQ ((E-2,2,3,3-tetramethyl-8-methylene-7-(oct-6-en-1-yloctahydro-1H-quinolizine showed growth-promoting activity around 164% and 139% at 10 μM, respectively. The hair cell proliferation induced by these compounds was significantly higher than that of minoxidil, a commercially available treatment for hair loss. Furthermore, the isolated compounds from alpinia exhibited anticancer activity against A549 lung cancer cells with IC50 in the range of 67–99 μM. Regarding the mechanism underlying their action, we hypothesized that the anti-alopecia and anticancer activities of these compounds could be attributed to the inhibition of the oncogenic/aging kinase PAK1.

  19. Synergistic antitumor effects of CDK inhibitor SNS-032 and an oncolytic adenovirus co-expressing TRAIL and Smac in pancreatic cancer

    Science.gov (United States)

    Ge, Yun; Lei, Wen; Ma, Yingyu; Wang, Yigang; Wei, Buyun; Chen, Xiaoyi; Ru, Guoqing; He, Xianglei; Mou, Xiaozhou; Wang, Shibing

    2017-01-01

    Gene therapy using oncolytic adenoviruses is a novel approach for human cancer therapeutics. The current study aimed to investigate whether the combined use of an adenovirus expressing tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and second mitochondria-derived activator of caspase (Smac) upon caspase activation (ZD55-TRAIL-IETD-Smac) and cyclin-dependent kinase (CDK) inhibitor SNS-032 will synergistically reinforce their anti-pancreatic cancer activities. The experiments in vitro demonstrated that SNS-032 enhances ZD55-TRAIL-IETD-Smac-induced apoptosis and causes marked pancreatic cancer cell death. Western blot assays suggested that the SNS-032 intensified ZD55-TRAIL-IETD-Smac-induced apoptosis of pancreatic cancer cells by affecting anti-apoptotic signaling elements, including CDK-2, CDK-9, Mcl-1 and XIAP. Additionally, animal experiments further confirmed that the combination of SNS-032 and ZD55-TRAIL-IETD-Smac significantly inhibited the growth of BxPC-3 pancreatic tumor xenografts. In conclusion, the present study demonstrated that SNS-032 sensitizes human pancreatic cancer cells to ZD55-TRAIL-IETD-Smac-induced cell death in vitro and in vivo. These findings indicate that combined treatment with SNS-032 and ZD55-TRAIL-IETD-Smac could represent a rational approach for anti-pancreatic cancer therapy. PMID:28440486

  20. 4-arylazo-3,5-diamino-1H-pyrazole CDK inhibitors: SAR study, crystal structure in complex with CDK2, selectivity, and cellular effects

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; Cankař, Petr; Fryšová, I.; Slouka, J.; Kontopidis, G.; Džubák, P.; Hajdúch, M.; Srovnal, J.; de Azevedo Jr., W.F.; Orság, Martin; Paprskářová, Martina; Rolčík, Jakub; Látr, Aleš; Fischer, P.M.; Strnad, Miroslav

    2006-01-01

    Roč. 49, č. 22 (2006), s. 6500-6509 ISSN 0022-2623 R&D Projects: GA ČR GA301/05/0418 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje Keywords : DEPENDENT KINASE INHIBITORS * POLYMERASE-II TRANSCRIPTION * TUMOR-NECROSIS-FACTOR Subject RIV: CE - Biochemistry Impact factor: 5.115, year: 2006

  1. The E3 Ligase CHIP Mediates p21 Degradation to Maintain Radioresistance.

    Science.gov (United States)

    Biswas, Kuntal; Sarkar, Sukumar; Du, Kangping; Brautigan, David L; Abbas, Tarek; Larner, James M

    2017-06-01

    Lung cancer resists radiotherapy, making it one of the deadliest forms of cancer. Here, we show that human lung cancer cell lines can be rendered sensitive to ionizing radiation (IR) by RNAi knockdown of C-terminus of Hsc70-interacting protein (CHIP/STUB1), a U-box-type E3 ubiquitin ligase that targets a number of stress-induced proteins. Mechanistically, ubiquitin-dependent degradation of the cyclin-dependent kinase (CDK) inhibitor, p21 protein, is reduced by CHIP knockdown, leading to enhanced senescence of cells in response to exposure to IR. Cellular senescence and sensitivity to IR is prevented by CRISPR/Cas9-mediated deletion of the p21 gene ( CDKN1A) in CHIP knockdown cells. Conversely, overexpression of CHIP potentiates p21 degradation and promotes greater radioresistance of lung cancer cells. In vitro and cell-based assays demonstrate that p21 is a novel and direct ubiquitylation substrate of CHIP that also requires the CHIP-associated chaperone HSP70. These data reveal that the inhibition of the E3 ubiquitin ligase CHIP promotes radiosensitivity, thus suggesting a novel strategy for the treatment of lung cancer. Implications: The CHIP-HSP70-p21 ubiquitylation/degradation axis identified here could be exploited to enhance the efficacy of radiotherapy in patients with non-small cell lung cancer. Mol Cancer Res; 15(6); 651-9. ©2017 AACR . ©2017 American Association for Cancer Research.

  2. Involvement of p21cip-1 and p27kip-1 in the molecular mechanisms of steel factor-induced proliferative synergy in vitro and of p21cip-1 in the maintenance of stem/progenitor cells in vivo.

    Science.gov (United States)

    Mantel, C; Luo, Z; Canfield, J; Braun, S; Deng, C; Broxmeyer, H E

    1996-11-15

    Steel factor (SLF) is a hematopoietic cytokine that synergizes with other growth factors to induce a greatly enhanced proliferative state of hematopoietic progenitor cells and factor-dependent cell lines. Even though the in vivo importance of SLF in the maintenance and responsiveness of stem and progenitor cells is well documented, the molecular mechanism involved in its synergistic effects are mainly unknown. Some factor-dependent myeloid cell lines respond to the synergistic proliferative effects of SLF plus other cytokines in a manner similar to that of normal myeloid progenitor cells from bone marrow and cord blood. We show here that SLF can synergize with granulocyte-macrophage colony-stimulating factor (GM-CSF) to induce an enhanced phosphorylation of the retinoblastoma gene product and a synergistic increase in the total intracellular protein level of the cyclin-dependent kinase inhibitor, p21cip-1, which is correlated with a simultaneous decrease in p27kip-1 in the human factor-dependent myeloid cell line, M07e. Moreover, these cytokines synergize to increase p21cip-1 binding and decrease p27kip-1 binding to cyclin-dependent kinase-2 (cdk2), an enzyme required for normal cell cycle progression; these inverse events correlated with increased cdk2 kinase activity. It is also shown that exogenous purified p21cip-1 can displace p27kip-1 already bound to cdk2 in vitro. These data implicate increased p21cip-1 and decreased p27kip-1 intracellular concentrations and their stoichiometric interplay in the enhanced proliferative status of cells stimulated by the combination of SLF and GM-CSF. In support of these findings, it is shown that hematopoietic progenitor cells from mice lacking p21cip-1 are defective in SLF synergistic proliferative response in vitro. Moreover, the cycling status of marrow and spleen progenitors and absolute numbers of marrow progenitors were significantly decreased in the p21cip-1 -/-, compared with the +/+ mice. We conclude that the cdk

  3. Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9.

    Science.gov (United States)

    Rye, Carl S; Chessum, Nicola E A; Lamont, Scott; Pike, Kurt G; Faulder, Paul; Demeritt, Julie; Kemmitt, Paul; Tucker, Julie; Zani, Lorenzo; Cheeseman, Matthew D; Isaac, Rosie; Goodwin, Louise; Boros, Joanna; Raynaud, Florence; Hayes, Angela; Henley, Alan T; de Billy, Emmanuel; Lynch, Christopher J; Sharp, Swee Y; Te Poele, Robert; Fee, Lisa O'; Foote, Kevin M; Green, Stephen; Workman, Paul; Jones, Keith

    2016-08-01

    Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit ( 1 ) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency ( 25 , 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM).

  4. Characterization of cells resistant to the potent histone deacetylase inhibitor spiruchostatin B (SP-B) and effect of overexpressed p21waf1/cip1 on the SP-B resistance or susceptibility of human leukemia cells.

    Science.gov (United States)

    Kanno, Syu-Ichi; Maeda, Naoyuki; Tomizawa, Ayako; Yomogida, Shin; Katoh, Tadashi; Ishikawa, Masaaki

    2012-09-01

    We previously showed that the B cell leukemia cell line NALM-6 had the highest susceptibility among a number of leukemia cell lines to spiruchostatin B (SP-B), a potent histone deacetylase (HDAC) inhibitor. We also showed that SP-B-induced cytotoxicity depended on induction of apoptosis that was mediated by p21waf1/cip1 expression. In the present study, we generated and characterized a stable, SP-B-resistant NALM-6 cell line (NALM-6/SP-B) by continuous exposure to SP-B, starting with a low SP-B concentration. NALM-6/SP-B cells were also more resistant to FK228, which has a similar chemical structure to SP-B, and were slightly more resistant to the P-gp substrates doxorubicin and vincristine than parental cells, but displayed similar susceptibility to other HDAC inhibitors and to paclitaxel as the parental cells. There was little change in the basal mRNA expression of HDAC1, p53, Bax, Bcl-2, Fas, caspase-3, c-Myc and MDR1 in NALM-6/SP-B compared to parental cells, but the mRNA expression of p21waf1/cip1 was decreased. The introduction of an exogenous p21waf1/cip1 expression vector restored SP-B induction of NALM-6/SP-B cell apoptosis. Moreover, overexpressed p21waf1/cip1 enhanced SP-B induction of the apoptosis of the human erythroleukemia leukemia cell line K562 which is less susceptible to SP-B than NALM-6 cells. These results suggest that downregulation of p21waf1/cip1, which is a characteristic feature of NALM-6/SP-B cells, was important for their resistance to SP-B, and that this SP-B resistance could be overcome by the introduction of exogenous p21waf1/cip1. Furthermore, introduction of p21waf1/cip1 to other leukemia cells such as K562 may enhance their susceptibility to SP-B. This is the first report of the characterization of SP-B-resistant cells and of the effect of overexpressed p21waf1/cip1 on the resistance or susceptibility of human leukemia cells to SP-B.

  5. Functional ablation of pRb activates Cdk2 and causes antiestrogen resistance in human breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Hemant Varma

    2007-12-01

    Full Text Available Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT, and cdk activity was inhibited using the cdk inhibitors p16(INK4A and p21(Waf1/Cip1. Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.

  6. Short Communication: The Broad-Spectrum Histone Deacetylase Inhibitors Vorinostat and Panobinostat Activate Latent HIV in CD4(+) T Cells In Part Through Phosphorylation of the T-Loop of the CDK9 Subunit of P-TEFb.

    Science.gov (United States)

    Jamaluddin, Md Saha; Hu, Pei-Wen; Jan, Yih; Siwak, Edward B; Rice, Andrew P

    2016-02-01

    Cessation of highly active antiretroviral therapy (HAART) in HIV-infected individual leads to a rebound of viral replication due to reactivation of a viral reservoir composed largely of latently infected memory CD4(+) T cells. Efforts to deplete this reservoir have focused on reactivation of transcriptionally silent latent proviruses. HIV provirus transcription depends critically on the positive transcription elongation factor b (P-TEFb), whose core components are cyclin-dependent kinase 9 (CDK9) and cyclin T1. In resting CD4(+) cells, the functional levels of P-TEFb are extremely low. Cellular activation upregulates cyclin T1 protein levels and CDK9 T-loop (T186) phosphorylation. The broad-spectrum histone deacetylase inhibitors (HDACis) vorinostat and panobinostat have been shown to reactivate latent virus in vivo in HAART-treated individuals. In this study, we have found that vorinostat and panobinostat activate P-TEFb in resting primary CD4(+) T cells through induction of CDK9 T-loop phosphorylation. In contrast, tacedinaline and romidepsin, HDAC 1 and 2 inhibitors, were unable to activate CDK9 T-loop phosphorylation. We used a CCL19 primary CD4(+) T-cell model HIV latency to assess the correlation between induction of CDK9 T-loop phosphorylation and reactivation of latent HIV virus by HDACis. Vorinostat and panobinostat treatment of cells harboring latent HIV increased CDK9 T-loop phosphorylation and reactivation of latent virus, whereas tacedinaline and romidepsin failed to induce T-loop phosphorylation or reactivate latent virus. We conclude that the ability of vorinostat and panobinostat to induce latent HIV is, in part, likely due to the ability of the broad-spectrum HDACis to upregulate P-TEFb through increased CDK9 T-loop phosphorylation.

  7. Transferable scoring function based on semiempirical quantum mechanical PM6-DH2 method: CDK2 with 15 structurally diverse inhibitors

    Czech Academy of Sciences Publication Activity Database

    Dobeš, Petr; Fanfrlík, Jindřich; Řezáč, Jan; Otyepka, M.; Hobza, Pavel

    2011-01-01

    Roč. 25, č. 3 (2011), s. 223-235 ISSN 0920-654X R&D Projects: GA MŠk LC512; GA ČR GAP208/11/0295 Grant - others:European Social Fund(XE) CZ.1.05/2.1.00/03.0058 Institutional research plan: CEZ:AV0Z40550506 Keywords : CDK2 * semiempirical quantum mechanical method PM6-DH2 * drug design Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.386, year: 2011

  8. PKCeta enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells.

    Science.gov (United States)

    Fima, E; Shtutman, M; Libros, P; Missel, A; Shahaf, G; Kahana, G; Livneh, E

    2001-10-11

    Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCeta in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCdelta, in MCF-7 cells had opposite effects and inhibited their growth. PKCeta expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21(WAF1) was also specifically elevated in PKCeta expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27(KIP1) were not altered by the induced expression of PKCeta, the cyclin E associated Cdk2 kinase activity was in correlation with the p27(KIP1) bound to the cyclin E complex and not by p21(WAF1) binding. PKCeta expression enhanced the removal of p27(KIP1) from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27(KIP1) to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21(WAF1) correlated with its increased activity in PKCeta-expressing cells. Thus, PKCeta induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

  9. 1α,25 dihydroxi-vitamin D{sub 3} modulates CDK4 and CDK6 expression and localization

    Energy Technology Data Exchange (ETDEWEB)

    Irazoqui, Ana P.; Heim, Nadia B.; Boland, Ricardo L.; Buitrago, Claudia G., E-mail: cbuitrag@criba.edu.ar

    2015-03-27

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH){sub 2}-vitamin D{sub 3} [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21{sup Waf1/Cip1} and p27{sup Kip1} expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D –induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and β isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs –dependent mechanism in hormone modulation of myogenesis. - Highlights: • 1,25D modulates CDKs 4 and 6 expression in skeletal muscle cells. • CDK4 co

  10. The Arabidopsis CDK inhibitor ICK3/KRP5 is rate limiting for primary root growth and promotes growth through cell elongation and endoreduplication.

    Science.gov (United States)

    Wen, Bo; Nieuwland, Jeroen; Murray, James A H

    2013-02-01

    The coordination of plant cell division and expansion controls plant morphogenesis, development, and growth. Cyclin-dependent kinases (CDKs) are not only key regulators of cell division but also play an important role in cell differentiation. In plants, CDK activity is modulated by the binding of INHIBITOR OF CDK/KIP-RELATED PROTEIN (ICK/KRP). Previously, ICK2/KRP2 has been shown to mediate auxin responses in lateral root initiation. Here are analysed the roles of all ICK/KRP genes in root growth. Analysis of ick/krp null-mutants revealed that only ick3/krp5 was affected in primary root growth. ICK3/KRP5 is strongly expressed in the root apical meristem (RAM), with lower expression in the expansion zone. ick3/krp5 roots grow more slowly than wildtype controls, and this results not from reduction of division in the proliferative region of the RAM but rather reduced expansion as cells exit the meristem. This leads to shorter final cell lengths in different tissues of the ick3/krp5 mutant root, particularly the epidermal non-hair cells, and this reduction in cell size correlates with reduced endoreduplication. Loss of ICK3/KRP5 also leads to delayed germination and in the mature embryo ICK3/KRP5 is specifically expressed in the transition zone between root and hypocotyl. Cells in the transition zone were smaller in the ick3/krp5 mutant, despite the absence of endoreduplication in the embryo suggesting a direct effect of ICK3/KRP5 on cell growth. It is concluded that ICK3/KRP5 is a positive regulator of both cell growth and endoreduplication.

  11. CDK2 differentially controls normal cell senescence and cancer cell proliferation upon exposure to reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Chae Young; Lee, Seung-Min; Park, Sung Sup [Laboratory of Cell Signaling, Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahangno, Yusong, Daejeon 305-806 (Korea, Republic of); Kwon, Ki-Sun, E-mail: kwonks@kribb.re.kr [Laboratory of Cell Signaling, Aging Research Center, Korea Research Institute of Bioscience and Biotechnology, 125 Gwahangno, Yusong, Daejeon 305-806 (Korea, Republic of)

    2012-08-17

    Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} differently adjusted senescence and proliferation in normal and cancer cells. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} exposure transiently decreased PCNA levels in normal cells. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} exposure transiently increased CDK2 activity in cancer cells. Black-Right-Pointing-Pointer p21{sup Cip1} is likely dispensable when H{sub 2}O{sub 2} induces senescence in normal cells. Black-Right-Pointing-Pointer Suggestively, CDK2 and PCNA play critical roles in H{sub 2}O{sub 2}-induced cell fate decision. -- Abstract: Reactive oxygen species modulate cell fate in a context-dependent manner. Sublethal doses of H{sub 2}O{sub 2} decreased the level of proliferating cell nuclear antigen (PCNA) in normal cells (including primary human dermal fibroblasts and IMR-90 cells) without affecting cyclin-dependent kinase 2 (CDK2) activity, leading to cell cycle arrest and subsequent senescence. In contrast, exposure of cancer cells (such as HeLa and MCF7 cells) to H{sub 2}O{sub 2} increased CDK2 activity with no accompanying change in the PCNA level, leading to cell proliferation. A CDK2 inhibitor, CVT-313, prevented H{sub 2}O{sub 2}-induced cancer cell proliferation. These results support the notion that the cyclin/CDK2/p21{sup Cip1}/PCNA complex plays an important role as a regulator of cell fate decisions.

  12. p21WAF1/CIP1 gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell

    International Nuclear Information System (INIS)

    Wei, Junxia; Zhao, Jiang; Long, Min; Han, Yuan; Wang, Xi; Lin, Fang; Ren, Jihong; He, Ting; Zhang, Huizhong

    2010-01-01

    Non-small-cell lung carcinomas (NSCLCs) exhibit poor prognosis and are usually resistant to conventional chemotherapy. Absence of p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor, has been linked to drug resistance in many in vitro cellular models. RNA activation (RNAa) is a transcriptional activation phenomena guided by double-strand RNA (dsRNA) targeting promoter region of target gene. In this study, we explored the effect of up-regulation of p21 gene expression on drug-resistance in A549 non-small-cell lung carcinoma cells by transfecting the dsRNA targeting the promoter region of p21 into A549 cells. Enhanced p21 expression was observed in A549 cells after transfection of dsRNA, which was correlated with a significant growth inhibition and enhancement of chemosensitivity to cisplatin in A549 cells in vitro. Moreover, in vivo experiment showed that saRNA targeting the promoter region of p21 could significantly inhibit A549 xenograft tumor growth. These results indicate that p21 plays a role in lung cancer drug-resistance process. In addition, this study also provides evidence for the usage of saRNA as a therapeutic option for up-regulating lower-expression genes in lung cancer

  13. p21WAF1/CIP1 gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell

    Directory of Open Access Journals (Sweden)

    Ren Jihong

    2010-11-01

    Full Text Available Abstract Background Non-small-cell lung carcinomas (NSCLCs exhibit poor prognosis and are usually resistant to conventional chemotherapy. Absence of p21WAF1/CIP1, a cyclin-dependent kinase (cdk inhibitor, has been linked to drug resistance in many in vitro cellular models. RNA activation (RNAa is a transcriptional activation phenomena guided by double-strand RNA (dsRNA targeting promoter region of target gene. Methods In this study, we explored the effect of up-regulation of p21 gene expression on drug-resistance in A549 non-small-cell lung carcinoma cells by transfecting the dsRNA targeting the promoter region of p21 into A549 cells. Results Enhanced p21 expression was observed in A549 cells after transfection of dsRNA, which was correlated with a significant growth inhibition and enhancement of chemosensitivity to cisplatin in A549 cells in vitro. Moreover, in vivo experiment showed that saRNA targeting the promoter region of p21 could significantly inhibit A549 xenograft tumor growth. Conclusions These results indicate that p21 plays a role in lung cancer drug-resistance process. In addition, this study also provides evidence for the usage of saRNA as a therapeutic option for up-regulating lower-expression genes in lung cancer.

  14. p21WAF1/CIP1 gene transcriptional activation exerts cell growth inhibition and enhances chemosensitivity to cisplatin in lung carcinoma cell.

    Science.gov (United States)

    Wei, Junxia; Zhao, Jiang; Long, Min; Han, Yuan; Wang, Xi; Lin, Fang; Ren, Jihong; He, Ting; Zhang, Huizhong

    2010-11-19

    Non-small-cell lung carcinomas (NSCLCs) exhibit poor prognosis and are usually resistant to conventional chemotherapy. Absence of p21WAF1/CIP1, a cyclin-dependent kinase (cdk) inhibitor, has been linked to drug resistance in many in vitro cellular models. RNA activation (RNAa) is a transcriptional activation phenomena guided by double-strand RNA (dsRNA) targeting promoter region of target gene. In this study, we explored the effect of up-regulation of p21 gene expression on drug-resistance in A549 non-small-cell lung carcinoma cells by transfecting the dsRNA targeting the promoter region of p21 into A549 cells. Enhanced p21 expression was observed in A549 cells after transfection of dsRNA, which was correlated with a significant growth inhibition and enhancement of chemosensitivity to cisplatin in A549 cells in vitro. Moreover, in vivo experiment showed that saRNA targeting the promoter region of p21 could significantly inhibit A549 xenograft tumor growth. These results indicate that p21 plays a role in lung cancer drug-resistance process. In addition, this study also provides evidence for the usage of saRNA as a therapeutic option for up-regulating lower-expression genes in lung cancer.

  15. Inhibition of Post-Transcriptional RNA Processing by CDK Inhibitors and Its Implication in Anti-Viral Therapy

    Czech Academy of Sciences Publication Activity Database

    Holčáková, J.; Müller, P.; Tomasec, P.; Hrstka, R.; Nekulová, M.; Kryštof, Vladimír; Strnad, Miroslav; Wilkinson, G. W. G.; Vojtěšek, B.

    2014-01-01

    Roč. 9, č. 2 (2014) E-ISSN 1932-6203 R&D Projects: GA ČR GBP206/12/G151 Institutional support: RVO:61389030 Keywords : IMMUNODEFICIENCY-VIRUS TYPE-1 * DEPENDENT KINASE INHIBITORS * LARGE T-ANTIGEN Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.234, year: 2014

  16. Age-specific functional epigenetic changes in p21 and p16 in injury-activated satellite cells

    Science.gov (United States)

    Li, Ju; Han, Suhyoun; Cousin, Wendy; Conboy, Irina M.

    2014-01-01

    The regenerative capacity of muscle dramatically decreases with age because old muscle stem cells fail to proliferate in response to tissue damage. Here we uncover key age-specific differences underlying this proliferative decline: namely, the genetic loci of CDK inhibitors (CDKI) p21 and p16 are more epigenetically silenced in young muscle stem cells, as compared to old, both in quiescent cells and those responding to tissue injury. Interestingly, phosphorylated ERK (pERK) induced in these cells by ectopic FGF-2 is found in association with p21 and p16 promoters, and moreover, only in the old cells. Importantly, in the old satellite cells FGF-2/pERK silences p21 epigenetically and transcriptionally, which leads to reduced p21 protein levels and enhanced cell proliferation. In agreement with the epigenetic silencing of the loci, young muscle stem cells do not depend as much as old on ectopic FGF/pERK for their myogenic proliferation. In addition, other CDKIs, such asp15INK4B and p27KIP1, become elevated in satellite cells with age, confirming and explaining the profound regenerative defect of old muscle. This work enhances our understanding of tissue aging, promoting strategies for combating age-imposed tissue degeneration. PMID:25447026

  17. In Silico Identification and In Vitro and In Vivo Validation of Anti-Psychotic Drug Fluspirilene as a Potential CDK2 Inhibitor and a Candidate Anti-Cancer Drug.

    Directory of Open Access Journals (Sweden)

    Xi-Nan Shi

    Full Text Available Hepatocellular carcinoma (HCC is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC's resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested in vitro. Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg. Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history

  18. Voruciclib, a Potent CDK4/6 Inhibitor, Antagonizes ABCB1 and ABCG2-Mediated Multi-Drug Resistance in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Pranav Gupta

    2018-02-01

    Full Text Available Background/Aims: The overexpression of ATP-Binding Cassette (ABC transporters has known to be one of the major obstacles impeding the success of chemotherapy in drug resistant cancers. In this study, we evaluated voruciclib, a CDK 4/6 inhibitor, for its chemo-sensitizing activity in ABCB1- and ABCG2- overexpressing cells. Methods: Cytotoxicity and reversal effect of voruciclib was determined by MTT assay. The intracellular accumulation and efflux of ABCB1 and ABCG2 substrates were measured by scintillation counter. The effects on expression and intracellular localization of ABCB1 and ABCG2 proteins were determined by Western blotting and immunofluorescence, respectively. Vanadate-sensitive ATPase assay was done to determine the effect of voruciclib on the ATPase activity of ABCB1 and ABCG2. Flow cytometric analysis was done to determine the effect of voruciclib on apoptosis of ABCB1 and ABCG2-overexpressing cells and docking analysis was done to determine the interaction of voruciclib with ABCB1 and ACBG2 protein. Results: Voruciclib significantly potentiated the effect of paclitaxel and doxorubicin in ABCB1-overexpressing cells, as well as mitoxantrone and SN-38 in ABCG2-overexpressing cells. Voruciclib moderately sensitized ABCC10- overexpressing cells to paclitaxel, whereas it did not alter the cytotoxicity of substrates of ABCC1. Furthermore, voruciclib increased the intracellular accumulation and decreased the efflux of substrate anti-cancer drugs from ABCB1- or ABCG2-overexpressing cells. However, voruciclib did not alter the expression or the sub-cellular localization of ABCB1 or ABCG2. Voruciclib stimulated the ATPase activity of both ABCB1 and ABCG2 in a concentration-dependent manner. Lastly, voruciclib exhibited a drug-induced apoptotic effect in ABCB1- or ABCG2- overexpressing cells. Conclusion: Voruciclib is currently a phase I clinical trial drug. Our findings strongly support its potential use in combination with conventional anti

  19. X-rays induce dose-dependent and cell cycle-independent accumulation of p21{sup sdi1/WAF1}

    Energy Technology Data Exchange (ETDEWEB)

    Tsuyama, Naohiro; Iwamoto, Keisuke S.; Mizuno, Terumi; Kyoizumi, Seishi; Seyama, Toshio [Radiation Effects Research Foundation, Hiroshima (Japan); Ide, Toshinori; Noda, Asao

    2001-03-01

    Cell cycle arrest at the G1 checkpoint is governed by a function of wild-type p53. We assessed the behavior of the sdi1 gene, which codes for a 21 kDa potent inhibitor of cdk/cyclins, after X-irradiation. X-irradiation induced sdi1 mRNA accumulation and G1 arrest only in cells possessing wild-type p53. Elevation of p21{sup sdi1/WAF1} was preceded by p53 accumulation, which occurred despite p53 mRNA constancy in normal cells growing in the log phase. The quantity of accumulated p53 and p21{sup sdi1/WAF1} was radiation dose dependent. A decrease in the S phase cell population in normal cells observed after irradiation reached a minimum at less-than-maximum levels of p53 and p21{sup sdi1/WAF1}. Furthermore, an accumulation of p53 and p21{sup sdi1/WAF1} was also observed when cells were synchronized in the G0, G1 and S phase and X-irradiated. These results indicated that an X-ray induced p53 and p21{sup sdi1/WAF1} accumulation mechanism exists throughout the cell cycle, and that the signal strength induced by X-irradiation is dose-dependent. (author)

  20. Targeting cyclin-dependent kinase 1 (CDK1) but not CDK4/6 or CDK2 is selectively lethal to MYC-dependent human breast cancer cells

    International Nuclear Information System (INIS)

    Kang, Jian; Sergio, C Marcelo; Sutherland, Robert L; Musgrove, Elizabeth A

    2014-01-01

    Although MYC is an attractive therapeutic target for breast cancer treatment, it has proven challenging to inhibit MYC directly, and clinically effective pharmaceutical agents targeting MYC are not yet available. An alternative approach is to identify genes that are synthetically lethal in MYC-dependent cancer. Recent studies have identified several cell cycle kinases as MYC synthetic-lethal genes. We therefore investigated the therapeutic potential of specific cyclin-dependent kinase (CDK) inhibition in MYC-driven breast cancer. Using small interfering RNA (siRNA), MYC expression was depleted in 26 human breast cancer cell lines and cell proliferation evaluated by BrdU incorporation. MYC-dependent and MYC-independent cell lines were classified based on their sensitivity to siRNA-mediated MYC knockdown. We then inhibited CDKs including CDK4/6, CDK2 and CDK1 individually using either RNAi or small molecule inhibitors, and compared sensitivity to CDK inhibition with MYC dependence in breast cancer cells. Breast cancer cells displayed a wide range of sensitivity to siRNA-mediated MYC knockdown. The sensitivity was correlated with MYC protein expression and MYC phosphorylation level. Sensitivity to siRNA-mediated MYC knockdown did not parallel sensitivity to the CDK4/6 inhibitor PD0332991; instead MYC-independent cell lines were generally sensitive to PD0332991. Cell cycle arrest induced by MYC knockdown was accompanied by a decrease in CDK2 activity, but inactivation of CDK2 did not selectively affect the viability of MYC-dependent breast cancer cells. In contrast, CDK1 inactivation significantly induced apoptosis and reduced viability of MYC-dependent cells but not MYC- independent cells. This selective induction of apoptosis by CDK1 inhibitors was associated with up-regulation of the pro-apoptotic molecule BIM and was p53-independent. Overall, these results suggest that further investigation of CDK1 inhibition as a potential therapy for MYC-dependent breast cancer

  1. Overview of CDK9 as a target in cancer research.

    Science.gov (United States)

    Morales, Fatima; Giordano, Antonio

    2016-01-01

    CDK9 is a protein in constant development in cancer therapy. Herein we present an overview of the enzyme as a target for cancer therapy. We provide data on its characteristics and mechanism of action. In recent years, CDK9 inhibitors that have been designed with molecular modeling have demonstrated good antitumoral activity in vitro. Clinical studies of the drugs flavopiridol, dinaciclib, seliciclib, SNS-032 and RGB-286638 used as CDK9 inhibitors are also reviewed, with their additional targets and their relative IC50 values. Unfortunately, treatment with these drugs remains unsuccessful and involves many adverse effects. We could conclude that there are many small molecules that bind to CDK9, but their lack of selectivity against other CDKs do not allow them to get to the clinical use. However, drug designers currently have the tools needed to improve the selectivity of CDK9 inhibitors and to make successful treatment available to patients.

  2. Residual Cdk1/2 activity after DNA damage promotes senescence

    Czech Academy of Sciences Publication Activity Database

    Müllers, E.; Cascales, H.S.; Burdová, Kamila; Macůrek, Libor; Lindqvist, A.

    2017-01-01

    Roč. 16, č. 3 (2017), s. 575-584 ISSN 1474-9726 R&D Projects: GA ČR GA13-18392S Institutional support: RVO:68378050 Keywords : Cdk1 * Cdk2 * cell cycle * checkpoint recovery * DNA damage response * G2phase * p21 * senescence Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology

  3. The pterocarpanquinone LQB-118 inhibits tumor cell proliferation by downregulation of c-Myc and cyclins D1 and B1 mRNA and upregulation of p21 cell cycle inhibitor expression.

    Science.gov (United States)

    Martino, Thiago; Magalhães, Fernanda C J; Justo, Graça A; Coelho, Marsen G P; Netto, Chaquip D; Costa, Paulo R R; Sabino, Kátia C C

    2014-06-15

    The incidence of cancer grows annually worldwide and in Brazil it is the second cause of death. The search for anti-cancer drugs has then become urgent. It depends on the studies of natural and chemical synthesis products. The antitumor action of LQB-118, a pterocarpanquinone structurally related to lapachol, has been demonstrated to induce mechanisms linked to leukemia cell apoptosis. This work investigated some mechanisms of the in vitro antitumor action of LQB-118 on prostate cancer cells. LQB-118 reduced the expression of the c-Myc transcription factor, downregulated the cyclin D1 and cyclin B1 mRNA levels and upregulated the p21 cell cycle inhibitor. These effects resulted in cell cycle arrest in the S and G2/M phases and inhibition of tumor cell proliferation. LQB-118 also induced programmed cell death of the prostate cancer cells, as evidenced by internucleosomal DNA fragmentation and annexin-V positive cells. Except the cell cycle arrest in the S phase and enhanced c-Myc expression, all the mechanisms observed here for the in vitro antitumor action of LQB-118 were also found for Paclitaxel, a traditional antineoplastic drug. These findings suggest new molecular mechanisms for the LQB-118 in vitro antitumor action. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Induction of Apoptosis and Cell Cycle Arrest by Flavokawain C on HT-29 Human Colon Adenocarcinoma via Enhancement of Reactive Oxygen Species Generation, Upregulation of p21, p27, and GADD153, and Inactivation of Inhibitor of Apoptosis Proteins.

    Science.gov (United States)

    Phang, Chung-Weng; Karsani, Saiful Anuar; Abd Malek, Sri Nurestri

    2017-07-01

    Chalcones have been shown to exhibit anti-cancer properties by targeting multiple molecular pathways. It was, therefore, of interest to investigate flavokawain C (FKC), a naturally occurring chalcone, which can be isolated from Kava ( Piper methysticum Forst) root extract. The aim of this study was to investigate the inhibitory effect of FKC on the growth of HT-29 cells and its underlying mechanism of action. Cell viability of HT-29 cells was assessed by Sulforhodamine B assay after FKC treatment. Induction of apoptosis was examined by established morphological and biochemical assays. ROS generation was determined by dichlorofluorescein fluorescence staining, and superoxide dismutase activity was measured using the spectrophotometric method. Western blotting was used to examine the changes in the protein levels. FKC markedly decreased the cell viability of HT-29 cells and the cells showed dramatic changes in cellular and nuclear morphologies with typical apoptotic features. The induction of apoptosis correlated well with the externalization of phosphatidylserine, DNA fragmentation, decreased mitochondrial membrane potential, activation of caspases, and PARP cleavage. This was associated with an increase in reactive oxygen species and a decrease in SOD activity. The protein levels of XIAP, c-IAP1, and c-IAP2 were downregulated, whereas the GADD153 was upregulated after FKC treatment. FKC induced cell cycle arrest at the G 1 and G 2 /M phases via upregulation of p21 and p27 in a p53-independent manner. Our results provide evidence that FKC has the potential to be developed into chemotherapeutic drug for the treatment of colon adenocarcinoma. Flavokawain C inhibited the growth of HT-29 human colon adenocarcinoma cellsFlavokawain C induced apoptosis in HT-29 cells, associated with an increase in reactive oxygen species and a decrease in SOD activityFlavokawain C induced cell cycle arrest at the G 1 and G 2 /M phases via upregulation of p21 and p27 in HT-29 cellsHT-29

  5. The cyclin-dependent kinase inhibitor flavopiridol disrupts sodium butyrate-induced p21WAF1/CIP1 expression and maturation while reciprocally potentiating apoptosis in human leukemia cells.

    Science.gov (United States)

    Rosato, Roberto R; Almenara, Jorge A; Cartee, Leanne; Betts, Vicki; Chellappan, Srikumar P; Grant, Steven

    2002-02-01

    Interactions between the cyclin-dependent kinase inhibitor flavopiridol (FP) and the histone deacetylase inhibitor sodium butyrate (SB) have been examined in human leukemia cells (U937) in relation to differentiation and apoptosis. Whereas 1 mM of SB or 100 nM of FP minimally induced apoptosis (4% and 10%, respectively) at 24 h, simultaneous exposure of U937 cells to these agents dramatically increased cell death (e.g., approximately 60%), reflected by both morphological and Annexin/propidium iodide-staining features, procaspase 3 activation, and poly(ADP-ribose) polymerase cleavage. Similar interactions were observed in human promyelocytic (HL-60), B-lymphoblastic (Raji), and T-lymphoblastic (Jurkat) leukemia cells. Coadministration of FP opposed SB-mediated accumulation of cells in G0G1 and differentiation, reflected by reduced CD11b expression, but instead dramatically increased procaspase-3, procaspase-8, Bid, and poly(ADP-ribose) polymerase cleavage, as well as mitochondrial damage (e.g., loss of mitochondrial membrane potential and cytochrome c release). FP also blocked SB-related p21WAF1-CIP1 induction through a caspase-independent mechanism and triggered the caspase-mediated cleavage of p27KIP1 and retinoblastoma protein. The latter event was accompanied by a marked reduction in retinoblastoma protein/E2F1 complex formation. However, FP did not modify the extent of SB-associated acetylation of histones H3 and H4. Treatment of cells with FP/SB also resulted in the caspase-mediated cleavage of Bcl-2 and caspase-independent down-regulation of Mcl-1. Levels of cyclins A, D1, and E, and X-linked inhibitor of apoptosis also declined in SB/FP-treated cells. Finally, FP/SB coexposure potently induced apoptosis in two primary acute myelogenous leukemia samples. Together, these findings demonstrate that FP, when combined with SB, induces multiple perturbations in cell cycle and apoptosis regulatory proteins, which oppose leukemic cell differentiation but instead

  6. Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells

    NARCIS (Netherlands)

    The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

    2015-01-01

    Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases,

  7. Evaluation and comparison of 3D-QSAR CoMSIA models for CDK1, CDK5, and GSK-3 inhibition by paullones

    DEFF Research Database (Denmark)

    Kunick, Conrad; Lauenroth, Kathrin; Wieking, Karen

    2004-01-01

    With a view to the rational design of selective GSK-3beta inhibitors, 3D-QSAR CoMSIA models were developed for the inhibition of the three serine/threonine kinases CDK1/cyclin B, CDK5/p25, and GSK-3beta by compounds from the paullone inhibitor family. The models are based on the kinase inhibition...

  8. An essential pathway links FLT3-ITD, HCK and CDK6 in acute myeloid leukemia

    Science.gov (United States)

    Lopez, Sophie; Voisset, Edwige; Tisserand, Julie C.; Mosca, Cyndie; Prebet, Thomas; Santamaria, David; Dubreuil, Patrice; Sepulveda, Paulo De

    2016-01-01

    CDK4/CDK6 and RB proteins drive the progression through the G1 phase of the cell cycle. In acute myeloid leukemia (AML), the activity of the CDK/Cyclin D complex is increased. The mechanism involved is unknown, as are the respective roles played by CDK4 or CDK6 in this process. Here, we report that AML cells carrying FLT3-ITD mutations are dependent on CDK6 for cell proliferation while CDK4 is not essential. We showed that FLT3-ITD signaling is responsible for CDK6 overexpression, through a pathway involving the SRC-family kinase HCK. Accordingly, FLT3-ITD failed to transform primary hematopoietic progenitor cells from Cdk6−/− mice. Our results demonstrate that CDK6 is the primary target of CDK4/CDK6 inhibitors in FLT3-ITD positive AML. Furthermore, we delineate an essential protein kinase pathway -FLT3/HCK/CDK6- in the context of AML with FLT3-ITD mutations. PMID:27323399

  9. Aminopurvalanol A, a Potent, Selective, and Cell Permeable Inhibitor of Cyclins/Cdk Complexes, Causes the Reduction of in Vitro Fertilizing Ability of Boar Spermatozoa, by Negatively Affecting the Capacitation-Dependent Actin Polymerization

    Directory of Open Access Journals (Sweden)

    Nicola Bernabò

    2017-12-01

    Full Text Available The adoption of high-througput technologies demonstrated that in mature spermatozoa are present proteins that are thought to be not present or active in sperm cells, such as those involved in control of cell cycle. Here, by using an in silico approach based on the application of networks theory, we found that Cyclins/Cdk complexes could play a central role in signal transduction active during capacitation. Then, we tested this hypothesis in the vitro model. With this approach, spermatozoa were incubated under capacitating conditions in control conditions (CTRL or in the presence of Aminopurvalanol A a potent, selective and cell permeable inhibitor of Cyclins/Cdk complexes at different concentrations (2, 10, and 20 μM. We found that this treatment caused dose-dependent inhibition of sperm fertilizing ability. We attribute this event to the loss of acrosome integrity due to the inhibition of physiological capacitation-dependent actin polymerization, rather than to a detrimental effect on membrane lipid remodeling or on other signaling pathways such as tubulin reorganization or MAPKs activation. In our opinion, these data could revamp the knowledge on biochemistry of sperm capacitation and could suggest new perspectives in studying male infertility.

  10. Searching for novel Cdk5 substrates in brain by comparative phosphoproteomics of wild type and Cdk5-/- mice.

    Directory of Open Access Journals (Sweden)

    Erick Contreras-Vallejos

    Full Text Available Protein phosphorylation is the most common post-translational modification that regulates several pivotal functions in cells. Cyclin-dependent kinase 5 (Cdk5 is a proline-directed serine/threonine kinase which is mostly active in the nervous system. It regulates several biological processes such as neuronal migration, cytoskeletal dynamics, axonal guidance and synaptic plasticity among others. In search for novel substrates of Cdk5 in the brain we performed quantitative phosphoproteomics analysis, isolating phosphoproteins from whole brain derived from E18.5 Cdk5+/+ and Cdk5-/- embryos, using an Immobilized Metal-Ion Affinity Chromatography (IMAC, which specifically binds to phosphorylated proteins. The isolated phosphoproteins were eluted and isotopically labeled for relative and absolute quantitation (iTRAQ and mass spectrometry identification. We found 40 proteins that showed decreased phosphorylation at Cdk5-/- brains. In addition, out of these 40 hypophosphorylated proteins we characterized two proteins, :MARCKS (Myristoylated Alanine-Rich protein Kinase C substrate and Grin1 (G protein regulated inducer of neurite outgrowth 1. MARCKS is known to be phosphorylated by Cdk5 in chick neural cells while Grin1 has not been reported to be phosphorylated by Cdk5. When these proteins were overexpressed in N2A neuroblastoma cell line along with p35, serine phosphorylation in their Cdk5 motifs was found to be increased. In contrast, treatments with roscovitine, the Cdk5 inhibitor, resulted in an opposite effect on serine phosphorylation in N2A cells and primary hippocampal neurons transfected with MARCKS. In summary, the results presented here identify Grin 1 as novel Cdk5 substrate and confirm previously identified MARCKS as a a bona fide Cdk5 substrate.

  11. Different domains of P21Cip1/waf1 regulate DNA replication and DNA repair-associated processes after UV

    International Nuclear Information System (INIS)

    Soria, Gaston; Speroni, Juliana; Podhajcer, Osvaldo L.; Gottifredi, Vanesa; Prives, Carol

    2007-01-01

    Full text: Many genotoxic insults result in p21 up-regulation and p21-dependent cell cycle arrest but UV irradiation triggers p21 proteolysis. The significance of the increased p21 turnover is unclear and might be associated to DNA repair. While the role of p21 in Nucleotide Excision Repair (NER) remains controversial, two recent reports explore its effect on Translesion DNA Synthesis (TLS), a process that avoids replication blockage during S phase. The first report shows that p21 degradation is required for efficient PCNA ubiquitination, a post transcriptional modification that is relevant for TLS. The second report demonstrates that p21 (-/-) cells have increased TLS-associated mutagenic rates. Herein we analyze the effect of p21 on different PCNA-driven processes including DNA replication, NER and TLS. Whereas only the CDK binding domain of p21 is required for cell cycle arrest in unstressed cells; neither the CDK- nor the PCNA-binding domains of p21 are able to block early and late steps of NER. Intriguingly, through its PCNA binding domain, p21 inhibited recruitment of the TLS-polymerase, polη to PCNA foci after UV. Moreover, this obstruction correlates with accumulation of γH2AX and increased apoptosis. Taking together, our data emphasizes the link between p21 turnover and efficient TLS. This might also suggest a potential effect of p21 on other activities of polζ, a DNA polymerase with central roles in other biological scenarios such as genetic conversion, homologous recombination and modulation of the cellular response to genotoxic agents [es

  12. Adenovirus-mediated gene transfer of MMAC1/PTEN to glioblastoma cells inhibits S phase entry by the recruitment of p27Kip1 into cyclin E/CDK2 complexes.

    Science.gov (United States)

    Cheney, I W; Neuteboom, S T; Vaillancourt, M T; Ramachandra, M; Bookstein, R

    1999-05-15

    Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.

  13. Pan-Cancer Analysis of the Mediator Complex Transcriptome Identifies CDK19 and CDK8 as Therapeutic Targets in Advanced Prostate Cancer.

    Science.gov (United States)

    Brägelmann, Johannes; Klümper, Niklas; Offermann, Anne; von Mässenhausen, Anne; Böhm, Diana; Deng, Mario; Queisser, Angela; Sanders, Christine; Syring, Isabella; Merseburger, Axel S; Vogel, Wenzel; Sievers, Elisabeth; Vlasic, Ignacija; Carlsson, Jessica; Andrén, Ove; Brossart, Peter; Duensing, Stefan; Svensson, Maria A; Shaikhibrahim, Zaki; Kirfel, Jutta; Perner, Sven

    2017-04-01

    Purpose: The Mediator complex is a multiprotein assembly, which serves as a hub for diverse signaling pathways to regulate gene expression. Because gene expression is frequently altered in cancer, a systematic understanding of the Mediator complex in malignancies could foster the development of novel targeted therapeutic approaches. Experimental Design: We performed a systematic deconvolution of the Mediator subunit expression profiles across 23 cancer entities ( n = 8,568) using data from The Cancer Genome Atlas (TCGA). Prostate cancer-specific findings were validated in two publicly available gene expression cohorts and a large cohort of primary and advanced prostate cancer ( n = 622) stained by immunohistochemistry. The role of CDK19 and CDK8 was evaluated by siRNA-mediated gene knockdown and inhibitor treatment in prostate cancer cell lines with functional assays and gene expression analysis by RNAseq. Results: Cluster analysis of TCGA expression data segregated tumor entities, indicating tumor-type-specific Mediator complex compositions. Only prostate cancer was marked by high expression of CDK19 In primary prostate cancer, CDK19 was associated with increased aggressiveness and shorter disease-free survival. During cancer progression, highest levels of CDK19 and of its paralog CDK8 were present in metastases. In vitro , inhibition of CDK19 and CDK8 by knockdown or treatment with a selective CDK8/CDK19 inhibitor significantly decreased migration and invasion. Conclusions: Our analysis revealed distinct transcriptional expression profiles of the Mediator complex across cancer entities indicating differential modes of transcriptional regulation. Moreover, it identified CDK19 and CDK8 to be specifically overexpressed during prostate cancer progression, highlighting their potential as novel therapeutic targets in advanced prostate cancer. Clin Cancer Res; 23(7); 1829-40. ©2016 AACR . ©2016 American Association for Cancer Research.

  14. Combination of PTEN and γ-Ionizing Radiation Enhances Cell Death and G2/M Arrest Through Regulation of AKT Activity and p21 Induction in Non-Small-Cell Lung Cancer Cells

    International Nuclear Information System (INIS)

    Park, Jong Kuk; Jung, Hae-Yun; Park, Seon Ho; Kang, Seung Yi; Yi, Mi-Rang; Um, Hong Duck; Hong, Sung Hee

    2008-01-01

    Purpose: To identify the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) during γ-ionizing radiation (γ-IR) treatment for non-small-cell lung cancer cells. Methods and Materials: Wild-type PTEN or mutant forms of PTEN plasmids were transfected to construct stable transfectants of the NCI-H1299 non-small-cell lung cancer cell line. Combined effects of PTEN expression and IR treatment were tested using immunoblot, clonogenic, and cell-counting assays. Related signaling pathways were studied with immunoblot and kinase assays. Results: At steady state, stable transfectants showed almost the same proliferation rate but had different AKT phosphorylation patterns. When treated with γ-IR, wild-type PTEN transfectants showed higher levels of cell death compared with mock vector or mutant transfectants, and showed increased G 2 /M cell-cycle arrest accompanied by p21 induction and CDK1 inactivation. NCI-H1299 cells were treated with phosphosinositide-3 kinase (PI3K)/AKT pathway inhibitor (LY29002), resulting in reduced AKT phosphorylation levels. Treatment of NCI-H1299 cells with LY29002 and γ-IR resulted in increased cell-cycle arrest and p21 induction. Endogenous wild-type PTEN-containing NCI-H460 cells were treated with PTEN-specific siRNA and then irradiated with γ-IR: however reduced PTEN levels did not induce cell-cycle arrest or p21 expression. Conclusions: Taken together, these findings indicate that PTEN may modulate cell death or the cell cycle via AKT inactivation by PTEN and γ-IR treatment. We also propose that a PTEN-PI3K/AKT-p21-CDK1 pathway could regulate cell death and the cell cycle by γ-IR treatment

  15. A selective chemical probe for exploring the role of CDK8 and CDK19 in human disease

    Science.gov (United States)

    Esdar, Christina; Waalboer, Dennis; Adeniji-Popoola, Olajumoke; Ortiz-Ruiz, Maria-Jesus; Mallinger, Aurélie; Samant, Rahul S.; Czodrowski, Paul; Musil, Djordje; Schwarz, Daniel; Schneider, Klaus; Stubbs, Mark; Ewan, Ken; Fraser, Elizabeth; TePoele, Robert; Court, Will; Box, Gary; Valenti, Melanie; de Haven Brandon, Alexis; Gowan, Sharon; Rohdich, Felix; Raynaud, Florence; Schneider, Richard; Poeschke, Oliver; Blaukat, Andree; Workman, Paul; Schiemann, Kai; Eccles, Suzanne A.; Wienke, Dirk; Blagg, Julian

    2015-01-01

    There is unmet need for chemical tools to explore the role of the Mediator complex in human pathologies ranging from cancer to cardiovascular disease. Here we determine that CCT251545, a small molecule WNT-pathway inhibitor discovered through cell-based screening, is a potent and selective chemical probe for the human Mediator complex-associated protein kinases CDK8 and CDK19 with >100-fold selectivity over 291 other kinases. X-ray crystallography demonstrates a Type 1 binding mode involving insertion of the CDK8 C-terminus into the ligand binding site. In contrast to Type II inhibitors of CDK8/19, CCT251545 displays potent cell-based activity. We show that CCT251545 and close analogues alter WNT-pathway regulated gene expression and other on-target effects of modulating CDK8/19 including genes regulated by STAT1. Consistent with this we find that phosphorylation of STAT1SER727 is a biomarker of CDK8 kinase activity in vitro and in vivo. Finally, we demonstrate in vivo activity of CCT251545 in WNT-dependent tumors. PMID:26502155

  16. Cytoplasmic p21 is a potential predictor for cisplatin sensitivity in ovarian cancer

    International Nuclear Information System (INIS)

    Xia, Xi; Weng, Yanjie; Liao, Shujie; Han, Zhiqiang; Liu, Ronghua; Zhu, Tao; Wang, Shixuan; Xu, Gang; Meng, Li; Zhou, Jianfeng; Ma, Ding; Ma, Quanfu; Li, Xiao; Ji, Teng; Chen, Pingbo; Xu, Hongbin; Li, Kezhen; Fang, Yong; Weng, Danhui

    2011-01-01

    P21 (WAF1/Cip1) binds to cyclin-dependent kinase complexes and inhibits their activities. It was originally described as an inhibitor of cancer cell proliferation. However, many recent studies have shown that p21 promotes tumor progression when accumulated in the cell cytoplasm. So far, little is known about the correlation between cytoplasmic p21 and drug resistance. This study was aimed to investigate the role of p21 in the cisplatin resistance of ovarian cancer. RT-PCR, western blot and immunofluorescence were used to detect p21 expression and location in cisplatin-resistant ovarian cancer cell line C13* and its parental line OV2008. Regulation of cytoplasmic p21 was performed through transfection of p21 siRNA, Akt2 shRNA and Akt2 constitutively active vector in the two cell lines; their effects on cisplatin-induced apoptosis were evaluated by flow cytometry. Tumor tissue sections of clinical samples were analyzed by immunohistochemistry. p21 predominantly localizes to the cytoplasm in C13* compared to OV2008. Persistent exposure to low dose cisplatin in OV2008 leads to p21 translocation from nuclear to cytoplasm, while it had not impact on p21 localization in C13*. Knockdown of cytoplasmic p21 by p21 siRNA transfection in C13* notably increased cisplatin-induced apoptosis through activation of caspase 3. Inhibition of p21 translocation into the cytoplasm by transfection of Akt2 shRNA into C13* cells significantly increased cisplatin-induced apoptosis, while induction of p21 translocation into the cytoplasm by transfection of constitutively active Akt2 in OV2008 enhanced the resistance to cisplatin. Immunohistochemical analysis of clinical ovarian tumor tissues demonstrated that cytoplasmic p21 was negatively correlated with the response to cisplatin based treatment. Cytoplasmic p21 is a novel biomarker of cisplatin resistance and it may represent a potential therapeutic target for ovarian tumors that are refractory to conventional treatment

  17. Knockdown of CDK2AP1 in primary human fibroblasts induces p53 dependent senescence.

    Directory of Open Access Journals (Sweden)

    Khaled N Alsayegh

    Full Text Available Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1 is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1 in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a no increase in senescence associated beta-galactosidase activity, (b decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1

  18. Synthesis, biological evaluation and molecular modeling of a novel series of 7-azaindole based tri-heterocyclic compounds as potent CDK2/Cyclin E inhibitors

    Czech Academy of Sciences Publication Activity Database

    Baltus, C.B.; Jorda, Radek; Marot, Ch.; Berka, K.; Bazgier, Václav; Kryštof, Vladimír; Prie, G.; Viaud-Massuard, M.C.

    2016-01-01

    Roč. 108, JAN 27 (2016), s. 701-719 ISSN 0223-5234 R&D Projects: GA MŠk(CZ) LO1204 Institutional support: RVO:61389030 Keywords : Cyclin-dependent kinase 2 * Kinase inhibitors * Anti-tumor agent Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.519, year: 2016

  19. p21WAF1/CIP1 interacts with protein kinase CK2

    DEFF Research Database (Denmark)

    Götz, C; Wagner, P; Issinger, O G

    1996-01-01

    p21WAF1/CIP1 which belongs to a class of regulatory proteins that interact with cyclin dependent kinases is a potent inhibitor of these kinases. The inhibition of the cyclin dependent kinases induces an arrest of cells in the G phase of the cell cycle. In addition p21WAF1/CIP1 associates with PCNA...

  20. Activation of CDK4 Triggers Development of Non-alcoholic Fatty Liver Disease

    Directory of Open Access Journals (Sweden)

    Jingling Jin

    2016-07-01

    Full Text Available The development of non-alcoholic fatty liver disease (NAFLD is a multiple step process. Here, we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and hepatocellular carcinoma (HCC. The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high-fat diet (HFD-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, this study reveals that activation of cdk4 triggers NAFLD and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD.

  1. Double-negative feedback between S-phase cyclin-CDK and CKI generates abruptness in the G1/S switch

    Directory of Open Access Journals (Sweden)

    Rainis eVenta

    2012-12-01

    Full Text Available The G1/S transition is a crucial decision point in the cell cycle. At G1/S, there is an abrupt switch from a state of high CDK inhibitor (CKI levels and low S-phase CDK activity to a state of high S-phase CDK activity and degraded CKI. In budding yeast, this transition is triggered by phosphorylation of the Cdk1 inhibitor Sic1 at multiple sites by G1-phase CDK (Cln1,2-Cdk1 and S-phase CDK (Clb5,6-Cdk1 complexes. Using mathematical modeling we demonstrate that the mechanistic basis for the abruptness and irreversibility of the G1/S transition is the highly specific phosphorylation of Sic1 by S-phase CDK complex. This switch is generated by a double negative feedback loop in which S-CDK1 phosphorylates Sic1, thus targeting it for destruction, and thereby liberating further S-CDK1 from the inhibitory Sic1-S-CDK1 complex. Our model predicts that the abruptness of the switch depends upon a strong binding affinity within the Sic1-S-CDK inhibitory complex. In vitro phosphorylation analysis using purified yeast proteins revealed that free Clb5-Cdk1 can create positive feedback by phosphorylating Sic1 that is bound in the inhibitory complex, and that Sic1 inhibits Clb5-Cdk1 with a sub-nanomolar inhibition constant. Our model also predicts that if the G1-phase CDK complex is too efficient at targeting Sic1 for destruction, then G1/S becomes a smooth and readily reversible transition. We propose that the optimal role for the G1-phase CDK in the switch would not be to act as a kinase activity directly responsible for abrupt degradation of CKI, but rather to act as a priming signal that initiates a positive feedback loop driven by emerging free S-phase CDK.

  2. Compound K, a Ginsenoside Metabolite, Inhibits Colon Cancer Growth via Multiple Pathways Including p53-p21 Interactions

    Directory of Open Access Journals (Sweden)

    Eugene B. Chang

    2013-01-01

    Full Text Available Compound K (20-O-beta-D-glucopyranosyl-20(S-protopanaxadiol, CK, an intestinal bacterial metabolite of ginseng protopanaxadiol saponins, has been shown to inhibit cell growth in a variety of cancers. However, the mechanisms are not completely understood, especially in colorectal cancer (CRC. A xenograft tumor model was used first to examine the anti-CRC effect of CK in vivo. Then, multiple in vitro assays were applied to investigate the anticancer effects of CK including antiproliferation, apoptosis and cell cycle distribution. In addition, a qPCR array and western blot analysis were executed to screen and validate the molecules and pathways involved. We observed that CK significantly inhibited the growth of HCT-116 tumors in an athymic nude mouse xenograft model. CK significantly inhibited the proliferation of human CRC cell lines HCT-116, SW-480, and HT-29 in a dose- and time-dependent manner. We also observed that CK induced cell apoptosis and arrested the cell cycle in the G1 phase in HCT-116 cells. The processes were related to the upregulation of p53/p21, FoxO3a-p27/p15 and Smad3, and downregulation of cdc25A, CDK4/6 and cyclin D1/3. The major regulated targets of CK were cyclin dependent inhibitors, including p21, p27, and p15. These results indicate that CK inhibits transcriptional activation of multiple tumor-promoting pathways in CRC, suggesting that CK could be an active compound in the prevention or treatment of CRC.

  3. Quantifying the CDK inhibitor VMY-1-103's activity and tissue levels in an in vivo tumor model by LC-MS/MS and by MRI.

    Science.gov (United States)

    Sirajuddin, Paul; Das, Sudeep; Ringer, Lymor; Rodriguez, Olga C; Sivakumar, Angiela; Lee, Yi-Chien; Üren, Aykut; Fricke, Stanley T; Rood, Brian; Ozcan, Alpay; Wang, Sean S; Karam, Sana; Yenugonda, Venkata; Salinas, Patricia; Petricoin, Emanuel; Pishvaian, Michael; Lisanti, Michael P; Wang, Yue; Schlegel, Richard; Moasser, Bahram; Albanese, Chris

    2012-10-15

    The development of new small molecule-based therapeutic drugs requires accurate quantification of drug bioavailability, biological activity and treatment efficacy. Rapidly measuring these endpoints is often hampered by the lack of efficient assay platforms with high sensitivity and specificity. Using an in vivo model system, we report a simple and sensitive liquid chromatography-tandem mass spectrometry assay to quantify the bioavailability of a recently developed novel cyclin-dependent kinase inhibitor VMY-1-103, a purvalanol B-based analog whose biological activity is enhanced via dansylation. We developed a rapid organic phase extraction technique and validated wide and functional VMY-1-103 distribution in various mouse tissues, consistent with its enhanced potency previously observed in a variety of human cancer cell lines. More importantly, in vivo MRI and single voxel proton MR-Spectroscopy further established that VMY-1-103 inhibited disease progression and affected key metabolites in a mouse model of hedgehog-driven medulloblastoma.

  4. Dopamine signaling negatively regulates striatal phosphorylation of Cdk5 at tyrosine 15 in mice.

    Directory of Open Access Journals (Sweden)

    Yukio eYamamura

    2013-02-01

    Full Text Available Striatal functions depend on the activity balance between the dopamine and glutamate neurotransmissions. Glutamate inputs activate cyclin-dependent kinase 5 (Cdk5, which inhibits postsynaptic dopamine signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa at Thr75 in the striatum. c-Abelson tyrosine kinase (c-Abl is known to phosphorylate Cdk5 at Tyr15 (Tyr15-Cdk5 and thereby facilitates the Cdk5 activity. We here report that Cdk5 with Tyr15 phosphorylation (Cdk5-pTyr15 is enriched in the mouse striatum, where dopaminergic stimulation inhibited phosphorylation of Tyr15-Cdk5 by acting through the D2 class dopamine receptors. Moreover, in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine mouse model, dopamine deficiency caused increased phosphorylation of both Tyr15-Cdk5 and Thr75-DARPP-32 in the striatum, which could be attenuated by administration of L-3,4-dihydroxyphenylalanine and imatinib (STI-571, a selective c-Abl inhibitor. Our results suggest a functional link of Cdk5-pTyr15 with postsynaptic dopamine and glutamate signals through the c-Abl kinase activity in the striatum.

  5. The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

    Science.gov (United States)

    Stivala, L A; Savio, M; Quarta, S; Scotti, C; Cazzalini, O; Rossi, L; Scovassi, I A; Pizzala, R; Melli, R; Bianchi, L; Vannini, V; Prosperi, E

    2000-04-01

    In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

  6. Discovery of N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7 H-pyrrolo[2,3- d]pyrimidin-2-yl)amino)phenyl)- N8-hydroxyoctanediamide as a Novel Inhibitor Targeting Cyclin-dependent Kinase 4/9 (CDK4/9) and Histone Deacetlyase1 (HDAC1) against Malignant Cancer.

    Science.gov (United States)

    Li, Yongtao; Luo, Xiaohe; Guo, Qingxiang; Nie, Yongwei; Wang, Tianqi; Zhang, Chao; Huang, Zhi; Wang, Xin; Liu, Yanhua; Chen, Yanan; Zheng, Jianyu; Yang, Shengyong; Fan, Yan; Xiang, Rong

    2018-04-12

    A series of novel, highly potent, selective inhibitors targeting both CDK4/9 and HDAC1 have been designed and synthesized. N1-(4-((7-Cyclopentyl-6-(dimethylcarbamoyl)-7 H-pyrrolo[2,3- d] pyrimidin-2-yl)amino)phenyl)- N8-hydroxyoctanediamide (6e) was discovered. The lead compound 6e with excellent CDK4/9 and HDAC1 inhibitory activity of IC 50 = 8.8, 12, and 2.2 nM, respectively, can effectively induce apoptosis of cancer cell lines. The kinase profiling of compound 6e showed excellent selectivity and specificity. Compound 6e induces G2/M arrest in high concentration and G0/G1 arrest in low concentration to prevent the proliferation and differentiation of cancer cells. Mice bared-breast cancer treated with 6e showed significant antitumor efficacy. The insight into mechanisms of 6e indicated that it could induce cancer cell death via cell apoptosis based on CDK4/9 and HDAC1 repression and phosphorylation of p53. Our data demonstrated the novel compound 6e could be a promising drug candidate for cancer therapy.

  7. Requirement for CDK6 in MLL-rearranged acute myeloid leukemia.

    Science.gov (United States)

    Placke, Theresa; Faber, Katrin; Nonami, Atsushi; Putwain, Sarah L; Salih, Helmut R; Heidel, Florian H; Krämer, Alwin; Root, David E; Barbie, David A; Krivtsov, Andrei V; Armstrong, Scott A; Hahn, William C; Huntly, Brian J; Sykes, Stephen M; Milsom, Michael D; Scholl, Claudia; Fröhling, Stefan

    2014-07-03

    Chromosomal rearrangements involving the H3K4 methyltransferase mixed-lineage leukemia (MLL) trigger aberrant gene expression in hematopoietic progenitors and give rise to an aggressive subtype of acute myeloid leukemia (AML). Insights into MLL fusion-mediated leukemogenesis have not yet translated into better therapies because MLL is difficult to target directly, and the identity of the genes downstream of MLL whose altered transcription mediates leukemic transformation are poorly annotated. We used a functional genetic approach to uncover that AML cells driven by MLL-AF9 are exceptionally reliant on the cell-cycle regulator CDK6, but not its functional homolog CDK4, and that the preferential growth inhibition induced by CDK6 depletion is mediated through enhanced myeloid differentiation. CDK6 essentiality is also evident in AML cells harboring alternate MLL fusions and a mouse model of MLL-AF9-driven leukemia and can be ascribed to transcriptional activation of CDK6 by mutant MLL. Importantly, the context-dependent effects of lowering CDK6 expression are closely phenocopied by a small-molecule CDK6 inhibitor currently in clinical development. These data identify CDK6 as critical effector of MLL fusions in leukemogenesis that might be targeted to overcome the differentiation block associated with MLL-rearranged AML, and underscore that cell-cycle regulators may have distinct, noncanonical, and nonredundant functions in different contexts. © 2014 by The American Society of Hematology.

  8. Combined Inhibition of CDK4/6 and PI3K/AKT/mTOR Pathways Induces a Synergistic Anti-Tumor Effect in Malignant Pleural Mesothelioma Cells.

    Science.gov (United States)

    Bonelli, Mara A; Digiacomo, Graziana; Fumarola, Claudia; Alfieri, Roberta; Quaini, Federico; Falco, Angela; Madeddu, Denise; La Monica, Silvia; Cretella, Daniele; Ravelli, Andrea; Ulivi, Paola; Tebaldi, Michela; Calistri, Daniele; Delmonte, Angelo; Ampollini, Luca; Carbognani, Paolo; Tiseo, Marcello; Cavazzoni, Andrea; Petronini, Pier Giorgio

    2017-08-01

    Malignant pleural mesothelioma (MPM) is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF, encoding for the cell cycle inhibitors p16 INK4a and p14 ARF , deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991), a highly selective inhibitor of cyclin-dependent kinase (CDK) 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  9. Combined Inhibition of CDK4/6 and PI3K/AKT/mTOR Pathways Induces a Synergistic Anti-Tumor Effect in Malignant Pleural Mesothelioma Cells

    Directory of Open Access Journals (Sweden)

    Mara A. Bonelli

    2017-08-01

    Full Text Available Malignant pleural mesothelioma (MPM is a progressive malignancy associated to the exposure of asbestos fibers. The most frequently inactivated tumor suppressor gene in MPM is CDKN2A/ARF, encoding for the cell cycle inhibitors p16INK4a and p14ARF, deleted in about 70% of MPM cases. Considering the high frequency of alterations of this gene, we tested in MPM cells the efficacy of palbociclib (PD-0332991, a highly selective inhibitor of cyclin-dependent kinase (CDK 4/6. The analyses were performed on a panel of MPM cell lines and on two primary culture cells from pleural effusion of patients with MPM. All the MPM cell lines, as well as the primary cultures, were sensitive to palbociclib with a significant blockade in G0/G1 phase of the cell cycle and with the acquisition of a senescent phenotype. Palbociclib reduced the phosphorylation levels of CDK6 and Rb, the expression of myc with a concomitant increased phosphorylation of AKT. Based on these results, we tested the efficacy of the combination of palbociclib with the PI3K inhibitors NVP-BEZ235 or NVP-BYL719. After palbociclib treatment, the sequential association with PI3K inhibitors synergistically hampered cell proliferation and strongly increased the percentage of senescent cells. In addition, AKT activation was repressed while p53 and p21 were up-regulated. Interestingly, two cycles of sequential drug administration produced irreversible growth arrest and senescent phenotype that were maintained even after drug withdrawal. These findings suggest that the sequential association of palbociclib with PI3K inhibitors may represent a valuable therapeutic option for the treatment of MPM.

  10. MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos.

    Science.gov (United States)

    Kisielewska, J; Philipova, R; Huang, J-Y; Whitaker, M

    2009-10-15

    Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca(2+) signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27(kip1) and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27(kip1) had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells.

  11. Immunohistochemical analysis of the D-type cyclin-dependent kinases Cdk4 and Cdk6, using a series of monoclonal antibodies.

    Science.gov (United States)

    Lukas, C; Jensen, S K; Bartkova, J; Lukas, J; Bartek, J

    1999-06-01

    Cellular signal transduction cascades triggered by mitogenic or antiproliferative cues eventually converge on a biochemical mechanism centered around the retinoblastoma tumor suppressor (pRb), the so-called RB pathway that governs G1-phase progression and guards the commitment to enter S phase. pRb, together with its immediate upstream regulators, the D-type cyclins, their partner cyclin-dependent kinases Cdk4 and Cdk6, and the Cdk inhibitors, form a functional unit that is involved in major decisions about cellular fate, and whose components, including the proto-oncogenic cyclin D-dependent kinases, are commonly deregulated in many types of cancer. We report here the production and characterization of a series of 12 monoclonal antibodies (MAbs) that specifically recognize either Cdk4 or Cdk6. These antibodies are proving to be invaluable molecular probes for defining abundance, subcellular localization, binding partners, and ultimately the function(s) of these cell cycle-regulatory kinases. Localization of the target epitopes was mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and two antibodies recognizing sequences adjacent to N-terminus of Cdk4 can discriminate between the wild-type protein and the oncogenic, melanoma-associated R24C mutant of this kinase. Individual antibodies of our panel recognize distinct pools of Cdk4/6, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, kinase assays, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Collectively, the antibodies described in this study provide the means for immunochemical analyses of the cyclin D-dependent kinases in human and animal cells, and represent useful molecular tools that should help better understand the biological roles of Cdk4 and Cdk6 in normal cell-cycle control, and their oncogenic activity in tumor cells.

  12. Rb and FZR1/Cdh1 determine CDK4/6-cyclin D requirement in C. elegans and human cancer cells.

    Science.gov (United States)

    The, Inge; Ruijtenberg, Suzan; Bouchet, Benjamin P; Cristobal, Alba; Prinsen, Martine B W; van Mourik, Tim; Koreth, John; Xu, Huihong; Heck, Albert J R; Akhmanova, Anna; Cuppen, Edwin; Boxem, Mike; Muñoz, Javier; van den Heuvel, Sander

    2015-01-06

    Cyclin-dependent kinases 4 and 6 (CDK4/6) in complex with D-type cyclins promote cell cycle entry. Most human cancers contain overactive CDK4/6-cyclin D, and CDK4/6-specific inhibitors are promising anti-cancer therapeutics. Here, we investigate the critical functions of CDK4/6-cyclin D kinases, starting from an unbiased screen in the nematode Caenorhabditis elegans. We found that simultaneous mutation of lin-35, a retinoblastoma (Rb)-related gene, and fzr-1, an orthologue to the APC/C co-activator Cdh1, completely eliminates the essential requirement of CDK4/6-cyclin D (CDK-4/CYD-1) in C. elegans. CDK-4/CYD-1 phosphorylates specific residues in the LIN-35 Rb spacer domain and FZR-1 amino terminus, resembling inactivating phosphorylations of the human proteins. In human breast cancer cells, simultaneous knockdown of Rb and FZR1 synergistically bypasses cell division arrest induced by the CDK4/6-specific inhibitor PD-0332991. Our data identify FZR1 as a candidate CDK4/6-cyclin D substrate and point to an APC/C(FZR1) activity as an important determinant in response to CDK4/6-inhibitors.

  13. LMW-E/CDK2 deregulates acinar morphogenesis, induces tumorigenesis, and associates with the activated b-Raf-ERK1/2-mTOR pathway in breast cancer patients.

    Directory of Open Access Journals (Sweden)

    MyLinh T Duong

    Full Text Available Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E isoforms exhibiting enhanced CDK2-associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E-expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor plus either rapamycin (mTOR inhibitor or sorafenib (a pan kinase inhibitor targeting b-Raf effectively prevented aberrant acinar formation in LMW-E-expressing cells by inducing G1/S cell cycle arrest

  14. Resveratrol inhibits Cdk5 activity through regulation of p35 expression

    Directory of Open Access Journals (Sweden)

    Kulkarni Ashok B

    2011-07-01

    Full Text Available Abstract Background We have previously reported that cyclin-dependent kinase 5 (Cdk5 participates in the regulation of nociceptive signaling. Through activation of the ERK1/2 pathway, Tumor Necrosis Factor-α (TNF-α induces expression of Egr-1. This results in the sustained and robust expression of p35, a coactivator of Cdk5, in PC12 cells, thereby increasing Cdk5 kinase activity. The aim of our present study was to test whether resveratrol, a polyphenolic compound with known analgesic activity, can regulate Cdk5/p35 activity. Results Here we used a cell-based assay in which a p35 promoter-luciferase construct was stably transfected in PC12 cells. Our studies demonstrate that resveratrol inhibits p35 promoter activity and also blocks the TNF-α mediated increase in Cdk5 activity in PC12 cells. Resveratrol also inhibits p35 expression and blocks the TNF-α mediated increase in Cdk5 activity in DRG neurons. In the presence of resveratrol, the MEK inhibitor decreased p35 promoter activity, whereas the inhibitors of p38 MAPK, JNK and NF-κB increased p35 promoter activity, indicating that these pathways regulate p35 expression differently. The TNF-α-mediated increase in Egr-1 expression was decreased by resveratrol treatment with a concomitant reduction in p35 expression and protein levels, resulting in reduced Cdk5 kinase activity. Conclusions We demonstrate here that resveratrol regulates p35 promoter activity in PC12 cells and DRG neurons. Most importantly, resveratrol blocks the TNF-α-mediated increase in p35 promoter activity, thereby reducing p35 expression and subsequent Cdk5 kinase activity. This new molecular mechanism adds to the known analgesic effects of resveratrol and confirms the need for identifying new analgesics based on their ability to inhibit Cdk5 activity for effective treatment of pain.

  15. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    International Nuclear Information System (INIS)

    Qiao, Lan; Paul, Pritha; Lee, Sora; Qiao, Jingbo; Wang, Yongsheng; Chung, Dai H.

    2013-01-01

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma

  16. Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

    2013-05-31

    Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

  17. Nitric oxide induces thioredoxin-1 nuclear translocation: Possible association with the p21Ras survival pathway

    International Nuclear Information System (INIS)

    Arai, Roberto J.; Masutani, H.; Yodoi, J.; Debbas, V.; Laurindo, Francisco R.; Stern, A.; Monteiro, Hugo P.

    2006-01-01

    One of the major redox-regulating molecules with thiol reducing activity is thioredoxin-1 (TRX-1). TRX-1 is a multifunctional protein that exists in the extracellular millieu, cytoplasm, and nucleus, and has a distinct role in each environment. It is well known that TRX-1 promptly migrates to the nuclear compartment in cells exposed to oxidants. However, the intracellular location of TRX-1 in cells exposed to nitrosothiols has not been investigated. Here, we demonstrated that the exposure of HeLa cells to increasing concentrations of the nitrosothiol S-nitroso-N-acetylpenicillamine (SNAP) promoted TRX-1 nuclear accumulation. The SNAP-induced TRX-1 translocation to the nucleus was inhibited by FPTIII, a selective inhibitor of p21Ras. Furthermore, TRX-1 migration was attenuated in cells stably transfected with NO insensitive p21Ras (p21 RasC118S ). Downstream to p21Ras, the MAP Kinases ERK1/2 were activated by SNAP under conditions that promote TRX-1 nuclear translocation. Inhibition of MEK prevented SNAP-stimulated ERK1/2 activation and TRX-1 nuclear migration. In addition, cells treated with p21Ras or MEK inhibitor showed increased susceptibility to cell death induced by SNAP. In conclusion, our observations suggest that the nuclear translocation of TRX-1 is induced by SNAP involving p21Ras survival pathway

  18. Insights on Structural Characteristics and Ligand Binding Mechanisms of CDK2

    Directory of Open Access Journals (Sweden)

    Yan Li

    2015-04-01

    Full Text Available Cyclin-dependent kinase 2 (CDK2 is a crucial regulator of the eukaryotic cell cycle. However it is well established that monomeric CDK2 lacks regulatory activity, which needs to be aroused by its positive regulators, cyclins E and A, or be phosphorylated on the catalytic segment. Interestingly, these activation steps bring some dynamic changes on the 3D-structure of the kinase, especially the activation segment. Until now, in the monomeric CDK2 structure, three binding sites have been reported, including the adenosine triphosphate (ATP binding site (Site I and two non-competitive binding sites (Site II and III. In addition, when the kinase is subjected to the cyclin binding process, the resulting structural changes give rise to a variation of the ATP binding site, thus generating an allosteric binding site (Site IV. All the four sites are demonstrated as being targeted by corresponding inhibitors, as is illustrated by the allosteric binding one which is targeted by inhibitor ANS (fluorophore 8-anilino-1-naphthalene sulfonate. In the present work, the binding mechanisms and their fluctuations during the activation process attract our attention. Therefore, we carry out corresponding studies on the structural characterization of CDK2, which are expected to facilitate the understanding of the molecular mechanisms of kinase proteins. Besides, the binding mechanisms of CDK2 with its relevant inhibitors, as well as the changes of binding mechanisms following conformational variations of CDK2, are summarized and compared. The summary of the conformational characteristics and ligand binding mechanisms of CDK2 in the present work will improve our understanding of the molecular mechanisms regulating the bioactivities of CDK2.

  19. Overexpression of DOC-1R inhibits cell cycle G1/S transition by repressing CDK2 expression and activation.

    Science.gov (United States)

    Liu, Qi; Liu, Xing; Gao, Jinlan; Shi, Xiuyan; Hu, Xihua; Wang, Shusen; Luo, Yang

    2013-01-01

    DOC-1R (deleted in oral cancer-1 related) is a novel putative tumor suppressor. This study investigated DOC-1R antitumor activity and the underlying molecular mechanisms. Cell phenotypes were assessed using flow cytometry, BrdU incorporation and CDK2 kinase assays in DOC-1R overexpressing HeLa cells. In addition, RT-PCR and Western blot assays were used to detect underlying molecular changes in these cells. The interaction between DOC-1R and CDK2 proteins was assayed by GST pull-down and immunoprecipitation-Western blot assays. The data showed that DOC-1R overexpression inhibited G1/S phase transition, DNA replication and suppressed CDK2 activity. Molecularly, DOC-1R inhibited CDK2 expression at the mRNA and protein levels, and there were decreased levels of G1-phase cyclins (cyclin D1 and E) and elevated levels of p21, p27, and p53 proteins. Meanwhile, DOC-1R associated with CDK2 and inhibited CDK2 activation by obstructing its association with cyclin E and A. In conclusion, the antitumor effects of DOC-1R may be mediated by negatively regulating G1 phase progression and G1/S transition through inhibiting CDK2 expression and activation.

  20. Cyclin A2 and CDK2 as Novel Targets of Aspirin and Salicylic Acid: A Potential Role in Cancer Prevention.

    Science.gov (United States)

    Dachineni, Rakesh; Ai, Guoqiang; Kumar, D Ramesh; Sadhu, Satya S; Tummala, Hemachand; Bhat, G Jayarama

    2016-03-01

    Data emerging from the past 10 years have consolidated the rationale for investigating the use of aspirin as a chemopreventive agent; however, the mechanisms leading to its anticancer effects are still being elucidated. We hypothesized that aspirin's chemopreventive actions may involve cell-cycle regulation through modulation of the levels or activity of cyclin A2/cyclin-dependent kinase-2 (CDK2). In this study, HT-29 and other diverse panel of cancer cells were used to demonstrate that both aspirin and its primary metabolite, salicylic acid, decreased cyclin A2 (CCNA2) and CDK2 protein and mRNA levels. The downregulatory effect of either drugs on cyclin A2 levels was prevented by pretreatment with lactacystin, an inhibitor of proteasomes, suggesting the involvement of 26S proteasomes. In-vitro kinase assays showed that lysates from cells treated with salicylic acid had lower levels of CDK2 activity. Importantly, three independent experiments revealed that salicylic acid directly binds to CDK2. First, inclusion of salicylic acid in naïve cell lysates, or in recombinant CDK2 preparations, increased the ability of the anti-CDK2 antibody to immunoprecipitate CDK2, suggesting that salicylic acid may directly bind and alter its conformation. Second, in 8-anilino-1-naphthalene-sulfonate (ANS)-CDK2 fluorescence assays, preincubation of CDK2 with salicylic acid dose-dependently quenched the fluorescence due to ANS. Third, computational analysis using molecular docking studies identified Asp145 and Lys33 as the potential sites of salicylic acid interactions with CDK2. These results demonstrate that aspirin and salicylic acid downregulate cyclin A2/CDK2 proteins in multiple cancer cell lines, suggesting a novel target and mechanism of action in chemoprevention. Biochemical and structural studies indicate that the antiproliferative actions of aspirin are mediated through cyclin A2/CDK2. ©2015 American Association for Cancer Research.

  1. Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization

    DEFF Research Database (Denmark)

    Petersen, B O; Lukas, J; Sørensen, Claus Storgaard

    1999-01-01

    by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo...

  2. CDK4/6 blockade in breast cancer: current experience and future perspectives.

    Science.gov (United States)

    Zardavas, Dimitrios; Pondé, Noam; Tryfonidis, Konstantinos

    2017-12-01

    Dysregulated cellular proliferation, one of the hallmarks of cancer, is mediated by aberrant activation of the cell cycle machinery through the biological effects of cyclin-dependent kinases (CDKs). The clinical development of non-selective CDK inhibitors failed due to combined lack of efficacy and excessive toxicity reported by clinical trials across different cancer types. The clinical development of second generation, CDK4/6-selective inhibitors, namely palbociclib, abemaciclib and ribociclib, led to practice-changing results in the setting of breast cancer. Areas covered: This review illustrates how CDK4/6-selective inhibitors got approval for the treatment of patients with either newly diagnosed or pretreated advanced hormone receptor positive, HER2-negative breast cancer. Furthermore, data about potential predictive biomarkers, as well as preclinical and preliminary clinical evidence for potential antitumor activity of CDK4/6 inhibition in other breast cancer subtypes is provided. Expert opinion: Future clinical development of CDK4/6 inhibitors in breast cancer will focus on the following aspects: i) optimization of treatment sequencing for patients with advanced disease, ii) early-stage disease, iii) other subtypes of breast cancer in rationally chosen therapeutic combinations and iv) the identification of predictive biomarkers.

  3. Molecular Dynamics Simulations and Classical Multidimensional Scaling Unveil New Metastable States in the Conformational Landscape of CDK2.

    Directory of Open Access Journals (Sweden)

    Pasquale Pisani

    Full Text Available Protein kinases are key regulatory nodes in cellular networks and their function has been shown to be intimately coupled with their structural flexibility. However, understanding the key structural mechanisms of large conformational transitions remains a difficult task. CDK2 is a crucial regulator of cell cycle. Its activity is finely tuned by Cyclin E/A and the catalytic segment phosphorylation, whereas its deregulation occurs in many types of cancer. ATP competitive inhibitors have failed to be approved for clinical use due to toxicity issues raised by a lack of selectivity. However, in the last few years type III allosteric inhibitors have emerged as an alternative strategy to selectively modulate CDK2 activity. In this study we have investigated the conformational variability of CDK2. A low dimensional conformational landscape of CDK2 was modeled using classical multidimensional scaling on a set of 255 crystal structures. Microsecond-scale plain and accelerated MD simulations were used to populate this landscape by using an out-of-sample extension of multidimensional scaling. CDK2 was simulated in the apo-form and in complex with the allosteric inhibitor 8-anilino-1-napthalenesulfonic acid (ANS. The apo-CDK2 landscape analysis showed a conformational equilibrium between an Src-like inactive conformation and an active-like form. These two states are separated by different metastable states that share hybrid structural features with both forms of the kinase. In contrast, the CDK2/ANS complex landscape is compatible with a conformational selection picture where the binding of ANS in proximity of the αC helix causes a population shift toward the inactive conformation. Interestingly, the new metastable states could enlarge the pool of candidate structures for the development of selective allosteric CDK2 inhibitors. The method here presented should not be limited to the CDK2 case but could be used to systematically unmask similar mechanisms

  4. Targeting CDK1 and MEK/ERK Overcomes Apoptotic Resistance in BRAF-Mutant Human Colorectal Cancer.

    Science.gov (United States)

    Zhang, Peng; Kawakami, Hisato; Liu, Weizhen; Zeng, Xiangyu; Strebhardt, Klaus; Tao, Kaixiong; Huang, Shengbing; Sinicrope, Frank A

    2018-03-01

    The BRAF V600E mutation occurs in approximately 8% of human colorectal cancers and is associated with therapeutic resistance that is due, in part, to reactivation of MEK/ERK signaling cascade. Recently, pathway analysis identified cyclin-dependent kinase 1 (CDK1) upregulation in a subset of human BRAF V600E colorectal cancers. Therefore, it was determined whether CDK1 antagonism enhances the efficacy of MEK inhibition in BRAF V600E colorectal cancer cells. BRAF V600E colorectal cancer cell lines expressing CDK1 were sensitized to apoptosis upon siRNA knockdown or small-molecule inhibition with RO-3306 (CDK1 inhibitor) or dinaciclib (CDK1, 2, 5, 9 inhibitors). Combination of RO-3306 or dinaciclib with cobimetinib (MEK inhibitor) cooperatively enhanced apoptosis and reduced clonogenic survival versus monotherapy. Cells isogenic or ectopic for BRAF V600E displayed resistance to CDK1 inhibitors, as did cells with ectopic expression of constitutively active MEK CDK1 inhibitors induced a CASP8 -dependent apoptosis shown by caspase-8 restoration in deficient NB7 cells that enhanced dinaciclib-induced CASP3 cleavage. CDK inhibitors suppressed pro-CASP8 phosphorylation at S387, as shown by drug withdrawal, which restored p-S387 and increased mitosis. In a colorectal cancer xenograft model, dinaciclib plus cobimetinib produced significantly greater tumor growth inhibition in association with a caspase-dependent apoptosis versus either drug alone. The Cancer Genome Atlas (TCGA) transcriptomic dataset revealed overexpression of CDK1 in human colorectal cancers versus normal colon. Together, these data establish CDK1 as a novel mediator of apoptosis resistance in BRAF V600E colorectal cancers whose combined targeting with a MEK/ERK inhibitor represents an effective therapeutic strategy. Implications: CDK1 is a novel mediator of apoptosis resistance in BRAF V600E colorectal cancers whose dual targeting with a MEK inhibitor may be therapeutically effective. Mol Cancer Res; 16

  5. Activation of CDK4 Triggers Development of Non-alcoholic Fatty Liver Disease.

    Science.gov (United States)

    Jin, Jingling; Valanejad, Leila; Nguyen, Thuy Phuong; Lewis, Kyle; Wright, Mary; Cast, Ashley; Stock, Lauren; Timchenko, Lubov; Timchenko, Nikolai A

    2016-07-19

    The development of non-alcoholic fatty liver disease (NAFLD) is a multiple step process. Here, we show that activation of cdk4 triggers the development of NAFLD. We found that cdk4 protein levels are elevated in mouse models of NAFLD and in patients with fatty livers. This increase leads to C/EBPα phosphorylation on Ser193 and formation of C/EBPα-p300 complexes, resulting in hepatic steatosis, fibrosis, and hepatocellular carcinoma (HCC). The disruption of this pathway in cdk4-resistant C/EBPα-S193A mice dramatically reduces development of high-fat diet (HFD)-mediated NAFLD. In addition, inhibition of cdk4 by flavopiridol or PD-0332991 significantly reduces development of hepatic steatosis, the first step of NAFLD. Thus, this study reveals that activation of cdk4 triggers NAFLD and that inhibitors of cdk4 may be used for the prevention/treatment of NAFLD. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  6. CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation.

    Science.gov (United States)

    Deng, Jiehui; Wang, Eric S; Jenkins, Russell W; Li, Shuai; Dries, Ruben; Yates, Kathleen; Chhabra, Sandeep; Huang, Wei; Liu, Hongye; Aref, Amir R; Ivanova, Elena; Paweletz, Cloud P; Bowden, Michaela; Zhou, Chensheng W; Herter-Sprie, Grit S; Sorrentino, Jessica A; Bisi, John E; Lizotte, Patrick H; Merlino, Ashley A; Quinn, Max M; Bufe, Lauren E; Yang, Annan; Zhang, Yanxi; Zhang, Hua; Gao, Peng; Chen, Ting; Cavanaugh, Megan E; Rode, Amanda J; Haines, Eric; Roberts, Patrick J; Strum, Jay C; Richards, William G; Lorch, Jochen H; Parangi, Sareh; Gunda, Viswanath; Boland, Genevieve M; Bueno, Raphael; Palakurthi, Sangeetha; Freeman, Gordon J; Ritz, Jerome; Haining, W Nicholas; Sharpless, Norman E; Arthanari, Haribabu; Shapiro, Geoffrey I; Barbie, David A; Gray, Nathanael S; Wong, Kwok-Kin

    2018-02-01

    Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo , due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies. Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR. See related commentary by Balko and Sosman, p. 143 See related article by Jenkins et al., p. 196 This article is highlighted in the In This Issue feature, p. 127 . ©2017 American Association for Cancer Research.

  7. CDK1 and CDK2 activity is a strong predictor of renal cell carcinoma recurrence.

    Science.gov (United States)

    Hongo, Fumiya; Takaha, Natsuki; Oishi, Masakatsu; Ueda, Takashi; Nakamura, Terukazu; Naitoh, Yasuyuki; Naya, Yoshio; Kamoi, Kazumi; Okihara, Koji; Matsushima, Tomoko; Nakayama, Satoshi; Ishihara, Hideki; Sakai, Toshiyuki; Miki, Tsuneharu

    2014-11-01

    In renal cell carcinoma (RCC), the prediction of metastasis via tumor prognostic markers remains a major problem. The objective of our study was to evaluate the efficacy of cyclin-dependent kinase (CDK)1 and CDK2 activity as a prognostic marker in human RCC. Surgical specimens were obtained from 125 patients with RCC without metastasis. Protein expression and kinase activity of CDKs were analyzed using a newly developed assay system named C2P (Sysmex, Kobe, Japan). We then examined the specific activities (SAs) of CDK1 and CDK2 and calculated CDK2SA-CDK1SA ratio in RCC. Also, risk score (RS) was examined. A total of 125 cases were tested, though 34 cases were excluded because of low sample quality (25 cases) and assay failure (9 cases). In total, 91 cases were analyzed. They included 68 male and 23 female patients, ranging in age from 19 to 83 years. At a median follow-up of 36 months (1-109M), tumor with low CDK2SA-CDK1SA ratio showed significantly better 5-year recurrence-free survival than those with high CDK2SA-CDK1SA ratio (88.7% vs. 54.7%, P = 0.00141). Also, RS enabled the classification of RCCs into high-risk and low-risk groups, and patients with tumors classified as low RS showed better recurrence-free survival than patients with tumors with high RS (88.7% vs. 54.7%, P = 0.0141). CDK1SA of tumors and the CDK2SA are both associated with recurrence and prognosis. CDK-based risk demonstrated is strongly associated with clinical outcome. CDK-based risk should be an accurate system for predicting recurrence and survival for planning follow-up. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Cyclin D-cdk6 complex is targeted by p21WAF in growth-arrested lymphoma cells

    Czech Academy of Sciences Publication Activity Database

    Tvrdík, Daniel; Djaborkhel, Rashed; Nagy, Alexandr; Eckschlager, T.; Raška, Ivan; Müller, Julius

    2002-01-01

    Roč. 140, - (2002), s. 49-56 ISSN 1047-8477 R&D Projects: GA ČR GA304/01/0564 Institutional research plan: CEZ:AV0Z5039906; CEZ:MSM 111100003 Keywords : cyclin-dependent kinase * follicular lymphoma * retinoblastoma protein Subject RIV: EA - Cell Biology Impact factor: 4.194, year: 2002

  9. Coordination between p21 and DDB2 in the cellular response to UV radiation.

    Directory of Open Access Journals (Sweden)

    Hao Li

    Full Text Available The tumor suppressor p53 guides the cellular response to DNA damage mainly by regulating expression of target genes. The cyclin-dependent kinase inhibitor p21, which is induced by p53, can both arrest the cell cycle and inhibit apoptosis. Interestingly, p53-inducible DDB2 (damaged-DNA binding protein 2 promotes apoptosis by mediating p21 degradation after ultraviolet (UV-induced DNA damage. Here, we developed an integrated model of the p53 network to explore how the UV-irradiated cell makes a decision between survival and death and how the activities of p21 and DDB2 are modulated. By numerical simulations, we found that p53 is activated progressively and the promoter selectivity of p53 depends on its concentration. For minor DNA damage, p53 settles at an intermediate level. p21 is induced by p53 to arrest the cell cycle via inhibiting E2F1 activity, allowing for DNA repair. The proapoptotic genes are expressed at low levels. For severe DNA damage, p53 undergoes a two-phase behavior and accumulates to high levels in the second phase. Consequently, those proapoptotic proteins accumulate remarkably. Bax activates the release of cytochrome c, while DDB2 promotes the degradation of p21, which leads to activation of E2F1 and induction of Apaf-1. Finally, the caspase cascade is activated to trigger apoptosis. We revealed that the downregulation of p21 is necessary for apoptosis induction and PTEN promotes apoptosis by amplifying p53 activation. This work demonstrates that how the dynamics of the p53 network can be finely regulated through feed-forward and feedback loops within the network and emphasizes the importance of p21 regulation in the DNA damage response.

  10. p21(WAF1/CIP1 RNA expression in highly HIV-1 exposed, uninfected individuals.

    Directory of Open Access Journals (Sweden)

    Joshua Herbeck

    Full Text Available Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN. However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior, p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80. Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year and lowest 40 LESC (median = 2 partners/year showed no difference between the groups (P = 0.84. There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12, but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.

  11. P21 Deficiency Delays Regeneration of Skeletal Muscular Tissue

    OpenAIRE

    Chinzei, Nobuaki; Hayashi, Shinya; Ueha, Takeshi; Fujishiro, Takaaki; Kanzaki, Noriyuki; Hashimoto, Shingo; Sakata, Shuhei; Kihara, Shinsuke; Haneda, Masahiko; Sakai, Yoshitada; Kuroda, Ryosuke; Kurosaka, Masahiro

    2015-01-01

    The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation....

  12. P21 deficiency delays regeneration of skeletal muscular tissue.

    Science.gov (United States)

    Chinzei, Nobuaki; Hayashi, Shinya; Ueha, Takeshi; Fujishiro, Takaaki; Kanzaki, Noriyuki; Hashimoto, Shingo; Sakata, Shuhei; Kihara, Shinsuke; Haneda, Masahiko; Sakai, Yoshitada; Kuroda, Ryosuke; Kurosaka, Masahiro

    2015-01-01

    The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO) mice and wild type (WT) mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

  13. P21 deficiency delays regeneration of skeletal muscular tissue.

    Directory of Open Access Journals (Sweden)

    Nobuaki Chinzei

    Full Text Available The potential relationship between cell cycle checkpoint control and tissue regeneration has been indicated. Despite considerable research being focused on the relationship between p21 and myogenesis, p21 function in skeletal muscle regeneration remains unclear. To clarify this, muscle injury model was recreated by intramuscular injection of bupivacaine hydrochloride in the soleus of p21 knockout (KO mice and wild type (WT mice. The mice were sacrificed at 3, 14, and 28 days post-operation. The results of hematoxylin-eosin staining and immunofluorescence of muscle membrane indicated that muscle regeneration was delayed in p21 KO mice. Cyclin D1 mRNA expression and both Ki-67 and PCNA immunohistochemistry suggested that p21 deficiency increased cell cycle and muscle cell proliferation. F4/80 immunohistochemistry also suggested the increase of immune response in p21 KO mice. On the other hand, both the mRNA expression and western blot analysis of MyoD, myogenin, and Pax7 indicated that muscular differentiation was delayed in p21KO mice. Considering these results, we confirmed that muscle injury causes an increase in cell proliferation. However, muscle differentiation in p21 KO mice was inhibited due to the low expression of muscular synthesis genes, leading to a delay in the muscular regeneration. Thus, we conclude that p21 plays an important role in the in vivo healing process in muscular injury.

  14. Inhibitors

    Science.gov (United States)

    ... Icon View public health webinars on blood disorders Inhibitors Language: English (US) Español (Spanish) Recommend on Facebook ... because treatment of bleeds becomes less effective. About Inhibitors People with hemophilia, and many with VWD type ...

  15. Pitx2 expression promotes p21 expression and cell cycle exit in neural stem cells.

    Science.gov (United States)

    Heldring, Nina; Joseph, Bertrand; Hermanson, Ola; Kioussi, Chrissa

    2012-11-01

    Cortical development is a complex process that involves many events including proliferation, cell cycle exit and differentiation that need to be appropriately synchronized. Neural stem cells (NSCs) isolated from embryonic cortex are characterized by their ability of self-renewal under continued maintenance of multipotency. Cell cycle progression and arrest during development is regulated by numerous factors, including cyclins, cyclin dependent kinases and their inhibitors. In this study, we exogenously expressed the homeodomain transcription factor Pitx2, usually expressed in postmitotic progenitors and neurons of the embryonic cortex, in NSCs with low expression of endogenous Pitx2. We found that Pitx2 expression induced a rapid decrease in proliferation associated with an accumulation of NSCs in G1 phase. A search for potential cell cycle inhibitors responsible for such cell cycle exit of NSCs revealed that Pitx2 expression caused a rapid and dramatic (≉20-fold) increase in expression of the cell cycle inhibitor p21 (WAF1/Cip1). In addition, Pitx2 bound directly to the p21 promoter as assessed by chromatin immunoprecipitation (ChIP) in NSCs. Surprisingly, Pitx2 expression was not associated with an increase in differentiation markers, but instead the expression of nestin, associated with undifferentiated NSCs, was maintained. Our results suggest that Pitx2 promotes p21 expression and induces cell cycle exit in neural progenitors.

  16. The CDK-APC/C Oscillator Predominantly Entrains Periodic Cell-Cycle Transcription

    Science.gov (United States)

    Rahi, Sahand Jamal; Pecani, Kresti; Ondracka, Andrej; Oikonomou, Catherine; Cross, Frederick R.

    2016-01-01

    Throughout cell cycle progression, the expression of multiple transcripts oscillate, and whether these are under the centralized control of the CDK-APC/C proteins or can be driven by a de-centralized transcription factor (TF) cascade is a fundamental question for understanding cell cycle regulation. In budding yeast, we find that the transcription of nearly all genes, as assessed by RNA-seq or fluorescence microscopy in single cells, is dictated by CDK-APC/C. Three exceptional genes are transcribed in a pulsatile pattern in a variety of CDK-APC/C arrests. Pursuing one of these transcripts, the SIC1 inhibitor of B-type cyclins, we use a combination of mathematical modeling and experimentation to provide evidence that, counter-intuitively, Sic1 provides a failsafe mechanism promoting nuclear division when levels of mitotic cyclins are low. PMID:27058667

  17. SKLB70326, a novel small-molecule inhibitor of cell-cycle progression, induces G{sub 0}/G{sub 1} phase arrest and apoptosis in human hepatic carcinoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Han, Yuanyuan; He, Haiyun [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Peng, Feng [Department of Thoracic Oncology of the Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Liu, Jiyan; Dai, Xiaoyun; Lin, Hongjun; Xu, Youzhi; Zhou, Tian; Mao, Yongqiu; Xie, Gang; Yang, Shengyong; Yu, Luoting; Yang, Li [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China); Zhao, Yinglan, E-mail: alancenxb@sina.com [State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041 (China)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer SKLB70326 is a novel compound and has activity of anti-HCC. Black-Right-Pointing-Pointer SKLB70326 induces cell cycle arrest and apoptosis in HepG2 cells. Black-Right-Pointing-Pointer SKLB70326 induces G{sub 0}/G{sub 1} phase arrest via inhibiting the activity of CDK2, CDK4 and CDK6. Black-Right-Pointing-Pointer SKLB70326 induces apoptosis through the intrinsic pathway. -- Abstract: We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro. We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro, and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G{sub 0}/G{sub 1} phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G{sub 0}/G{sub 1} phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

  18. Expression of p21 and p27 in gallbladder cancer

    International Nuclear Information System (INIS)

    Alsheyab, Fawzi M.; Ziadeh, Moroug T.; Bani-Hani, Kamal E.

    2007-01-01

    To investigate the expression of p21 and p27 factors in gallbladder cancer (GBC), and to correlate their expression with clinicopathological parameters: age, gender, stage, invasion and grade. Thirty-two surgically resected specimens were collected between 1994-2001 from different health centers in north Jordan. Tissues belong to 25 females and 7 males were examined immunohistochemically. The study took place in the Pathology Department, Jordan University of Science and Technology, Jordan. Levels of p21 were found in 75% and p27 in 25%. Furthermore, p21 was expressed in 50% of the specimens which belong to patients with ages 64 years have p 21WAF1/CIP1 expression (p=0.001). The expression of p21 between advanced stages (stages III and IV) was 89.5% and early stages (stages I and II) was 53.8% (p=0.031). The p27 expression was markedly decreased in GBC cases (25%) and there were no significant correlation between p27KIP1 expression and all clinicopathological parameters including gender, World health Organization grades, stages and invasion, whereas expression of p21 was 75% and there was a significant correlation between p21 and clinicopathological parameters including gender, stages and invasion. (author)

  19. An ERK/Cdk5 axis controls the diabetogenic actions of PPARγ.

    Science.gov (United States)

    Banks, Alexander S; McAllister, Fiona E; Camporez, João Paulo G; Zushin, Peter-James H; Jurczak, Michael J; Laznik-Bogoslavski, Dina; Shulman, Gerald I; Gygi, Steven P; Spiegelman, Bruce M

    2015-01-15

    Obesity-linked insulin resistance is a major precursor to the development of type 2 diabetes. Previous work has shown that phosphorylation of PPARγ (peroxisome proliferator-activated receptor γ) at serine 273 by cyclin-dependent kinase 5 (Cdk5) stimulates diabetogenic gene expression in adipose tissues. Inhibition of this modification is a key therapeutic mechanism for anti-diabetic drugs that bind PPARγ, such as the thiazolidinediones and PPARγ partial agonists or non-agonists. For a better understanding of the importance of this obesity-linked PPARγ phosphorylation, we created mice that ablated Cdk5 specifically in adipose tissues. These mice have both a paradoxical increase in PPARγ phosphorylation at serine 273 and worsened insulin resistance. Unbiased proteomic studies show that extracellular signal-regulated kinase (ERK) kinases are activated in these knockout animals. Here we show that ERK directly phosphorylates serine 273 of PPARγ in a robust manner and that Cdk5 suppresses ERKs through direct action on a novel site in MAP kinase/ERK kinase (MEK). Importantly, pharmacological inhibition of MEK and ERK markedly improves insulin resistance in both obese wild-type and ob/ob mice, and also completely reverses the deleterious effects of the Cdk5 ablation. These data show that an ERK/Cdk5 axis controls PPARγ function and suggest that MEK/ERK inhibitors may hold promise for the treatment of type 2 diabetes.

  20. p21(WAF1/CIP1 upregulation through the stress granule-associated protein CUGBP1 confers resistance to bortezomib-mediated apoptosis.

    Directory of Open Access Journals (Sweden)

    Cristina Gareau

    Full Text Available p21(WAF1/CIP1 is a well known cyclin-dependent kinase inhibitor induced by various stress stimuli. Depending on the stress applied, p21 upregulation can either promote apoptosis or prevent against apoptotic injury. The stress-mediated induction of p21 involves not only its transcriptional activation but also its posttranscriptional regulation, mainly through stabilization of p21 mRNA levels. We have previously reported that the proteasome inhibitor MG132 induces the stabilization of p21 mRNA, which correlates with the formation of cytoplasmic RNA stress granules. The mechanism underlying p21 mRNA stabilization, however, remains unknown.We identified the stress granules component CUGBP1 as a factor required for p21 mRNA stabilization following treatment with bortezomib ( =  PS-341/Velcade. This peptide boronate inhibitor of the 26S proteasome is very efficient for the treatment of myelomas and other hematological tumors. However, solid tumors are sometimes refractory to bortezomib treatment. We found that depleting CUGBP1 in cancer cells prevents bortezomib-mediated p21 upregulation. FISH experiments combined to mRNA stability assays show that this effect is largely due to a mistargeting of p21 mRNA in stress granules leading to its degradation. Altering the expression of p21 itself, either by depleting CUGBP1 or p21, promotes bortezomib-mediated apoptosis.We propose that one key mechanism by which apoptosis is inhibited upon treatment with chemotherapeutic drugs might involve upregulation of the p21 protein through CUGBP1.

  1. Familial partial duplication (1)(p21p31)

    Energy Technology Data Exchange (ETDEWEB)

    Hoechstetter, L.; Soukup, S.; Schorry, E.K. [Children`s Hospital Research Foundation, Cincinnati, OH (United States)

    1995-11-20

    A partial duplication (1)(p21p31), resulting from a maternal direct insertion (13,1) (q22p21p31), was found in a 30-year-old woman with mental retardation, cleft palate, and multiple minor anomalies. Two other affected and deceased relatives were presumed to have the same chromosome imbalance. Duplication 1p cases are reviewed. 8 refs., 5 figs., 1 tab.

  2. Cell Cycle Regulating Kinase Cdk4 as a Potential Target for Tumor Cell Treatment and Tumor Imaging

    Directory of Open Access Journals (Sweden)

    Franziska Graf

    2009-01-01

    Full Text Available The cyclin-dependent kinase (Cdk-cyclin D/retinoblastoma (pRb/E2F cascade, which controls the G1/S transition of cell cycle, has been found to be altered in many neoplasias. Inhibition of this pathway by using, for example, selective Cdk4 inhibitors has been suggested to be a promising approach for cancer therapy. We hypothesized that appropriately radiolabeled Cdk4 inhibitors are suitable probes for tumor imaging and may be helpful studying cell proliferation processes in vivo by positron emission tomography. Herein, we report the synthesis and biological, biochemical, and radiopharmacological characterizations of two I124-labeled small molecule Cdk4 inhibitors (8-cyclopentyl-6-iodo-5-methyl-2-(4-piperazin-1-yl-phenylamino-8H-pyrido[2,3-d]-pyrimidin-7-one (CKIA and 8-cyclopentyl-6-iodo-5-methyl-2-(5-(piperazin-1-yl-pyridin-2-yl-amino-8H-pyrido[2,3-d]pyrimidin-7-one (CKIB. Our data demonstrate a defined and specific inhibition of tumor cell proliferation through CKIA and CKIB by inhibition of the Cdk4/pRb/E2F pathway emphasizing potential therapeutic benefit of CKIA and CKIB. Furthermore, radiopharmacological properties of [I124]CKIA and [I124]CKIB observed in human tumor cells are promising prerequisites for in vivo biodistribution and imaging studies.

  3. Co-expression of CDC2 and CDK4 in human cancer cells is disrupted following γ-irradiation

    International Nuclear Information System (INIS)

    Warenius, Hilmar; Seabra, Laurence

    1995-01-01

    Purpose/Objective: Radiation delays progress through both the G1/S and G2/M checkpoints and also causes proliferation-related death in eukaryotic cells. The transition from G2 to mitosis is known to be controlled by the p 34 cdc2/cyclin B complex. Progress from G1 to S-phase is less well understood but is believed to involve, in part, the formation of a quaternary complex of PCNA, p21, cyclin D and cdk4. Here we examine putative relationships between the cdc2 and cdc4 cyclin-dependent kinases and exposure to γ-irradiation. Materials and Methods: Expression of the cdc2, cdk4, cyclin B and cyclin D genes was measured by Western blotting in 20 human in vitro cancer cell lines covering a wide range of histology and intrinsic cellular radiosensitivity. In order to examine whether the relationship between cdc2 and cdk4 expression was influenced by radiation we chose seven (HX142, 1407, 2780, MGHU, RT112, OAW42 and HRT18) of the human in vitro cell lines which we have previously shown to cover a wide range of intrinsic cellular radiosensitivities and which also showed a correlation between cdc2 and cdk4 expression of similar strength to the full twenty cell lines. The CDK protein levels were then followed by Western blots at 1, 24 and 48 hours after 2Gy of γ-irradiation from a 137 Cs unit. Results: Whilst no obvious relationship was apparent between the expression of cyclin B and cyclin D in the 20 cell lines, cdc2 and cdk4 appeared to be expressed to similar degrees in each cell line. Quantitation of cdc2 and cdk4 proteins by photodensitometry confirmed that there was a strong correlation (r=0.87, p=0.000006) between the two CDKs though only a weak relationship (r=0.491), between the respective cyclins with which cdc2 and cdk4 are known to complex in normal cells. We also compared protein levels of 10 proliferation-related gene produces including cdc2, cdk4 and cyclins B and D. Only cdc2 and cdk4 expression was completely disrupted at 1 hour. Sham irradiated cell lines

  4. Up-Regulation of P21 Inhibits TRAIL-Mediated Extrinsic Apoptosis, Contributing Resistance to SAHA in Acute Myeloid Leukemia Cells

    Directory of Open Access Journals (Sweden)

    Xing Wu

    2014-08-01

    Full Text Available Background/Aim: P21, a multifunctional cell cycle-regulatory molecule, regulates apoptotic cell death. In this study we examined the effect of altered p21 expression on the sensitivity of acute myeloid leukemia cells in response to HDAC inhibitor SAHA treatment and investigated the underlying mechanism. Methods: Stably transfected HL60 cell lines were established in RPMI-1640 with supplementation of G-418. Cell viability was measured by MTT assay. Western blot was applied to assess the protein expression levels of target genes. Cell apoptosis was monitored by AnnexinV-PE/7AAD assay. Results: We showed HL60 cells that that didn't up-regulate p21 expression were more sensitive to SAHA-mediated apoptosis than NB4 and U937 cells that had increased p21 level. Enforced expression of p21 in HL60 cells reduced sensitivity to SAHA and blocked TRAIL-mediated apoptosis. Conversely, p21 silencing in NB4 cells enhanced SAHA-mediated apoptosis and lethality. Finally, we found that combined treatment with SAHA and rapamycin down-regulated p21 and enhanced apoptosis in AML cells. Conclusion: We conclude that up-regulated p21 expression mediates resistance to SAHA via inhibition of TRAIL apoptotic pathway. P21 may serve as a candidate biomarker to predict responsiveness or resistance to SAHA-based therapy in AML patients. In addition, rapamycin may be an effective agent to override p21-mediated resistance to SAHA in AML patients.

  5. Phosphorylation of Rad9 at serine 328 by cyclin A-Cdk2 triggers apoptosis via interfering Bcl-xL.

    Directory of Open Access Journals (Sweden)

    Zhuo Zhan

    Full Text Available Cyclin A-Cdk2, a cell cycle regulated Ser/Thr kinase, plays important roles in a variety of apoptoticprocesses. However, the mechanism of cyclin A-Cdk2 regulated apoptosis remains unclear. Here, we demonstrated that Rad9, a member of the BH3-only subfamily of Bcl-2 proteins, could be phosphorylated by cyclin A-Cdk2 in vitro and in vivo. Cyclin A-Cdk2 catalyzed the phosphorylation of Rad9 at serine 328 in HeLa cells during apoptosis induced by etoposide, an inhibitor of topoisomeraseII. The phosphorylation of Rad9 resulted in its translocation from the nucleus to the mitochondria and its interaction with Bcl-xL. The forced activation of cyclin A-Cdk2 in these cells by the overexpression of cyclin A,triggered Rad9 phosphorylation at serine 328 and thereby promoted the interaction of Rad9 with Bcl-xL and the subsequent initiation of the apoptotic program. The pro-apoptotic effects regulated by the cyclin A-Cdk2 complex were significantly lower in cells transfected with Rad9S328A, an expression vector that encodes a Rad9 mutant that is resistant to cyclin A-Cdk2 phosphorylation. These findings suggest that cyclin A-Cdk2 regulates apoptosis through a mechanism that involves Rad9phosphorylation.

  6. AbetaPP induces cdk5-dependent tau hyperphosphorylation in transgenic mice Tg2576.

    Science.gov (United States)

    Otth, Carola; Concha, Ilona I; Arendt, Thomas; Stieler, Jens; Schliebs, Reinhard; González-Billault, Christian; Maccioni, Ricardo B

    2002-10-01

    Previous studies of Abeta-induced neuronal damage of hippocampal cells in culture have provided strong evidence that deregulation of the Cdk5/p35 kinase system is involved in the neurodegeneration pathway. Cdk5 inhibitors and antisense probes neuroprotected hippocampal cells against the neurotoxic action of Abeta. To further investigate the mechanisms underlying the participation of Cdk5 in neuronal degeneration, the transgenic mouse containing the Swedish mutations, Tg2576, was used as an animal model. Immunocytochemical studies using anti-Abeta(1-17) antibody evidenced the presence of labeled small-clustered core plaques in the hippocampus and cortex of 18-month-old transgenic mice brains. The loss of granular cells without a compressed appearance was detected in the vicinity of the cores in the dentate gyrus of the hippocampus. Immunostaining of Tg2576 brain sections with antibodies AT8, PHF1 and GFAP labeled punctuate dystrophic neurites in and around the amyloid core. Reactive astrogliosis around the plaques in the hippocampus was also observed. Studies at the molecular level showed differences in the expression of the truncated Cdk5 activator p25 in the transgenic animal, as compared with wild type controls. However no differences in Cdk5 levels were detected, thus corroborating previous cellular findings. Interestingly, hyperphosphorylated tau epitopes were substantially increased as assessed with the AT8 and PHF1 antibodies, in agreement with the observation of a p25 increase in the transgenic animal. These observations strongly suggest that the increased exposure of Alzheimer's type tau phosphoepitopes in the transgenic mice correlated with deregulation of Cdk5 leading to an increase in p25 levels. These studies also provide further evidence on the links between extraneuronal amyloid deposition and tau pathology.

  7. CDK5 as a Therapeutic Target in Prostate Cancer Metastasis

    National Research Council Canada - National Science Library

    Nelkin, Barry

    2007-01-01

    .... We also proposed to examine the role of CDK5 activity in growth of prostate cancer metastatic to bone, using PC3 based bioluminescent cell clones, and to explore the potential for CDK5 inhibition...

  8. Virtual Screening for Potential Allosteric Inhibitors of Cyclin-Dependent Kinase 2 from Traditional Chinese Medicine

    Directory of Open Access Journals (Sweden)

    Fang Lu

    2016-09-01

    Full Text Available Cyclin-dependent kinase 2 (CDK2, a member of Cyclin-dependent kinases (CDKs, plays an important role in cell division and DNA replication. It is regarded as a desired target to treat cancer and tumor by interrupting aberrant cell proliferation. Compared to lower subtype selectivity of CDK2 ATP-competitive inhibitors, CDK2 allosteric inhibitor with higher subtype selectivity has been used to treat CDK2-related diseases. Recently, the first crystal structure of CDK2 with allosteric inhibitor has been reported, which provides new opportunities to design pure allosteric inhibitors of CDK2. The binding site of the ATP-competition inhibitors and the allosteric inhibitors are partially overlapped in space position, so the same compound might interact with the two binding sites. Thus a novel screening strategy was essential for the discovery of pure CDK2 allosteric inhibitors. In this study, pharmacophore and molecular docking were used to screen potential CDK2 allosteric inhibitors and ATP-competition inhibitors from Traditional Chinese Medicine (TCM. In the docking result of the allosteric site, the compounds which can act with the CDK2 ATP site were discarded, and the remaining compounds were regarded as the potential pure allosteric inhibitors. Among the results, prostaglandin E1 and nordihydroguaiaretic acid (NDGA were available and their growth inhibitory effect on human HepG2 cell lines was determined by MTT assay. The two compounds could substantially inhibit the growth of HepG2 cell lines with an estimated IC50 of 41.223 μmol/L and 45.646 μmol/L. This study provides virtual screening strategy of allosteric compounds and a reliable method to discover potential pure CDK2 allosteric inhibitors from TCM. Prostaglandin E1 and NDGA could be regarded as promising candidates for CDK2 allosteric inhibitors.

  9. Study of P21 Expression in Oral Lichen Planus and Oral Squamous Cell Carcinoma by Immunohistochemical Technique.

    Science.gov (United States)

    Baghaei, Fahimeh; Shojaei, Setareh; Afshar-Moghaddam, Noushin; Zargaran, Massoumeh; Rastin, Verisheh; Nasr, Mohsen; Moghimbeigi, Abbas

    2015-09-01

    Lichen planus is a mucocutaneous disease that is relatively common in middle aged individuals. Some studies have shown that oral lichen planus has a potential to progress to squamous cell carcinoma.p21 is a cyclin-dependent kinase inhibitor that regulates the cell cycle, thus it acts as an inhibitor in cell proliferation. This study was aimed to evaluate and compare the immunostaining of p21 (as a proliferation inhibitory factor) in oral lichen planus (OLP) and oral squamous cell carcinoma (OSCC). In this descriptive cross-sectional study, p21expression was investigated in 24 samples of oral lichen planus (OLP), 24 samples of oral squamous cell carcinoma (OSCC) and 24 samples of oral epithelial hyperplasia (OEH) by employing immunohistochemical staining. The mean percentage of p21-positive cells in OSCC (54.5±6.6) was significantly higher than that in OLP (32.8±6.08) and OEH (9.4±3.8). Moreover, OLP samples expressed p21 significantly higher than the OEH. Kruskal Wallis test revealed a statistically significant difference between the groups regarding the intensity of staining (plichen planus to SCC. Therefore, continuous follow-up periods for OLP are recommended for diagnosis of the malignant transformations in early stages.

  10. A novel WISp39 protein links Hsp90 and p21 stability to the G2/M checkpoint.

    Science.gov (United States)

    Benzeno, Sharon; Diehl, J Alan

    2005-04-01

    Transcriptional regulation of the p21(Cip1) cyclin-dependent kinase inhibitor is a well-established mechanism by which the cell orchestrates a proper spatial and temporal cell cycle progression. Now, in the January 2005 issue of Molecular Cell (2005; Vol 17, 237-49), a study by Jascur et al identifies a novel multi-protein complex that is critical in contributing to a p53-dependent G2 cell cycle checkpoint. The authors demonstrate the significance of stabilizing the p21 protein in the context of this complex.

  11. Quantifying the CDK inhibitor VMY-1-103’s activity and tissue levels in an in vivo tumor model by LC-MS/MS and by MRI

    Science.gov (United States)

    Sirajuddin, Paul; Das, Sudeep; Ringer, Lymor; Rodriguez, Olga C.; Sivakumar, Angiela; Lee, Yi-Chien; Üren, Aykut; Fricke, Stanley T.; Rood, Brian; Ozcan, Alpay; Wang, Sean S.; Karam, Sana; Yenugonda, Venkata; Salinas, Patricia; Petricoin III, Emanuel; Pishvaian, Michael; Lisanti, Michael P.; Wang, Yue; Schlegel, Richard; Moasser, Bahram; Albanese, Chris

    2012-01-01

    The development of new small molecule-based therapeutic drugs requires accurate quantification of drug bioavailability, biological activity and treatment efficacy. Rapidly measuring these endpoints is often hampered by the lack of efficient assay platforms with high sensitivity and specificity. Using an in vivo model system, we report a simple and sensitive liquid chromatography-tandem mass spectrometry assay to quantify the bioavailability of a recently developed novel cyclin-dependent kinase inhibitor VMY-1-103, a purvalanol B-based analog whose biological activity is enhanced via dansylation. We developed a rapid organic phase extraction technique and validated wide and functional VMY-1-103 distribution in various mouse tissues, consistent with its enhanced potency previously observed in a variety of human cancer cell lines. More importantly, in vivo MRI and single voxel proton MR-Spectroscopy further established that VMY-1-103 inhibited disease progression and affected key metabolites in a mouse model of hedgehog-driven medulloblastoma. PMID:22983062

  12. Retinoic Acid Induces Apoptosis of Prostate Cancer DU145 Cells through Cdk5 Overactivation

    Directory of Open Access Journals (Sweden)

    Mei-Chih Chen

    2012-01-01

    Full Text Available Retinoic acid (RA has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP. Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and Annexin V staining could also be blocked by CP treatment. Furthermore, RA-triggered caspase 3 activation and following Cdk5 over-activation were destroyed by treatments of both CP and Cdk5 knockdown. In conclusion, we report a new mechanism in which RA could cause apoptosis of androgen-independent prostate cancer cells through p35 cleavage and Cdk5 over-activation. This finding may contribute to constructing a clearer image of RA function and bring RA as a valuable chemoprevention agent for prostate cancer patients.

  13. p21WAF1 expression induced by MEK/ERK pathway activation or inhibition correlates with growth arrest, myogenic differentiation and onco-phenotype reversal in rhabdomyosarcoma cells

    Directory of Open Access Journals (Sweden)

    Giacinti Cristina

    2005-12-01

    Full Text Available Abstract Background p21WAF1, implicated in the cell cycle control of both normal and malignant cells, can be induced by p53-dependent and independent mechanisms. In some cells, MEKs/ERKs regulate p21WAF1 transcriptionally, while in others they also affect the post-transcriptional processes. In myogenic differentiation, p21WAF1 expression is also controlled by the myogenic transcription factor MyoD. We have previously demonstrated that the embryonal rhabdomyosarcoma cell line undergoes growth arrest and myogenic differentiation following treatments with TPA and the MEK inhibitor U0126, which respectively activate and inhibit the ERK pathway. In this paper we attempt to clarify the mechanism of ERK-mediated and ERK-independent growth arrest and myogenic differentiation of embryonal and alveolar rhabdomyosarcoma cell lines, particularly as regards the expression of the cell cycle inhibitor p21WAF1. Results p21WAF1 expression and growth arrest are induced in both embryonal (RD and alveolar (RH30 rhabdomyosarcoma cell lines following TPA or MEK/ERK inhibitor (U0126 treatments, whereas myogenic differentiation is induced in RD cells alone. Furthermore, the TPA-mediated post-transcriptional mechanism of p21WAF1-enhanced expression in RD cells is due to activation of the MEK/ERK pathway, as shown by transfections with constitutively active MEK1 or MEK2, which induces p21WAF1 expression, and with ERK1 and ERK2 siRNA, which prevents p21WAF1 expression. By contrast, U0126-mediated p21WAF1 expression is controlled transcriptionally by the p38 pathway. Similarly, myogenin and MyoD expression is induced both by U0126 and TPA and is prevented by p38 inhibition. Although MyoD and myogenin depletion by siRNA prevents U0126-mediated p21WAF1 expression, the over-expression of these two transcription factors is insufficient to induce p21WAF1. These data suggest that the transcriptional mechanism of p21WAF1 expression in RD cells is rescued when MEK/ERK inhibition

  14. Analysis list: Cdk9 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Cdk9 Blood,Embryonic fibroblast,Pluripotent stem cell + mm9 http://dbarchive.biosci...encedbc.jp/kyushu-u/mm9/target/Cdk9.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk9.5.tsv h...ttp://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk9.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk9....Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk9....Embryonic_fibroblast.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk9.Pluripotent_stem_cell.tsv

  15. Loss of p12CDK2-AP1 Expression in Human Oral Squamous Cell Carcinoma with Disrupted Transforming Growth Factor-β-Smad Signaling Pathway

    Directory of Open Access Journals (Sweden)

    Hui Peng

    2006-12-01

    Full Text Available We examined correlations between TGF-β1, TβR-I and TβR-II, p12CDK2-AP1 p21WAF1 p27KIP1 Smad2, and p-Smad2 in 125 cases of human oral squamous cell carcinoma (OSCC to test the hypothesis that resistance to TGF-β1-induced growth suppression is due to the disruption of its signaling pathway as a consequence of reduced or lost p12CDK2-AP1. Immunoreactivity for TβR-II decreased in OSCC with increasing disease aggressiveness; however, no differences were observed for TβR-I and TGF-β1. The expression of TβR-II significantly correlated with p12CDK2-AP1 and p27KIP1 (P<.001 and P<.01, respectively. Furthermore, there was a significant relationship between TβR-II expression and p-Smad2 (P < .001. The in vivo correlation of the levels of TβR-II, p12CDK2-AP1 and p27 KIP1 was confirmed in normal and OSCC cell lines. Additionally, in vitro analysis of TGF-β-treated cells showed that TGF-β1 treatment of normal keratinocytes suppressed cell growth with upregulation of p-Smad2, p12CDK2-API and p21WAF1 expression, whereas there was no effect on OSCC cell lines. These results provide evidence of a link between a disrupted TGF-β-Smad signaling pathway and loss of induction of cell cycle-inhibitory proteins, especially p12CDK2-AP1 in OSCC, which may lead to the resistance of TGF-β1 growth-inhibitory effect on OSCC.

  16. Cdk7 Is Required for Activity-Dependent Neuronal Gene Expression, Long-Lasting Synaptic Plasticity and Long-Term Memory

    Directory of Open Access Journals (Sweden)

    Guiqin He

    2017-11-01

    Full Text Available In the brain, de novo gene expression driven by learning-associated neuronal activities is critical for the formation of long-term memories. However, the signaling machinery mediating neuronal activity-induced gene expression, especially the rapid transcription of immediate-early genes (IEGs remains unclear. Cyclin-dependent kinases (Cdks are a family of serine/threonine kinases that have been firmly established as key regulators of transcription processes underling coordinated cell cycle entry and sequential progression in nearly all types of proliferative cells. Cdk7 is a subunit of transcriptional initiation factor II-H (TFIIH and the only known Cdk-activating kinase (CAK in metazoans. Recent studies using a novel Cdk7 specific covalent inhibitor, THZ1, revealed important roles of Cdk7 in transcription regulation in cancer cells. However, whether Cdk7 plays a role in the regulation of transcription in neurons remains unknown. In this study, we present evidence demonstrating that, in post-mitotic neurons, Cdk7 activity is positively correlated with neuronal activities in cultured primary neurons, acute hippocampal slices and in the brain. Cdk7 inhibition by THZ1 significantly suppressed mRNA levels of IEGs, selectively impaired long-lasting synaptic plasticity induced by 4 trains of high frequency stimulation (HFS and prevented the formation of long-term memories.

  17. Wilms' tumor 1-associating protein promotes renal cell carcinoma proliferation by regulating CDK2 mRNA stability.

    Science.gov (United States)

    Tang, Jingyuan; Wang, Feng; Cheng, Gong; Si, Shuhui; Sun, Xi; Han, Jie; Yu, Hao; Zhang, Wei; Lv, Qiang; Wei, Ji-Fu; Yang, Haiwei

    2018-02-27

    Wilms' tumor 1-associating protein (WTAP) plays an important role in physiological processes and the development of tumor such as cell cycle regulation. The regulation of cell cycle is mainly dependent on cyclins and cyclin-dependent protein kinases (CDKs). Recent studies have shown that CDKs are closely related to the tumor diagnosis, progression and response to treatment. However, their specific biological roles and related mechanism in renal cell carcinoma (RCC) remain unknown. Quantitative real-time PCR, western blotting and immunohistochemistry were used to detect the expression of WTAP and CDK2. The survival analysis was adopted to explore the association between WTAP expression and the prognosis of RCC. Cells were stably transfected with lentivirus approach and cell proliferation and cell cycle, as well as tumorigenesis in nude mice were performed to assess the effect of WTAP in RCC. RNA immunoprecipitation, Luciferase reporter assay and siRNA were employed to identify the direct binding sites of WTAP with CDK2 transcript. Colony formation assay was conducted to confirm the function of CDK2 in WTAP-induced growth promoting. In RCC cell lines and tissues, WTAP was significantly over-expressed. Compared with patients with low expression of WTAP, patients with high expression of WTAP had lower overall survival rate. Additionally, cell function test indicated that cell proliferation abilities in WTAP over-expressed group were enhanced, while WTAP knockdown showed the opposite results. Subcutaneous xenograft tumor model displayed that knockdown of WTAP could impede tumorigenesis in vivo. Mechanism study exhibited that CDK2 expression was positively associated with the expression of WTAP. Moreover, WTAP stabilized CDK2 transcript to enhance CDK2 expression via binding to 3'-UTR of CDK2 transcript. Additionally, specific inhibitors of CDK2 activity and small interfering RNA (siRNA) of CDK2 expression inhibited WTAP-mediated promotion of proliferation. These

  18. Phosphorylation of the centrosomal protein, Cep169, by Cdk1 promotes its dissociation from centrosomes in mitosis.

    Science.gov (United States)

    Mori, Yusuke; Inoue, Yoko; Taniyama, Yuki; Tanaka, Sayori; Terada, Yasuhiko

    2015-12-25

    Cep169 is a centrosomal protein conserved among vertebrates. In our previous reports, we showed that mammalian Cep169 interacts and collaborates with CDK5RAP2 to regulate microtubule (MT) dynamics and stabilization. Although Cep169 is required for MT regulation, its precise cellular function remains largely elusive. Here we show that Cep169 associates with centrosomes during interphase, but dissociates from these structures from the onset of mitosis, although CDK5RAP2 (Cep215) is continuously located at the centrosomes throughout cell cycle. Interestingly, treatment with purvalanol A, a Cdk1 inhibitor, nearly completely blocked the dissociation of Cep169 from centrosomes during mitosis. In addition, mass spectrometry analyses identified 7 phosphorylated residues of Cep169 corresponding to consensus phosphorylation sequence for Cdk1. These data suggest that the dissociation of Cep169 from centrosomes is controlled by Cdk1/Cyclin B during mitosis, and that Cep169 might regulate MT dynamics of mitotic spindle. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Analysis list: CDK9 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available CDK9 Blood,Liver + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CDK9....1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/CDK9.5.tsv http://dbarchive.biosciencedbc.jp/k...yushu-u/hg19/target/CDK9.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/CDK9.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/CDK9.Liver.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Liver.gml ...

  20. Role of hTERT in apoptosis of cervical cancer induced by histone deacetylase inhibitor

    International Nuclear Information System (INIS)

    Wu, Peng; Meng, Li; Wang, Hui; Zhou, Jianfeng; Xu, Gang; Wang, Shixuan; Xi, Ling; Chen, Gang; Wang, Beibei; Zhu, Tao; Lu, Yunping; Ma, Ding

    2005-01-01

    Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase holoenzyme as well as the rate-limiting component of the telomerase enzyme complex. However, the role of the hTERT in apoptosis induced by histone deacetylase inhibitor has only been marginally addressed. For the first time, our study demonstrated that trichostatin A (TSA) briefly activated the proliferation of cervical cancer cell lines, HeLa and SiHa, within 12 h, but then inhibited cell growth after that time point. In response to TSA, hTERT expression, telomerase activity, and telomere length also underwent similar changes during the same time frame. Furthermore, the data in our study showed that cells transfected with dominant negative hTERT were more likely to undergo apoptosis induced by TSA than cells transfected with wild-type hTERT. The cyclin/cdk inhibitor p21 waf1 was down-regulated by hTERT without changing the expression of p53. Results from this study suggest that the hTERT might be a primary target of TSA and the anti-apoptosis effect of hTERT might be carried out through a p21 waf1 -dependent and p53-independent pathway

  1. Targets downstream of Cdk8 in Dictyostelium development

    Directory of Open Access Journals (Sweden)

    Skelton Jason

    2011-01-01

    Full Text Available Abstract Background Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. Results Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2 with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD and one transcriptionally (short chain dehydrogenase/reductase (SDR1. Conclusions This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

  2. Insertional mutagenesis in mice deficient for p15Ink4b, p16Ink4a, p21Cip1, and p27Kip1 reveals cancer gene interactions and correlations with tumor phenotypes

    DEFF Research Database (Denmark)

    Kool, Jaap; Uren, Anthony G; Martins, Carla P

    2010-01-01

    -throughput murine leukemia virus insertional mutagenesis screens in mice that are deficient for one or two CDK inhibitors. We retrieved 9,117 retroviral insertions from 476 lymphomas to define hundreds of loci that are mutated more frequently than expected by chance. Many of these loci are skewed toward a specific...

  3. Vorinostat enhances protein stability of p27 and p21 through negative regulation of Skp2 and Cks1 in human breast cancer cells.

    Science.gov (United States)

    Uehara, Norihisa; Yoshizawa, Katsuhiko; Tsubura, Airo

    2012-07-01

    Vorinostat is a histone deacetylase inhibitor that blocks cancer cell proliferation through the regulation of cyclin-dependent kinase inhibitors. We, herein, examined the involvement of S-phase kinase-associated protein 2 (Skp2) and cyclin-dependent kinase subunit 1 (Cks1), the components of the SCFSkp2-Cks1 (Skp1/Cul1/F-box protein) ubiquitin ligase complex, in the regulation of p27 and p21 during vorinostat-induced growth arrest of MDA-MB-231 and MCF-7 human breast cancer cells. Vorinostat significantly reduced BrdU incorporation in MDA-MB-231 and MCF-7 cells, which was associated with increased p27 and p21 protein levels without concomitant induction of p27 mRNA. Vorinostat-induced accumulation of p27 and p21 proteins was inversely correlated with the mRNA and protein levels of Skp2 and Cks1. Cycloheximide chase analysis revealed that vorinostat increased the half-life of p27 and p21 proteins. The accumulation of p27 and p21 proteins was attenuated by forced expression of Skp2 and Cks1, which conferred resistance to the vorinostat-induced S-phase reduction. These results suggest that vorinostat-induced growth arrest may be in part due to the enhanced protein stability of p27 and p21 through the downregulation of Skp2 and Cks1.

  4. Knockdown of MBP-1 in human foreskin fibroblasts induces p53-p21 dependent senescence.

    Directory of Open Access Journals (Sweden)

    Asish K Ghosh

    Full Text Available MBP-1 acts as a general transcriptional repressor. Overexpression of MBP-1 induces cell death in a number of cancer cells and regresses tumor growth. However, the function of endogenous MBP-1 in normal cell growth regulation remains unknown. To unravel the role of endogenous MBP-1, we knocked down MBP-1 expression in primary human foreskin fibroblasts (HFF by RNA interference. Knockdown of MBP-1 in HFF (HFF-MBPsi-4 resulted in an induction of premature senescence, displayed flattened cell morphology, and increased senescence-associated beta-galactosidase activity. FACS analysis of HFF-MBPsi-4 revealed accumulation of a high number of cells in the G1-phase. A significant upregulation of cyclin D1 and reduction of cyclin A was detected in HFF-MBPsi-4 as compared to control HFF. Senescent fibroblasts exhibited enhanced expression of phosphorylated and acetylated p53, and cyclin-dependent kinase inhibitor, p21. Further analysis suggested that promyolocytic leukemia protein (PML bodies are dramatically increased in HFF-MBPsi-4. Together, these results demonstrated that knockdown of endogenous MBP-1 is involved in cellular senescence of HFF through p53-p21 pathway.

  5. Phosphorylation of Ubc9 by Cdk1 enhances SUMOylation activity.

    Directory of Open Access Journals (Sweden)

    Yee-Fun Su

    Full Text Available Increasing evidence has pointed to an important role of SUMOylation in cell cycle regulation, especially for M phase. In the current studies, we have obtained evidence through in vitro studies that the master M phase regulator CDK1/cyclin B kinase phosphorylates the SUMOylation machinery component Ubc9, leading to its enhanced SUMOylation activity. First, we show that CDK1/cyclin B, but not many other cell cycle kinases such as CDK2/cyclin E, ERK1, ERK2, PKA and JNK2/SAPK1, specifically enhances SUMOylation activity. Second, CDK1/cyclin B phosphorylates the SUMOylation machinery component Ubc9, but not SAE1/SAE2 or SUMO1. Third, CDK1/cyclin B-phosphorylated Ubc9 exhibits increased SUMOylation activity and elevated accumulation of the Ubc9-SUMO1 thioester conjugate. Fourth, CDK1/cyclin B enhances SUMOylation activity through phosphorylation of Ubc9 at serine 71. These studies demonstrate for the first time that the cell cycle-specific kinase CDK1/cyclin B phosphorylates a SUMOylation machinery component to increase its overall SUMOylation activity, suggesting that SUMOylation is part of the cell cycle program orchestrated by CDK1 through Ubc9.

  6. Sequential Cdk1 and Plk1 phosphorylation of caspase-8 triggers apoptotic cell death during mitosis.

    Science.gov (United States)

    Matthess, Yves; Raab, Monika; Knecht, Rainald; Becker, Sven; Strebhardt, Klaus

    2014-05-01

    Caspase-8 is crucial for cell death induction, especially via the death receptor pathway. The dysregulated expression or function of caspase-8 can promote tumor formation, progression and treatment resistance in different human cancers. Here, we show procaspase-8 is regulated during the cell cycle through the concerted inhibitory action of Cdk1/cyclin B1 and polo-like kinase 1 (Plk1). By phosphorylating S387 in procaspase-8 Cdk1/cyclin B1 generates a phospho-epitope for the binding of the PBD of Plk1. Subsequently, S305 in procaspase-8 is phosphorylated by Plk1 during mitosis. Using an RNAi-based strategy we could demonstrate that the extrinsic cell death is increased upon Fas-stimulation when endogenous caspase-8 is replaced by a mutant (S305A) mimicking the non-phosphorylated form. Together, our data show that sequential phosphorylation by Cdk1/cyclin B1 and Plk1 decreases the sensitivity of cells toward stimuli of the extrinsic pathway during mitosis. Thus, the clinical Plk1 inhibitor BI 2536 decreases the threshold of different cancer cell types toward Fas-induced cell death. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. LincRNA-p21 inhibits invasion and metastasis of hepatocellular carcinoma through miR-9/E-cadherin cascade signaling pathway molecular mechanism

    Directory of Open Access Journals (Sweden)

    Ding G

    2017-06-01

    Full Text Available Gangqiang Ding, Zhen Peng, Jia Shang, Yi Kang, Huibin Ning, Chongshan Mao Department of Infectious Diseases, People’s Hospital of Zhengzhou University, Henan Provincial People’s Hospital, Zhengzhou, China Abstract: In the previous study, it was found that long intergenic noncoding RNA-p21 (lincRNA-p21 was downregulated in hepatocellular carcinoma (HCC and lincRNA-p21 overexpression inhibited tumor invasion through inducing epithelial–mesenchymal transition. However, the underlying mechanism was not fully elaborated. In this study, lincRNA-p21 expression was measured in 12 paired HCC and nontumor adjacent normal tissues by ­quantitative real-time polymerase chain reaction. The effects of lincRNA-p21 on HCC cells were studied using lentivirus expressing lincRNA-p21 vector in vitro. The association between lincRNA-p21 level and miR-9 level was tested with the Spearman rank correlation. The effects of miR-9 on HCC cells were studied by using miR-9 inhibitor in vitro. Luciferase assay was used to validate the target of miR-9. The results showed that lincRNA-p21 was downregulated in human HCC tissues and cell lines. LincRNA-p21 overexpression significantly inhibited HCC cell migration and invasion in vitro. Besides, lincRNA-p21 negatively regulated miR-9 expression level, and miR-9 was upregulated in human HCC tissues and cells. MiR-9 knockdown inhibited HCC cell migration and invasion in vitro. Finally, the luciferase assay results showed that E-cadherin was a direct target of miR-9. The expression level of E-cadherin was found to be regulated by lincRNA-p21 and miR-9. Altogether, the results suggested that lincRNA-p21 inhibits migration and invasion of HCC cells through regulating miR-9-mediated E-cadherin cascade signaling pathway. Keywords: hepatocellular carcinoma, lincRNA-p21, miR-9, E-cadherin, epithelial–mesenchymal transition

  8. Gene expression of cyclin-dependent kinase inhibitors and effect of heparin on their expression in mice with hypoxia-induced pulmonary hypertension

    International Nuclear Information System (INIS)

    Yu Lunyin; Quinn, Deborah A.; Garg, Hari G.; Hales, Charles A.

    2006-01-01

    The balance between cell proliferation and cell quiescence is regulated delicately by a variety of mediators, in which cyclin-dependent kinases (CDK) and CDK inhibitors (CDKI) play a very important role. Heparin which inhibits pulmonary artery smooth muscle cell (PASMC) proliferation increases the levels of two CDKIs, p21 and p27, although only p27 is important in inhibition of PASMC growth in vitro and in vivo. In the present study we investigated the expression profile of all the cell cycle regulating genes, including all seven CDKIs (p21, p27, p57, p15, p16, p18, and p19), in the lungs of mice with hypoxia-induced pulmonary hypertension. A cell cycle pathway specific gene microarray was used to profile the 96 genes involved in cell cycle regulation. We also observed the effect of heparin on gene expression. We found that (a) hypoxic exposure for two weeks significantly inhibited p27 expression and stimulated p18 activity, showing a 98% decrease in p27 and 81% increase in p18; (b) other CDKIs, p21, p57, p15, p16, and p19 were not affected significantly in response to hypoxia; (c) heparin treatment restored p27 expression, but did not influence p18; (d) ERK1/2 and p38 were mediators in heparin upregulation of p27. This study provides an expression profile of cell cycle regulating genes under hypoxia in mice with hypoxia-induced pulmonary hypertension and strengthens the previous finding that p27 is the only CDKI involved in heparin regulation of PASMC proliferation and hypoxia-induced pulmonary hypertension

  9. MYC Modulation around the CDK2/p27/SKP2 Axis

    Science.gov (United States)

    Hydbring, Per; Castell, Alina; Larsson, Lars-Gunnar

    2017-01-01

    MYC is a pleiotropic transcription factor that controls a number of fundamental cellular processes required for the proliferation and survival of normal and malignant cells, including the cell cycle. MYC interacts with several central cell cycle regulators that control the balance between cell cycle progression and temporary or permanent cell cycle arrest (cellular senescence). Among these are the cyclin E/A/cyclin-dependent kinase 2 (CDK2) complexes, the CDK inhibitor p27KIP1 (p27) and the E3 ubiquitin ligase component S-phase kinase-associated protein 2 (SKP2), which control each other by forming a triangular network. MYC is engaged in bidirectional crosstalk with each of these players; while MYC regulates their expression and/or activity, these factors in turn modulate MYC through protein interactions and post-translational modifications including phosphorylation and ubiquitylation, impacting on MYC’s transcriptional output on genes involved in cell cycle progression and senescence. Here we elaborate on these network interactions with MYC and their impact on transcription, cell cycle, replication and stress signaling, and on the role of other players interconnected to this network, such as CDK1, the retinoblastoma protein (pRB), protein phosphatase 2A (PP2A), the F-box proteins FBXW7 and FBXO28, the RAS oncoprotein and the ubiquitin/proteasome system. Finally, we describe how the MYC/CDK2/p27/SKP2 axis impacts on tumor development and discuss possible ways to interfere therapeutically with this system to improve cancer treatment. PMID:28665315

  10. Downregulation of p21(WAF1/CIP1) and estrogen receptor alpha in MCF-7 cells by antisense oligonucleotides containing locked nucleic acid (LNA)

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Pfundheller, Henrik M; Lykkesfeldt, Anne E

    2004-01-01

    of phosphorothioate oligonucleotides (PS AONs). The antisense efficiency of LNA-containing oligonucleotides was systematically compared with standard PS AONs targeting expression of two endogenous proteins in the human breast cancer cell line MCF-7, namely, the cyclin-dependent kinase inhibitor p21(WAF1/CIP1...

  11. Genetic and pharmacological inhibition of CDK9 drives neutrophil apoptosis to resolve inflammation in zebrafish in vivo

    Science.gov (United States)

    Hoodless, Laura J.; Lucas, Christopher D.; Duffin, Rodger; Denvir, Martin A.; Haslett, Christopher; Tucker, Carl S.; Rossi, Adriano G.

    2016-01-01

    Neutrophilic inflammation is tightly regulated and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro and promote resolution of inflammation in rodent models. Here we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation. Using live cell imaging in zebrafish with labelled neutrophils and macrophages, we show that pharmacological inhibition, morpholino-mediated knockdown and CRISPR/cas9-mediated knockout of CDK9 enhances inflammation resolution by reducing neutrophil numbers via induction of apoptosis after tailfin injury. Importantly, knockdown of the negative regulator La-related protein 7 (LaRP7) increased neutrophilic inflammation. Our data show that CDK9 is a possible target for controlling resolution of inflammation. PMID:27833165

  12. Palbociclib synergizes with BRAF and MEK inhibitors in treatment naïve melanoma but not after the development of BRAF inhibitor resistance.

    Science.gov (United States)

    Martin, Claire A; Cullinane, Carleen; Kirby, Laura; Abuhammad, Shatha; Lelliott, Emily J; Waldeck, Kelly; Young, Richard J; Brajanovski, Natalie; Cameron, Donald P; Walker, Rachael; Sanij, Elaine; Poortinga, Gretchen; Hannan, Ross D; Pearson, Richard B; Hicks, Rodney J; McArthur, Grant A; Sheppard, Karen E

    2017-12-15

    Increased CDK4 activity occurs in the majority of melanomas and CDK4/6 inhibitors in combination with BRAF and MEK inhibitors are currently in clinical trials for the treatment of melanoma. We hypothesize that the timing of the addition of CDK4/6 inhibitors to the current BRAF and MEK inhibitor regime will impact on the efficacy of this triplet drug combination. The efficacy of BRAF, MEK and CDK4/6 inhibitors as single agents and in combination was assessed in human BRAF mutant cell lines that were treatment naïve, BRAF inhibitor tolerant or had acquired resistance to BRAF inhibitors. Xenograft studies were then performed to test the in vivo efficacy of the BRAF and CDK4/6 inhibitor combination. Melanoma cells that had developed early reversible tolerance or acquired resistance to BRAF inhibition remained sensitive to palbociclib. In drug-tolerant cells, the efficacy of the combination of palbociclib with BRAF and/or MEK inhibitors was equivalent to single agent palbociclib. Similarly, acquired BRAF inhibitor resistance cells lost efficacy to the palbociclib and BRAF combination. In contrast, upfront treatment of melanoma cells with palbociclib in combination with BRAF and/or MEK inhibitors induced either cell death or senescence and was superior to a BRAF plus MEK inhibitor combination. In vivo palbociclib plus BRAF inhibitor induced rapid and sustained tumor regression without the development of therapy resistance. In summary, upfront dual targeting of CDK4/6 and mutant BRAF signaling enables tumor cells to evade resistance to monotherapy and is required for robust and sustained tumor regression. Melanoma patients whose tumors have acquired resistance to BRAF inhibition are less likely to have favorable responses to subsequent treatment with the triplet combination of BRAF, MEK and CDK4/6 inhibitors. © 2017 UICC.

  13. Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

    DEFF Research Database (Denmark)

    Galanos, Panagiotis; Vougas, Konstantinos; Walter, David

    2016-01-01

    cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near...

  14. RNF115/BCA2 E3 Ubiquitin Ligase Promotes Breast Cancer Cell Proliferation through Targeting p21Waf1/Cip1 for Ubiquitin-Mediated Degradation

    Directory of Open Access Journals (Sweden)

    Zehua Wang

    2013-09-01

    Full Text Available The E3 ubiquitin ligase RING finger protein 115 (RNF115, also known as breast cancer-associated gene 2 (BCA2, has previously been reported to be overexpressed in estrogen receptor α (ERα-positive breast tumors and to promote breast cell proliferation; however, its mechanism is unknown. In this study, we demonstrated that silencing of BCA2 by small interfering RNAs (siRNAs in two ERα-positive breast cancer cell lines, MCF-7 and T47D, decreases cell proliferation and increases the protein levels of the cyclin-dependent kinase inhibitor p21Waf/Cip1. The protein stability of p21 was negatively regulated by BCA2. BCA2 directly interacts with p21 and promotes p21 ubiquitination and proteasomal degradation. Knockdown of p21 partially rescues the cell growth arrest induced by the BCA2 siRNA. These results suggest that BCA2 promotes ERα-positive breast cancer cell proliferation at least partially through downregulating the expression of p21.

  15. Translational Upregulation of an Individual p21Cip1 Transcript Variant by GCN2 Regulates Cell Proliferation and Survival under Nutrient Stress.

    Directory of Open Access Journals (Sweden)

    Stacey L Lehman

    2015-06-01

    Full Text Available Multiple transcripts encode for the cell cycle inhibitor p21(Cip1. These transcripts produce identical proteins but differ in their 5' untranslated regions (UTRs. Although several stresses that induce p21 have been characterized, the mechanisms regulating the individual transcript variants and their functional significance are unknown. Here we demonstrate through (35S labeling, luciferase reporter assays, and polysome transcript profiling that activation of the Integrated Stress Response (ISR kinase GCN2 selectively upregulates the translation of a p21 transcript variant containing 5' upstream open reading frames (uORFs through phosphorylation of the eukaryotic translation initiation factor eIF2α. Mutational analysis reveals that the uORFs suppress translation under basal conditions, but promote translation under stress. Functionally, ablation of p21 ameliorates G1/S arrest and reduces cell survival in response to GCN2 activation. These findings uncover a novel mechanism of p21 post-transcriptional regulation, offer functional significance for the existence of multiple p21 transcripts, and support a key role for GCN2 in regulating the cell cycle under stress.

  16. The 3p21.1-p21.3 hereditary vascular retinopathy locus increases the risk for Raynaud's phenomenon and migraine

    NARCIS (Netherlands)

    Hottenga, J. J.; Vanmolkot, K. R. J.; Kors, E. E.; Kheradmand Kia, S.; de Jong, P. T. V. M.; Haan, J.; Terwindt, G. M.; Frants, R. R.; Ferrari, M. D.; van den Maagdenberg, A. M. J. M.

    2005-01-01

    Previously, we described a large Dutch family with hereditary vascular retinopathy (HVR), Raynaud's phenomenon and migraine. A locus for HVR was mapped on chromosome 3p21.1-p21.3, but the gene has not yet been identified. The fact that all three disorders share a vascular aetiology prompted us to

  17. The Role of Cyclins and Cyclins Inhibitors in the Multistep Process of HPV-Associated Cervical Carcinoma

    International Nuclear Information System (INIS)

    Bahnassy, A.A.; Mokhtar, N.M.; Zekri, A.; Alam El-Din, H.M.; Aboubaker, A.A.; Kamel, K.; El-Sabah, M.T.

    2006-01-01

    Background: Human papillomavirus (HPV) types 16 and 18 are associated with cervical carcinogenesis. This is possibly achieved through an interaction between HPV oncogenic proteins and some cell cycle regulatory genes. However, the exact pathogenetic mechanisms are not well defined yet. Methods: We investigated 110 subjects (43 invasive squamous cell carcinoma [ISCC], 38 CIN Ill, II CIN II, 18 CIN I) confirmed to be positive for HPV 16 and/or 18 as well as 20 normal cervical tissue (NCT) samples for abnormal expression of cyclin DJ, cyclin E, CDK4, cyclin inhibitors (p2Jwa/; p27, pI6/NK4A) and Ki-67 using immunohistochemistry and differential PCR techniques. Results: There was a significant increase in the expression of Ki-67, cyclin E, CDK4, pJ6/NK4A (p=0003, 0.001,0.001) and a significant decrease in p27K1P/ from NCT to ISCC (p=0.003). There was a significant correlation between altered expression of p27K1P I and p 161NK4A (p KIpl (ρ=0.011) in all studied groups In ISCC, there was significant relationship between standard clinico-pathological prognostic factors and high Ki-67 index, increased cyclin D J and cyclin E, reduced p2 7Kip / and p21 waf Conclusion: I) Aberrations involving p27K/P 1, cyclin E, CDK4 and pJ6/NK4A are considered early events in HPV 16 and IS-associated cervical carcinogenesis (CINI and lI), whereas cyclin DI aberrations are late events (CINIII and ISCC). 2) immunohistochemical tests for pJ61NK4A and cyclin E could help in early diagnosis of cervical carcinoma. 3) Only FIGO stage, cyclin DI, p27K1P1 and Ki-67 are independent prognostic factors that might help in predicting outcome of cervical cancer palients

  18. IL-1-induced ERK1/2 activation up-regulates p21{sup Waf1/Cip1} protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    Energy Technology Data Exchange (ETDEWEB)

    Arakawa, Tomohiro [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Hayashi, Hidetoshi [Department of Drug Metabolism and Disposition, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Itoh, Saotomo; Takii, Takemasa [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan); Onozaki, Kikuo, E-mail: konozaki@phar.nagoya-cu.ac.jp [Department of Molecular Health Sciences, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603 (Japan)

    2010-02-12

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21{sup Waf1/Cip1} (p21) protein. Here, we demonstrate that IL-1 induces the stabilization of p21 protein via ERK1/2 pathway. The degradation of p21 was inhibited by IL-1, however the ubiquitination level of p21 was not affected. In addition, the degradation of non-ubiquitinated form of lysine less mutant p21-K6R was also inhibited by IL-1, suggesting that IL-1 stabilized p21 protein via ubiquitin-independent pathway. Furthermore, the inhibition of p21 protein degradation was prevented by a selective inhibitor of ERK1/2 pathway, PD98059. These results suggest that IL-1-induced ERK1/2 activation leads to the up-regulation of p21 by inhibiting degradation via ubiquitin-independent pathway in human melanoma cells A375.

  19. Molecular Mechanism of Enhanced Anticancer Effect of Nanoparticle Formulated LY2835219 via p16-CDK4/6-pRb Pathway in Colorectal Carcinoma Cell Line

    Directory of Open Access Journals (Sweden)

    Xu Tang

    2016-01-01

    Full Text Available LY2835219 is a dual inhibitor to CDK4 and CDK6. This study was to prepare LY2835219-loaded chitosan nanoparticles (CNP/LY and LY2835219-loaded hyaluronic acid-conjugated chitosan nanoparticles (HACNP/LY and revealed their anticancer effect and influence on p16-CDK4/6-pRb pathway against colon cell line. The nanoparticle sizes of CNP/LY and HACNP/LY were approximately 195±39.6 nm and 217±31.1 nm, respectively. The zeta potentials of CNP/LY and HACNP/LY were 37.3±1.5 mV and 30.3±2.2 mV, respectively. And the preparation process showed considerable drug encapsulation efficiency and loading efficiency. LY2835219, CNP/LY, and HACNP/LY inhibited HT29 cell proliferation with 0.68, 0.54, and 0.30 μM of IC50, respectively. G1 phase was arrested by LY2835219 and its formulations. Furthermore, inhibition of CDK4/6 by LY2835219 formulations induced CDK4, CDK6, cyclin D1, and pRb decrease and p16 increase at both protein and mRNA levels. Overall, nanoparticle formulated LY2835219 could enhance the cytotoxicity and cell cycle arrest, and HACNP/LY strengthened the trend furtherly compared to CNP/LY. It is the first time to demonstrate the anticancer effect and mechanism against HT29 by LY2835219 and its nanoparticles. The drug and its nanoparticle formulations delay the cell growth and arrest cell cycle through p16-CDK4/6-pRb pathway, while the nanoparticle formulated LY2835219 could strengthen the process.

  20. 2-Phenylquinazolinones as dual-activity tankyrase-kinase inhibitors.

    Science.gov (United States)

    Nkizinkiko, Yves; Desantis, Jenny; Koivunen, Jarkko; Haikarainen, Teemu; Murthy, Sudarshan; Sancineto, Luca; Massari, Serena; Ianni, Federica; Obaji, Ezeogo; Loza, Maria I; Pihlajaniemi, Taina; Brea, Jose; Tabarrini, Oriana; Lehtiö, Lari

    2018-01-26

    Tankyrases (TNKSs) are enzymes specialized in catalyzing poly-ADP-ribosylation of target proteins. Several studies have validated TNKSs as anti-cancer drug targets due to their regulatory role in Wnt/β-catenin pathway. Recently a lot of effort has been put into developing more potent and selective TNKS inhibitors and optimizing them towards anti-cancer agents. We noticed that some 2-phenylquinazolinones (2-PQs) reported as CDK9 inhibitors were similar to previously published TNKS inhibitors. In this study, we profiled this series of 2-PQs against TNKS and selected kinases that are involved in the Wnt/β-catenin pathway. We found that they were much more potent TNKS inhibitors than they were CDK9/kinase inhibitors. We evaluated the compound selectivity to tankyrases over the ARTD enzyme family and solved co-crystal structures of the compounds with TNKS2. Comparative structure-based studies of the catalytic domain of TNKS2 with selected CDK9 inhibitors and docking studies of the inhibitors with two kinases (CDK9 and Akt) revealed important structural features, which could explain the selectivity of the compounds towards either tankyrases or kinases. We also discovered a compound, which was able to inhibit tankyrases, CDK9 and Akt kinases with equal µM potency.

  1. Discovery of 4,6-disubstituted pyrimidines as potent inhibitors of the heat shock factor 1 (HSF1) stress pathway and CDK9† †The authors declare the following competing financial interest(s): C. Rye, N. Chessum, L. Zani, M. Cheeseman, F. Raynaud, A. Hayes, A. Henley, E. de Billy, C. Lynch, S. Sharp, R. te Poele, L. O'Fee, P. Workman, and K. Jones are or have been employees of The Institute of Cancer Research which has a commercial interest in the development of HSF1 inhibitors. Authors who are or have been employed by The Institute of Cancer Research are subject to a “Rewards to Discoverers Scheme” which may reward contributors to a program that is subsequently licensed. ‡ ‡Electronic supplementary information (ESI) available: Experimental and assay procedures, crystallographic data, compound characterisation data. See DOI: 10.1039/c6md00159a Click here for additional data file.

    Science.gov (United States)

    Rye, Carl S.; Chessum, Nicola E. A.; Lamont, Scott; Pike, Kurt G.; Faulder, Paul; Demeritt, Julie; Kemmitt, Paul; Tucker, Julie; Zani, Lorenzo; Cheeseman, Matthew D.; Isaac, Rosie; Goodwin, Louise; Boros, Joanna; Raynaud, Florence; Hayes, Angela; Henley, Alan T.; de Billy, Emmanuel; Lynch, Christopher J.; Sharp, Swee Y.; te Poele, Robert; Fee, Lisa O’; Foote, Kevin M.; Green, Stephen

    2016-01-01

    Heat shock factor 1 (HSF1) is a transcription factor that plays key roles in cancer, including providing a mechanism for cell survival under proteotoxic stress. Therefore, inhibition of the HSF1-stress pathway represents an exciting new opportunity in cancer treatment. We employed an unbiased phenotypic screen to discover inhibitors of the HSF1-stress pathway. Using this approach we identified an initial hit (1) based on a 4,6-pyrimidine scaffold (2.00 μM). Optimisation of cellular SAR led to an inhibitor with improved potency (25, 15 nM) in the HSF1 phenotypic assay. The 4,6-pyrimidine 25 was also shown to have high potency against the CDK9 enzyme (3 nM). PMID:27746890

  2. Short communication: SAHA (vorinostat) induces CDK9 Thr-186 (T-loop) phosphorylation in resting CD4+ T cells: implications for reactivation of latent HIV.

    Science.gov (United States)

    Ramakrishnan, Rajesh; Liu, Hongbing; Rice, Andrew P

    2015-01-01

    The histone deacetylase inhibitor (HDACi) suberoylanilide hydroxyamic acid (SAHA), also known as vorinostat, has recently been reported to activate latent HIV-1 in patients undergoing antiretroviral therapy. It is possible that SAHA reactivation of latent viruses may involve effects on cellular transcription factors such as positive transcription elongation factor b (P-TEFb), a protein kinase whose core is composed of CDK9 and Cyclin T1. P-TEFb is recruited by the HIV-1 Tat protein to activate productive RNA polymerase II elongation of the integrated provirus. We found that SAHA treatment of isolated resting CD4(+) T cells induced CDK9 Thr-186 (T-loop) phosphorylation in six of eight healthy donors and increased Cyclin T1 expression in one donor; Thr-186 phosphorylation is required for P-TEFb function. Disulfiram, another small molecule currently under evaluation in clinical trials for reactivation of latent HIV-1, was also found capable of inducing CDK9 Thr-186 phosphorylation and Cyclin T1 levels in resting CD4(+) T cells from healthy donors. In a Jurkat CD4(+) T cells HIV-1 latency system, disulfiram reactivated the latent provirus and induced CDK9 Thr-186 phosphorylation. Our findings suggest that small molecules capable of reactivating latent HIV-1 in resting CD4(+) T cells may function in part by increasing CDK9 Thr-186 phosphorylation and perhaps Cyclin T1 expression, thereby up-regulating P-TEFb function.

  3. Targeting the AKT/GSK3β/cyclin D1/Cdk4 survival signaling pathway for eradication of tumor radioresistance acquired by fractionated radiotherapy.

    Science.gov (United States)

    Shimura, Tsutomu; Kakuda, Satoshi; Ochiai, Yasushi; Kuwahara, Yoshikazu; Takai, Yoshihiro; Fukumoto, Manabu

    2011-06-01

    Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3β-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3β/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3β/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor eradication in combination with chemotherapy. Copyright © 2011

  4. A recombinant protein based on Trypanosoma cruzi P21 enhances phagocytosis.

    Science.gov (United States)

    Rodrigues, Adele A; Clemente, Tatiana M; Dos Santos, Marlus A; Machado, Fabrício C; Gomes, Rafael G B; Moreira, Heline Hellen T; Cruz, Mário C; Brígido, Paula C; Dos Santos, Paulo C F; Martins, Flávia A; Bahia, Diana; Maricato, Juliana T; Janini, Luiz M R; Reboredo, Eduardo H; Mortara, Renato A; da Silva, Claudio V

    2012-01-01

    P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6)) on inflammatory macrophages during phagocytosis. Our results showed that P21-His(6) acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6) represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

  5. A recombinant protein based on Trypanosoma cruzi P21 enhances phagocytosis.

    Directory of Open Access Journals (Sweden)

    Adele A Rodrigues

    Full Text Available BACKGROUND: P21 is a secreted protein expressed in all developmental stages of Trypanosoma cruzi. The aim of this study was to determine the effect of the recombinant protein based on P21 (P21-His(6 on inflammatory macrophages during phagocytosis. FINDINGS: Our results showed that P21-His(6 acts as a phagocytosis inducer by binding to CXCR4 chemokine receptor and activating actin polymerization in a way dependent onthe PI3-kinase signaling pathway. CONCLUSIONS: Thus, our results shed light on the notion that native P21 is a component related to T. cruzi evasion from the immune response and that CXCR4 may be involved in phagocytosis. P21-His(6 represents an important experimental control tool to study phagocytosis signaling pathways of different intracellular parasites and particles.

  6. Flow cytometric analysis of p21 protein expression on irradiated human lymphocytes; Analise por citometria de fluxo da expressao da proteina p21 em linfocitos humanos irradiados

    Energy Technology Data Exchange (ETDEWEB)

    Santos, N.F.G.; Amaral, A., E-mail: neyliane@gmail.com [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Departamento de Energia Nuclear. Laboratorio de Modelagem e Biodosimetria Aplicada; Freitas-Silva, R. [Universidade Federal de Pernambuco (UFPE), Garanhuns, PE (Brazil). Departamento de Ciencias Naturais e Exatas; Pereira, V.R.A. [Fundacao Oswaldo Cruz (FIOCRUZ), Recife, PE (Brazil). Centro de Pesquisas Aggeu Magalhaes. Departamento de Imunologia. Lab. de Imunoparasitologia; Tasat, D.R. [Universidad Nacional de General San Martin, Buenos Aires (Argentina). Escuela de Ciencia y Tecnologia. Laboratorio de Biologia Celular del Pulmon

    2013-08-15

    Cell cycle blockage in G1 is a mechanism p21 protein-regulated and coupled to DNA damage response to permit genetic content analysis, damage repair and cell death. Analysis of proteins that participates of this response has progressed with new analytic tools, and data contributes to comprehension of radioinduced molecular events as well as to new approaches on practices that employ ionizing radiation. On this perspective, the aim of this research was to evaluate, by flow cytometry, p21 expression on irradiated human lymphocytes, maintained under different experimental conditions. Peripheral blood samples from 10 healthy subjects were irradiated with doses of 0 (non-irradiated), 1, 2 and 4 Gy. Lymphocytes were processed to analysis on ex vivo (no cultured) condition and after 24; 48 and 72 hours culture, with and without phytohemagglutinin stimulation. p21 protein expression levels were measured by flow cytometry, as percentage values. Results indicate that flow cytometric assay allows detection of changes on p21 expression, since it was detected significant increase on phytohemagglutinin-stimulated samples, for all times, against basal expression (ex vivo). However, it was not observed significant alterations on p21 protein radioinduced levels, for all doses, times and culture conditions analyzed. These results not indicate so p21 protein as bioindicator of ionizing radiation exposure. Nevertheless, data confirmation may to require analysis of a more numerous population. (author)

  7. Purification, characterization, and kinetic mechanism of cyclin D1. CDK4, a major target for cell cycle regulation.

    Science.gov (United States)

    Konstantinidis, A K; Radhakrishnan, R; Gu, F; Rao, R N; Yeh, W K

    1998-10-09

    The cyclin D1.CDK4-pRb (retinoblastoma protein) pathway plays a central role in the cell cycle, and its deregulation is correlated with many types of cancers. As a major drug target, we purified dimeric cyclin D1.CDK4 complex to near-homogeneity by a four-step procedure from a recombinant baculovirus-infected insect culture. We optimized the kinase activity and stability and developed a reproducible assay. We examined several catalytic and kinetic properties of the complex and, via steady-state kinetics, derived a kinetic mechanism with a peptide (RbING) and subsequently investigated the mechanistic implications with a physiologically relevant protein (Rb21) as the phosphoacceptor. The complex bound ATP 130-fold tighter when Rb21 instead of RbING was used as the phosphoacceptor. By using staurosporine and ADP as inhibitors, the kinetic mechanism of the complex appeared to be a "single displacement or Bi-Bi" with Mg2+.ATP as the leading substrate and phosphorylated RbING as the last product released. In addition, we purified a cyclin D1-CDK4 fusion protein to homogeneity by a three-step protocol from another recombinant baculovirus culture and observed similar kinetic properties and mechanisms as those from the complex. We attempted to model staurosporine in the ATP-binding site of CDK4 according to our kinetic data. Our biochemical and modeling data provide validation of both the complex and fusion protein as highly active kinases and their usefulness in antiproliferative inhibitor discovery.

  8. PUMA Cooperates with p21 to Regulate Mammary Epithelial Morphogenesis and Epithelial-To-Mesenchymal Transition.

    Directory of Open Access Journals (Sweden)

    Yanhong Zhang

    Full Text Available Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT markers. Interestingly, we found that knockdown of ΔNp73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT.

  9. PUMA Cooperates with p21 to Regulate Mammary Epithelial Morphogenesis and Epithelial-To-Mesenchymal Transition

    Science.gov (United States)

    Jung, Yong Sam; Chen, Xinbin

    2013-01-01

    Lumen formation is essential for mammary morphogenesis and requires proliferative suppression and apoptotic clearance of the inner cells within developing acini. Previously, we showed that knockdown of p53 or p73 leads to aberrant mammary acinus formation accompanied with decreased expression of p53 family targets PUMA and p21, suggesting that PUMA, an inducer of apoptosis, and p21, an inducer of cell cycle arrest, directly regulate mammary morphogenesis. To address this, we generated multiple MCF10A cell lines in which PUMA, p21, or both were stably knocked down. We found that morphogenesis of MCF10A cells was altered modestly by knockdown of either PUMA or p21 alone but markedly by knockdown of both PUMA and p21. Moreover, we found that knockdown of PUMA and p21 leads to loss of E-cadherin expression along with increased expression of epithelial-to-mesenchymal transition (EMT) markers. Interestingly, we found that knockdown of ΔNp73, which antagonizes the ability of wide-type p53 and TA isoform of p73 to regulate PUMA and p21, mitigates the abnormal morphogenesis and EMT induced by knockdown of PUMA or p21. Together, our data suggest that PUMA cooperates with p21 to regulate normal acinus formation and EMT. PMID:23805223

  10. RNA-Binding Protein FXR1 Regulates p21 and TERC RNA to Bypass p53-Mediated Cellular Senescence in OSCC.

    Directory of Open Access Journals (Sweden)

    Mrinmoyee Majumder

    2016-09-01

    Full Text Available RNA-binding proteins (RBP regulate numerous aspects of co- and post-transcriptional gene expression in cancer cells. Here, we demonstrate that RBP, fragile X-related protein 1 (FXR1, plays an essential role in cellular senescence by utilizing mRNA turnover pathway. We report that overexpressed FXR1 in head and neck squamous cell carcinoma targets (G-quadruplex (G4 RNA structure within both mRNA encoding p21 (Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A, Cip1 and the non-coding RNA Telomerase RNA Component (TERC, and regulates their turnover to avoid senescence. Silencing of FXR1 in cancer cells triggers the activation of Cyclin-Dependent Kinase Inhibitors, p53, increases DNA damage, and ultimately, cellular senescence. Overexpressed FXR1 binds and destabilizes p21 mRNA, subsequently reduces p21 protein expression in oral cancer cells. In addition, FXR1 also binds and stabilizes TERC RNA and suppresses the cellular senescence possibly through telomerase activity. Finally, we report that FXR1-regulated senescence is irreversible and FXR1-depleted cells fail to form colonies to re-enter cellular proliferation. Collectively, FXR1 displays a novel mechanism of controlling the expression of p21 through p53-dependent manner to bypass cellular senescence in oral cancer cells.

  11. Phosphorylation of XIAP by CDK1–cyclin-B1 controls mitotic cell death

    Science.gov (United States)

    Hou, Ying; Allan, Lindsey A.

    2017-01-01

    ABSTRACT Regulation of cell death is crucial for the response of cancer cells to drug treatments that cause arrest in mitosis, and is likely to be important for protection against chromosome instability in normal cells. Prolonged mitotic arrest can result in cell death by activation of caspases and the induction of apoptosis. Here, we show that X-linked inhibitor of apoptosis (XIAP) plays a key role in the control of mitotic cell death. Ablation of XIAP expression sensitises cells to prolonged mitotic arrest caused by a microtubule poison. XIAP is stable during mitotic arrest, but its function is controlled through phosphorylation by the mitotic kinase CDK1–cyclin-B1 at S40. Mutation of S40 to a phosphomimetic residue (S40D) inhibits binding to activated effector caspases and abolishes the anti-apoptotic function of XIAP, whereas a non-phosphorylatable mutant (S40A) blocks apoptosis. By performing live-cell imaging, we show that phosphorylation of XIAP reduces the threshold for the onset of cell death in mitosis. This work illustrates that mitotic cell death is a form of apoptosis linked to the progression of mitosis through control by CDK1–cyclin-B1. PMID:27927753

  12. Non-CDK-bound p27 (p27{sup NCDK}) is a marker for cell stress and is regulated through the Akt/PKB and AMPK-kinase pathways

    Energy Technology Data Exchange (ETDEWEB)

    Bjoerklund, Mia A. [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Vaahtomeri, Kari [Genome-Scale Biology Program and Institute of Biotechnology, 00014 University of Helsinki, Helsinki (Finland); Peltonen, Karita [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Viollet, Benoit [Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), 75014 Paris (France); INSERM U567, 75014 Paris (France); Maekelae, Tomi P. [Genome-Scale Biology Program and Institute of Biotechnology, 00014 University of Helsinki, Helsinki (Finland); Band, Arja M. [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Laiho, Marikki, E-mail: mlaiho1@jhmi.edu [Molecular Cancer Biology Program, Biomedicum Helsinki and Haartman Institute, University of Helsinki, Helsinki (Finland); Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD (United States)

    2010-03-10

    p27Kip1 (p27) tumour suppressor protein is regulated by multiple mechanisms including its turnover, localization and complex formation with its key targets, cyclin-dependent kinases (CDK) and cyclins. We have earlier shown that p27 exists in cells in a form that lacks cyclin/CDK interactions (hence non-CDK, p27{sup NCDK}) but the nature of p27{sup NCDK} has remained unresolved. Here we demonstrate that the epitope recognized by the p27{sup NCDK}-specific antibody resides in the p27 CDK-interaction domain and that p27{sup NCDK} is regulated by the balance of CDK inhibitors and cyclin-CDK complexes. We find that signalling by cellular growth promoting pathways, like phosphoinositol 3-kinase (PI3K) and specifically Akt/PKB kinase, inversely correlates with p27{sup NCDK} levels whereas total p27 levels are unaffected. p27{sup NCDK}, but not total p27, is increased by cellular perturbations such as hyperosmotic and metabolic stress and activation of AMP-activated protein kinase (AMPK). By using AMPK catalytic subunit proficient and deficient cells we further demonstrate that the AMPK pathway governs p27{sup NCDK} responses to metabolic stress and PI3K inhibition. These results indicate that p27{sup NCDK} is a sensitive marker for both cell stress and proliferation over and above p27 and is regulated by Akt/PKB and AMPK pathways.

  13. Multisite phosphorylation networks as signal processors for Cdk1.

    Science.gov (United States)

    Kõivomägi, Mardo; Ord, Mihkel; Iofik, Anna; Valk, Ervin; Venta, Rainis; Faustova, Ilona; Kivi, Rait; Balog, Eva Rose M; Rubin, Seth M; Loog, Mart

    2013-12-01

    The order and timing of cell-cycle events is controlled by changing substrate specificity and different activity thresholds of cyclin-dependent kinases (CDKs). However, it is not understood how a single protein kinase can trigger hundreds of switches in a sufficiently time-resolved fashion. We show that cyclin-Cdk1-Cks1-dependent phosphorylation of multisite targets in Saccharomyces cerevisiae is controlled by key substrate parameters including distances between phosphorylation sites, distribution of serines and threonines as phosphoacceptors and positioning of cyclin-docking motifs. The component mediating the key interactions in this process is Cks1, the phosphoadaptor subunit of the cyclin-Cdk1-Cks1 complex. We propose that variation of these parameters within networks of phosphorylation sites in different targets provides a wide range of possibilities for differential amplification of Cdk1 signals, thus providing a mechanism to generate a wide range of thresholds in the cell cycle.

  14. Novel Alternative Splice Variants of Mouse Cdk5rap2.

    Directory of Open Access Journals (Sweden)

    Nadine Kraemer

    Full Text Available Autosomal recessive primary microcephaly (MCPH is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

  15. Expression of p21/waf1 in oral squamous cell carcinomas--correlation with p53 and mdm2 and cellular proliferation index.

    Science.gov (United States)

    Ng, I O; Lam, K Y; Ng, M; Regezi, J A

    1999-01-01

    The cyclin-dependent kinase inhibitor p21/waf1 is regulated by p53-dependent and p53-independent pathways. In addition, mdm2 is an oncogene which forms an auto-regulatory loop with the normal p53 protein and its role has been implicated in oncogenesis. To determine whether a correlation exists between the expression of these gene products, tumor differentiation, tumor staging and radiation therapy, we investigated the expression of p21, p53 and mdm2, and cellular proliferation by Ki-67 (MIB1) labeling index using immunohistochemistry in 88 human oral squamous cell carcinoma (SCC) samples from 56 patients. Tumor expression of all nuclear proteins was scored according to the percentage of positive cancer nuclei, both with the cancer tissue as a whole as well as in different epithelial compartments of differentiation. Positive p21, p53, mdm2 and MIB1 staining was present in 82.4, 67.8, 25.9 and 98.8% of the SCC samples. The staining in different epithelial compartments of differentiation varied: those of p21 and mdm2 present predominantly in suprabasal and upper regions of the tumors: those of p53 and MIB1 in basal and suprabasal regions. Higher levels of p21 expression were seen in actively proliferating tumors (P = 0.025). p21 expression positively correlated with mdm2 expression but not with p53 expression. Moreover, the level of p21 expression was higher in older patients (P = 0.024) and female patients (P = 0.008). There was no significant association among p53, mdm2 and MIB1. Expression of p53 was higher in tumors with poorer cellular differentiation and in younger patients (P = 0.038 and 0.028). There was no association between tumor stage by TNM classification and the expression of any of these gene products or proliferation index. Radiation therapy did not alter the expression of any of these. To conclude, p21 protein was overexpressed in oral SCCs, and this overexpression was related to cell proliferation index and mdm2 expression but independent of p53

  16. Correlation between expression of p53, p21/WAF1, and MDM2 proteins and their prognostic significance in primary hepatocellular carcinoma

    Directory of Open Access Journals (Sweden)

    Fu Jia

    2009-12-01

    Full Text Available Abstract Background Tumor Protein p53 (p53, cyclin-dependent kinase inhibitor 1A (p21/WAF1, and murine double minute 2 (MDM2 participate in the regulation of cell growth. Altered expression of these gene products has been found in malignant tumors and has been associated with poor prognosis. Our aim was to investigate the expression of the 3 proteins in hepatocellular carcinoma (HCC and their prognostic significance. Methods We examined p53, p21/WAF1, and MDM2 expression in 181 pairs of HCC tissues and the adjacent hepatic tissues by performing immunohistochemistry and examined the expression of the 3 proteins in 7 pairs of HCC tissues and the adjacent hepatic tissues by using western blot analysis. Results The expression of p53, p21/WAF1, and MDM2 in the HCC tissues was significantly higher than those in the adjacent hepatic tissues (P P = 0.008. A statistical correlation was observed between expression of p53 and p21/WAF1 (R = 0.380, P = 0.000, p53 and MDM2 (R = 0.299, P = 0.000, p21/WAF1 and MDM2 (R = 0.285, P = 0.000 in 181 liver tissues adjacent to the tumor. Patients with a low pathologic grade HCC (I+II had a higher tendency to express p53 on tumor cells than the patients with high pathologic grade HCC (III+IV (P = 0.007. Survival analysis showed that positive p21/WAF1 expression or/and negative MDM2 expression in HCC was a predictor of better survival of patients after tumor resection (P Conclusions The proteins p53, p21/WAF1, and MDM2 were overexpressed in all the HCC cases in this study, and p53 and p21/WAF1 overexpression were positively correlated. The expression of p21/WAF1 and MDM2 can be considered as 2 useful indicators for predicting the prognosis of HCC.

  17. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

    Directory of Open Access Journals (Sweden)

    Wei-Ru Huang

    Full Text Available Avian reovirus (ARV protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128 of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

  18. Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

    Science.gov (United States)

    Huang, Wei-Ru; Chiu, Hung-Chuan; Liao, Tsai-Ling; Chuang, Kuo-Pin; Shih, Wing-Ling; Liu, Hung-Jen

    2015-01-01

    Avian reovirus (ARV) protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128) of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

  19. MDM2 Promotes Proteasomal Degradation of p21Waf1 via a Conformation Change*

    Science.gov (United States)

    Xu, Hongxia; Zhang, Zhuo; Li, Mao; Zhang, Ruiwen

    2010-01-01

    MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21Waf1. The present study was designed to characterize the mechanism(s) by which MDM2 induces p21Waf1 degradation. We first determined the regions of MDM2 required for p21Waf1 degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21Waf1 interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21Waf1 degradation. Specifically, amino acids 226–250 of MDM2 were required for p21Waf1 binding and degradation, and amino acids 251–260 were necessary for p21Waf1 degradation. The latter region induced a conformation change in p21Waf1, increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226–250 or aa 251–260), its capacity to promote p21Waf1 degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21Waf1) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties. PMID:20308078

  20. Induction of p21CIP1 protein and cell cycle arrest after inhibition of Aurora B kinase is attributed to aneuploidy and reactive oxygen species.

    Science.gov (United States)

    Kumari, Geeta; Ulrich, Tanja; Krause, Michael; Finkernagel, Florian; Gaubatz, Stefan

    2014-06-06

    Cell cycle progression requires a series of highly coordinated events that ultimately lead to faithful segregation of chromosomes. Aurora B is an essential mitotic kinase, which is involved in regulation of microtubule-kinetochore attachments and cytokinesis. Inhibition of Aurora B results in stabilization of p53 and induction of p53-target genes such as p21 to inhibit proliferation. We have previously demonstrated that induction of p21 by p53 after inhibition of Aurora B is dependent on the p38 MAPK, which promotes transcriptional elongation of p21 by RNA Pol II. In this study, we show that a subset of p53-target genes are induced in a p38-dependent manner upon inhibition of Aurora B. We also demonstrate that inhibition of Aurora B results in down-regulation of E2F-mediated transcription and that the cell cycle arrest after Aurora B inhibition depends on p53 and pRB tumor suppressor pathways. In addition, we report that activation of p21 after inhibition of Aurora B is correlated with increased chromosome missegregation and aneuploidy but not with binucleation or tetraploidy. We provide evidence that p21 is activated in aneuploid cells by reactive oxygen species (ROS) and p38 MAPK. Finally, we demonstrate that certain drugs that act on aneuploid cells synergize with inhibitors of Aurora B to inhibit colony formation and oncogenic transformation. These findings provide an important link between aneuploidy and the stress pathways activated by Aurora B inhibition and also support the use of Aurora B inhibitors in combination therapy for treatment of cancer. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Beryllium sulfate induces p21 CDKN1A expression and a senescence-like cell cycle arrest in susceptible cancer cell types.

    Science.gov (United States)

    Gorjala, Priyatham; Gary, Ronald K

    2010-12-01

    In fibroblasts, beryllium salt causes activation of the p53 transcription factor and induction of a senescence-like state. It is not known whether Be(2+) can affect the proliferation of cancer cells, which are generally unsusceptible to senescence. A172 glioblastoma and RKO colon carcinoma cell lines each have wildtype p53, so these cell types have the potential to be responsive to agents that activate p53. In A172 cells, BeSO(4) produced a G(0)/G(1)-phase cell cycle arrest and increased expression of senescence-associated β-galactosidase, an enzymatic marker of senescence. BeSO(4) caused phosphorylation of serine-15 of p53, accumulation of p53 protein, and expression of p21, the cyclin-dependent kinase inhibitor that is prominent during senescence. BeSO(4) inhibited A172 growth with an IC(50) = 4.7 μM in a 6-day proliferation assay. In contrast, BeSO(4) had no effect on RKO cells, even though Be(2+) uptake was similar for the two cell types. This differential responsiveness marks BeSO(4) as a reagent capable of activating a separable branch of the p53 signaling network. A172 and RKO cells are known to exhibit p53-dependent upregulation of p21 in response to DNA damage. The RKO cells produced high levels of p21 when exposed to DNA damaging agents, yet failed to express p21 when treated with BeSO(4). Conversely, BeSO(4) did not cause DNA damage in A172 cells, yet it was a potent inducer of p21 expression. These observations indicate that the growth control pathway affected by BeSO(4) is distinct from the DNA damage response pathway, even though both ultimately converge on p53 and p21.

  2. S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

    Directory of Open Access Journals (Sweden)

    AGM Mostofa

    2018-04-01

    Full Text Available Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

  3. File list: Oth.PSC.05.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.05.Cdk9.AllCell mm9 TFs and others Cdk9 Pluripotent stem cell SRX236483,SRX...104410 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Cdk9.AllCell.bed ...

  4. File list: Oth.Bld.50.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.50.Cdk9.AllCell mm9 TFs and others Cdk9 Blood SRX277329,SRX020973,SRX020972...,SRX020974,SRX020971 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.50.Cdk9.AllCell.bed ...

  5. Cdk2 is required for p53-independent G2/M checkpoint control.

    Directory of Open Access Journals (Sweden)

    Jon H Chung

    2010-02-01

    Full Text Available The activation of phase-specific cyclin-dependent kinases (Cdks is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G(2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G(2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G(2/M checkpoint activation.

  6. Diverse models for the prediction of CDK4 inhibitory activity of ...

    Indian Academy of Sciences (India)

    which form in a cyclical fashion.2 The CDKs are a family of heterodimeric Ser/Thr protein kinases each ... to proliferation. The major Cdk4/cyclin D substrate is the product of the retinoblastoma gene (pRB).8 Dur- ... whose products are required for S phase.9,10 Alter- ations in the cascade involving CDK4, CDK6, cyclin D,.

  7. Specific up-regulation of p21 by a small active RNA sequence suppresses human colorectal cancer growth

    Science.gov (United States)

    Wang, Lu-Lu; Guo, Hui-Hui; Zhan, Yun; Feng, Chen-Lin; Huang, Shuai; Han, Yan-Xing; Zheng, Wen-Sheng; Jiang, Jian-Dong

    2017-01-01

    The double stranded small active RNA (saRNA)- p21-saRNA-322 inhibits tumor growth by stimulating the p21 gene expression. We focused our research of p21-saRNA-322 on colorectal cancer because 1) p21 down-regulation is a signature abnormality of the cancer, and 2) colorectal cancer might be a suitable target for in situ p21-saRNA-322 delivery. The goal of the present study is to learn the activity of p21-saRNA-322 in colorectal cancer. Three human colorectal cancer cell lines, HCT-116, HCT-116 (p53–/−) and HT-29 were transfected with the p21-saRNA-322. The expression of P21 protein and p21 mRNA were measured using the Western blot and reverse transcriptase polymerase chain reaction (RT-PCR). The effect of p21-saRNA-322 on cancer cells was evaluated in vitro; and furthermore, a xenograft colorectal tumor mode in mice was established to estimate the tumor suppressing ability of p21-saRNA-322 in vivo. The results showed that in all three colorectal cancer cell lines, the expression of p21 mRNA and P21 protein were dramatically elevated after p21-saRNA-322 transfection. Transfection of p21-saRNA-322 caused apoptosis and cell cycle arrest at the G0/G1. Furthermore, anti-proliferation effect, reduction of colonies formation and cell senescence were observed in p21-saRNA-322 treated cells. Animal studies showed that p21-saRNA-322 treatment significantly inhibited the HT-29 tumor growth and facilitated p21 activation in vivo. These results indicated that, p21-saRNA-322-induceded up-regulation of p21 might be a promising therapeutic option for the treatment of colorectal cancer. PMID:28445988

  8. Increased expression of p21WAF1/CIP1 in kidney proximal tubules mediates fibrosis.

    Science.gov (United States)

    Megyesi, Judit; Tarcsafalvi, Adel; Li, Shenyang; Hodeify, Rawad; Seng, Nang San Hti Lar; Portilla, Didier; Price, Peter M

    2015-01-15

    Tissue fibrosis is a major cause of death in developed countries. It commonly occurs after either acute or chronic injury and affects diverse organs, including the heart, liver, lung, and kidney. Using the renal ablation model of chronic kidney disease, we previously found that the development of progressive renal fibrosis was dependent on p21(WAF1/Cip1) expression; the genetic knockout of the p21 gene greatly alleviated this disease. In the present study, we expanded on this observation and report that fibrosis induced by two different acute injuries to the kidney is also dependent on p21. In addition, when p21 expression was restricted only to the proximal tubule, fibrosis after injury was induced in the whole organ. One molecular fibrogenic switch we describe is transforming growth factor-β induction, which occurred in vivo and in cultured kidney cells exposed to adenovirus expressing p21. Our data suggests that fibrosis is p21 dependent and that preventing p21 induction after stress could be a novel therapeutic target.

  9. Phosphorylation of AIB1 at Mitosis Is Regulated by CDK1/CYCLIN B

    Science.gov (United States)

    Ferrero, Macarena; Ferragud, Juan; Orlando, Leonardo; Valero, Luz; Sánchez del Pino, Manuel; Farràs, Rosa; Font de Mora, Jaime

    2011-01-01

    Background Although the AIB1 oncogene has an important role during the early phase of the cell cycle as a coactivator of E2F1, little is known about its function during mitosis. Methodology/Principal Findings Mitotic cells isolated by nocodazole treatment as well as by shake-off revealed a post-translational modification occurring in AIB1 specifically during mitosis. This modification was sensitive to the treatment with phosphatase, suggesting its modification by phosphorylation. Using specific inhibitors and in vitro kinase assays we demonstrate that AIB1 is phosphorylated on Ser728 and Ser867 by Cdk1/cyclin B at the onset of mitosis and remains phosphorylated until exit from M phase. Differences in the sensitivity to phosphatase inhibitors suggest that PP1 mediates dephosphorylation of AIB1 at the end of mitosis. The phosphorylation of AIB1 during mitosis was not associated with ubiquitylation or degradation, as confirmed by western blotting and flow cytometry analysis. In addition, luciferase reporter assays showed that this phosphorylation did not alter the transcriptional properties of AIB1. Importantly, fluorescence microscopy and sub-cellular fractionation showed that AIB1 phosphorylation correlated with the exclusion from the condensed chromatin, thus preventing access to the promoters of AIB1-dependent genes. Phospho-specific antibodies developed against Ser728 further demonstrated the presence of phosphorylated AIB1 only in mitotic cells where it was localized preferentially in the periphery of the cell. Conclusions Collectively, our results describe a new mechanism for the regulation of AIB1 during mitosis, whereby phosphorylation of AIB1 by Cdk1 correlates with the subcellular redistribution of AIB1 from a chromatin-associated state in interphase to a more peripheral localization during mitosis. At the exit of mitosis, AIB1 is dephosphorylated, presumably by PP1. This exclusion from chromatin during mitosis may represent a mechanism for governing the

  10. CDK5 A Novel Role in Prostate Cancer Immunotherapy

    Science.gov (United States)

    2016-10-01

    combination with immunotherapies, including immune checkpoint blockers, a prostate cancer vaccine , and other agents will be conducted and optimized...combination with immunotherapies, including immune checkpoint blockers, a prostate cancer vaccine , and other agents based on our findings in Specific...KEYWORDS: Prostate cancer, CDK5, immunotherapy, vaccine , tumor microenvironment 3. ACCOMPLISHMENTS: What were the major goals of the project? Major

  11. Specific CDK4/6 inhibition in breast cancer

    DEFF Research Database (Denmark)

    Polk, Anne; Kolmos, Ida Lykke; Kümler, Iben

    2016-01-01

    BACKGROUND: Loss of cell cycle control is a hallmark of cancer, and aberrations in the cyclin-dependent kinase-retinoblastoma (CDK-Rb) pathway are common in breast cancer (BC). Consequently, inhibition of this pathway is an attractive therapeutic strategy. The present review addresses efficacy...

  12. Cdk5 is essential for synaptic vesicle endocytosis

    DEFF Research Database (Denmark)

    Tan, Timothy C; Valova, Valentina A; Malladi, Chandra S

    2003-01-01

    Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin...

  13. CDK1 Inhibition Targets the p53-NOXA-MCL1 Axis, Selectively Kills Embryonic Stem Cells, and Prevents Teratoma Formation

    Directory of Open Access Journals (Sweden)

    Noelle E. Huskey

    2015-03-01

    Full Text Available Embryonic stem cells (ESCs have adopted an accelerated cell-cycle program with shortened gap phases and precocious expression of cell-cycle regulatory proteins, including cyclins and cyclin-dependent kinases (CDKs. We examined the effect of CDK inhibition on the pathways regulating proliferation and survival of ESCs. We found that inhibiting cyclin-dependent kinase 1 (CDK1 leads to activation of the DNA damage response, nuclear p53 stabilization, activation of a subset of p53 target genes including NOXA, and negative regulation of the anti-apoptotic protein MCL1 in human and mouse ESCs, but not differentiated cells. We demonstrate that MCL1 is highly expressed in ESCs and loss of MCL1 leads to ESC death. Finally, we show that clinically relevant CDK1 inhibitors prevent formation of ESC-derived tumors and induce necrosis in established ESC-derived tumors. Our data demonstrate that ES cells are uniquely sensitive to CDK1 inhibition via a p53/NOXA/MCL1 pathway.

  14. Cell cycle control by a minimal Cdk network.

    Directory of Open Access Journals (Sweden)

    Claude Gérard

    2015-02-01

    Full Text Available In present-day eukaryotes, the cell division cycle is controlled by a complex network of interacting proteins, including members of the cyclin and cyclin-dependent protein kinase (Cdk families, and the Anaphase Promoting Complex (APC. Successful progression through the cell cycle depends on precise, temporally ordered regulation of the functions of these proteins. In light of this complexity, it is surprising that in fission yeast, a minimal Cdk network consisting of a single cyclin-Cdk fusion protein can control DNA synthesis and mitosis in a manner that is indistinguishable from wild type. To improve our understanding of the cell cycle regulatory network, we built and analysed a mathematical model of the molecular interactions controlling the G1/S and G2/M transitions in these minimal cells. The model accounts for all observed properties of yeast strains operating with the fusion protein. Importantly, coupling the model's predictions with experimental analysis of alternative minimal cells, we uncover an explanation for the unexpected fact that elimination of inhibitory phosphorylation of Cdk is benign in these strains while it strongly affects normal cells. Furthermore, in the strain without inhibitory phosphorylation of the fusion protein, the distribution of cell size at division is unusually broad, an observation that is accounted for by stochastic simulations of the model. Our approach provides novel insights into the organization and quantitative regulation of wild type cell cycle progression. In particular, it leads us to propose a new mechanistic model for the phenomenon of mitotic catastrophe, relying on a combination of unregulated, multi-cyclin-dependent Cdk activities.

  15. p21 is Responsible for Ionizing Radiation-induced Bypass of Mitosis.

    Science.gov (United States)

    Zhang, Xu Rui; Liu, Yong Ai; Sun, Fang; Li, He; Lei, Su Wen; Wang, Ju Fang

    2016-07-01

    To explore the role of p21 in ionizing radiation-induced changes in protein levels during the G2/M transition and long-term G2 arrest. Protein expression levels were assessed by western blot in the human uveal melanoma 92-1 cells after treatment with ionizing radiation. Depletion of p21 was carried out by employing the siRNA technique. Cell cycle distribution was determined by flow cytometry combined with histone H3 phosphorylation at Ser28, an M-phase marker. Senescence was assessed by senescence- associated-β-galactosidase (SA-β-gal) staining combined with Ki67 staining, a cell proliferation marker. Accompanying increased p21, the protein levels of G2/M transition genes declined significantly in 92-1 cells irradiated with 5 Gy of X-rays. Furthermore, these irradiated cells were blocked at the G2 phase followed by cellular senescence. Depletion of p21 rescued radiation-induced G2 arrest as demonstrated by the upregulation of G2/M transition kinases, as well as the high expression of histone H3 phosphorylated at Ser28. Knockdown of p21 resulted in entry into mitosis of irradiated 92-1 cells. However, cells with serious DNA damage failed to undergo cytokinesis, leading to the accumulation of multinucleated cells. Our results indicated that p21 was responsible for the downregulation of G2/M transition regulatory proteins and the bypass of mitosis induced by irradiation. Downregulation of p21 by siRNA resulted in G2-arrested cells entering into mitosis with serious DNA damage. This is the first report on elucidating the role of p21 in the bypass of mitosis. Copyright © 2016 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

  16. Trisubstituted pyrazolopyrimidines as novel angiogenesis inhibitors.

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    Sabine B Weitensteiner

    Full Text Available Current inhibitors of angiogenesis comprise either therapeutic antibodies (e.g. bevacicumab binding to VEGF-A or small molecular inhibitors of receptor tyrosin kinases like e.g. sunitinib, which inhibits PDGFR and VEGFR. We have recently identified cyclin-dependent kinase 5 (Cdk5 as novel alternative and pharmacologically accessible target in the context of angiogenesis. In the present work we demonstrate that trisubstituted pyrazolo[4,3-d]pyrimidines constitute a novel class of compounds which potently inhibit angiogenesis. All seven tested compounds inhibited endothelial cell proliferation with IC(50 values between 1 and 18 µM. Interestingly, this seems not to be due to cytotoxicity, since none of them showed acute cytotoxic effects on endothelial cells at a concentration of 10 µM,. The three most potent compounds (LGR1404, LGR1406 and LGR1407 also inhibited cell migration (by 27, 51 and 31%, resp., chemotaxis (by 50, 70 and 60% in accumulative distance, resp., and tube formation (by 25, 60 and 30% of total tube length, resp. at the non-toxic concentration of 10 µM. Furthermore, angiogenesis was reduced in vivo in the CAM assay by these three compounds. A kinase selectivity profiling revealed that the compounds prevalently inhibit Cdk2, Cdk5 and Cdk9. The phenotype of the migrating cells (reduced formation of lamellipodia, loss of Rac-1 translocation to the membrane resembles the previously described effects of silencing of Cdk5 in endothelial cells. We conclude that especially LGR1406 and LGR1407 are highly attractive anti-angiogenic compounds, whose effects seem to largely depend on their Cdk5 inhibiting properties.

  17. Growth Hormone Menurunkan Ekspresi Protein p53 dan p21 Sel Endotel Tikus Jantan (GROWTH HORMONE REDUCES P53 AND P21 ENDOTHELIAL PROTEIN EXPRESSION IN MALE RATS

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    I Gusti Ayu Dewi Ratnayanti

    2016-11-01

    Full Text Available The use of growth hormone (GH treatment in aging related condition such as atherosclerosis is stillcontroversial. Previous study showed GH reduce atherosclerotic plaque and prevent endothelial cellsenescence. This study was aimed to understand the mechanism of GH effect to endothelial senescencethrough p53/p21 pathway. A randomized posttest only control group design study was conducted. Twentymale Wistar rats were randomized into five groups; negative control (P0, positive control (P1, and GHtreated group (P2, P3, P4. Negative control group was fed with standard diet, and others were fed withatherogenic diet for 20 weeks. After 10 weeks, subjects were injected subcutaneously (0,1 mL with aquadest(P0 and P1 and increasing dose of GH (0,02 IU, 0,04 IU, and 0,08 IU for P2, P3, P4 once a day respectivelyfor 10 weeks. In the end of the study all subjects were examined for p53 and p21 endothelial proteinexpressions. Immunohistochemistry of endothelial p53 showed reduce expression in treated groups (P0:7.28 ± 0.36; P1: 39.51 ± 1.18; P2: 32.70 ± 1.10; P3: 16.98 ± 0.78; and P4: 14.29 ± 0.38. The reduction was also observed in p21 expression (P0: 5.38 ± 0.49; P1: 37.81 ± 0.76; P2: 26.02 ± 1.54; P3: 16.37 ± 1.24; andP4: 4.82 ± 0.61. One way analysis of variance and post hoc test (LSD analysis showed significant differencesbetween all groups (p<0.05. In conclusion, GH reduces endothelial expression of p53 and p21 and thispathway may contribute to GH effect on atherosclerotic plaque and endothelial senescence.

  18. Interferon regulatory factor-1 together with reactive oxygen species promotes the acceleration of cell cycle progression by up-regulating the cyclin E and CDK2 genes during high glucose-induced proliferation of vascular smooth muscle cells.

    Science.gov (United States)

    Zhang, Xi; Liu, Long; Chen, Chao; Chi, Ya-Li; Yang, Xiang-Qun; Xu, Yan; Li, Xiao-Tong; Guo, Shi-Lei; Xiong, Shao-Hu; Shen, Man-Ru; Sun, Yu; Zhang, Chuan-Sen; Hu, Kai-Meng

    2013-10-14

    The high glucose-induced proliferation of vascular smooth muscle cells (VSMCs) plays an important role in the development of diabetic vascular diseases. In a previous study, we confirmed that Interferon regulatory factor-1 (Irf-1) is a positive regulator of the high glucose-induced proliferation of VSMCs. However, the mechanisms remain to be determined. The levels of cyclin/CDK expression in two cell models involving Irf-1 knockdown and overexpression were quantified to explore the relationship between Irf-1 and its downstream effectors under normal or high glucose conditions. Subsequently, cells were treated with high glucose/NAC, normal glucose/H₂O₂, high glucose/U0126 or normal glucose/H₂O₂/U0126 during an incubation period. Then proliferation, cyclin/CDK expression and cell cycle distribution assays were performed to determine whether ROS/Erk1/2 signaling pathway was involved in the Irf-1-induced regulation of VSMC growth under high glucose conditions. We found that Irf-1 overexpression led to down-regulation of cyclin D1/CDK4 and inhibited cell cycle progression in VSMCs under normal glucose conditions. In high glucose conditions, Irf-1 overexpression led to an up-regulation of cyclin E/CDK2 and an acceleration of cell cycle progression, whereas silencing of Irf-1 suppressed the expression of both proteins and inhibited the cell cycle during the high glucose-induced proliferation of VSMCs. Treatment of VSMCs with antioxidants prevented the Irf-1 overexpression-induced proliferation of VSMCs, the up-regulation of cyclin E/CDK2 and the acceleration of cell cycle progression in high glucose conditions. In contrast, under normal glucose conditions, H₂O₂ stimulation and Irf-1 overexpression induced cell proliferation, up-regulated cyclin E/CDK2 expression and promoted cell cycle acceleration. In addition, overexpression of Irf-1 promoted the activation of Erk1/2 and when VSMCs overexpressing Irf-1 were treated with U0126, the specific Erk1/2 inhibitor

  19. Expression and significance of VEGF, CD34, Ki-67 and p21 in pterygium

    Directory of Open Access Journals (Sweden)

    Li-Bo Wang

    2014-07-01

    Full Text Available AIM: To investigate the expression of VEGF, CD34, Ki-67 and p21 in pterygium as well as the correlation between their expression and clinical pathological characteristics; explore its pathogenesis. METHODS: Immunohistochemical S-P staining method was adopted in detecting the expression of VEGF, CD34, Ki-67 and p21 in 62 cases of pterygia and 20 cases of normal conjunctival tissues. Relationship between these markers and clinical pathological characteristics was analyzed. RESULTS:(1The positive expression of VEGF, CD34, Ki-67 and p21 in 62 cases of pterygia was 74.2%(46/62, 77.4%(48/62, 66.1%(41/62and 40.3%(25/62respectively. The differences were statistically significant compared with normal conjunctival tissues(PPP>0.05; the expression of Ki-67 was correlated with clinical stages(PP>0.05; the expression of p21 was correlated with clinical stages and pterygium characters(PP>0.05.(3Spearman correlation showed that there was a positive correlation between VEGF and Ki-67(r=0.279, Pr=0.299, Pr=-0.267, PP>0.05.CONCLUSION:(1Overexpression of VEGF, Ki-67, CD34 and low expression of p21 suggest that these markers are concerned with the development and progression of pterygium.(2Expression of VEGF and CD34 increases along with the increase of clinical types and stages, expression of Ki-67 increases along with the increase of clinical stages, and expression of p21 decreases along with the improvement of clinical types or stages; they suggest that these markers may play important roles in the development and recurrence of pterygium.(3There is positive correlation between VEGF and Ki-67, VEGF and CD34 as well as negative correlation between VEGF and p21. They suggest that there may be synergistic action between two factors during the development and progression of pterygium.

  20. CDK2 and mTOR are direct molecular targets of isoangustone A in the suppression of human prostate cancer cell growth

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Eunjung; Son, Joe Eun; Byun, Sanguine; Lee, Seung Joon; Kim, Yeong A [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Liu, Kangdong [The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912 (United States); Kim, Jiyoung [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Lim, Soon Sung; Park, Jung Han Yoon [Department of Food Science and Nutrition, College of Natural Science, Hallym University, Chuncheon, 200-702 (Korea, Republic of); Dong, Zigang [The Hormel Institute, University of Minnesota, 801 16th Avenue NE, Austin, MN 55912 (United States); Lee, Ki Won, E-mail: kiwon@snu.ac.kr [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270 (Korea, Republic of); Lee, Hyong Joo, E-mail: leehyjo@snu.ac.kr [WCU Biomodulation Major, Department of Agricultural Biotechnology and Center for Food and Bioconvergence, Seoul National University, Seoul 151-921 (Korea, Republic of); Advanced Institutes of Convergence Technology, Seoul National University, Suwon 443-270 (Korea, Republic of)

    2013-10-01

    Licorice extract which is used as a natural sweetener has been shown to possess inhibitory effects against prostate cancer, but the mechanisms responsible are poorly understood. Here, we report a compound, isoangustone A (IAA) in licorice that potently suppresses the growth of aggressive prostate cancer and sought to clarify its mechanism of action. We analyzed its inhibitory effects on the growth of PTEN-deleted human prostate cancer cells, in vitro and in vivo. Administration of IAA significantly attenuated the growth of prostate cancer cell cultures and xenograft tumors. These effects were found to be attributable to inhibition of the G1/S phase cell cycle transition and the accumulation of p27{sup kip1}. The elevated p27{sup kip1} expression levels were concurrent with the decrease of its phosphorylation at threonine 187 through suppression of CDK2 kinase activity and the reduced phosphorylation of Akt at Serine 473 by diminishing the kinase activity of the mammalian target of rapamycin (mTOR). Further analysis using recombinant proteins and immunoprecipitated cell lysates determined that IAA exerts suppressive effects against CDK2 and mTOR kinase activity by direct binding with both proteins. These findings suggested that the licorice compound IAA is a potent molecular inhibitor of CDK2 and mTOR, with strong implications for the treatment of prostate cancer. Thus, licorice-derived extracts with high IAA content warrant further clinical investigation for nutritional sources for prostate cancer patients. - Highlights: • Isoangustone A suppresses growth of PC3 and LNCaP prostate cancer cells. • Administration of isoangustone A inhibits tumor growth in mice. • Treatment of isoangustone A induces cell cycle arrest and accumulation of p27{sup kip1}. • Isoangustone A inhibits CDK2 and mTOR activity. • Isoangustone A directly binds with CDK2 and mTOR complex in prostate cancer cells.

  1. Structural basis for CDK6 activation by a virus-encoded cyclin

    Energy Technology Data Exchange (ETDEWEB)

    Schulze-Gahmen, Ursula; Kim, Sung-Hou

    2002-01-17

    Cyclin from herpesvirus saimiri (Vcyclin) preferentially forms complexes with cyclin-dependent kinase 6 (CDK6) from primate host cells. These complexes show higher kinase activity than host cell CDK complexes with cellular cyclins and are resistant to cyclin-dependent inhibitory proteins (CDKIs). The crystal structure of human CDK6-Vcyclin in an active state was determined to 3.1 Angstrom resolution to get a better understanding of the structural basis of CDK6 activation by viral cyclins. The unphosphorylated CDK6 complexed to Vcyclin has many features characteristic of cyclinA-activated, phosphorylated CDK2. There are, however, differences in the conformation at the tip of the T-loop and its interactions with Vcyclin. Residues in the N-terminal extension of Vcyclin wrap around the tip of the CDK6 T-loop and form a short b-sheet with the T-loop backbone. These interactions lead to a 20 percent larger buried surface in the CDK6-Vcyclin interface than in the CDK2-cyclinA complex and are probably largely responsible for Vcyclin specificity for CDK6 and resistance of the complex to inhibition by INK-typeCDKIs.

  2. Anti-inflammatory activity of chloroquine and amodiaquine through p21-mediated suppression of T cell proliferation and Th1 cell differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Oh, Sera; Shin, Ji Hyun; Jang, Eun Jung; Won, Hee Yeon; Kim, Hyo Kyeong; Jeong, Mi- Gyeong [College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Kim, Kwang Soo [Molecular Neurobiology Laboratory, Department of Psychiatry, McLean Hospital, Harvard Medical School, Belmont, MA 02478 (United States); Hwang, Eun Sook, E-mail: eshwang@ewha.ac.kr [College of Pharmacy and Graduate School of Pharmaceutical Sciences, Ewha Womans University, Seoul 120-750 (Korea, Republic of)

    2016-05-27

    Chloroquine (CQ) and amodiaquine (AQ) have been used for treating or preventing malaria for decades, and their application has expanded into treating inflammatory disease in humans. CQ and AQ are applicable for controlling rheumatoid arthritis, but their molecular mechanisms of anti-inflammatory activity remain to be elucidated. In this study, we examined the effects of CQ and AQ on T cell activation and T cell-mediated immune response. CQ had no significant effect on T cell numbers, but decreased the population of T cells with a high division rate. However, AQ treatment significantly increased the number of cells with low division rates and eliminated cells with high division rates, resulting in the inhibition of T cell proliferation triggered by T cell receptor stimulation, of which inhibition occurred in developing effector T helper and regulatory T cells, regardless of the different exogenous cytokines. Interestingly, the cyclin-dependent kinase inhibitor p21 was significantly and dose-dependently increased by CQ, and more potently by AQ, while other cell cycle regulators were unchanged. Both CQ and AQ elevated the transcription level of p21 though the activation of p53, but also blocked p21 protein degradation in the presence of cycloheximide, causing p21 protein accumulation mainly in the nucleus. Sustained treatment of developing T cells with either CQ or AQ suppressed IFN-γ production in a dose dependent manner and potently inhibited the differentiation of IFN-γ-producing Th1 cells. These results demonstrate that CQ and AQ increase the expression level of p21 and inhibit T cell proliferation and the development of IFN-γ-producing Th1 cells, thereby revealing beneficial roles in treating a wide range of chronic inflammatory diseases mediated by inflammatory T cells. -- Highlights: •T cell division rates are suppressed by chloroquine and amodiaquine treatment. •Chloroquine and amodiaquine potently increased the p21 expression. •The p21 induction is

  3. p21/Cyclin E pathway modulates anticlastogenic function of Bmi-1 in cancer cells

    Science.gov (United States)

    Deng, Wen; Zhou, Yuan; Tiwari, Agnes FY; Su, Hang; Yang, Jie; Zhu, Dandan; Lau, Victoria Ming Yi; Hau, Pok Man; Yip, Yim Ling; Cheung, Annie LM; Guan, Xin-Yuan; Tsao, Sai Wah

    2015-01-01

    Apart from regulating stem cell self-renewal, embryonic development and proliferation, Bmi-1 has been recently reported to be critical in the maintenance of genome integrity. In searching for novel mechanisms underlying the anticlastogenic function of Bmi-1, we observed, for the first time, that Bmi-1 positively regulates p21 expression. We extended the finding that Bmi-1 deficiency induced chromosome breaks in multiple cancer cell models. Interestingly, we further demonstrated that knockdown of cyclin E or ectopic overexpression of p21 rescued Bmi-1 deficiency-induced chromosome breaks. We therefore conclude that p21/cyclin E pathway is crucial in modulating the anticlastogenic function of Bmi-1. As it is well established that the overexpression of cyclin E potently induces genome instability and p21 suppresses the function of cyclin E, the novel and important implication from our findings is that Bmi-1 plays an important role in limiting genomic instability in cylin E-overexpressing cancer cells by positive regulation of p21. PMID:25131797

  4. Histone deacetylase inhibitors SAHA and sodium butyrate block G1-to-S cell cycle progression in neurosphere formation by adult subventricular cells

    Directory of Open Access Journals (Sweden)

    Doughty Martin L

    2011-05-01

    Full Text Available Abstract Background Histone deacetylases (HDACs are enzymes that modulate gene expression and cellular processes by deacetylating histones and non-histone proteins. While small molecule inhibitors of HDAC activity (HDACi are used clinically in the treatment of cancer, pre-clinical treatment models suggest they also exert neuroprotective effects and stimulate neurogenesis in neuropathological conditions. However, the direct effects of HDACi on cell cycle progression and proliferation, two properties required for continued neurogenesis, have not been fully characterized in adult neural stem cells (NSCs. In this study, we examined the effects of two broad class I and class II HDACi on adult mouse NSCs, the hydroxamate-based HDACi suberoylanilide hydroxamic acid (vorinostat, SAHA and the short chain fatty acid HDACi sodium butyrate. Results We show that both HDACi suppress the formation of neurospheres by adult mouse NSCs grown in proliferation culture conditions in vitro. DNA synthesis is significantly inhibited in adult mouse NSCs exposed to either SAHA or sodium butyrate and inhibition is associated with an arrest in the G1 phase of the cell cycle. HDACi exposure also resulted in transcriptional changes in adult mouse NSCs. Cdk inhibitor genes p21 and p27 transcript levels are increased and associated with elevated H3K9 acetylation levels at proximal promoter regions of p21 and p27. mRNA levels for notch effector Hes genes and Spry-box stem cell transcription factors are downregulated, whereas pro-neural transcription factors Neurog1 and Neurod1 are upregulated. Lastly, we show HDAC inhibition under proliferation culture conditions leads to long-term changes in cell fate in adult mouse NSCs induced to differentiate in vitro. Conclusion SAHA and sodium butyrate directly regulate cdk inhibitor transcription to control cell cycle progression in adult mouse NSCs. HDAC inhibition results in G1 arrest in adult mouse NSCs and transcriptional changes

  5. Rising cyclin-CDK levels order cell cycle events.

    Directory of Open Access Journals (Sweden)

    Catherine Oikonomou

    Full Text Available Diverse mitotic events can be triggered in the correct order and time by a single cyclin-CDK. A single regulator could confer order and timing on multiple events if later events require higher cyclin-CDK than earlier events, so that gradually rising cyclin-CDK levels can sequentially trigger responsive events: the "quantitative model" of ordering.This 'quantitative model' makes predictions for the effect of locking cyclin at fixed levels for a protracted period: at low cyclin levels, early events should occur rapidly, while late events should be slow, defective, or highly variable (depending on threshold mechanism. We titrated the budding yeast mitotic cyclin Clb2 within its endogenous expression range to a stable, fixed level and measured time to occurrence of three mitotic events: growth depolarization, spindle formation, and spindle elongation, as a function of fixed Clb2 level. These events require increasingly more Clb2 according to their normal order of occurrence. Events occur efficiently and with low variability at fixed Clb2 levels similar to those observed when the events normally occur. A second prediction of the model is that increasing the rate of cyclin accumulation should globally advance timing of all events. Moderate (<2-fold overexpression of Clb2 accelerates all events of mitosis, resulting in consistently rapid sequential cell cycles. However, this moderate overexpression also causes a significant frequency of premature mitoses leading to inviability, suggesting that Clb2 expression level is optimized to balance the fitness costs of variability and catastrophe.We conclude that mitotic events are regulated by discrete cyclin-CDK thresholds. These thresholds are sequentially triggered as cyclin increases, yielding reliable order and timing. In many biological processes a graded input must be translated into discrete outputs. In such systems, expression of the central regulator is likely to be tuned to an optimum level, as we

  6. Ras activation by insulin and epidermal growth factor through enhanced exchange of guanine nucleotides on p21ras

    NARCIS (Netherlands)

    Medema, R.H.; Vries-Smits, A.M. de; Zon, G.C.M. van der; Maassen, J.A.; Bos, J.L.

    1993-01-01

    A number of growth factors, including insulin and epidermal growth factor (EGF), induce accumulation of the GTP-bound form of p21ras. This accumulation could be caused either by an increase in guanine nucleotide exchange on p21ras or by a decrease in the GTPase activity of p21ras. To investigate

  7. CDK-Taverna: an open workflow environment for cheminformatics

    Directory of Open Access Journals (Sweden)

    Zielesny Achim

    2010-03-01

    Full Text Available Abstract Background Small molecules are of increasing interest for bioinformatics in areas such as metabolomics and drug discovery. The recent release of large open access chemistry databases generates a demand for flexible tools to process them and discover new knowledge. To freely support open science based on these data resources, it is desirable for the processing tools to be open source and available for everyone. Results Here we describe a novel combination of the workflow engine Taverna and the cheminformatics library Chemistry Development Kit (CDK resulting in a open source workflow solution for cheminformatics. We have implemented more than 160 different workers to handle specific cheminformatics tasks. We describe the applications of CDK-Taverna in various usage scenarios. Conclusions The combination of the workflow engine Taverna and the Chemistry Development Kit provides the first open source cheminformatics workflow solution for the biosciences. With the Taverna-community working towards a more powerful workflow engine and a more user-friendly user interface, CDK-Taverna has the potential to become a free alternative to existing proprietary workflow tools.

  8. Efficacy of cyclin dependent kinase 4 inhibitors as potent neuroprotective agents against insults relevant to Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Priyankar Sanphui

    Full Text Available Alzheimer's disease (AD is a progressive neurodegenerative disease with no cure till today. Aberrant activation of cell cycle regulatory proteins is implicated in neurodegenerative diseases including AD. We and others have shown that Cyclin dependent kinase 4 (Cdk4 is activated in AD brain and is required for neuron death. In this study, we tested the efficiency of commercially available Cdk4 specific inhibitors as well as a small library of synthetic molecule inhibitors targeting Cdk4 as neuroprotective agents in cellular models of neuron death. We found that several of these inhibitors significantly protected neuronal cells against death induced by nerve growth factor (NGF deprivation and oligomeric beta amyloid (Aβ that are implicated in AD. These neuroprotective agents inhibit specifically Cdk4 kinase activity, loss of mitochondrial integrity, induction of pro-apoptotic protein Bim and caspase3 activation in response to NGF deprivation. The efficacies of commercial and synthesized inhibitors are comparable. The synthesized molecules are either phenanthrene based or naphthalene based and they are synthesized by using Pschorr reaction and Buchwald coupling respectively as one of the key steps. A number of molecules of both kinds block neurodegeneration effectively. Therefore, we propose that Cdk4 inhibition would be a therapeutic choice for ameliorating neurodegeneration in AD and these synthetic Cdk4 inhibitors could lead to development of effective drugs for AD.

  9. Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.

    Directory of Open Access Journals (Sweden)

    Lara Wohlbold

    2012-08-01

    Full Text Available The cyclin-dependent kinases (CDKs that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS Cdk2 after exposure to ionizing radiation (IR enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.

  10. Anti-angiogenic effects of purine inhibitors of cyclin dependent kinases

    Czech Academy of Sciences Publication Activity Database

    Liebl, J.; Kryštof, Vladimír; Vereb, G.; Takacs, L.; Strnad, Miroslav; Pechan, P.; Havlíček, Libor; Zatloukal, Marek; Fuerst, R.; Vollmar, A. M.; Zahler, S.

    2011-01-01

    Roč. 14, č. 3 (2011), s. 281-291 ISSN 0969-6970 R&D Projects: GA ČR GA204/08/0511; GA ČR GA301/08/1649 Institutional research plan: CEZ:AV0Z50380511 Keywords : Angiogenesis * Cdk * Small molecule Cdk inhibitors * Roscovitine Subject RIV: CE - Biochemistry Impact factor: 6.063, year: 2011

  11. Cdk1, PKCδ and calcineurin-mediated Drp1 pathway contributes to mitochondrial fission-induced cardiomyocyte death

    Energy Technology Data Exchange (ETDEWEB)

    Zaja, Ivan [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bai, Xiaowen, E-mail: xibai@mcw.edu [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Liu, Yanan; Kikuchi, Chika; Dosenovic, Svjetlana; Yan, Yasheng; Canfield, Scott G. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Bosnjak, Zeljko J. [Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States); Department of Physiology, Medical College of Wisconsin, Milwaukee, WI 53226 (United States)

    2014-10-31

    Highlights: • Drp1-mediated increased mitochondrial fission but not fusion is involved the cardiomyocyte death during anoxia-reoxygenation injury. • Reactive oxygen species are upstream initiators of mitochondrial fission. • Increased mitochondrial fission is resulted from Cdk1-, PKCδ-, and calcineurin-mediated Drp1 pathways. - Abstract: Myocardial ischemia–reperfusion (I/R) injury is one of the leading causes of death and disability worldwide. Mitochondrial fission has been shown to be involved in cardiomyocyte death. However, molecular machinery involved in mitochondrial fission during I/R injury has not yet been completely understood. In this study we aimed to investigate molecular mechanisms of controlling activation of dynamin-related protein 1 (Drp1, a key protein in mitochondrial fission) during anoxia-reoxygenation (A/R) injury of HL1 cardiomyocytes. A/R injury induced cardiomyocyte death accompanied by the increases of mitochondrial fission, reactive oxygen species (ROS) production and activated Drp1 (pSer616 Drp1), and decrease of inactivated Drp1 (pSer637 Drp1) while mitochondrial fusion protein levels were not significantly changed. Blocking Drp1 activity with mitochondrial division inhibitor mdivi1 attenuated cell death, mitochondrial fission, and Drp1 activation after A/R. Trolox, a ROS scavenger, decreased pSer616 Drp1 level and mitochondrial fission after A/R. Immunoprecipitation assay further indicates that cyclin dependent kinase 1 (Cdk1) and protein kinase C isoform delta (PKCδ) bind Drp1, thus increasing mitochondrial fission. Inhibiting Cdk1 and PKCδ attenuated the increases in pSer616 Drp1, mitochondrial fission, and cardiomyocyte death. FK506, a calcineurin inhibitor, blocked the decrease in expression of inactivated pSer637 Drp1 and mitochondrial fission. Our findings reveal the following novel molecular mechanisms controlling mitochondrial fission during A/R injury of cardiomyocytes: (1) ROS are upstream initiators of

  12. p53, p21, Rb, and MDM2 proteins in tongue carcinoma from patients 75 years.

    Science.gov (United States)

    Regezi, J A; Dekker, N P; McMillan, A; Ramirez-Amador, V; Meneses-Garcia, A; Ruiz-Godoy Rivera, L M; Chrysomali, E; Ng, I O

    1999-07-01

    Relatively rare squamous cell carcinomas of the tongue in young patients may be associated with different etiologic factors and pathogenetic mechanisms than carcinomas from the same site in older patients. Alterations in cell cycle proteins likely contribute to the biologic behavior of these neoplasms. The purpose of this investigation was to evaluate cell cycle proteins (p53, p21, Rb, MDM2) in lateral tongue cancers from patients at the two ends of the age spectrum. All available archived lateral tongue carcinomas from patients 75 years (23 males and 13 females) were stained and compared. Positive p53 staining was seen in 18/36 of the 75-year group (p = 0.149). Increased p21 staining (both percent of positive cells and intensity) was evident in 25/32 of the 75-year group (p = 1.0). Increased p21 expression was seen in both p53-positive and -negative cases in both age groups. Rb protein was increased in 16/29 of the 75-year group (p = 0.58). Fourteen cases (4/35 vs 10/36, p = 0.135) showed positive MDM2 staining; MDM2-positive cases were also p53 positive in 4/4 younger and 8/10 older patients. We conclude that p53, p21, Rb, and MDM2 are over-expressed in lateral tongue cancers, and that immunohistochemical profiles are heterogeneous. A p53-independent pathway of p21 induction is supported by the results; p53 suppression may be associated with MDM2 protein expression in a subset of cancers. Significant differences in the expression of p53, p21, Rb, and MDM2 proteins are not evident in lateral tongue carcinomas from patients 75 years.

  13. p21 promotes oncolytic adenoviral activity in ovarian cancer and is a potential biomarker

    Directory of Open Access Journals (Sweden)

    Lockley Michelle

    2010-07-01

    Full Text Available Abstract The oncolytic adenovirus dl922-947 replicates selectively within and lyses cells with a dysregulated Rb pathway, a finding seen in > 90% human cancers. dl922-947 is more potent than wild type adenovirus and the E1B-deletion mutant dl1520 (Onyx-015. We wished to determine which host cell factors influence cytotoxicity. SV40 large T-transformed MRC5-VA cells are 3-logs more sensitive to dl922-947 than isogenic parental MRC5 cells, confirming that an abnormal G1/S checkpoint increases viral efficacy. The sensitivity of ovarian cancer cells to dl922-947 varied widely: IC50 values ranged from 51 (SKOV3ip1 to 0.03 pfu/cell (TOV21G. Cells sensitive to dl922-947 had higher S phase populations and supported earlier E1A expression. Cytotoxicity correlated poorly with both infectivity and replication, but well with expression of p21 by microarray and western blot analyses. Matched p21+/+ and -/- Hct116 cells confirmed that p21 influences dl922-947 activity in vitro and in vivo. siRNA-mediated p21 knockdown in sensitive TOV21G cells decreases E1A expression and viral cytotoxicity, whilst expression of p21 in resistant A2780CP cells increases virus activity in vitro and in intraperitoneal xenografts. These results highlight that host cell factors beyond simple infectivity can influence the efficacy of oncolytic adenoviruses. p21 expression may be an important biomarker of response in clinical trials.

  14. Correlation between chromosome 9p21 locus deletion and prognosis in clinically localized prostate cancer.

    Science.gov (United States)

    Barros, Érika Aparecida Felix de; Pontes-Junior, José; Reis, Sabrina Thalita; Lima, Amanda Eunice Ramos; Souza, Isida C; Salgueiro, Jose Lucas; Fontes, Douglas; Dellê, Humberto; Coelho, Rafael Ferreira; Viana, Nayara Izabel; Leite, Kátia Ramos Moreira; Nahas, William C; Srougi, Miguel

    2017-05-04

    Some studies have reported that deletions at chromosome arm 9p occur frequently and represent a critical step in carcinogenesis of some neoplasms. Our aim was to evaluate the deletion of locus 9p21 and chromosomes 3, 7 and 17 in localized prostate cancer (PC) and correlate these alterations with prognostic factors and biochemical recurrence after surgery. We retrospectively evaluated surgical specimens from 111 patients with localized PC who underwent radical prostatectomy. Biochemical recurrence was defined as a prostate-specific antigen (PSA) >0.2 ng/mL and the mean postoperative follow-up was 123 months. The deletions were evaluated using fluorescence in situ hybridization with centromeric and locus-specific probes in a tissue microarray containing 2 samples from each patient. We correlated the occurrence of any deletion with pathological stage, Gleason score, ISUP grade group, PSA and biochemical recurrence. We observed a loss of any probe in only 8 patients (7.2%). The most common deletion was the loss of locus 9p21, which occurred in 6.4% of cases. Deletions of chromosomes 3, 7 and 17 were observed in 2.3%, 1.2% and 1.8% patients, respectively. There was no correlation between chromosome loss and Gleason score, ISUP, PSA or stage. Biochemical recurrence occurred in 83% cases involving 9p21 deletions. Loss of 9p21 locus was significantly associated with time to recurrence (p = 0.038). We found low rates of deletion in chromosomes 3, 7 and 17 and 9p21 locus. We observed that 9p21 locus deletion was associated with worse prognosis in localized PC treated by radical prostatectomy.

  15. p21{sup WAF1/CIP1} deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ae Jeong; Jee, Hye Jin; Song, Naree; Kim, Minjee [Department of Biochemistry, College of Medicine, Dong-A University, Busan (Korea, Republic of); Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan (Korea, Republic of); Jeong, Seon-Young [Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan (Korea, Republic of); Department of Medical Genetics, Ajou University School of Medicine (Korea, Republic of); Yun, Jeanho, E-mail: yunj@dau.ac.kr [Department of Biochemistry, College of Medicine, Dong-A University, Busan (Korea, Republic of); Mitochondria Hub Regulation Center, College of Medicine, Dong-A University, Busan (Korea, Republic of)

    2013-01-11

    Highlights: Black-Right-Pointing-Pointer p21{sup -/-} HCT116 cells exhibited an increase in mitochondrial mass. Black-Right-Pointing-Pointer The expression levels of PGC-1{alpha} and AMPK were upregulated in p21{sup -/-} HCT116 cells. Black-Right-Pointing-Pointer The proliferation of p21{sup -/-} HCT116 cells in galactose medium was significantly impaired. Black-Right-Pointing-Pointer p21 may play a role in maintaining proper mitochondrial mass and respiratory function. -- Abstract: p21{sup WAF1/CIP1} is a critical regulator of cell cycle progression. However, the role of p21 in mitochondrial function remains poorly understood. In this study, we examined the effect of p21 deficiency on mitochondrial function in HCT116 human colon cancer cells. We found that there was a significant increase in the mitochondrial mass of p21{sup -/-} HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53{sup -/-} cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1{alpha} and TFAM and AMPK activity were also elevated in p21{sup -/-} cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1{alpha} axis. However, the increase in mitochondrial biogenesis in p21{sup -/-} cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21{sup -/-} cells exhibited significant proliferation impairment in galactose medium, suggesting that p21 deficiency induces a defect in the mitochondrial respiratory chain in HCT116 cells. Taken together, our results suggest that the loss of p21 results in an aberrant increase in the mitochondrial mass and in mitochondrial dysfunction in HCT116 cells, indicating that p21 is required to maintain proper mitochondrial mass and respiratory function.

  16. Effects of p21 deletion in mouse models of premature aging

    Science.gov (United States)

    Benson, Erica K.; Zhao, Bo; Sassoon, David A.; Lee, Sam W.; Aaronson, Stuart A.

    2017-01-01

    An approach to investigate the role of cellular senescence in organismal aging has been to abrogate signaling pathways known to induce cellular senescence and to assess the effects in mouse models of premature aging. Recently, we reported the effect of loss of function of p21, a gene implicated in p53-induced cellular senescence, in the background of the Ku80−/− premature aging mouse (Zhao et al., EMBO Rep 2009). Here, we provide an overview of the effects of p21 deletion in different models of premature aging. PMID:19535900

  17. p21(WAF1) is component of a positive feedback loop that maintains the p53 transcriptional program.

    Science.gov (United States)

    Pang, Lisa Y; Scott, Mary; Hayward, Richard L; Mohammed, Hisham; Whitelaw, C Bruce A; Smith, Graeme C M; Hupp, Ted R

    2011-03-15

    The regulation of p53 activity through the MDM2 negative feedback loop is driven in part by an extrinsic ATM-pulse that maintains p53 oscillations in response to DNA damage. We report here that the p53 pathway has evolved an intrinsic positive feedback loop that is maintained by the p53-inducible gene product p21(WAF1). p21-null cancer cells have defects in p53 protein turnover, reductions in MDM2-mediated degradation of p53, and reduced DNA damage-induced ubiquitination of p53. TLR3-IRF1 or ATM-dependent signaling to p53 is defective in p21-null cells and complementation of the p21 gene in p21-null cancer cells restores the p53 transcriptional response. The mechanism of p53 inactivity in p21-null cells is linked to a p53 protein equilibrium shift from chromatin into cytosolic fractions and complementation of the p21 gene into p21-null cells restores the nuclear localization of p53. A loss of p53 transcriptional function in murine B-cells heterozygous or homozygous null for p21 highlights a p21-gene dosage effect that maintains the full p53 transcriptional response. ATM inhibition results in nuclear exclusion of p53 highlighting a positive genetic interaction between ATM and p21. P21 protein oscillates in undamaged proliferating cells, and reductions of p21 protein using siRNA eliminate the DNA damage-induced p53 pulse. The p53 transcription program has evolved a negative feedback loop maintained by MDM2 that is counteracted by a positive feedback loop maintained by ATM-p21 the balance of which controls the specific activity of p53 as a transcription factor.

  18. Polyphyllin VII increases sensitivity to gefitinib by modulating the elevation of P21 in acquired gefitinib resistant non-small cell lung cancer

    Directory of Open Access Journals (Sweden)

    Honggang Wang

    2017-07-01

    Full Text Available Blockade of EGFR with reversible EGFR tyrosine kinase inhibitors (TKIs is considered the frontline strategy for advanced NSCLC with EGFR mutations. However, acquired resistance to EGFR-TKI has been observed, resulting in disease progression and limited clinical benefit. Polyphyllin VII is the main member of polyphyllin family, which has been demonstrated to show strong anticancer activity against carcinomas. The sensitizing effect and underlying mechanism of Polyphyllin VII against acquired EGFR-TKI resistant NSCLC are still unexplored. In the present study, we aim to examined the sensitizing effect of Polyphyllin VII to gefitinib by modulating P21 signaling pathway in gefitinib acquired resistant NSCLC in vitro and in vivo. Gefitinib sensitive PC-9 cells and gefitinib acquired resistant H1975 cells were used. Cell proliferation and Clonogenic assay, Cell cycle analysis, Western blotting analysis and xenograft treatment were carried out. Polyphyllin VII enhanced the anti-proliferative effects of gefitinib and gefitinib-induced G1 phase arrest by modulation of P21 signaling pathway in acquired gefitinib resistant cells in vitro and in vivo. Polyphyllin VII elevated sensitization of gefitinib acquired resistant NSCLC cells to gefitinib through G1 phase arrest and modulation of P21 signaling pathway. It provides a potential new strategy to overcome gefitinib acquired resistance for EGFR-TKI resistant NSCLC.

  19. Apoptosis Induced by Piroxicam plus Cisplatin Combined Treatment Is Triggered by p21 in Mesothelioma

    Science.gov (United States)

    Baldi, Alfonso; Piccolo, Maria Teresa; Boccellino, Maria Rosaria; Donizetti, Aldo; Cardillo, Irene; La Porta, Raffaele; Quagliuolo, Lucio; Spugnini, Enrico P.; Cordero, Francesca; Citro, Gennaro; Menegozzo, Massimo; Calogero, Raffaele A.; Crispi, Stefania

    2011-01-01

    Background Malignant mesothelioma (MM) is a rare, highly aggressive tumor, associated to asbestos exposure. To date no chemotherapy regimen for MM has proven to be definitively curative, and new therapies for MM treatment need to be developed. We have previously shown in vivo that piroxicam/cisplatin combined treatment in MM, specifically acts on cell cycle regulation triggering apoptosis, with survival increase. Methodology/Principal Findings We analyzed, at molecular level, the apoptotic increase caused by piroxicam/cisplatin treatment in MM cell lines. By means of genome wide analyses, we analyzed transcriptional gene deregulation both after the single piroxicam or cisplatin and the combined treatment. Here we show that apoptotic increase following combined treatment is mediated by p21, since apoptotic increase in piroxicam/cisplatin combined treatment is abolished upon p21 silencing. Conclusions/Significance Piroxicam/cisplatin combined treatment determines an apoptosis increase in MM cells, which is dependent on the p21 expression. The results provided suggest that piroxicam/cisplatin combination might be tested in clinical settings in tumor specimens that express p21. PMID:21858171

  20. Apoptosis induced by piroxicam plus cisplatin combined treatment is triggered by p21 in mesothelioma.

    Directory of Open Access Journals (Sweden)

    Alfonso Baldi

    Full Text Available BACKGROUND: Malignant mesothelioma (MM is a rare, highly aggressive tumor, associated to asbestos exposure. To date no chemotherapy regimen for MM has proven to be definitively curative, and new therapies for MM treatment need to be developed. We have previously shown in vivo that piroxicam/cisplatin combined treatment in MM, specifically acts on cell cycle regulation triggering apoptosis, with survival increase. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed, at molecular level, the apoptotic increase caused by piroxicam/cisplatin treatment in MM cell lines. By means of genome wide analyses, we analyzed transcriptional gene deregulation both after the single piroxicam or cisplatin and the combined treatment. Here we show that apoptotic increase following combined treatment is mediated by p21, since apoptotic increase in piroxicam/cisplatin combined treatment is abolished upon p21 silencing. CONCLUSIONS/SIGNIFICANCE: Piroxicam/cisplatin combined treatment determines an apoptosis increase in MM cells, which is dependent on the p21 expression. The results provided suggest that piroxicam/cisplatin combination might be tested in clinical settings in tumor specimens that express p21.

  1. Exploiting Chemical Libraries, Structure, and Genomics in the Search for Kinase Inhibitors

    NARCIS (Netherlands)

    Gray, Nathanael S.; Wodicka, Lisa; Thunnissen, Andy-Mark W.H.; Norman, Thea C.; Kwon, Soojin; Espinoza, F. Hernan; Morgan, David O.; Barnes, Georjana; LeClerc, Sophie; Meijer, Laurent; Kim, Sung-Hou; Lockhart, David J.; Schultz, Peter G.

    1998-01-01

    Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening, potent inhibitors

  2. Explicit treatment of active-site waters enhances quantum mechanical/implicit solvent scoring: Inhibition of CDK2 by new pyrazolo[1,5-a]pyrimidines.

    Science.gov (United States)

    Hylsová, Michaela; Carbain, Benoit; Fanfrlík, Jindřich; Musilová, Lenka; Haldar, Susanta; Köprülüoğlu, Cemal; Ajani, Haresh; Brahmkshatriya, Pathik S; Jorda, Radek; Kryštof, Vladimír; Hobza, Pavel; Echalier, Aude; Paruch, Kamil; Lepšík, Martin

    2017-01-27

    We present comprehensive testing of solvent representation in quantum mechanics (QM)-based scoring of protein-ligand affinities. To this aim, we prepared 21 new inhibitors of cyclin-dependent kinase 2 (CDK2) with the pyrazolo[1,5-a]pyrimidine core, whose activities spanned three orders of magnitude. The crystal structure of a potent inhibitor bound to the active CDK2/cyclin A complex revealed that the biphenyl substituent at position 5 of the pyrazolo[1,5-a]pyrimidine scaffold was located in a previously unexplored pocket and that six water molecules resided in the active site. Using molecular dynamics, protein-ligand interactions and active-site water H-bond networks as well as thermodynamics were probed. Thereafter, all the inhibitors were scored by the QM approach utilizing the COSMO implicit solvent model. Such a standard treatment failed to produce a correlation with the experiment (R 2  = 0.49). However, the addition of the active-site waters resulted in significant improvement (R 2  = 0.68). The activities of the compounds could thus be interpreted by taking into account their specific noncovalent interactions with CDK2 and the active-site waters. In summary, using a combination of several experimental and theoretical approaches we demonstrate that the inclusion of explicit solvent effects enhance QM/COSMO scoring to produce a reliable structure-activity relationship with physical insights. More generally, this approach is envisioned to contribute to increased accuracy of the computational design of novel inhibitors. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. Cinnamon and Its Metabolite Sodium Benzoate Attenuate the Activation of p21rac and Protect Memory and Learning in an Animal Model of Alzheimer's Disease.

    Directory of Open Access Journals (Sweden)

    Khushbu K Modi

    Full Text Available This study underlines the importance of cinnamon, a commonly used natural spice and flavoring material, and its metabolite sodium benzoate (NaB in attenuating oxidative stress and protecting memory and learning in an animal model of Alzheimer's disease (AD. NaB, but not sodium formate, was found to inhibit LPS-induced production of reactive oxygen species (ROS in mouse microglial cells. Similarly, NaB also inhibited fibrillar amyloid beta (Aβ- and 1-methyl-4-phenylpyridinium(+-induced microglial production of ROS. Although NaB reduced the level of cholesterol in vivo in mice, reversal of the inhibitory effect of NaB on ROS production by mevalonate, and geranylgeranyl pyrophosphate, but not cholesterol, suggests that depletion of intermediates, but not end products, of the mevalonate pathway is involved in the antioxidant effect of NaB. Furthermore, we demonstrate that an inhibitor of p21rac geranylgeranyl protein transferase suppressed the production of ROS and that NaB suppressed the activation of p21rac in microglia. As expected, marked activation of p21rac was observed in the hippocampus of subjects with AD and 5XFAD transgenic (Tg mouse model of AD. However, oral feeding of cinnamon (Cinnamonum verum powder and NaB suppressed the activation of p21rac and attenuated oxidative stress in the hippocampus of Tg mice as evident by decreased dihydroethidium (DHE and nitrotyrosine staining, reduced homocysteine level and increased level of reduced glutathione. This was accompanied by suppression of neuronal apoptosis, inhibition of glial activation, and reduction of Aβ burden in the hippocampus and protection of memory and learning in transgenic mice. Therefore, cinnamon powder may be a promising natural supplement in halting or delaying the progression of AD.

  4. Suppression of c-Myc enhances p21WAF1/CIP1 -mediated G1 cell cycle arrest through the modulation of ERK phosphorylation by ascochlorin.

    Science.gov (United States)

    Jeong, Yun-Jeong; Hoe, Hyang-Sook; Cho, Hyun-Ji; Park, Kwan-Kyu; Kim, Dae-Dong; Kim, Cheorl-Ho; Magae, Junji; Kang, Dong Wook; Lee, Sang-Rae; Chang, Young-Chae

    2018-02-01

    Numerous anti-cancer agents inhibit cell cycle progression via a p53-dependent mechanism; however, other genes such as the proto-oncogene c-Myc are promising targets for anticancer therapy. In the present study, we provide evidence that ascochlorin, an isoprenoid antibiotic, is a non-toxic anti-cancer agent that induces G1 cell cycle arrest and p21 WAF1/CIP1 expression by downregulating of c-Myc protein expression. Ascochlorin promoted the G1 arrest, upregulated p53 and p21 WAF1/CIP1 , and downregulated c-Myc in HCT116 cells. In p53-deficient cells, ascochlorin enhanced the expression of G1 arrest-related genes except p53. Small interfering RNA (siRNA) mediated c-Myc silencing indicated that the transcriptional repression of c-Myc was related to ascochlorin-mediated modulation of p21 WAF1/CIP1 expression. Ascochlorin suppressed the stabilization of the c-Myc protein by inhibiting ERK and P70S6K/4EBP1 phosphorylation, whereas it had no effect on c-Myc degradation mediated by PI3K/Akt/GSK3β. The ERK inhibitor PD98059 and siRNA-mediated ERK silencing induced G1 arrest and p21 WAF1/CIP1 expression by downregulating c-Myc in p53-deficient cells. These results indicated that ascochlorin-induced G1 arrest is associated with the repression of ERK phosphorylation and c-Myc expression. Thus, we reveal a role for ascochlorin in inhibiting tumor growth via G1 arrest, and identify a novel regulatory mechanism for ERK/c-Myc. © 2017 Wiley Periodicals, Inc.

  5. Curcumin suppresses proliferation of colon cancer cells by targeting CDK2.

    Science.gov (United States)

    Lim, Tae-Gyu; Lee, Sung-Young; Huang, Zunnan; Lim, Do Young; Chen, Hanyong; Jung, Sung Keun; Bode, Ann M; Lee, Ki Won; Dong, Zigang

    2014-04-01

    Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

  6. Cdk5 phosphorylates non-genotoxically overexpressed p53 following inhibition of PP2A to induce cell cycle arrest/apoptosis and inhibits tumor progression

    Directory of Open Access Journals (Sweden)

    Kumari Ratna

    2010-07-01

    Full Text Available Abstract Background p53 is the most studied tumor suppressor and its overexpression may or may not cause cell death depending upon the genetic background of the cells. p53 is degraded by human papillomavirus (HPV E6 protein in cervical carcinoma. Several stress activated kinases are known to phosphorylate p53 and, among them cyclin dependent kinase 5 (Cdk5 is one of the kinase studied in neuronal cell system. Recently, the involvement of Cdk5 in phosphorylating p53 has been shown in certain cancer types. Phosphorylation at specific serine residues in p53 is essential for it to cause cell growth inhibition. Activation of p53 under non stress conditions is poorly understood. Therefore, the activation of p53 and detection of upstream kinases that phosphorylate non-genotoxically overexpressed p53 will be of therapeutic importance for cancer treatment. Results To determine the non-genotoxic effect of p53; Tet-On system was utilized and p53 inducible HPV-positive HeLa cells were developed. p53 overexpression in HPV-positive cells did not induce cell cycle arrest or apoptosis. However, we demonstrate that overexpressed p53 can be activated to upregulate p21 and Bax which causes G2 arrest and apoptosis, by inhibiting protein phosphatase 2A. Additionally, we report that the upstream kinase cyclin dependent kinase 5 interacts with p53 to phosphorylate it at Serine20 and Serine46 residues thereby promoting its recruitment on p21 and bax promoters. Upregulation and translocation of Bax causes apoptosis through intrinsic mitochondrial pathway. Interestingly, overexpressed activated p53 specifically inhibits cell-growth and causes regression in vivo tumor growth as well. Conclusion Present study details the mechanism of activation of p53 and puts forth the possibility of p53 gene therapy to work in HPV positive cervical carcinoma.

  7. CDK2 Phosphorylation on Threonine39 by AKT and Its Implication on Cyclin Binding, Cellular Localization, and Cell Cycle Progression

    National Research Council Canada - National Science Library

    Da Silva, Thiago

    2007-01-01

    .... Our hypothesis is that Cdk2 exists in two freely exchangeable conformations: that seen in the active, cyclin bound crystal and that of the inactive monomeric Cdk2, with the latter predominating in the absence of cyclin...

  8. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    International Nuclear Information System (INIS)

    Liu, Hongbing; Herrmann, Christine H.; Chiang, Karen; Sung, Tzu-Ling; Moon, Sung-Hwan; Donehower, Lawrence A.; Rice, Andrew P.

    2010-01-01

    Positive elongation factor b (P-TEFb) is a cellular protein kinase that is required for RNA polymerase II (RNAP II) transcriptional elongation of protein coding genes. P-TEFb is a set of different molecular complexes, each containing CDK9 as the catalytic subunit. There are two isoforms of the CDK9 protein - the major 42 KDa CDK9 isoform and the minor 55KDa isoform that is translated from an in-frame mRNA that arises from an upstream transcriptional start site. We found that shRNA depletion of the 55K CDK9 protein in HeLa cells induces apoptosis and double-strand DNA breaks (DSBs). The levels of apoptosis and DSBs induced by the depletion were reduced by expression of a 55K CDK9 protein variant resistant to the shRNA, indicating that these phenotypes are the consequence of depletion of the 55K protein and not off-target effects. We also found that the 55K CDK9 protein, but not the 42K CDK9 protein, specifically associates with Ku70, a protein involved in DSB repair. Our findings suggest that the 55K CDK9 protein may function in repair of DNA through an association with Ku70.

  9. Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

    Science.gov (United States)

    Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

    2015-02-01

    Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

  10. CDK4 amplification predicts recurrence of well-differentiated liposarcoma of the abdomen.

    Directory of Open Access Journals (Sweden)

    Sanghoon Lee

    Full Text Available The absence of CDK4 amplification in liposarcomas is associated with favorable prognosis. We aimed to identify the factors associated with tumor recurrence in patients with well-differentiated (WD and dedifferentiated (DD liposarcomas.From 2000 to 2010, surgical resections for 101 WD and DD liposarcomas were performed. Cases in which complete surgical resections with curative intent were carried out were selected. MDM2 and CDK4 gene amplification were analyzed by quantitative real-time polymerase chain reaction (Q-PCR.There were 31 WD and 17 DD liposarcomas. Locoregional recurrence was observed in 11 WD and 3 DD liposarcomas. WD liposarcomas showed better patient survival compared to DD liposarcomas (P<0.05. Q-PCR analysis of the liposarcomas revealed the presence of CDK4 amplification in 44 cases (91.7% and MDM2 amplification in 46 cases (95.8%. WD liposarcomas with recurrence after surgical resection had significantly higher levels of CDK4 amplification compared to those without recurrence (P = 0.041. High level of CDK4 amplification (cases with CDK4 amplification higher than the median 7.54 was associated with poor recurrence-free survival compared to low CDK4 amplification in both univariate (P = 0.012 and multivariate analyses (P = 0.020.Level of CDK4 amplification determined by Q-PCR was associated with the recurrence of WD liposarcomas after surgical resection.

  11. A CDK-independent metabolic oscillator orchestrates the budding yeast cell cycle

    NARCIS (Netherlands)

    Papagiannakis, A.; Niebel, B.; Wit, E.; Heinemann, M.

    2017-01-01

    Eukaryotic cell division is known to be controlled by the cyclin/ CDK machinery. However, eukaryotes have evolved prior to CDKs, and cells can divide in the absence of major cyclin/CDK components. We hypothesized that an autonomous metabolic oscillator provides dynamic triggers for cell cycle

  12. Chemoprevention utility of silibinin and Cdk4 pathway inhibition in Apc−/+ mice

    International Nuclear Information System (INIS)

    Karim, Baktiar O; Rhee, Ki-Jong; Liu, Guosheng; Zheng, Dongfeng; Huso, David L

    2013-01-01

    Colorectal cancer (CRC) is the second leading cause of death from cancer in the United States. Colorectal cancers have a prolonged latency following initiation that may span decades providing ample time for implementing a chemoprevention strategy that could block or reverse the progression to CRC. Cdk4 pathway alterations have been linked to a number of cancers including CRC. In these experiments we focused on the Cdk4 pathway and its role in intestinal tumorigenesis as a possible target in chemoprevention strategies. We evaluated the effect of Cdk4 blockade on the prevention of intestinal tumor formation by crossing Cdk4 −/− mice to Apc −/+ mice. In addition, we tested the effect of the dietary compound silibinin on the Cdk4 pathway in Apc −/+ mice and HT-29 colon cancer cells in culture. Cdk4 −/− mice backcrossed to Apc −/+ mice reduced intestinal adenoma formation compared to Apc −/+ controls. Silibinin effectively targeted the Cdk4 pathway causing hypophosphorylation of the retinoblastoma protein, inhibited cell growth, and induced apoptosis. As a result silibinin blocked the development of intestinal adenomas by 52% in this genetic model (Apc −/+ mice) of early events in colorectal cancer formation. No toxic abnormalities were detected in mice which received silibinin. Modification of the Cdk4 pathway using a natural plant-derived compound such as silibinin may be a useful chemopreventive strategy for colorectal carcinomas

  13. p21-Activated kinases are required for transformation in a cell-based model of neurofibromatosis type 2.

    Directory of Open Access Journals (Sweden)

    Hoi Yee Chow

    2010-11-01

    Full Text Available NF2 is an autosomal dominant disease characterized by development of bilateral vestibular schwannomas and other benign tumors in central nervous system. Loss of the NF2 gene product, Merlin, leads to aberrant Schwann cell proliferation, motility, and survival, but the mechanisms by which this tumor suppressor functions remain unclear. One well-defined target of Merlin is the group I family of p21-activated kinases, which are allosterically inhibited by Merlin and which, when activated, stimulate cell cycle progression, motility, and increased survival. Here, we examine the effect of Pak inhibition on cells with diminished Merlin function.Using a specific peptide inhibitor of group I Paks, we show that loss of Pak activity restores normal cell movement in cells lacking Merlin function. In addition, xenografts of such cells form fewer and smaller tumors than do cells without Pak inhibition. However, in tumors, loss of Pak activity does not reduce Erk or Akt activity, two signaling proteins that are thought to mediate Pak function in growth factor pathways.These results suggest that Pak functions in novel signaling pathways in NF2, and may serve as a useful therapeutic target in this disease.

  14. p21-LacZ reporter mice reflect p53-dependent toxic insult

    International Nuclear Information System (INIS)

    Vasey, Douglas B.; Wolf, C. Roland; MacArtney, Thomas; Brown, Ken; Whitelaw, C. Bruce A.

    2008-01-01

    There is an urgent need to discover less toxic and more selective drugs to treat disease. The use of transgenic mice that report on toxic insult-induced transcription can provide a valuable tool in this regard. To exemplify this strategy, we have generated transgenic mice carrying a p21-LacZ transgene. Transgene activity reflected endogenous p21 gene activation in various tissues, displayed compound-specific spatial expression signatures in the brain and immune tissues and enabled p53-dependent and p53-independent responses to be identified. We discuss the application of these mice in delineating the molecular events in normal cellular growth and disease and for the evaluation of drug toxicity

  15. Radiosensitivity modulating factors: Role of PARP-1, PARP-2 and Cdk5 proteins and chromatin implication

    International Nuclear Information System (INIS)

    Boudra, M.T.

    2011-12-01

    The post-translational modifications of DNA repair proteins and histone remodeling factors by poly(ADP-ribose)ylation and phosphorylation are essential for the maintenance of DNA integrity and chromatin structure, and in particular in response to DNA damaging produced by ionizing radiation (IR). Amongst the proteins implicated in these two processes are the poly(ADP-ribose) polymerase -1 (PARP-1) and PARP-2, and the cyclin-dependent kinase Cdk5: PARP-1 and 2 are involved in DNA single strand break (SSB) repair (SSBR) and Cdk5 depletion has been linked with increased cell sensitivity to PARP inhibition. We have shown by using HeLa cells stably depleted for either CdK5 or PARP-2, that the recruitment profile of PARP-1 and XRCC-1, two proteins involved in the short-patch (SP) SSBR sub-pathway, to DNA damage sites is sub-maximal and that of PCNA, a protein involved in the long-patch (LP) repair pathway, is increased in the absence of Cdk5 and decreased in the absence of PARP-2 suggesting that both Cdk5 and PARP-2 are involved in both SSBR sub-pathways. PARP-2 and Cdk5 also impact on the poly(ADP-ribose) levels in cells as in the absence of Cdk5 a hyper-activation of PARP-1 was found and in the absence of PARP-2 a reduction in poly(ADP-ribose) glyco-hydrolase (PARG) activity was seen. However, in spite of these changes no impact on the repair of SSBs induced by IR was seen in either the Cdk5 or PARP-2 depleted cells (Cdk5 KD or PARP-2 KD cells) but, interestingly, increased radiation sensitivity in terms of cell killing was noted in the Cdk5 depleted cells. We also found that Cdk5, PARP-2 and PARG were all implicated in the regulation of the recruitment and the dissociation of the chromatin-remodeling factor ALC1 from DNA damage sites suggesting a role for these three proteins in changes in chromatin structure after DNA photo-damage. These results, taken together with the observation that PARP-1 recruitment is sub-optimal in both Cdk5 KD and PARP-2 KD cells, show that

  16. Analysis of p21-Activated Kinase Function in Neurofibromatosis Type 2

    Science.gov (United States)

    2010-01-01

    mediated activation ( Abo et al., 1998; Cau et al., 2001; Lee et al., 2002), the basal kinase activity of the group II Paks was not stimulated by Cdc42...Received: November 15, 2007 Revised: February 25, 2008 Accepted: March 3, 2008 Published: April 18, 2008 REFERENCES Abo , A., Qu, J., Cammarano, M.S...et al. Role of p21-activated kinase pathway defects in the cognitive deficits of Alzheimer disease. Nat Neurosci 2006;9(2):234-42. 53. Reutershan J

  17. Species-Specific Expression of Full-Length and Alternatively Spliced Variant Forms of CDK5RAP2.

    Directory of Open Access Journals (Sweden)

    John S Y Park

    Full Text Available CDK5RAP2 is one of the primary microcephaly genes that are associated with reduced brain size and mental retardation. We have previously shown that human CDK5RAP2 exists as a full-length form (hCDK5RAP2 or an alternatively spliced variant form (hCDK5RAP2-V1 that is lacking exon 32. The equivalent of hCDK5RAP2-V1 has been reported in rat and mouse but the presence of full-length equivalent hCDK5RAP2 in rat and mouse has not been examined. Here, we demonstrate that rat expresses both a full length and an alternatively spliced variant form of CDK5RAP2 that are equivalent to our previously reported hCDK5RAP2 and hCDK5RAP2-V1, repectively. However, mouse expresses only one form of CDK5RAP2 that is equivalent to the human and rat alternatively spliced variant forms. Knowledge of this expression of different forms of CDK5RAP2 in human, rat and mouse is essential in selecting the appropriate model for studies of CDK5RAP2 and primary microcephaly but our findings further indicate the evolutionary divergence of mouse from the human and rat species.

  18. CDK5-mediated phosphorylation of p19INK4d avoids DNA damage-induced neurodegeneration in mouse hippocampus and prevents loss of cognitive functions.

    Science.gov (United States)

    Ogara, María Florencia; Belluscio, Laura M; de la Fuente, Verónica; Berardino, Bruno G; Sonzogni, Silvina V; Byk, Laura; Marazita, Mariela; Cánepa, Eduardo T

    2014-07-01

    DNA damage, which perturbs genomic stability, has been linked to cognitive decline in the aging human brain, and mutations in DNA repair genes have neurological implications. Several studies have suggested that DNA damage is also increased in brain disorders such as Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. However, the precise mechanisms connecting DNA damage with neurodegeneration remain poorly understood. CDK5, a critical enzyme in the development of the central nervous system, phosphorylates a number of synaptic proteins and regulates dendritic spine morphogenesis, synaptic plasticity and learning. In addition to these physiological roles, CDK5 has been involved in the neuronal death initiated by DNA damage. We hypothesized that p19INK4d, a member of the cell cycle inhibitor family INK4, is involved in a neuroprotective mechanism activated in response to DNA damage. We found that in response to genotoxic injury or increased levels of intracellular calcium, p19INK4d is transcriptionally induced and phosphorylated by CDK5 which provides it with greater stability in postmitotic neurons. p19INK4d expression improves DNA repair, decreases apoptosis and increases neuronal survival under conditions of genotoxic stress. Our in vivo experiments showed that decreased levels of p19INK4d rendered hippocampal neurons more sensitive to genotoxic insult resulting in the loss of cognitive abilities that rely on the integrity of this brain structure. We propose a feedback mechanism by which the neurotoxic effects of CDK5-p25 activated by genotoxic stress or abnormal intracellular calcium levels are counteracted by the induction and stabilization of p19INK4d protein reducing the adverse consequences on brain functions. Copyright © 2014 Elsevier B.V. All rights reserved.

  19. p21(Waf1/Cip1) expression and the p53/MDM2 feedback loop in gastric carcinogenesis

    NARCIS (Netherlands)

    Craanen, M. E.; Blok, P.; Offerhaus, G. J.; Meijer, G. A.; Dekker, W.; Kuipers, E. J.; Meuwissen, S. G.

    1999-01-01

    Data are non-existent regarding coincidental alterations in the expression of p53 and its downstream target genes MDM2 and p21(Waf1/Cip1) in gastric carcinogenesis. An immunohistochemical study was therefore performed to examine the interrelationships of p53, MDM2, and p21(Waf1/Cip1) expression in a

  20. The Prognostic Impact of p53 Expression on Sporadic Colorectal Cancer Is Dependent on p21 Status

    International Nuclear Information System (INIS)

    Kruschewski, Martin; Mueller, Kathrin; Lipka, Sybille; Budczies, Jan; Noske, Aurelia; Buhr, Heinz Johannes; Elezkurtaj, Sefer

    2011-01-01

    The prognostic value of p53 and p21 expression in colorectal cancer is still under debate. We hypothesize that the prognostic impact of p53 expression is dependent on p21 status. The expression of p53 and p21 was immunohistochemically investigated in a prospective cohort of 116 patients with UICC stage II and III sporadic colorectal cancer. The results were correlated with overall and recurrence-free survival. The mean observation period was 51.8 ± 2.5 months. Expression of p53 was observed in 72 tumors (63%). Overall survival was significantly better in patients with p53-positive carcinomas than in those without p53 expression (p = 0.048). No differences were found in recurrence-free survival (p = 0.161). The p53+/p21− combination was seen in 68% (n = 49), the p53+/p21+ combination in 32% (n = 23). Patients with p53+/p21− carcinomas had significantly better overall and recurrence-free survival than those with p53+/p21+ (p < 0.0001 resp. p = 0.003). Our data suggest that the prognostic impact of p53 expression on sporadic colorectal cancer is dependent on p21 status

  1. Preclinical Therapeutic Synergy of MEK1/2 and CDK4/6 Inhibition in Neuroblastoma.

    Science.gov (United States)

    Hart, Lori S; Rader, JulieAnn; Raman, Pichai; Batra, Vandana; Russell, Mike R; Tsang, Matthew; Gagliardi, Maria; Chen, Lucy; Martinez, Daniel; Li, Yimei; Wood, Andrew; Kim, Sunkyu; Parasuraman, Sudha; Delach, Scott; Cole, Kristina A; Krupa, Shiva; Boehm, Markus; Peters, Malte; Caponigro, Giordano; Maris, John M

    2017-04-01

    Purpose: Neuroblastoma is treated with aggressive multimodal therapy, yet more than 50% of patients experience relapse. We recently showed that relapsed neuroblastomas frequently harbor mutations leading to hyperactivated ERK signaling and sensitivity to MEK inhibition therapy. Here we sought to define a synergistic therapeutic partner to potentiate MEK inhibition. Experimental Design: We first surveyed 22 genetically annotated human neuroblastoma-derived cell lines (from 20 unique patients) for sensitivity to the MEK inhibitor binimetinib. After noting an inverse correlation with sensitivity to ribociclib (CDK4/6 inhibitor), we studied the combinatorial effect of these two agents using proliferation assays, cell-cycle analysis, Ki67 immunostaining, time-lapse microscopy, and xenograft studies. Results: Sensitivity to binimetinib and ribociclib was inversely related ( r = -0.58, P = 0.009). MYCN amplification status and expression were associated with ribociclib sensitivity and binimetinib resistance, whereas increased MAPK signaling was the main determinant of binimetinib sensitivity and ribociclib resistance. Treatment with both compounds resulted in synergistic or additive cellular growth inhibition in all lines tested and significant inhibition of tumor growth in three of four xenograft models of neuroblastoma. The augmented growth inhibition was attributed to diminished cell-cycle progression that was reversible upon removal of drugs. Conclusions: Here we demonstrate that combined binimetinib and ribociclib treatment shows therapeutic synergy across a broad panel of high-risk neuroblastoma preclinical models. These data support testing this combination therapy in relapsed high-risk neuroblastoma patients, with focus on cases with hyperactivated RAS-MAPK signaling. Clin Cancer Res; 23(7); 1785-96. ©2016 AACR . ©2016 American Association for Cancer Research.

  2. Snail regulates p21WAF/CIP1 expression in cooperation with E2 A and Twist

    International Nuclear Information System (INIS)

    Takahashi, Eishi; Funato, Noriko; Higashihori, Norihisa; Hata, Yuiro; Gridley, Thomas; Nakamura, Masataka

    2004-01-01

    Snail, a zinc-finger transcriptional repressor, is essential for mesoderm and neural crest cell formation and epithelial-mesenchymal transition. The basic helix-loop-helix transcription factors E2A and Twist have been linked with Snail during embryonic development. In this study, we examined the role of Snail in cellular differentiation through regulation of p21 WAF/CIP1 expression. A reporter assay with the p21 promoter demonstrated that Snail inhibited expression of p21 induced by E2A. Co-expression of Snail with Twist showed additive inhibitory effects. Deletion mutants of the p21 promoter revealed that sequences between -270 and -264, which formed a complex with unidentified nuclear factor(s), were critical for E2A and Snail function. The E2A-dependent expression of the endogenous p21 gene was also inhibited by Snail

  3. Changes in neuronal CycD/Cdk4 activity affect aging, neurodegeneration, and oxidative stress.

    Science.gov (United States)

    Icreverzi, Amalia; de la Cruz, Aida Flor A; Walker, David W; Edgar, Bruce A

    2015-10-01

    Mitochondrial dysfunction has been implicated in human diseases, including cancer, and proposed to accelerate aging. The Drosophila Cyclin-dependent protein kinase complex cyclin D/cyclin-dependent kinase 4 (CycD/Cdk4) promotes cellular growth by stimulating mitochondrial biogenesis. Here, we examine the neurodegenerative and aging consequences of altering CycD/Cdk4 function in Drosophila. We show that pan-neuronal loss or gain of CycD/Cdk4 increases mitochondrial superoxide, oxidative stress markers, and neurodegeneration and decreases lifespan. We find that RNAi-mediated depletion of the mitochondrial transcription factor, Tfam, can abrogate CycD/Cdk4's detrimental effects on both lifespan and neurodegeneration. This indicates that CycD/Cdk4's pathological consequences are mediated through altered mitochondrial function and a concomitant increase in reactive oxygen species. In support of this, we demonstrate that CycD/Cdk4 activity levels in the brain affect the expression of a set of 'oxidative stress' genes. Our results indicate that the precise regulation of neuronal CycD/Cdk4 activity is important to limit mitochondrial reactive oxygen species production and prevent neurodegeneration. © 2015 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

  4. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    Energy Technology Data Exchange (ETDEWEB)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India); Choudhuri, Tathagata [Institute of Life Sciences, Nalco Square, Bhubaneswar, Orissa 751023 (India); Department of Biotechnology, Visva Bharati University, Santiniketan, West Bengal (India); Kundu, Chanakya Nath, E-mail: cnkundu@gmail.com [Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Orissa 751024 (India)

    2014-03-15

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  5. Resveratrol mediated cell death in cigarette smoke transformed breast epithelial cells is through induction of p21Waf1/Cip1 and inhibition of long patch base excision repair pathway

    International Nuclear Information System (INIS)

    Mohapatra, Purusottam; Satapathy, Shakti Ranjan; Das, Dipon; Siddharth, Sumit; Choudhuri, Tathagata; Kundu, Chanakya Nath

    2014-01-01

    Cigarette smoking is a key factor for the development and progression of different cancers including mammary tumor in women. Resveratrol (Res) is a promising natural chemotherapeutic agent that regulates many cellular targets including p21, a cip/kip family of cyclin kinase inhibitors involved in DNA damage-induced cell cycle arrest and blocking of DNA replication and repair. We have recently shown that cigarette smoke condensate (CSC) prepared from commercially available Indian cigarette can cause neoplastic transformation of normal breast epithelial MCF-10A cell. Here we studied the mechanism of Res mediated apoptosis in CSC transformed (MCF-10A-Tr) cells in vitro and in vivo. Res mediated apoptosis in MCF-10A-Tr cells was a p21 dependent event. It increased the p21 protein expression in MCF-10A-Tr cells and MCF-10A-Tr cells-mediated tumors in xenograft mice. Res treatment reduced the tumor size(s) and expression of anti-apoptotic proteins (e.g. PI3K, AKT, NFκB) in solid tumor. The expressions of cell cycle regulatory (Cyclins, CDC-2, CDC-6, etc.), BER associated (Pol-β, Pol-δ, Pol-ε, Pol-η, RPA, Fen-1, DNA-Ligase-I, etc.) proteins and LP-BER activity decreased in MCF-10A-Tr cells but remain significantly unaltered in isogenic p21 null MCF-10A-Tr cells after Res treatment. Interestingly, no significant changes were noted in SP-BER activity in both the cell lines after Res exposure. Finally, it was observed that increased p21 blocks the LP-BER in MCF-10A-Tr cells by increasing its interaction with PCNA via competing with Fen-1 after Res treatment. Thus, Res caused apoptosis in CSC-induced cancer cells by reduction of LP-BER activity and this phenomenon largely depends on p21. - Highlights: • Resveratrol (Res) caused reduction of MCF-10A-Tr cell growth by inducing apoptosis. • Res caused cell cycle arrest and DNA damage in p21 dependent manner. • Res mediated LP-BER reduction in MCF-10A-Tr cells was a p21 dependent phenomenon. • Res inhibits BER and PI

  6. Tbx1 regulates progenitor cell proliferation in the dental epithelium by modulating Pitx2 activation of p21.

    Science.gov (United States)

    Cao, Huojun; Florez, Sergio; Amen, Melanie; Huynh, Tuong; Skobe, Ziedonis; Baldini, Antonio; Amendt, Brad A

    2010-11-15

    Tbx1(-/-) mice present with phenotypic effects observed in DiGeorge syndrome patients however, the molecular mechanisms of Tbx1 regulating craniofacial and tooth development are unclear. Analyses of the Tbx1 null mice reveal incisor microdontia, small cervical loops and BrdU labeling reveals a defect in epithelial cell proliferation. Furthermore, Tbx1 null mice molars are lacking normal cusp morphology. Interestingly, p21 (associated with cell cycle arrest) is up regulated in the dental epithelium of Tbx1(-/-) embryos. These data suggest that Tbx1 inhibits p21 expression to allow for cell proliferation in the dental epithelial cervical loop, however Tbx1 does not directly regulate p21 expression. A new molecular mechanism has been identified where Tbx1 inhibits Pitx2 transcriptional activity and decreases the expression of Pitx2 target genes, p21, Lef-1 and Pitx2c. p21 protein is increased in PITX2C transgenic mouse embryo fibroblasts (MEF) and chromatin immunoprecipitation assays demonstrate endogenous Pitx2 binding to the p21 promoter. Tbx1 attenuates PITX2 activation of endogenous p21 expression and Tbx1 null MEFs reveal increased Pitx2a and activation of Pitx2c isoform expression. Tbx1 physically interacts with the PITX2 C-terminus and represses PITX2 transcriptional activation of the p21, LEF-1, and Pitx2c promoters. Tbx1(-/+)/Pitx2(-/+) double heterozygous mice present with an extra premolar-like tooth revealing a genetic interaction between these factors. The ability of Tbx1 to repress PITX2 activation of p21 may promote cell proliferation. In addition, PITX2 regulation of p21 reveals a new role for PITX2 in repressing cell proliferation. These data demonstrate new functional mechanisms for Tbx1 in tooth morphogenesis and provide a molecular basis for craniofacial defects in DiGeorge syndrome patients. Copyright © 2010 Elsevier Inc. All rights reserved.

  7. p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Ming-Yong Cao

    2011-10-01

    Full Text Available We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatal rat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21 in hypoxia/reoxygenation-induced senescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing small hairpin RNA (shRNA targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21 expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber with N2 for 6 h (hypoxia 6 h, then 21% oxygen for 8 h (reoxygenation 8 h. Cell cycle was examined using flow cytometry. Senescence was assessed using β-galactosidase staining. The expression of p53, p21, p16INK4a, and cyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution, stronger β-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the opposite results and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silenced cells had a smaller percentage of G1 cells, weaker β-galactosidase activity and lower 16INK4a expression, and higher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data implies that p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescence process. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451

  8. p21WAF1 and hypoxia/reoxygenation-induced premature senescence of H9c2 cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Dan Wang

    2011-10-01

    Full Text Available We have previously reported on hypoxia/reoxygenation-induced premature senescence in neonatalrat cardiomyocytes. In this research, we investigated the effects of p21WAF1 (p21 in hypoxia/reoxygenation-inducedsenescence, using H9c2 cells. A plasmid overexpressing wild type p21WAF1 and a plasmid expressing smallhairpin RNA (shRNA targeting p21WAF1 were constructed, and transfected into H9c2 cells to control the p21expression. Hypoxia/reoxygenation conditions were 1% O2 and 5% CO2, balancing the incubator chamber withN2 for 6 h (hypoxia 6 h, then 21% oxygen for 8 h (reoxygenation 8 h. Cell cycle was examined using flowcytometry. Senescence was assessed using b-galactosidase staining. The expression of p53, p21, p16INK4a, andcyclin D1 was assayed using Western blotting. At hypoxia 6 h, cells overexpressing p21 had a larger G1 distribution,stronger b-galactosidase activity, and lower cyclin D1 expression compared to control cells, while the oppositeresults and higher p53 expression were obtained in p21-knockdown cells. At reoxygenation 8 h, p21-silencedcells had a smaller percentage of G1 cells, weaker b-galactosidase activity and lower 16INK4a expression, andhigher cyclin D1 expression, but the overexpression group showed no difference. Taken together, this data impliesthat p21WAF1 is important for the hypoxia phase, but not the reoxygenation phase, in the H9c2 senescenceprocess. (Folia Histochemica et Cytobiologica 2011, Vol. 49, No. 3, 445–451

  9. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    Energy Technology Data Exchange (ETDEWEB)

    Lafaye, Céline; Barbier, Ewa; Miscioscia, Audrey; Saint-Pierre, Christine [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Kraut, Alexandra; Couté, Yohann [Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S_1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France); Plo, Isabelle [INSERM, U1009, Institut Gustave Roussy, Université Paris 11, 114 rue Edouard Vaillant, Villejuif F-94805 (France); Gasparutto, Didier; Ravanat, Jean-Luc [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France); Breton, Jean, E-mail: jean.breton@cea.fr [Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E_3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France)

    2014-03-28

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis.

  10. DNA binding of the p21 repressor ZBTB2 is inhibited by cytosine hydroxymethylation

    International Nuclear Information System (INIS)

    3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Lafaye, Céline; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Barbier, Ewa; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Miscioscia, Audrey; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Saint-Pierre, Christine; 1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" >Kraut, Alexandra; 1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Etude de la Dynamique des Protéomes, Biologie à Grande Echelle, UMR S1038 CEA/INSERM/UJF-Grenoble 1, iRTSV, 17 rue des Martyrs, Grenoble F-38054 (France))" >Couté, Yohann; Plo, Isabelle; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Gasparutto, Didier; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Ravanat, Jean-Luc; 3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" data-affiliation=" (Laboratoire Lésions des Acides Nucléiques, Service de Chimie Inorganique et Biologique, UMR E3 CEA/UJF-Grenoble 1, INAC, 17 rue des Martyrs, Grenoble F-38054 (France))" >Breton, Jean

    2014-01-01

    Highlights: • 5-hmC epigenetic modification is measurable in HeLa, SH-SY5Y and UT7-MPL cell lines. • ZBTB2 binds to DNA probes containing 5-mC but not to sequences containing 5-hmC. • This differential binding is verified with DNA sequences involved in p21 regulation. - Abstract: Recent studies have demonstrated that the modified base 5-hydroxymethylcytosine (5-hmC) is detectable at various rates in DNA extracted from human tissues. This oxidative product of 5-methylcytosine (5-mC) constitutes a new and important actor of epigenetic mechanisms. We designed a DNA pull down assay to trap and identify nuclear proteins bound to 5-hmC and/or 5-mC. We applied this strategy to three cancerous cell lines (HeLa, SH-SY5Y and UT7-MPL) in which we also measured 5-mC and 5-hmC levels by HPLC-MS/MS. We found that the putative oncoprotein Zinc finger and BTB domain-containing protein 2 (ZBTB2) is associated with methylated DNA sequences and that this interaction is inhibited by the presence of 5-hmC replacing 5-mC. As published data mention ZBTB2 recognition of p21 regulating sequences, we verified that this sequence specific binding was also alleviated by 5-hmC. ZBTB2 being considered as a multifunctional cell proliferation activator, notably through p21 repression, this work points out new epigenetic processes potentially involved in carcinogenesis

  11. Expression of cdk4 and p16 in Oral Lichen Planus.

    Science.gov (United States)

    Goel, Sinny; Khurana, Nita; Marwah, Akanksha; Gupta, Sunita

    2015-01-01

    The purpose of this study was to evaluate the expression of cdk4 and p16, the proteins implicated in hyperproliferation and arrest in oral lichen planus and to compare their expression in erosive and non-erosive oral lichen planus and with normal mucosa and oral squamous cell carcinoma. Analysis of cdk4 and p16 expression was done in 43 erosive oral lichen planus (EOLP) and 17 non-erosive oral lichen planus (NOLP) cases, 10 normal mucosa and 10 oral squamous cell carcinoma (OSCC) cases with immunohistochemistry. This study demonstrated a significantly increased expression of cytoplasmic cdk4 (80% cases, cells stained - 19.6%), and cytoplasmic p16 (68.3% cases, cells stained - 16.4%) in oral lichen planus (OLP) compared to normal mucosa. cdk4 was much higher in OSCC in both cytoplasm and nuclei compared to normal mucosa. Also, while comparing OLP with positive control, significant difference was noted for cdk4 and p16, with expression being more in OSCC. While comparing EOLP with NOLP; significant differences were seen for cdk4 cytoplasmic staining only, for number of cases with positive staining as well as number of cells stained. Overexpression of cytoplasmic cdk4 and p16 was registered in oral lichen planus, however considerably lower than in squamous cell carcinoma. Erosive oral lichen planus demonstrated overexpression of cytoplasmic cdk4 and premalignant nature compared to non-erosive lesion. Therefore there is an obvious possibility for cytoplasmic expression of cdk4 and p16 to predict malignant potential of oral lichen planus lesions.

  12. [Microsatellite alterations on chromosome 9p21-22 in sporadic colorectal cancer].

    Science.gov (United States)

    Zhang, Y; Lai, M

    1999-12-01

    To study whether alteration of p16 plays an important role in the development of colorectal carcinomas and the relationship between the molecular changes of 9p21-22 chromosome subregion in sporadic colorectal cancers. To detect microsatellite instability (MSI) and loss of heterozygosity (LOH) by PCR, denatured-polyacrylamide gel-electrophoresis and silver staining (microsatellite DNA-PCR-silver staining method) and to compare the results with the clinopathological parameters. Between MSI positive and negative cases and the clinopathological findings, some evidences found in the MSI positive group were as follows: (1) tendency towards younger patients (usually < 50 years in age, P < 0.05); (2) more frequently seen in mucoid carcinomas (P < 0.01). Microsatellite DNA-PCR-silver staining method is very sensitive in detecting even a tiny change of a single base. MSI occured in the selected microsatellite loci of different subregions and different chromosomes might be different in significance, therefore, a right choice of the suitable loci for studying the microsatellite changes is important. Since the frequency of loss of heterozygosity at 9p21-22 is low (merely 8.42%), it is considered that p16 is not closely associated with the development of sporadic colorectal cancer.

  13. Cell Cycle Checkpoint Proteins p21 and Hus1 Regulating Intercellular Signaling Induced By Alpha Particle Irradiation

    Science.gov (United States)

    Wu, Lijun; Zhao, Ye; Wang, Jun; Hang, Haiying

    In recent years, the attentions for radiation induced bystander effects (RIBE) have been paid on the intercellular signaling events connecting the irradiated and non-irradiated cells. p21 is a member of the Cip/Kip family and plays essential roles in cell cycle progression arrest after cellular irradiation. DNA damage checkpoint protein Hus1 is a member of the Rad9-Rad1-Hus1 complex and functions as scaffold at the damage sites to facilitate the activation of downstream effectors. Using the medium trasfer method and the cells of MEF, MEF (p21-/-), MEF (p21-/-Hus1-/-) as either medium donor or receptor cells, it was found that with 5cGy alpha particle irradiation, the bystander cells showed a significant induction of -H2AX for normal MEFs (p¡0.05). However, the absence of p21 resulted in deficiency in inducing bystander effects. Further results indicated p21 affected the intercellular DNA damage signaling mainly through disrupting the production or release of the damage signals from irradiated cells. When Hus1 and p21 were both knocked out, an obvious induction of -H2AX recurred in bystander cells and the induction of -H2AX was GJIC (gap junction-mediated intercellular communication) dependent, indicating the interrelationship between p21 and Hus1 regulated the production and relay of DNA damage signals from irradiated cells to non-irradiated bystander cells.

  14. Protective role of p21(Waf1/Cip1) against prostaglandin A2-mediated apoptosis of human colorectal carcinoma cells.

    Science.gov (United States)

    Gorospe, M; Wang, X; Guyton, K Z; Holbrook, N J

    1996-01-01

    Prostaglandin A2 (PGA2) suppresses tumor growth in vivo, is potently antiproliferative in vitro, and is a model drug for the study of the mammalian stress response. Our previous studies using breast carcinoma MCF-7 cells suggested that p21(Waf1/Cip1) induction enabled cells to survive PGA2 exposure. Indeed, the marked sensitivity of human colorectal carcinoma RKO cells to the cytotoxicity of PGA2 is known to be associated with a lack of a PGA2-mediated increase in p21(Waf1/Cip1) expression, inhibition of cyclin-dependent kinase activity, and growth arrest. To determine if cell death following exposure to PGA2 could be prevented by forcing the expression of p21(Waf1/Cip1) in RKO cells, we utilized an adenoviral vector-based expression system. We demonstrate that ectopic expression of p21(Waf1/Cip1) largely rescued RKO cells from PGA2-induced apoptotic cell death, directly implicating p21(Waf1/Cip1) as a determinant of the cellular outcome (survival versus death) following exposure to PGA2. To discern whether p21(Waf1/Cip1)-mediated protection operates through the implementation of cellular growth arrest, other growth-inhibitory treatments were studied for the ability to attenuate PGA2-induced cell death. Neither serum depletion nor suramin (a growth factor receptor antagonist) protected RKO cells against PGA2 cytotoxicity, and neither induced p21(Waf1/Cip1) expression. Mimosine, however, enhanced p21(Waf1/Cip1) expression, completely inhibited RKO cell proliferation, and exerted marked protection against a subsequent PGA2 challenge. Taken together, our results directly demonstrate a protective role for p21(Waf1/Cip1) during PGA2 cellular stress and provide strong evidence that the implementation of cellular growth arrest contributes to this protective influence. PMID:8943319

  15. Neuroendocrine prostate cancer (NEPCa) increased the neighboring PCa chemo-resistance via altering the PTHrP/p38/Hsp27/androgen receptor (AR)/p21 signals

    Science.gov (United States)

    Cui, Yun; Sun, Yin; Hu, Shuai; Luo, Jie; Li, Lei; Li, Xin; Yeh, Shuyuan; Jin, Jie; Chang, Chawnshang

    2016-01-01

    Prostatic neuroendocrine cells (NE) are an integral part of prostate cancer (PCa) that are associated with PCa progression. As the current androgen-deprivation therapy (ADT) with anti-androgens may promote the neuroendocrine PCa (NEPCa) development, and few therapies can effectively suppress NEPCa, understanding the impact of NEPCa on PCa progression may help us to develop better therapies to battle PCa. Here we found NEPCa cells could increase the docetaxel-resistance of their neighboring PCa cells. Mechanism dissection revealed that through secretion of PTHrP, NEPCa cells could alter the p38/MAPK/Hsp27 signals in their neighboring PCa cells that resulted in increased androgen receptor (AR) activity via promoting AR nuclear translocation. The consequences of increased AR function might then increase docetaxel-resistance via increasing p21 expression. In vivo xenograft mice experiments also confirmed NEPCa could increase the docetaxel-resistance of neighboring PCa, and targeting this newly identified PTHrP/p38/Hsp27/AR/p21 signaling pathway with either p38 inhibitor (SB203580) or sh-PTHrP may result in improving/restoring the docetaxel sensitivity to better suppress PCa. PMID:27375022

  16. Effect of RNAi p21 gene on uncoupling of EL-4 cells induced by X-irradiation

    International Nuclear Information System (INIS)

    Ju Guizhi; Yan Fengqin; Fu Shibo; Shen Bo; Sun Shilong; Yang Ying; Li Pengwu

    2008-01-01

    Objective: To investigate the effect of RNAi p21 gene on uncoupling of EL-4 cells induced by X-irradiation. Methods: Construction of RNAi p21 plasmid of pSileneer3.1-H1 neo-p21 was performed. Lipofectamine transfection assay was used to transfer the p21siBNA into EL-4 cells. Fluorescent staining and flow cytometry (FCM) analysis were employed for measurement of protein expression. Fluorescent staining of propidium iodide (PI) and FCM were used for measurement of potyploid cells. Results: In dose-effect experiment it was found that the expression of P21 protein of EL-4 cells increased significantly 24 h after X- irradiation with different doses compared with sham-inadiated control. In time course experiment it was found that the expression of P21 protein of EL-4 cells increased significantly at 8 h to 72 h after 4.0 Gy X-irradiation compared with sham-irradiated control. The results showed that the number of polyploid cells in EL-4 cells was not changed markedly after X-irradiation with doses of 0.5-6.0 Gy. After RNA interference with p21 gene, the expression of P21 protein of EL-4 cells decreased significantly 24 h and 48 h after 4.0 Gy X-irradiation in transfection of plasmid of pSilencer3.1-H1 neo-p21 compared with transfection of plasmid of pSilencer3.1-H1 nco control. And at the same time, the number of polyploid cells in EL-4 cells was increased significantly in transfection of plasmid of pSilencer3.1-H1 neo-p21 compared with transfection of plasmid of pSilencer3.1-H1 nco control. Conclusions: Uncoupling could be induced by X-irradiation in EL-4 cells following BNAi p21 gene, suggesting that P21 protein may play an important role in uncoupling induced by X-rays. (authors)

  17. Differential effects of chromosome 9p21 variation on subphenotypes of intracranial aneurysm: site distribution.

    Science.gov (United States)

    Nakaoka, Hirofumi; Takahashi, Tomoko; Akiyama, Koichi; Cui, Tailin; Tajima, Atsushi; Krischek, Boris; Kasuya, Hidetoshi; Hata, Akira; Inoue, Ituro

    2010-08-01

    Recently, a genome-wide association study identified associations between single nucleotide polymorphisms on chromosome 9p21 and risk of harboring intracranial aneurysm (IA). Aneurysm characteristics or subphenotypes of IAs, such as history of subarachnoid hemorrhage, presence of multiple IAs and location of IAs, are clinically important. We investigated whether the association between 9p21 variation and risk of IA varied among these subphenotypes. We conducted a case-control study of 981 cases and 699 controls in Japanese. Four single nucleotide polymorphisms tagging the 9p21 risk locus were genotyped. The OR and 95% CI were estimated using logistic regression analyses. Among the 4 single nucleotide polymorphisms, rs1333040 showed the strongest evidence of association with IA (P=1.5x10(-6); per allele OR, 1.43; 95% CI, 1.24-1.66). None of the patient characteristics (gender, age, smoking, and hypertension) was a significant confounder or effect modifier of the association. Subgroup analyses of IA subphenotypes showed that among the most common sites of IAs, the association was strongest for IAs of the posterior communicating artery (OR, 1.69; 95% CI, 1.26-2.26) and not significant for IAs in the anterior communicating artery (OR, 1.22; 95% CI, 0.96-1.57). When dichotomizing IA sites, the association was stronger for IAs of the posterior circulation-posterior communicating artery group (OR, 1.73; 95% CI, 1.32-2.26) vs the anterior circulation group (OR, 1.28; 95% CI, 1.07-1.53). Heterogeneity in these ORs was significant (P=0.032). The associations did not vary when stratifying by history of subarachnoid hemorrhage (OR, 1.42; 95% CI, 1.18-1.71 for ruptured IA; OR, 1.27; 95% CI, 1.00-1.62 for unruptured IA) or by multiplicity of IA (OR, 1.57; 95% CI, 1.21-2.03 for multiple IAs; OR, 1.36; 95% CI, 1.15-1.61 for single IA). Our results suggest that genetic influence on formation may vary between IA subphenotypes.

  18. Association between 9p21 genomic markers and heart disease: a meta-analysis.

    Science.gov (United States)

    Palomaki, Glenn E; Melillo, Stephanie; Bradley, Linda A

    2010-02-17

    Associations between chromosome 9p21 single-nucleotide polymorphisms (SNPs) and heart disease have been reported and replicated. If testing improves risk assessments using traditional factors, it may provide opportunities to improve public health. To perform a targeted systematic review of published literature for effect size, heterogeneity, publication bias, and strength of evidence and to consider whether testing might provide clinical utility. Electronic search via HuGE Navigator through January 2009 and review of reference lists from included articles. English-language articles that tested for 9p21 SNPs with coronary heart/artery disease or myocardial infarction as primary outcomes. Included articles also provided race, numbers of participants, and data to compute an odds ratio (OR). Articles were excluded if reporting only intermediate outcomes (eg, atherosclerosis) or if all participants had existing disease. Twenty-five articles were initially identified and 16 were included. A follow-up search identified 6 additional articles. Independent extraction was performed by 2 reviewers and consensus was reached. Credibility of evidence was assessed using published Venice criteria. Forty-seven distinct data sets from the 22 articles were analyzed, including 35 872 cases and 95 837 controls. The summary OR for heart disease among individuals with 2 vs 1 at-risk alleles was 1.25 (95% confidence interval [CI], 1.21-1.29), with low to moderate heterogeneity. Age at disease diagnosis was a significant covariate, with ORs of 1.35 (95% CI, 1.30-1.40) for age 55 years or younger and 1.21 (95% CI, 1.16-1.25) for age 75 years or younger. For a 65-year-old man, the 10-year heart disease risk for 2 vs 1 at-risk alleles would be 13.2% vs 11%. For a 40-year-old woman, the 10-year heart disease risk for 2 vs 1 at-risk alleles would be 2.4% vs 2.0%. Nearly identical but inverse results were found when comparing 1 vs 0 at-risk alleles. Three studies showed net reclassification

  19. Inactivation of the CRL4-CDT2-SET8/p21 ubiquitylation and degradation axis underlies the therapeutic efficacy of pevonedistat in melanoma

    Directory of Open Access Journals (Sweden)

    Mouadh Benamar

    2016-08-01

    Research in Context: The identification of new molecular targets and effective inhibitors is of utmost significance for the clinical management of melanoma. This study identifies CDT2, a substrate receptor for the CRL4 ubiquitin ligase, as a prognostic marker and therapeutic target in melanoma. CDT2 is required for melanoma cell proliferation and inhibition of CRL4CDT2 by pevonedistat suppresses melanoma in vitro and in vivo through the induction of DNA rereplication and senescence through the stabilization of the CRL4CDT2 substrates p21 and SET8. Pevonedistat also synergizes with vemurafenib in vivo and suppresses vemurafenib-resistant melanoma cells. These findings show a significant promise for targeting CRL4CDT2 therapeutically.

  20. Substance P induces rapid and transient membrane blebbing in U373MG cells in a p21-activated kinase-dependent manner.

    Directory of Open Access Journals (Sweden)

    John Meshki

    Full Text Available U373MG astrocytoma cells endogenously express the full-length neurokinin 1 receptor (NK1R. Substance P (SP, the natural ligand for NK1R, triggers rapid and transient membrane blebbing and we report that these morphological changes have different dynamics and intracellular signaling as compared to the changes that we have previously described in HEK293-NK1R cells. In both cell lines, the SP-induced morphological changes are Gq-independent, and they require the Rho, Rho-associated coiled-coil kinase (ROCK signaling pathway. Using confocal microscopy we have demonstrated that tubulin is phosphorylated subsequent to cell stimulation with SP and that tubulin accumulates inside the blebs. Colchicine, a tubulin polymerization inhibitor, blocked SP-induced blebbing in U373MG but not in HEK293-NK1R cells. Although p21-activated kinase (PAK is expressed in both cell lines, SP induced rapid phosphorylation of PAK in U373MG, but failed to phosphorylate PAK in HEK293-NK1R cells. The cell-permeable Rho inhibitor C3 transferase inhibited SP-induced PAK phosphorylation, but the ROCK inhibitor Y27632 had no effect on PAK phosphorylation, suggesting that Rho activates PAK in a ROCK-independent manner. Our study demonstrates that SP triggers rapid changes in cell morphology mediated by distinct intracellular signaling mechanisms in U373MG versus HEK293-NK1R cells.

  1. A novel role for the cell cycle regulatory complex cyclin D1?CDK4 in gluconeogenesis

    OpenAIRE

    Hosooka, Tetsuya; Ogawa, Wataru

    2015-01-01

    Dysregulation of gluconeogenesis is a key pathological feature of type 2 diabetes. However, the molecular mechanisms underlying the regulation of gluconeogenesis remain unclear. Bhalla et?al. recently reported that cyclin D1 suppresses hepatic gluconeogenesis through CDK4?dependent phosphorylation of PGC1alpha and consequent inhibition of its activity. The cyclin D1?CDK4 might thus serve as an important link between the cell cycle and control of energy metabolism through modulation of PGC1alp...

  2. A novel role for the cell cycle regulatory complex cyclin D1-CDK4 in gluconeogenesis.

    Science.gov (United States)

    Hosooka, Tetsuya; Ogawa, Wataru

    2016-01-01

    Dysregulation of gluconeogenesis is a key pathological feature of type 2 diabetes. However, the molecular mechanisms underlying the regulation of gluconeogenesis remain unclear. Bhalla et al. recently reported that cyclin D1 suppresses hepatic gluconeogenesis through CDK4-dependent phosphorylation of PGC1alpha and consequent inhibition of its activity. The cyclin D1-CDK4 might thus serve as an important link between the cell cycle and control of energy metabolism through modulation of PGC1alpha activity.

  3. CDK5 knockdown prevents hippocampal degeneration and cognitive dysfunction produced by cerebral ischemia

    OpenAIRE

    Gutiérrez-Vargas, Johana A; Múnera, Alejandro; Cardona-Gómez, Gloria P

    2015-01-01

    Acute ischemic stroke is a cerebrovascular accident and it is the most common cause of physical disabilities around the globe. Patients may present with repeated ictuses, experiencing mental consequences, such as depression and cognitive disorders. Cyclin-dependent kinase 5 (CDK5) is a kinase that is involved in neurotransmission and plasticity, but its dysregulation contributes to cognitive disorders and dementia. Gene therapy targeting CDK5 was administered to the right hippocampus of ische...

  4. Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway

    International Nuclear Information System (INIS)

    Han, Seula; Woo, Jong Kyu; Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani; Oh, Seung Hyun; Ryu, Jae-Ha; Kim, Woo-Young

    2016-01-01

    In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. - Highlights: • Evodiamine selectively kills breast cancer stem like cells at G1 phase. • Evodiamine utilizes different mechanism of cell cycle modulation in CSLC and in bulk cancer cells. • Evodiamine activate the p53, p21 and Rb pathway.

  5. Evodiamine selectively targets cancer stem-like cells through the p53-p21-Rb pathway

    Energy Technology Data Exchange (ETDEWEB)

    Han, Seula [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of); Woo, Jong Kyu [College of Pharmacy, Gachon University, Incheon (Korea, Republic of); Jung, Yuchae; Jeong, Dawoon; Kang, Minsook; Yoo, Young-Ji; Lee, Hani [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of); Oh, Seung Hyun [College of Pharmacy, Gachon University, Incheon (Korea, Republic of); Ryu, Jae-Ha [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of); Kim, Woo-Young, E-mail: wykim@sookmyung.ac.kr [The Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul (Korea, Republic of)

    2016-01-22

    In spite of the recent improvements, the resistance to chemotherapy/radiotherapy followed by relapse is the main hurdle for the successful treatment of breast cancer, a leading cause of death in women. A small population of breast cancer cells that have stem-like characteristics (cancer stem-like cells; CSLC) may contribute to this resistance and relapse. Here, we report on a component of a traditional Chinese medicine, evodiamine, which selectively targets CSLC of breast cancer cell lines MCF7 and MDAMB 231 at a concentration that does show a little or no cytotoxic effect on bulk cancer cells. While evodiamine caused the accumulation of bulk cancer cells at the G2/M phase, it did not hold CSLC in a specific cell cycle phase but instead, selectively killed CSLC. This was not due to the culture of CSLC in suspension or without FBS. A proteomic analysis and western blotting revealed that evodiamine changed the expression of cell cycle regulating molecules more efficiently in CSLC cells than in bulk cancer cells. Surprisingly, evodiamine selectively activated p53 and p21 and decreased inactive Rb, the master molecules in G1/S checkpoint. These data collectively suggest a novel mechanism involving CSLC-specific targeting by evodiamine and its possible use to the therapy of breast cancer. - Highlights: • Evodiamine selectively kills breast cancer stem like cells at G1 phase. • Evodiamine utilizes different mechanism of cell cycle modulation in CSLC and in bulk cancer cells. • Evodiamine activate the p53, p21 and Rb pathway.

  6. Urodynamic evaluation of patients with autosomal dominant pure spastic paraplegia linked to chromosome 2p21-p24

    DEFF Research Database (Denmark)

    Jensen, L N; Gerstenberg, T; Kallestrup, E B

    1998-01-01

    , measurements of the cutaneous perception threshold, bulbocavernosus reflex latency, and somatosensory evoked potentials (SSEPs) of the pudendal nerve were performed. RESULTS: All patients experienced urinary urgency or urge incontinence. Rectal urgency and sexual dysfunction were reported by most patients...... bowel and sexual dysfunction in patients with ADPSP linked to chromosome 2p21-p24 are due to a combination of somatic and autonomic nervous system involvement which support the proposed multisystem affection in ADPSP linked to chromosome 2p21-p24....

  7. ASPL-TFE3 Oncoprotein Regulates Cell Cycle Progression and Induces Cellular Senescence by Up-Regulating p21

    Directory of Open Access Journals (Sweden)

    Naoko Ishiguro

    2016-10-01

    Full Text Available Alveolar soft part sarcoma is an extremely rare soft tissue sarcoma with poor prognosis. It is characterized by the unbalanced recurrent chromosomal translocation der(17t(X;17(p11;q25, resulting in the generation of an ASPL-TFE3 fusion gene. ASPL-TFE3 oncoprotein functions as an aberrant transcriptional factor and is considered to play a crucial role in the tumorigenesis of alveolar soft part sarcoma. However, the underlying molecular mechanisms are poorly understood. In this study, we identified p21 (p21WAF1/CIP1 as a direct transcriptional target of ASPL-TFE3. Ectopic ASPL-TFE3 expression in 293 cells resulted in cell cycle arrest and significant increases in protein and mRNA levels of p21. ASPL-TFE3 activated p21 expression in a p53-independent manner through direct transcriptional interactions with the p21 promoter region. When ASPL-TFE3 was expressed in human bone marrow–derived mesenchymal stem cells in a tetracycline-inducible manner, we observed the up-regulation of p21 expression and the induction of senescence-associated β-galactosidase activity. Suppression of p21 significantly decreased the induction of ASPL-TFE3-mediated cellular senescence. Furthermore, ASPL-TFE3 expression in mesenchymal stem cells resulted in a significant up-regulation of proinflammatory cytokines associated with senescence-associated secretory phenotype (SASP. These results show that ASPL-TFE3 regulates cell cycle progression and induces cellular senescence by up-regulating p21 expression. In addition, our data suggest a potential mechanism by which ASPL-TFE3-induced senescence may play a role in tumorigenesis by inducing SASP, which could promote the protumorigenic microenvironment.

  8. Blockade of protein geranylgeranylation inhibits Cdk2-dependent p27Kip1 phosphorylation on Thr187 and accumulates p27Kip1 in the nucleus: implications for breast cancer therapy.

    Science.gov (United States)

    Kazi, Aslamuzzaman; Carie, Adam; Blaskovich, Michelle A; Bucher, Cynthia; Thai, Van; Moulder, Stacy; Peng, Hairuo; Carrico, Dora; Pusateri, Erin; Pledger, Warren J; Berndt, Norbert; Hamilton, Andrew; Sebti, Saïd M

    2009-04-01

    We describe the design of a potent and selective peptidomimetic inhibitor of geranylgeranyltransferase I (GGTI), GGTI-2418, and its methyl ester GGTI-2417, which increases the levels of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) and induces breast tumor regression in vivo. Experiments with p27(Kip1) small interfering RNA in breast cancer cells and p27(Kip1) null murine embryonic fibroblasts demonstrate that the ability of GGTI-2417 to induce cell death requires p27(Kip1). GGTI-2417 inhibits the Cdk2-mediated phosphorylation of p27(Kip1) at Thr187 and accumulates p27(Kip1) in the nucleus. In nude mouse xenografts, GGTI-2418 suppresses the growth of human breast tumors. Furthermore, in ErbB2 transgenic mice, GGTI-2418 increases p27(Kip1) and induces significant regression of breast tumors. We conclude that GGTIs' antitumor activity is, at least in part, due to inhibiting Cdk2-dependent p27(Kip1) phosphorylation at Thr187 and accumulating nuclear p27(Kip1). Thus, GGTI treatment might improve the poor prognosis of breast cancer patients with low nuclear p27(Kip1) levels.

  9. Blockade of Protein Geranylgeranylation Inhibits Cdk2-Dependent p27Kip1 Phosphorylation on Thr187 and Accumulates p27Kip1 in the Nucleus: Implications for Breast Cancer Therapy▿ §

    Science.gov (United States)

    Kazi, Aslamuzzaman; Carie, Adam; Blaskovich, Michelle A.; Bucher, Cynthia; Thai, Van; Moulder, Stacy; Peng, Hairuo; Carrico, Dora; Pusateri, Erin; Pledger, Warren J.; Berndt, Norbert; Hamilton, Andrew; Sebti, Saïd M.

    2009-01-01

    We describe the design of a potent and selective peptidomimetic inhibitor of geranylgeranyltransferase I (GGTI), GGTI-2418, and its methyl ester GGTI-2417, which increases the levels of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 and induces breast tumor regression in vivo. Experiments with p27Kip1 small interfering RNA in breast cancer cells and p27Kip1 null murine embryonic fibroblasts demonstrate that the ability of GGTI-2417 to induce cell death requires p27Kip1. GGTI-2417 inhibits the Cdk2-mediated phosphorylation of p27Kip1 at Thr187 and accumulates p27Kip1 in the nucleus. In nude mouse xenografts, GGTI-2418 suppresses the growth of human breast tumors. Furthermore, in ErbB2 transgenic mice, GGTI-2418 increases p27Kip1 and induces significant regression of breast tumors. We conclude that GGTIs' antitumor activity is, at least in part, due to inhibiting Cdk2-dependent p27Kip1 phosphorylation at Thr187 and accumulating nuclear p27Kip1. Thus, GGTI treatment might improve the poor prognosis of breast cancer patients with low nuclear p27Kip1 levels. PMID:19204084

  10. The investigation of the antiapoptotic effect of TAT-p21 in doxorubicin-induced cardiomyopathy in mice

    OpenAIRE

    Nowak, Marek Tadeusz

    2011-01-01

    Cardiomyocytes are terminally differentiated postmitotic cells. Therefore, the adult myocardium is not able to compensate the loss of cardiomyocytes by proliferation. Cardiomyocyte apoptosis is observed in the development of heart failure of various origins. Interestingly, there is a close connection between the regulatory mechanisms of apoptosis and the cell cycle. Cyclin/cyclin-dependent kinase (cdk) complexes required for cell cycle progression are essential for apoptosis as well. Their...

  11. Immunohistochemical study of p21 and Bcl-2 in leukoplakia, oral submucous fibrosis and oral squamous cell carcinoma.

    Science.gov (United States)

    Sutariya, Rakesh V; Manjunatha, Bhari Sharanesha

    2016-11-01

    Oral Squamous cell carcinoma (OSCC) results from genetic damage, leading to uncontrolled cell proliferation of damaged cells and the cell death. In the course of its progression, visible changes are taking place at the cellular level (atypical) and the resultant at the tissue level (epithelial dysplasia). The Aim of the present study was to evaluate and compare the expressions of intensity of p21 and Bcl-2 in Leukoplakia, oralsubmucous fibrosis (OSMF) and oral squamous cell carcinoma. Total 60 cases, 30 cases of oral squamous cell carcinoma, 15 cases of oral submucous fibrosis and 15 cases of Leukoplakia were evaluated immunohistochemically for p21 and Bcl-2 expression. p21 showed positive expression in 13 (86.67%) cases out of 15 cases of OSMF, 12 (80%) cases of leukoplakia out of 15 cases and 24 (80%) cases out of 30 cases of OSCC. The Bcl-2 expression was positive in 13 (86.67%) cases of OSMF, all cases of Leukoplakia and 25 (83.33%) cases of OSCC. No statistical significance was noted in the expression of p21 and Bcl-2 positive expression between OSMF, Leukoplakia and OSCC. Statistical analysis for comparison of intensity of p21 expression in different grades of OSCC showed no significance. Statistical significance difference was found between the expressions of Bcl-2 in moderately and poorly differentiated SCC. The intensity of p21 and Bcl-2 expressions in different grades of OSCC indicates a key role in progression of oral neoplasia.

  12. Role of p53 in cdk Inhibitor VMY-1-103-Induced Apoptosis in Prostate Cancer

    Science.gov (United States)

    2012-09-01

    Cancer Center; Thomas Jefferson University; philadelphia, pA UsA ‡Current address: Marshall University Medical school; huntington , WV UsA †These authors...underlying genetic or molecular abnormality, these tumors are highly proliferative, and advances in clinical treatment will require developing...ultimately resulted in disease recurrence.22 We9 and others23 have recently explored the possibility that inhibit- ing the transcription factor, Gli

  13. Early Intervention with Cdk9 Inhibitors to Prevent Post-Traumatic Osteoarthritis

    Science.gov (United States)

    2015-10-01

    Series. o Arresting Cartilage Degradation after Injury: Focus on Primary Response Genes, UC Davis Dean’s Symposium, Translating Basic Science to...apoptosis and cell cycle arrest by FTY720 in gastric cancer cells and nude mice. J Cell Biochem 111: 218-228. Zhou Q, Yik JH (2006) The Yin and Yang of P...biomarkers for early intestinal cancer using 15N metabolic labeling and quantitative proteomics in the ApcMin/+ mouse, J. Proteome Res. 12 (2013) 4152

  14. Targeting Transcriptional Addictions in Small Cell Lung Cancer with a Covalent CDK7 Inhibitor

    DEFF Research Database (Denmark)

    Christensen, Camilla L; Kwiatkowski, Nicholas; Abraham, Brian J

    2014-01-01

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality, and the identification of effective pharmacological strategies to target SCLC biology represents an urgent need. Using a high-throughput cellular screen of a diverse chemical library, we observe that SCLC is sensitive...

  15. Role of Cyclin D1 and cdk Inhibitors in Breast Cancer Pathogenesis

    National Research Council Canada - National Science Library

    Roovers, Kristin

    2002-01-01

    Cyclin Dl is frequently overexpressed in breast cancer tumors. Thus, an understanding of cyclin Dl regulation in normal and malignant cells may prove beneficial in the search for therapeutic targets...

  16. The Prozone Effect Accounts for the Paradoxical Function of the Cdk-Binding Protein Suc1/Cks

    Directory of Open Access Journals (Sweden)

    Sang Hoon Ha

    2016-02-01

    Full Text Available Previous work has shown that Suc1/Cks proteins can promote the hyperphosphorylation of primed Cdk1 substrates through the formation of ternary Cdk1-Cks-phosphosubstrate complexes. This raises the possibility that Cks proteins might be able to both facilitate and interfere with hyperphosphorylation through a mechanism analogous to the prozone effect in antigen-antibody interactions, with substoichiometric Cks promoting the formation of Cdk1-Cks-phosphosubstrate complexes and suprastoichiometric Cks instead promoting the formation of Cdk1-Cks and Cks-phosphosubstrate complexes. We tested this hypothesis through a combination of theory, proof-of-principle experiments with oligonucleotide annealing, and experiments on the interaction of Xenopus cyclin B1-Cdk1-Cks2 with Wee1A in vitro and in Xenopus extracts. Our findings help explain why both Cks under-expression and overexpression interfere with cell-cycle progression and provide insight into the regulation of the Cdk1 system.

  17. Role of the p53/p21 system in the response of human colon carcinoma cells to Doxorubicin

    Directory of Open Access Journals (Sweden)

    Passarelli Laura

    2004-12-01

    Full Text Available Abstract Background Colon adenocarcinomas are refractory to a number of widely used anticancer agents. Multifactorial mechanisms have been implicated in this intrinsically resistant phenotype, including deregulation of cell death pathways. In this regard, the p53 protein has a well established role in the control of tumor cell response to DNA damaging agents; however, the relationship between p53-driven genes and drug sensitivity remains controversial. The present study investigates the role of the p53/p21 system in the response of human colon carcinoma cells to treatment with the cytotoxic agent doxorubicin (DOX and the possibility to modify the therapeutic index of DOX by modulation of p53 and/or p21 protein levels. Methods The relationship between p53 and p21 protein levels and the cytotoxic effect of DOX was investigated, by MTT assay and western blot analysis, in HCT116 (p53-positive and HT29 (p53-negative colon cancer cells. We then assessed the effects of DOX in two isogenic cell lines derived from HCT116 by abrogating the expression and/or function of p53 and p21 (HCT116-E6 and HCT116 p21-/-, respectively. Finally, we evaluated the effect of pre-treatment with the piperidine nitroxide Tempol (TPL, an agent that was reported to induce p21 expression irrespective of p53 status, on the cytotoxicity of DOX in the four cell lines. Comparisons of IC50 values and apoptotic cell percentages were performed by ANOVA and Bonferroni's test for independent samples. C.I. calculations were performed by the combination Index method. Results Our results indicate that, in the colon carcinoma cell lines tested, sensitivity to DOX is associated with p21 upregulation upon drug exposure, and DOX cytotoxicity is potentiated by pre-treatment with TPL, but only in those cell lines in which p21 can be upregulated. Conclusions p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can

  18. Altered expression of the Cdk5 activator-like protein, Cdk5α, causes neurodegeneration, in part by accelerating the rate of aging

    Directory of Open Access Journals (Sweden)

    Joshua Spurrier

    2018-03-01

    Full Text Available Aging is the greatest risk factor for neurodegeneration, but the connection between the two processes remains opaque. This is in part for want of a rigorous way to define physiological age, as opposed to chronological age. Here, we develop a comprehensive metric for physiological age in Drosophila, based on genome-wide expression profiling. We applied this metric to a model of adult-onset neurodegeneration, increased or decreased expression of the activating subunit of the Cdk5 protein kinase, encoded by the gene Cdk5α, the ortholog of mammalian p35. Cdk5α-mediated degeneration was associated with a 27-150% acceleration of the intrinsic rate of aging, depending on the tissue and genetic manipulation. Gene ontology analysis and direct experimental tests revealed that affected age-associated processes included numerous core phenotypes of neurodegeneration, including enhanced oxidative stress and impaired proteostasis. Taken together, our results suggest that Cdk5α-mediated neurodegeneration results from accelerated aging, in combination with cell-autonomous neuronal insults. These data fundamentally recast our picture of the relationship between neurodegeneration and its most prominent risk factor, natural aging.

  19. Preclinical activity of selinexor, an inhibitor of XPO1, in sarcoma.

    Science.gov (United States)

    Nakayama, Robert; Zhang, Yi-Xiang; Czaplinski, Jeffrey T; Anatone, Alex J; Sicinska, Ewa T; Fletcher, Jonathan A; Demetri, George D; Wagner, Andrew J

    2016-03-29

    Selinexor is an orally bioavailable selective inhibitor of nuclear export that has been demonstrated to have preclinical activity in various cancer types and that is currently in Phase I and II clinical trials for advanced cancers. In this study, we evaluated the effects of selinexor in several preclinical models of various sarcoma subtypes. The efficacy of selinexor was investigated in vitro and in vivo using 17 cell lines and 9 sarcoma xenograft models including gastrointestinal stromal tumor (GIST), liposarcoma (LPS), leiomyosarcoma, rhabdomyosarcoma, undifferentiated sarcomas, and alveolar soft part sarcoma (ASPS). Most sarcoma cell lines were sensitive to selinexor with IC50s ranging from 28.8 nM to 218.2 nM (median: 66.1 nM). Selinexor suppressed sarcoma tumor xenograft growth, including models of ASPS that were resistant in vitro. In GIST cells with KIT mutations, selinexor induced G1- arrest without attenuation of phosphorylation of KIT, AKT, or MAPK, in contrast to imatinib. In LPS cell lines with MDM2 and CDK4 amplification, selinexor induced G1-arrest and apoptosis irrespective of p53 expression or mutation and irrespective of RB expression. Selinexor increased p53 and p21 expression at the protein but not RNA level, indicating a post-transcriptional effect. These results indicate that selinexor has potent in vitro and in vivo activity against a wide variety of sarcoma models by inducing G1-arrest independent of known molecular mechanisms in GIST and LPS. These studies further justify the exploration of selinexor in clinical trials targeting various sarcoma subtypes.

  20. CDK activity provides temporal and quantitative cues for organizing genome duplication.

    Directory of Open Access Journals (Sweden)

    Anthony Perrot

    2018-02-01

    Full Text Available In eukaryotes, the spatial and temporal organization of genome duplication gives rise to distinctive profiles of replication origin usage along the chromosomes. While it has become increasingly clear that these programs are important for cellular physiology, the mechanisms by which they are determined and modulated remain elusive. Replication initiation requires the function of cyclin-dependent kinases (CDKs, which associate with various cyclin partners to drive cell proliferation. Surprisingly, although we possess detailed knowledge of the CDK regulators and targets that are crucial for origin activation, little is known about whether CDKs play a critical role in establishing the genome-wide pattern of origin selection. We have addressed this question in the fission yeast, taking advantage of a simplified cell cycle network in which cell proliferation is driven by a single cyclin-CDK module. This system allows us to precisely control CDK activity in vivo using chemical genetics. First, in contrast to previous reports, our results clearly show that distinct cyclin-CDK pairs are not essential for regulating specific subsets of origins and for establishing a normal replication program. Importantly, we then demonstrate that the timing at which CDK activity reaches the S phase threshold is critical for the organization of replication in distinct efficiency domains, while the level of CDK activity at the onset of S phase is a dose-dependent modulator of overall origin efficiencies. Our study therefore implicates these different aspects of CDK regulation as versatile mechanisms for shaping the architecture of DNA replication across the genome.

  1. Loss of keratinocytic RXRα combined with activated CDK4 or oncogenic NRAS generates UVB-induced melanomas via loss of p53 and PTEN in the tumor microenvironment.

    Science.gov (United States)

    Coleman, Daniel J; Chagani, Sharmeen; Hyter, Stephen; Sherman, Anna M; Löhr, Christiane V; Liang, Xiaobo; Ganguli-Indra, Gitali; Indra, Arup K

    2015-01-01

    Understanding the molecular mechanisms behind formation of melanoma, the deadliest form of skin cancer, is crucial for improved diagnosis and treatment. One key is to better understand the cross-talk between epidermal keratinocytes and pigment-producing melanocytes. Here, using a bigenic mouse model system combining mutant oncogenic NRAS(Q61K) (constitutively active RAS) or mutant activated CDK4(R24C/R24C) (prevents binding of CDK4 by kinase inhibitor p16(INK4A)) with an epidermis-specific knockout of the nuclear retinoid X receptor alpha (RXRα(ep-/-)) results in increased melanoma formation after chronic ultraviolet-B (UVB) irradiation compared with control mice with functional RXRα. Melanomas from both groups of bigenic RXRα(ep-/-) mice are larger in size with higher proliferative capacity, and exhibit enhanced angiogenic properties and increased expression of malignant melanoma markers. Analysis of tumor adjacent normal skin from these mice revealed altered expression of several biomarkers indicative of enhanced melanoma susceptibility, including reduced expression of tumor suppressor p53 and loss of PTEN, with concomitant increase in activated AKT. Loss of epidermal RXRα in combination with UVB significantly enhances invasion of melanocytic cells to draining lymph nodes in bigenic mice expressing oncogenic NRAS(Q61K) compared with controls with functional RXRα. These results suggest a crucial role of keratinocytic RXRα to suppress formation of UVB-induced melanomas and their progression to malignant cancers in the context of driver mutations such as activated CDK4(R24C/R24C) or oncogenic NRAS(Q61K). These findings suggest that RXRα may serve as a clinical diagnostic marker and therapeutic target in melanoma progression and metastasis. ©2014 American Association for Cancer Research.

  2. Essential role of endogenous prolactin and CDK7 in estrogen-induced upregulation of the prolactin receptor in breast cancer cells.

    Science.gov (United States)

    Kavarthapu, Raghuveer; Dufau, Maria L

    2017-04-18

    Our early studies have shown that Estradiol (E2)/Estrogen Receptor α (ER) in a non-DNA dependent manner through complex formation with C/EBPβ/SP1 induced transcriptional activation of the generic hPIII promoter and expression of the Prolactin Receptor (PRLR) receptor in MCF-7 cells. Subsequent studies demonstrated effects of unliganded ERα with requisite participation of endogenous PRL on the activation of PRLR transcription. Also, EGF/ERBB1 in the absence of PRL and E2 effectively induced upregulation of the PRLR. In this study we have delineated the transcriptional mechanism of upregulation of PRLR receptor induced by E2 incorporating knowledge of the various transcriptional upregulation modalities from our previous studies. Here, we demonstrate an essential requirement of STAT5a induced by PRL via PRLR receptor which associates at the promoter and its interaction with phoshoERα S118. Knock-down of PRL by siRNA significantly reduced E2-induced PRLR promoter activity, mRNA and protein expression, recruitment of ERα to the complex at promoter, C/EBPβ association to its DNA site and productive complex formation at hPIII promoter. The specific CDK7 inhibitor (THZ1) that attenuates E2-induced ERα phosphorylation at S118 abrogated E2-induced PRLR promoter activation. Further studies demonstrated that E2 induced cell migration was inhibited by PRL siRNA and THZ1 indicating its dependence on PRL/PRLR and CDK7, respectively. Our studies have demonstrated the essential role of endogenous PRL and CDK7 in the upregulation of PRLR by E2 and provide insights for therapeutic approaches that will mitigate the transcription/expression of PRLR and its participation in breast cancer progression fueled by E2 and PRL via their cognate receptors.

  3. CDK2 and PKA mediated-sequential phosphorylation is critical for p19INK4d function in the DNA damage response.

    Directory of Open Access Journals (Sweden)

    Mariela C Marazita

    Full Text Available DNA damage triggers a phosphorylation-based signaling cascade known as the DNA damage response. p19INK4d, a member of the INK4 family of CDK4/6 inhibitors, has been reported to participate in the DNA damage response promoting DNA repair and cell survival. Here, we provide mechanistic insight into the activation mechanism of p19INK4d linked to the response to DNA damage. Results showed that p19INK4d becomes phosphorylated following UV radiation, β-amyloid peptide and cisplatin treatments. ATM-Chk2/ATR-Chk1 signaling pathways were found to be differentially involved in p19INK4d phosphorylation depending on the type of DNA damage. Two sequential phosphorylation events at serine 76 and threonine 141 were identified using p19INK4d single-point mutants in metabolic labeling assays with (32P-orthophosphate. CDK2 and PKA were found to participate in p19INK4d phosphorylation process and that they would mediate serine 76 and threonine 141 modifications respectively. Nuclear translocation of p19INK4d induced by DNA damage was shown to be dependent on serine 76 phosphorylation. Most importantly, both phosphorylation sites were found to be crucial for p19INK4d function in DNA repair and cell survival. In contrast, serine 76 and threonine 141 were dispensable for CDK4/6 inhibition highlighting the independence of p19INK4d functions, in agreement with our previous findings. These results constitute the first description of the activation mechanism of p19INK4d in response to genotoxic stress and demonstrate the functional relevance of this activation following DNA damage.

  4. Association of increased radiocurability of murine carcinomas with low constitutive expression of p21WAF1/CIP1 protein

    International Nuclear Information System (INIS)

    Akimoto, Tetsuo; Seong, Jinsil; Hunter, Nancy R.; Buchmiller, Lara; Mason, Kathy; Milas, Luka

    1999-01-01

    Purpose: The study investigated whether basal, constitutive levels of p21 WAF1/CIP1 protein in murine carcinomas are related to in vivo tumor radioresponse. The study is based on recent observations demonstrating that in vitro cancer cell lines are resistant to cytotoxic drugs when they express high basal levels of p21 WAF1/CIP1 protein, and that the loss of the p21 gene in the HCT116 human colorectal cancer cell line results in increased radioresponse of xenografts derived from that cell line. Methods and Materials: Protein levels of p21 WAF1/CIP1 , p53, bax, and bcl-2 were determined in 8 carcinomas (3 mammary carcinomas designated MCa-4, MCa-29, and MCa-35, 2 squamous cell carcinomas designated SCC-IV and SCC-VII, ovarian adenocarcinoma OCa-I, hepatocarcinoma HCa-I, and adenosquamous carcinoma ACa-SG) syngeneic to C3Hf/Kam mice using Western blot analysis. The tumors, growing in the right hind legs of mice, were 8 mm in diameter at the time of analysis. These tumors greatly differ in their radioresponse, assessed by TCD50 assay, and in their susceptibility to radiation-induced apoptosis. Results: Protein levels of these oncogenes varied among tumors, with p21 WAF1/CIP1 showing the greatest variation: its mean densitometric value ranged from 1 to 19. Bcl-2 levels also showed broad variation in densitometric values, from 1 to 10. In comparison, bax and p53 (7 of 8 tumors contained wild-type p53) varied much less among different tumor types; their variation was within a 5-fold range, and the level of p53 was similar in 6 of 8 tumors. Tumor radioresponse correlated significantly (R = 0.77, p = 0.02) only with the magnitude of p21 WAF1/CIP1 expression: tumors with high levels of p21 WAF1/CIP1 were less radiocurable than those with lower levels. Tumor radiocurability showed a significant positive correlation (p = 0.02) with the extent of radiation-induced apoptosis, indicating that tumors that responded to radiation with higher percentages of apoptosis were more

  5. [Isolation and identification of a novel phosphate-dissolving strain P21].

    Science.gov (United States)

    Yang, Hui; Fan, Bingquan; Gong, Mingbo; Li, Quanxia

    2008-01-01

    Phosphate-dissolving microorganisms can be applied for better use of insoluble phosphorus as fertilizer., A phosphate-dissolving strain P21 was isolated from soil samples in China. The isolate was identified as Erwinia herbicola var. ananas, based on its 16Sr DNA sequence and physiological characteristics. Its activity was measured in solid media as well as liquid media using different phosphate sources including tricalium phosphate, hydroxyapatite, ferric phosphate, aluminium phosphate, zinc phosphate, and rock phosphates. E. herbicola could strongly dissolve 1206.20 mg tricalium phosphate and 529.67 mg hydroxyapatite in per liter liquid media. The strain showed high phosphate-dissolving ability for rock phosphates from Jinning and Kunyang in Yunnan province, Yaan in Sichuan province and Jinping in Jiangsu province with the capacity of 6.64 mg, 78.46 mg, 67.07 mg and 65.24 mg soluble phosphate respectively per liter medium, whereas the phosphate-dissolving ability to the rest of the eight rock phosphates was weak. According to the experiments, the phosphate-dissolving ability of E. herbicola was specific to different rock phosphates, and phosphate-dissolving ability of E. herbicola was not directly related to pH reduction of liquid media.

  6. Genetic Variant rs10757278 on Chromosome 9p21 Contributes to Myocardial Infarction Susceptibility

    Directory of Open Access Journals (Sweden)

    Guangyuan Chen

    2015-05-01

    Full Text Available Large-scale genome-wide association studies (GWAS have revealed that rs10757278 polymorphism (or its proxy rs1333049 on chromosome 9p21 is associated with myocardial infarction (MI susceptibility in individuals of Caucasian ancestry. Following studies in other populations investigated this association. However, some of these studies reported weak or no significant association. Here, we reevaluated this association using large-scale samples by searching PubMed and Google Scholar databases. Our results showed significant association between rs10757278 polymorphism and MI with p = 6.09 × 10−22, odds ratio (OR = 1.29, 95% confidence interval (CI 1.22–1.36 in pooled population. We further performed a subgroup analysis, and found significant association between rs10757278 polymorphism and MI in Asian and Caucasian populations. We identified that the association between rs10757278 polymorphism and MI did not vary substantially by excluding any one study. However, the heterogeneity among the selected studies varies substantially by excluding the study from the Pakistan population. We found even more significant association between rs10757278 polymorphism and MI in pooled population, p = 3.55 × 10−53, after excluding the study from the Pakistan population. In summary, previous studies reported weak or no significant association between rs10757278 polymorphism and MI. Interestingly, our analysis suggests that rs10757278 polymorphism is significantly associated with MI susceptibility by analyzing large-scale samples.

  7. A norovirus GII.P21 outbreak in a boarding school, Austria 2014.

    Science.gov (United States)

    Lin, Yung-Ching; Hipfl, Elisabeth; Lederer, Ingeborg; Allerberger, Franz; Schmid, Daniela

    2015-08-01

    An Austrian boarding school reported a cluster of gastroenteritis on January 10, 2014. Environmental swabs from the school cafeteria and a nearby kebab restaurant tested positive for norovirus. The outbreak was investigated to identify its source(s). An outbreak case was defined as a student or staff member with diarrhoea or vomiting that developed between January 7 and 13. Details on food exposure were collected via a self-administered questionnaire; risk ratios (RR) and 95% confidence intervals (CI) were calculated. Norovirus from the stool specimens of cases and asymptomatic kebab restaurant workers were genotyped. Twenty-eight cases were identified among 144 persons (attack rate 19%). The outbreak emerged and peaked on January 9, and ended on January 12. Compared to those who did not eat kebab, those who ate kebab on 7, 8, and 9 January were respectively 11 (95% CI 4.2-28), 6.7 (95% CI 3.4-13), and 9.3 (95% CI 4.0-22) times more likely to develop disease within the following 2 days. Stool specimens from three cases and three restaurant workers were positive for norovirus GII.P21. The kebab prepared by norovirus-positive restaurant workers was the most likely source of the outbreak. It is recommended that food handlers comply strictly with hand hygiene and avoid bare-handed contact with ready-to-eat food to minimize the risk of food-borne infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. HDAC4 regulates satellite cell proliferation and differentiation by targeting P21 and Sharp1 genes.

    Science.gov (United States)

    Marroncelli, Nicoletta; Bianchi, Marzia; Bertin, Marco; Consalvi, Silvia; Saccone, Valentina; De Bardi, Marco; Puri, Pier Lorenzo; Palacios, Daniela; Adamo, Sergio; Moresi, Viviana

    2018-02-22

    Skeletal muscle exhibits a high regenerative capacity, mainly due to the ability of satellite cells to replicate and differentiate in response to appropriate stimuli. Epigenetic control is effective at different stages of this process. It has been shown that the chromatin-remodeling factor HDAC4 is able to regulate satellite cell proliferation and commitment. However, its molecular targets are still uncovered. To explain the signaling pathways regulated by HDAC4 in satellite cells, we generated tamoxifen-inducible mice with conditional inactivation of HDAC4 in Pax7 + cells (HDAC4 KO mice). We found that the proliferation and differentiation of HDAC4 KO satellite cells were compromised, although similar amounts of satellite cells were found in mice. Moreover, we found that the inhibition of HDAC4 in satellite cells was sufficient to block the differentiation process. By RNA-sequencing analysis we identified P21 and Sharp1 as HDAC4 target genes. Reducing the expression of these target genes in HDAC4 KO satellite cells, we also defined the molecular pathways regulated by HDAC4 in the epigenetic control of satellite cell expansion and fusion.

  9. Inca: a novel p21-activated kinase-associated protein required for cranial neural crest development.

    Science.gov (United States)

    Luo, Ting; Xu, Yanhua; Hoffman, Trevor L; Zhang, Tailin; Schilling, Thomas; Sargent, Thomas D

    2007-04-01

    Inca (induced in neural crest by AP2) is a novel protein discovered in a microarray screen for genes that are upregulated in Xenopus embryos by the transcriptional activator protein Tfap2a. It has no significant similarity to any known protein, but is conserved among vertebrates. In Xenopus, zebrafish and mouse embryos, Inca is expressed predominantly in the premigratory and migrating neural crest (NC). Knockdown experiments in frog and fish using antisense morpholinos reveal essential functions for Inca in a subset of NC cells that form craniofacial cartilage. Cells lacking Inca migrate successfully but fail to condense into skeletal primordia. Overexpression of Inca disrupts cortical actin and prevents formation of actin "purse strings", which are required for wound healing in Xenopus embryos. We show that Inca physically interacts with p21-activated kinase 5 (PAK5), a known regulator of the actin cytoskeleton that is co-expressed with Inca in embryonic ectoderm, including in the NC. These results suggest that Inca and PAK5 cooperate in restructuring cytoskeletal organization and in the regulation of cell adhesion in the early embryo and in NC cells during craniofacial development.

  10. P21-activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells.

    Science.gov (United States)

    Choi, Jinyong; Pease, David Randall; Chen, Siqi; Zhang, Bin; Phee, Hyewon

    2018-01-03

    The p21-activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus-derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T-cell subsets including Foxp3 + Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3 + Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3 + Treg cells upon T-cell receptor and interleukin-2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3 + Treg cells. © 2018 The Authors. Immunology Published by John Wiley & Sons Ltd.

  11. Integrated in vivo multiomics analysis identifies p21-activated kinase signaling as a driver of colitis.

    Science.gov (United States)

    Lyons, Jesse; Brubaker, Douglas K; Ghazi, Phaedra C; Baldwin, Katherine R; Edwards, Amanda; Boukhali, Myriam; Strasser, Samantha Dale; Suarez-Lopez, Lucia; Lin, Yi-Jang; Yajnik, Vijay; Kissil, Joseph L; Haas, Wilhelm; Lauffenburger, Douglas A; Haigis, Kevin M

    2018-02-27

    Inflammatory bowel disease (IBD) is a chronic disorder of the gastrointestinal tract that has limited treatment options. To gain insight into the pathogenesis of chronic colonic inflammation (colitis), we performed a multiomics analysis that integrated RNA microarray, total protein mass spectrometry (MS), and phosphoprotein MS measurements from a mouse model of the disease. Because we collected all three types of data from individual samples, we tracked information flow from RNA to protein to phosphoprotein and identified signaling molecules that were coordinately or discordantly regulated and pathways that had complex regulation in vivo. For example, the genes encoding acute-phase proteins were expressed in the liver, but the proteins were detected by MS in the colon during inflammation. We also ascertained the types of data that best described particular facets of chronic inflammation. Using gene set enrichment analysis and trans-omics coexpression network analysis, we found that each data set provided a distinct viewpoint on the molecular pathogenesis of colitis. Combining human transcriptomic data with the mouse multiomics data implicated increased p21-activated kinase (Pak) signaling as a driver of colitis. Chemical inhibition of Pak1 and Pak2 with FRAX597 suppressed active colitis in mice. These studies provide translational insights into the mechanisms contributing to colitis and identify Pak as a potential therapeutic target in IBD. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

  12. 8-Azapurines as new inhibitors of cyclin-dependent kinases

    Czech Academy of Sciences Publication Activity Database

    Havlíček, Libor; Fuksová, Květoslava; Kryštof, Vladimír; Orság, Martin; Vojtěšek, B.; Strnad, Miroslav

    2005-01-01

    Roč. 13, č. 8 (2005), s. 5399-5407 ISSN 0968-0896 R&D Projects: GA AV ČR KJB6137301; GA ČR GA301/05/0418 Institutional research plan: CEZ:AV0Z50380511 Source of funding: V - iné verejné zdroje ; V - iné verejné zdroje Keywords : CDK2 * Inhibitor * Anticancer drug Subject RIV: FD - Oncology ; Hematology Impact factor: 2.286, year: 2005

  13. Cyclin-dependent kinase inhibitors as anticancer drugs

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; Uldrijan, S.

    2010-01-01

    Roč. 11, č. 3 (2010), s. 291-302 ISSN 1389-4501 R&D Projects: GA ČR GA204/08/0511; GA ČR GA301/08/1649; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z50380511 Keywords : CDK * protein kinase * inhibitor Subject RIV: CE - Biochemistry Impact factor: 3.061, year: 2010

  14. CDK6 mediates the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.

    Science.gov (United States)

    Yuan, Weiwei; Tang, Chunmei; Zhu, Wensi; Zhu, Jiening; Lin, Qiuxiong; Fu, Yongheng; Deng, Chunyu; Xue, Yumei; Yang, Min; Wu, Shulin; Shan, Zhixin

    2016-01-01

    MicroRNA-1 (miR-1) is approved involved in cardiac hypertrophy, but the underlying molecular mechanisms of miR-1 in cardiac hypertrophy are not well elucidated. The present study aimed to investigate the potential role of miR-1 in modulating CDKs-Rb pathway during cardiomyocyte hypertrophy. A rat model of hypertrophy was established with abdominal aortic constriction, and a cell model of hypertrophy was also achieved based on PE-promoted neonatal rat ventricular cardiomyocytes (NRVCs). We demonstrated that miR-1 expression was markedly decreased in hypertrophic myocardium and hypertrophic cardiomyocytes. Dual luciferase reporter assays revealed that miR-1 interacted with the 3'UTR of CDK6, and miR-1 was verified to inhibit CDK6 expression at the posttranscriptional level. CDK6 protein expression was observed increased in hypertrophic myocardium and hypertrophic cardiomyocytes. Morover, miR-1 mimic, in parallel to CDK6 siRNA, could inhibit PE-induced hypertrophy of NRVCs, with decreases in cell size, newly transcribed RNA, expressions of ANF and β-MHC, and the phosphorylated pRb. Taken together, our results reveal that derepression of CDK6 and activation of Rb pathway contributes to the effect of attenuation of miR-1 on provoking cardiomyocyte hypertrophy.

  15. Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

    Science.gov (United States)

    Pan, Q.; Qiao, F.; Gao, C.; Norman, B.; Optican, L.

    2011-01-01

    The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75). Here we show that this phosphorylation promotes the ubiquitin-dependent degradation of Src, thus restricting the availability of active Src. We demonstrate that Src(S75) phosphorylation occurs in vivo in epithelial cells, and like ubiquitinylation, is associated only with active Src. Preventing Cdk5-dependent phosphorylation of Src(S75), by site-specific mutation of S75 or by Cdk5 inhibition or suppression, increases Src(Y419) phosphorylation and kinase activity, resulting in Src-dependent cytoskeletal changes. In transfected cells, ubiquitinylation of Src(S75A) is about 35% that of wild-type Src-V5, and its half-life is approximately 2.5-fold greater. Cdk5 suppression leads to a comparable decrease in the ubiquitinylation of endogenous Src and a similar increase in Src stability. Together, these findings demonstrate that Cdk5-dependent phosphorylation of Src(S75) is a physiologically significant mechanism of regulating intracellular Src activity. PMID:21442427

  16. Folding of the Cerebral Cortex Requires Cdk5 in Upper-Layer Neurons in Gyrencephalic Mammals

    Directory of Open Access Journals (Sweden)

    Yohei Shinmyo

    2017-08-01

    Full Text Available Folds in the cerebral cortex in mammals are believed to be key structures for accommodating increased cortical neurons in the cranial cavity. However, the mechanisms underlying cortical folding remain largely unknown, mainly because genetic manipulations for the gyrencephalic brain have been unavailable. By combining in utero electroporation and the CRISPR/Cas9 system, we succeeded in efficient gene knockout of Cdk5, which is mutated in some patients with classical lissencephaly, in the gyrencephalic brains of ferrets. We show that Cdk5 knockout in the ferret cerebral cortex markedly impaired cortical folding. Furthermore, the results obtained from the introduction of dominant-negative Cdk5 into specific cortical layers suggest that Cdk5 function in upper-layer neurons is more important for cortical folding than that in lower-layer neurons. Cdk5 inhibition induced severe migration defects in cortical neurons. Taken together, our findings suggest that the appropriate positioning of upper-layer neurons is critical for cortical folding.

  17. CDK5 knockdown prevents hippocampal degeneration and cognitive dysfunction produced by cerebral ischemia.

    Science.gov (United States)

    Gutiérrez-Vargas, Johana A; Múnera, Alejandro; Cardona-Gómez, Gloria P

    2015-12-01

    Acute ischemic stroke is a cerebrovascular accident and it is the most common cause of physical disabilities around the globe. Patients may present with repeated ictuses, experiencing mental consequences, such as depression and cognitive disorders. Cyclin-dependent kinase 5 (CDK5) is a kinase that is involved in neurotransmission and plasticity, but its dysregulation contributes to cognitive disorders and dementia. Gene therapy targeting CDK5 was administered to the right hippocampus of ischemic rats during transient cerebral middle artery occlusion. Physiologic parameters (blood pressure, pH, pO2, and pCO2) were measured. The CDK5 downregulation resulted in neurologic and motor improvement during the first week after ischemia. Cyclin-dependent kinase 5 RNA interference (RNAi) prevented dysfunctions in learning, memory, and reversal learning at 1 month after ischemia. These observations were supported by the prevention of neuronal loss, the reduction of microtubule-associated protein 2 (MAP2) immunoreactivity, and a decrease in astroglial and microglia hyperreactivities and tauopathy. Additionally, CDK5 silencing led to an increase in the expression of brain-derived neurotrophic factor (BDNF), its Tropomyosin Receptor kinase B (TRKB) receptor, and activation of cyclic AMP response element-binding protein (CREB) and extracellular signal-regulated kinase (ERK), which are important targets in neuronal plasticity. Together, our findings suggest that gene therapy based on CDK5 silencing prevents cerebral ischemia-induced neurodegeneration and motor and cognitive deficits.

  18. The investigational Aurora kinase A inhibitor alisertib (MLN8237) induces cell cycle G2/M arrest, apoptosis, and autophagy via p38 MAPK and Akt/mTOR signaling pathways in human breast cancer cells.

    Science.gov (United States)

    Li, Jin-Ping; Yang, Yin-Xue; Liu, Qi-Lun; Pan, Shu-Ting; He, Zhi-Xu; Zhang, Xueji; Yang, Tianxin; Chen, Xiao-Wu; Wang, Dong; Qiu, Jia-Xuan; Zhou, Shu-Feng

    2015-01-01

    Alisertib (ALS) is an investigational potent Aurora A kinase inhibitor currently undergoing clinical trials for the treatment of hematological and non-hematological malignancies. However, its antitumor activity has not been tested in human breast cancer. This study aimed to investigate the effect of ALS on the growth, apoptosis, and autophagy, and the underlying mechanisms in human breast cancer MCF7 and MDA-MB-231 cells. In the current study, we identified that ALS had potent growth-inhibitory, pro-apoptotic, and pro-autophagic effects in MCF7 and MDA-MB-231 cells. ALS arrested the cells in G2/M phase in MCF7 and MDA-MB-231 cells which was accompanied by the downregulation of cyclin-dependent kinase (CDK)1/cell division cycle (CDC) 2, CDK2, and cyclin B1 and upregulation of p21 Waf1/Cip1, p27 Kip1, and p53, suggesting that ALS induces G2/M arrest through modulation of p53/p21/CDC2/cyclin B1 pathways. ALS induced mitochondria-mediated apoptosis in MCF7 and MDA-MB-231 cells; ALS significantly decreased the expression of B-cell lymphoma 2 (Bcl-2), but increased the expression of B-cell lymphoma 2-associated X protein (Bax) and p53-upregulated modulator of apoptosis (PUMA), and increased the expression of cleaved caspases 3 and 9. ALS significantly increased the expression level of membrane-bound microtubule-associated protein 1 light chain 3 (LC3)-II and beclin 1 and induced inhibition of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and p38 mitogen-activated protein kinase (MAPK) pathways in MCF7 and MDA-MB-231 cells as indicated by their altered phosphorylation, contributing to the pro-autophagic activities of ALS. Furthermore, treatment with wortmannin markedly downregulated ALS-induced p38 MAPK activation and LC3 conversion. In addition, knockdown of the p38 MAPK gene by ribonucleic acid interference upregulated Akt activation and resulted in LC3-II accumulation. These findings indicate that ALS promotes cellular

  19. Short Communication: p21/CDKN1A Expression Shows Broad Interindividual Diversity in a Subset of HIV-1 Elite Controllers.

    Science.gov (United States)

    de Pablo, Alicia; Bogoi, Roberta; Bejarano, Irene; Toro, Carlos; Valencia, Eulalia; Moreno, Victoria; Martín-Carbonero, Luz; Gómez-Hernando, César; Rodés, Berta

    2016-03-01

    The p21/CDKN1A protein has been described in vitro as well as in a small subset of patients as a restriction factor for HIV infection. We evaluated p21/CDKN1A mRNA expression on CD4(+) T cells from HIV-infected individuals with two outcomes (18 elite controllers and 28 viremic progressors). Our results show broad interindividual variation in this factor, which is unrelated to the patient's phenotype. Considering the gene's genetic surroundings in chromosome 6, such as HLA genotype and single nucleotide polymorphisms (SNPs), there was a positive association with carrying HLA-B2705 alleles and the rs733590 SNP. Thus, this natural variation of p21/CDKN1A alone does not appear to be a prognostic indicator of effective viral control in vivo and other factors must be considered.

  20. Validated context-dependent associations of coronary heart disease risk with genotype variation in the chromosome 9p21 region

    DEFF Research Database (Denmark)

    Lusk, Christine M; Dyson, Greg; Clark, Andrew G

    2014-01-01

    variations marking the 9p21 region for explaining variation in CHD risk? We analyzed a sample of 7,589 (3,869 females and 3,720 males) European American participants of the Atherosclerosis Risk in Communities study. We confirmed CHD-SNP genotype associations for two 9p21 region marker SNPs previously......Markers of the chromosome 9p21 region are regarded as the strongest and most reliably significant genome-wide association study (GWAS) signals for Coronary heart disease (CHD) risk; this was recently confirmed by the CARDIoGRAMplusC4D Consortium meta-analysis. However, while these associations...... are significant at the population level, they may not be clinically relevant predictors of risk for all individuals. We describe here the results of a study designed to address the question: What is the contribution of context defined by traditional risk factors in determining the utility of DNA sequence...

  1. Biaryl purine derivatives as potent antiproliferative agents: inhibitors of cyclin dependent kinases. Part I.

    Science.gov (United States)

    Trova, Michael P; Barnes, Keith D; Barford, Curt; Benanti, Travis; Bielaska, Mark; Burry, Lori; Lehman, John M; Murphy, Christine; O'Grady, Harold; Peace, Denise; Salamone, Susan; Smith, Jennifer; Snider, Patricia; Toporowski, Joseph; Tregay, Steven; Wilson, Alison; Wyle, Michael; Zheng, Xiaozhang; Friedrich, Thomas D

    2009-12-01

    The introduction of an aryl ring onto the 4-position of the C-6 benzyl amino group of the Cdk inhibitor roscovitine (2), maintained the potent Cdk inhibition demonstrated by roscovitine (2) as well as greatly improving the antiproliferative activity. A series of C-6 biarylmethylamino derivatives was prepared addressing modifications on the C-6 biaryl rings, N-9 and C-2 positions to provide compounds that displayed potent cytotoxic activity against tumor cell lines. In particular, derivative 21h demonstrated a >750-fold improvement in the growth inhibition of HeLa cells compared to roscovitine (2).

  2. CDKN2B expression and subcutaneous adipose tissue expandability: Possible influence of the 9p21 atherosclerosis locus

    Energy Technology Data Exchange (ETDEWEB)

    Svensson, Per-Arne; Wahlstrand, Björn; Olsson, Maja [Institute of Medicine, The Sahlgrenska Academy at University of Gothenburg (Sweden); Froguel, Philippe; Falchi, Mario [Department of Genomics of Common Disease, School of Public Health, Imperial College London (United Kingdom); Bergman, Richard N. [Diabetes and Obesity Research Institute, Cedars-Sinai Medical Center, Los Angeles, CA (United States); McTernan, Philip G. [Division of Metabolic and Vascular Health, Warwick Medical School, University of Warwick, Coventry (United Kingdom); Hedner, Thomas; Carlsson, Lena M.S. [Institute of Medicine, The Sahlgrenska Academy at University of Gothenburg (Sweden); Jacobson, Peter, E-mail: peter.jacobson@medfak.gu.se [Institute of Medicine, The Sahlgrenska Academy at University of Gothenburg (Sweden)

    2014-04-18

    Highlights: • The tumor suppressor gene CDKN2B is highly expressed in human adipose tissue. • Risk alleles at the 9p21 locus modify CDKN2B expression in a BMI-dependent fashion. • There is an inverse relationship between expression of CDKN2B and adipogenic genes. • CDKN2B expression influences to postprandial triacylglycerol clearance. • CDKN2B expression in adipose tissue is linked to markers of hepatic steatosis. - Abstract: Risk alleles within a gene desert at the 9p21 locus constitute the most prevalent genetic determinant of cardiovascular disease. Previous research has demonstrated that 9p21 risk variants influence gene expression in vascular tissues, yet the biological mechanisms by which this would mediate atherosclerosis merits further investigation. To investigate possible influences of this locus on other tissues, we explored expression patterns of 9p21-regulated genes in a panel of multiple human tissues and found that the tumor suppressor CDKN2B was highly expressed in subcutaneous adipose tissue (SAT). CDKN2B expression was regulated by obesity status, and this effect was stronger in carriers of 9p21 risk alleles. Covariation between expression of CDKN2B and genes implemented in adipogenesis was consistent with an inhibitory effect of CDKN2B on SAT proliferation. Moreover, studies of postprandial triacylglycerol clearance indicated that CDKN2B is involved in down-regulation of SAT fatty acid trafficking. CDKN2B expression in SAT correlated with indicators of ectopic fat accumulation, including markers of hepatic steatosis. Among genes regulated by 9p21 risk variants, CDKN2B appears to play a significant role in the regulation of SAT expandability, which is a strong determinant of lipotoxicity and therefore might contribute to the development of atherosclerosis.

  3. On the Wegener granulomatosis associated region on chromosome 6p21.3

    Directory of Open Access Journals (Sweden)

    Csernok Elena

    2006-03-01

    Full Text Available Abstract Background Wegener granulomatosis (WG belongs to the heterogeneous group of systemic vasculitides. The multifactorial pathophysiology of WG is supposedly caused by yet unknown environmental influence(s on the basis of genetic predisposition. The presence of anti-neutrophil cytoplasmic antibodies (ANCA in the plasma of patients and genetic involvement of the human leukocyte antigen system reflect an autoimmune background of the disease. Strong associations were revealed with WG by markers located in the major histocompatibility complex class II (MHC II region in the vicinity of human leukocyte antigen (HLA-DPB1 and the retinoid X receptor B (RXRB loci. In order to define the involvement of the 6p21.3 region in WG in more detail this previous population-based association study was expanded here to the respective 3.6 megabase encompassing this region on chromosome 6. The RXRB gene was analysed as well as a splice-site variation of the butyrophilin-like (BTNL2 gene which is also located within the respective region. The latter polymorphism has been evaluated here as it appears as a HLA independent susceptibility factor in another granulomatous disorder, sarcoidosis. Methods 150–180 German WG patients and a corresponding cohort of healthy controls (n = 100–261 were used in a two-step study. A panel of 94 microsatellites was designed for the initial step using a DNA pooling approach. Markers with significantly differing allele frequencies between patient and control pools were individually genotyped. The RXRB gene was analysed for single strand conformation polymorphisms (SSCP and restriction fragment length polymorphisms (RFLP. The splice-site polymorphism in the BTNL2 gene was also investigated by RFLP analysis. Results A previously investigated microsatellite (#1.0.3.7, Santa Cruz genome browser (UCSC May 2004 Freeze localisation: chr6:31257596-34999883, which was used as a positive control, remained associated throughout the whole two

  4. On the Wegener granulomatosis associated region on chromosome 6p21.3

    Science.gov (United States)

    Szyld, Paweł; Jagiello, Peter; Csernok, Elena; Gross, Wolfgang L; Epplen, Joerg T

    2006-01-01

    Background Wegener granulomatosis (WG) belongs to the heterogeneous group of systemic vasculitides. The multifactorial pathophysiology of WG is supposedly caused by yet unknown environmental influence(s) on the basis of genetic predisposition. The presence of anti-neutrophil cytoplasmic antibodies (ANCA) in the plasma of patients and genetic involvement of the human leukocyte antigen system reflect an autoimmune background of the disease. Strong associations were revealed with WG by markers located in the major histocompatibility complex class II (MHC II) region in the vicinity of human leukocyte antigen (HLA)-DPB1 and the retinoid X receptor B (RXRB) loci. In order to define the involvement of the 6p21.3 region in WG in more detail this previous population-based association study was expanded here to the respective 3.6 megabase encompassing this region on chromosome 6. The RXRB gene was analysed as well as a splice-site variation of the butyrophilin-like (BTNL2) gene which is also located within the respective region. The latter polymorphism has been evaluated here as it appears as a HLA independent susceptibility factor in another granulomatous disorder, sarcoidosis. Methods 150–180 German WG patients and a corresponding cohort of healthy controls (n = 100–261) were used in a two-step study. A panel of 94 microsatellites was designed for the initial step using a DNA pooling approach. Markers with significantly differing allele frequencies between patient and control pools were individually genotyped. The RXRB gene was analysed for single strand conformation polymorphisms (SSCP) and restriction fragment length polymorphisms (RFLP). The splice-site polymorphism in the BTNL2 gene was also investigated by RFLP analysis. Results A previously investigated microsatellite (#1.0.3.7, Santa Cruz genome browser (UCSC) May 2004 Freeze localisation: chr6:31257596-34999883), which was used as a positive control, remained associated throughout the whole two-step approach

  5. Herpes simplex virus internalization into epithelial cells requires Na+/H+ exchangers and p21-activated kinases but neither clathrin- nor caveolin-mediated endocytosis.

    Science.gov (United States)

    Devadas, Deepika; Koithan, Thalea; Diestel, Randi; Prank, Ute; Sodeik, Beate; Döhner, Katinka

    2014-11-01

    Herpes simplex virus 1 (HSV-1) is an alphaherpesvirus that has been reported to infect some epithelial cell types by fusion at the plasma membrane but others by endocytosis. To determine the molecular mechanisms of productive HSV-1 cell entry, we perturbed key endocytosis host factors using specific inhibitors, RNA interference (RNAi), or overexpression of dominant negative proteins and investigated their effects on HSV-1 infection in the permissive epithelial cell lines Vero, HeLa, HEp-2, and PtK2. HSV-1 internalization required neither endosomal acidification nor clathrin- or caveolin-mediated endocytosis. In contrast, HSV-1 gene expression and internalization were significantly reduced after treatment with 5-(N-ethyl-N-isopropyl)amiloride (EIPA). EIPA blocks the activity of Na(+)/H(+) exchangers, which are plasma membrane proteins implicated in all forms of macropinocytosis. HSV-1 internalization furthermore required the function of p21-activated kinases that contribute to macropinosome formation. However, in contrast to some forms of macropinocytosis, HSV-1 did not enlist the activities of protein kinase C (PKC), tyrosine kinases, C-terminal binding protein 1, or dynamin to activate its internalization. These data suggest that HSV-1 depends on Na(+)/H(+) exchangers and p21-activated kinases either for macropinocytosis or for local actin rearrangements required for fusion at the plasma membrane or subsequent passage through the actin cortex underneath the plasma membrane. After initial replication in epithelial cells, herpes simplex viruses (HSVs) establish latent infections in neurons innervating these regions. Upon primary infection and reactivation from latency, HSVs cause many human skin and neurological diseases, particularly in immunocompromised hosts, despite the availability of effective antiviral drugs. Many viruses use macropinocytosis for virus internalization, and many host factors mediating this entry route have been identified, although the

  6. Structurally related antitumor effects of flavanones in vitro and in vivo: involvement of caspase 3 activation, p21 gene expression, and reactive oxygen species production

    International Nuclear Information System (INIS)

    Shen, S.-C.; Ko, C.H.; Tseng, S.-W.; Tsai, S.-H.; Chen, Y.-C.

    2004-01-01

    Flavonoids exist extensively in plants and Chinese herbs, and several biological effects of flavonoids have been demonstrated. The antitumor effects in colorectal carcinoma cells (HT29, COLO205, and COLO320HSR) of eight flavanones including flavanone, 2'-OH flavanone, 4'-OH flavanone, 6-OH flavanone, 7-OH flavanone, naringenin, nargin, and taxifolin were investigated. Results of the MTT assay indicate that 2'-OH flavanone showed the most potent cytotoxic effect on these three cells, and cell death induced by 2'-OH flavanone was via the occurrence of DNA ladders, apoptotic bodies, and hypodiploid cells, all characteristics of apoptosis. Induction of caspase 3 protein processing and enzyme activity associated with cleavage of poly(ADP-ribose) polymerase (PARP) was identified in 2'-OH flavanone-treated cells, and a peptidyl inhibitor (Ac-DEVD-FMK) of caspase 3 attenuated the cytotoxicity of 2'-OH flavanone in COLO205 and HT-29 cells. Elevation of p21 (but not p53) and a decrease in Mcl-1 protein were found in 2'-OH flavanone-treated COLO205 and HT-29 cells. Elevation of intracellular reactive oxygen species (ROS) was detected in 2'-OH flavanone-treated cells by the 2',7'-dichlorodihydrofluorescein diacetate (DCHF-DA) assay, and ROS scavengers including 4,5-dihydro-1,3-benzene disulfonic acid (tiron), catalase, superoxide dismutase (SOD), and pyrrolidine dithiocarbamate (PDTC) suppressed the 2'-OH flavanone-induced cytotoxic effect. Subcutaneous injection of COLO205 induced tumor formation in nude mice, and 2'-OH flavanone showed a significant inhibitory effect on tumor formation. The appearance of apoptotic cells with H and E staining, and an increase in p21, but not p53, protein by immunohistochemistry were observed in tumor tissues under 2'-OH flavanone treatment. Primary tumor cells (COLO205-X) derived from a tumor specimen elicited by COLO205 were established, and 2'-OH flavanone showed an significant apoptotic effect in COLO205-X cells in accordance with the

  7. Targeted deletion of the 9p21 noncoding coronary artery disease risk interval in mice

    Energy Technology Data Exchange (ETDEWEB)

    Visel, Axel; Zhu, Yiwen; May, Dalit; Afzal, Veena; Gong, Elaine; Attanasio, Catia; Blow, Matthew J.; Cohen, Jonathan C.; Rubin, Edward M.; Pennacchio, Len A.

    2010-01-01

    Sequence polymorphisms in a 58kb interval on chromosome 9p21 confer a markedly increased risk for coronary artery disease (CAD), the leading cause of death worldwide 1,2. The variants have a substantial impact on the epidemiology of CAD and other life?threatening vascular conditions since nearly a quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein?coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70kb noncoding interval on mouse chromosome 4 affects cardiac expression of neighboring genes, as well as proliferation properties of vascular cells. Chr4delta70kb/delta70kb mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the noncoding interval, Cdkn2a and Cdkn2b, is severely reduced in chr4delta70kb/delta70kb mice, indicating that distant-acting gene regulatory functions are located in the noncoding CAD risk interval. Allelespecific expression of Cdkn2b transcripts in heterozygous mice revealed that the deletion affects expression through a cis-acting mechanism. Primary cultures of chr4delta70kb/delta70kb aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval plays a pivotal role in regulation of cardiac Cdkn2a/b expression and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation.

  8. Cdk5 regulates accurate maturation of newborn granule cells in the adult hippocampus.

    Directory of Open Access Journals (Sweden)

    Sebastian Jessberger

    2008-11-01

    Full Text Available Newborn granule cells become functionally integrated into the synaptic circuitry of the adult dentate gyrus after a morphological and electrophysiological maturation process. The molecular mechanisms by which immature neurons and the neurites extending from them find their appropriate position and target area remain largely unknown. Here we show that single-cell-specific knockdown of cyclin-dependent kinase 5 (cdk5 activity in newborn cells using a retrovirus-based strategy leads to aberrant growth of dendritic processes, which is associated with an altered migration pattern of newborn cells. Even though spine formation and maturation are reduced in cdk5-deficient cells, aberrant dendrites form ectopic synapses onto hilar neurons. These observations identify cdk5 to be critically involved in the maturation and dendrite extension of newborn neurons in the course of adult neurogenesis. The data presented here also suggest a mechanistic dissociation between accurate dendritic targeting and subsequent synapse formation.

  9. Isolation of Proteins Interacting with the Cyclin D1-CDK6 Complex from Normal and Tumorigenic Human Breast Cells Using a Novel Yeast Three-Hybrid System

    National Research Council Canada - National Science Library

    Nichols, Michael

    1998-01-01

    ...% of human breast cancer cases. Cyclin D1, through its association with the catalytic subunit CDK6 and CDK4, controls G1 cell cycle progression, presumably by phosphorylating a substrate protein...

  10. The PAF complex and Prf1/Rtf1 delineate distinct Cdk9-dependent pathways regulating transcription elongation in fission yeast.

    Science.gov (United States)

    Mbogning, Jean; Nagy, Stephen; Pagé, Viviane; Schwer, Beate; Shuman, Stewart; Fisher, Robert P; Tanny, Jason C

    2013-01-01

    Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.

  11. The PAF complex and Prf1/Rtf1 delineate distinct Cdk9-dependent pathways regulating transcription elongation in fission yeast.

    Directory of Open Access Journals (Sweden)

    Jean Mbogning

    Full Text Available Cyclin-dependent kinase 9 (Cdk9 promotes elongation by RNA polymerase II (RNAPII, mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.

  12. Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression

    DEFF Research Database (Denmark)

    Corsino, P.; Davis, B.; Law, M.

    2007-01-01

    Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMITV...

  13. Downregulation of β1,4-galactosyltransferase 1 inhibits CDK11p58-mediated apoptosis induced by cycloheximide

    International Nuclear Information System (INIS)

    Li Zejuan; Wang Hanzhou; Zong Hongliang; Sun Qing; Kong Xiangfei; Jiang Jianhai; Gu Jianxin

    2005-01-01

    Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34 cdc2 -related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11 p58 induces apoptosis is not clear. Some evidences suggested β1,4-galactosyltransferase 1 (β1,4-GT 1) might participate in apoptosis induced by CDK11 p58 . In this study, we demonstrated that ectopically expressed β1,4-GT 1 increased CDK11 p58 -mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of β1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11 p58 -overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of β1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11 p58 by caspase-3 was reduced. We proposed that β1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11 p58 . This may represent a new mechanism of β1,4-GT 1 in CHX-induced apoptosis of CDK11 p58 -overexpressing cells

  14. [Effects of polydatin on learning and memory and Cdk5 kinase activity in the hippocampus of rats with chronic alcoholism].

    Science.gov (United States)

    Li, Xin-juan; Zhang, Yan; Xu, Chun-yang; Li, Shuang; Du, Ai-lin; Zhang, Li-bin; Zhang, Rui-ling

    2015-03-01

    To observe the effects of polydatin on learning and memory and cyclin-dependent kinase 5 (Cdk5) kinase activity in the hippocampus of rats with chronic alcoholism. Forty rats were randomly divided into 4 groups: control group, chronic alcoholism group, low and high polydatin group. The rat chronic alcoholism model was established by ethanol 3.0 g/(kg · d) (intragastric administration). The abstinence scoring was used to evaluate the rats withdrawal symptoms; cognitive function was measured by Morris water maze experiment; Cdk5 protein expression in the hippocampus was detected by immunofluorescence; Cdk5 kinase activity in the hippocampus was detected by liquid scintillation counting method. The abstinence score, escape latency, Cdk5 kinase activity in chronic alcoholism group rats were significantly higher than those of control group (P alcoholism group (P alcoholism group( P alcoholism group were significantly increased compared with control group (P alcoholism group ( P alcoholism damage may interrelate with regulation of Cdk5 kinase activity.

  15. Chromosome 9p21 genetic variants are associated with myocardial infarction but not with ischemic stroke in a Taiwanese population.

    Science.gov (United States)

    Lin, Hsiu-Fen; Tsai, Pei-Chien; Liao, Yi-Chu; Lin, Tsung-Hsien; Tai, Chih-Ta; Juo, Suh-Hang Hank; Lin, Ruey-Tay

    2011-08-01

    Genetic variants on chromosome 9p21 confer a robust risk for coronary artery disease but inconsistent risk for stroke. This study investigated whether such genetic variants exert differential risks on myocardial infarction (MI) and ischemic stroke in a Taiwanese population. The study recruited 425 MI patients, 687 patients with ischemic stroke, and 1377 healthy controls. Four key single nucleotide polymorphisms (SNPs) on chromosome 9p21 were genotyped. Multivariate permutation analyses demonstrated that the risk T allele of rs1333040 and G allele of rs2383207 were associated with MI (P = 0.045 and 0.002, respectively). Subjects with the rs2383207 GG genotype had a 1.85-fold (P = 0.021) risk for MI when compared with the subjects with the AA genotype. Further analysis showed that significant results only exist in the young MI group (stroke (adjusted P ranged from 0.097 to 0.540). Haplotype analysis showed global P values of 0.032 for MI and 0.290 for stroke. Genetic variations in the 9p21 region are associated with MI but not with stroke in a Taiwanese population. Early-onset MI was more likely to carry the risk genotypes of 9p21 SNPs.

  16. Mutational signatures reveal the role of RAD52 in p53-independent p21-driven genomic instability

    DEFF Research Database (Denmark)

    Galanos, Panagiotis; Pappas, George; Polyzos, Alexander

    2018-01-01

    BACKGROUND: Genomic instability promotes evolution and heterogeneity of tumors. Unraveling its mechanistic basis is essential for the design of appropriate therapeutic strategies. In a previous study, we reported an unexpected oncogenic property of p21WAF1/Cip1, showing that its chronic expressio...

  17. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus

    DEFF Research Database (Denmark)

    Willumsen, B M; Norris, K; Papageorge, A G

    1984-01-01

    Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane...

  18. Immunodetection of rasP21 and c-myc oncogenes in oral mucosal swab preparation from clove cigarette smokers

    Directory of Open Access Journals (Sweden)

    Silvi Kintawati

    2008-12-01

    Full Text Available Background: Smoking is the biggest factor for oral cavity malignancy. Some carcinogens found in cigar will stimulate epithel cell in oral cavity and cause mechanism disturbance on tissue resistance and produce abnormal genes (oncogenes. Oncogenes ras and myc are found on malignant tumor in oral cavity which are associated with smoking. Purpose: This research is to find the expression of oncogenes rasP21 and c-myc in oral mucosa epithelial of smoker with immunocytochemistry reaction. Methods: An oral mucosal swab was performed to 30 smokers categorized as light, moderate, and chain, and 10 non smokers which was followed by immunocytochemistry reaction using antibody towards oncogene rasP21 and c-myc is reacted to identify the influence of smoking towards malignant tumor in oral cavity. The result is statistically analyzed using Kruskal-Wallis test. Result: Based on the observation result of oncogene rasP21reaction, it shows that there is significant difference between non smoker group and light smoker, compared to moderate and chain smoker group (p < 0.01. On the other side, the observation result of oncogene c-myc indicates that there is no significant difference between the group of non smokers and the group of light, moderate, and chain smokers (p > 0.05. Conclusion: The higher the possibility of oral cavity malignancy and that the antibody for rasP21 oncogene can be used as a marker for early detection of oral cavity malignancy caused by smoking.

  19. IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: Regulation by SOCS3

    International Nuclear Information System (INIS)

    Sun Rui; Jaruga, Barbara; Kulkarni, Shailin; Sun Haoyu; Gao Bin

    2005-01-01

    The precise role of IL-6 in liver regeneration and hepatocyte proliferation is controversial and the role of SOCS3 in liver regeneration remains unknown. Here we show that in vitro treatment with IL-6 inhibited primary mouse hepatocyte proliferation. IL-6 induced p21 cip1 protein expression in primary mouse hepatocytes. Disruption of the p21 cip1 gene abolished the inhibitory effect of IL-6 on cell proliferation. Co-culture with nonparenchymal liver cells diminished IL-6 inhibition of hepatocyte proliferation, which was likely due to IL-6 stimulation of nonparenchymal cells to produce HGF. Finally, IL-6 induced higher levels of p21 cip1 protein expression and a slightly stronger inhibition of cell proliferation in SOCS3 +/- mouse hepatocytes compared to wild-type hepatocytes, while liver regeneration was enhanced and prolonged in SOCS3 +/- mice. Our findings suggest that IL-6 directly inhibits hepatocyte proliferation via a p21 cip1 -dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration

  20. Overexpression of KLF4 promotes cell senescence through microRNA-203-survivin-p21 pathway

    Science.gov (United States)

    Xu, Qing; Liu, Mei; Zhang, Ju; Xue, Liyan; Zhang, Guo; Hu, Chenfei; Wang, Zaozao; He, Shun; Chen, Lechuang; Ma, Kai; Liu, Xianghe; Zhao, Yahui; Lv, Ning; Liang, Shufang; Zhu, Hongxia; Xu, Ningzhi

    2016-01-01

    Krüppel-like factor 4 (KLF4) is a transcription factor and functions as a tumor suppressor or tumor promoter in different cancer types. KLF4 regulates many gene expression, thus affects the process of cell proliferation, differentiation, and apoptosis. Recently, KLF4 was reported to induce senescence during the generation of induced pluripotent stem (iPS) cells, but the exact mechanism is still unclear. In this study, we constructed two doxycycline-inducing KLF4 cell models, and demonstrated overexpression of KLF4 could promote cell senescence, detected by senescence-associated β-galactosidase activity assay. Then we confirmed that p21, a key effector of senescence, was directly induced by KLF4. KLF4 could also inhibit survivin, which could indirectly induce p21. By miRNA microarray, we found a series of miRNAs regulated by KLF4 and involved in senescence. We demonstrated that KLF4 could upregulate miR-203, and miR-203 contributed to senescence through miR-203-survivin-p21 pathway. Our results suggest that KLF4 could promote cell senescence through a complex network: miR-203, survivin, and p21, which were all regulated by overexpression of KLF4 and contributed to cell senescence. PMID:27531889

  1. Melanoma prone families with CDK4 germline mutation: phenotypic profile and associations with MC1R variants.

    Science.gov (United States)

    Puntervoll, Hanne Eknes; Yang, Xiaohong R; Vetti, Hildegunn Høberg; Bachmann, Ingeborg M; Avril, Marie Françoise; Benfodda, Meriem; Catricalà, Caterina; Dalle, Stéphane; Duval-Modeste, Anne B; Ghiorzo, Paola; Grammatico, Paola; Harland, Mark; Hayward, Nicholas K; Hu, Hui-Han; Jouary, Thomas; Martin-Denavit, Tanguy; Ozola, Aija; Palmer, Jane M; Pastorino, Lorenza; Pjanova, Dace; Soufir, Nadem; Steine, Solrun J; Stratigos, Alexander J; Thomas, Luc; Tinat, Julie; Tsao, Hensin; Veinalde, Ruta; Tucker, Margaret A; Bressac-de Paillerets, Brigitte; Newton-Bishop, Julia A; Goldstein, Alisa M; Akslen, Lars A; Molven, Anders

    2013-04-01

    CDKN2A and CDK4 are high risk susceptibility genes for cutaneous malignant melanoma. Melanoma families with CDKN2A germline mutations have been extensively characterised, whereas CDK4 families are rare and lack a systematic investigation of their phenotype. All known families with CDK4 germline mutations (n=17) were recruited for the study by contacting the authors of published papers or by requests via the Melanoma Genetics Consortium (GenoMEL). Phenotypic data related to primary melanoma and pigmentation characteristics were collected. The CDK4 exon 2 and the complete coding region of the MC1R gene were sequenced. Eleven families carried the CDK4 R24H mutation whereas six families had the R24C mutation. The total number of subjects with verified melanoma was 103, with a median age at first melanoma diagnosis of 39 years. Forty-three (41.7%) subjects had developed multiple primary melanomas (MPM). A CDK4 mutation was found in 89 (including 62 melanoma cases) of 209 tested subjects. CDK4 positive family members (both melanoma cases and unaffected subjects) were more likely to have clinically atypical nevi than CDK4 negative family members (pMC1R red hair colour variants compared with subjects with one tumour (p=0.010). Our study shows that families with CDK4 germline mutations cannot be distinguished phenotypically from CDKN2A melanoma families, which are characterised by early onset of disease, increased occurrence of clinically atypical nevi, and development of MPM. In a clinical setting, the CDK4 gene should therefore always be examined when a melanoma family tests negative for CDKN2A mutation.

  2. Involvement of cyclin D1/CDK4 and pRb mediated by PI3K/AKT pathway activation in Pb2+-induced neuronal death in cultured hippocampal neurons

    International Nuclear Information System (INIS)

    Li Chenchen; Xing Tairan; Tang Mingliang; Yong Wu; Yan Dan; Deng Hongmin; Wang Huili; Wang Ming; Chen Jutao; Ruan Diyun

    2008-01-01

    Lead (Pb) is widely recognized as a neurotoxicant. One of the suggested mechanisms of lead neurotoxicity is apoptotic cell death. And the mechanism by which Pb 2+ causes neuronal death is not well understood. The present study sought to examine the obligate nature of cyclin D1/cyclin-dependent kinase 4 (CDK4), phosphorylation of its substrate retinoblastoma protein (pRb) and its select upstream signal phosphoinositide 3-kinase (PI3K)/AKT pathway in the death of primary cultured rat hippocampal neurons evoked by Pb 2+ . Our data showed that lead treatment of primary hippocampal cultures results in dose-dependent cell death. Inhibition of CDK4 prevented Pb 2+ -induced neuronal death significantly but was incomplete. In addition, we demonstrated that the levels of cyclin D1 and pRb/p107 were increased during Pb 2+ treatment. These elevated expression persisted up to 48 h, returning to control levels after 72 h. We also presented pharmacological and morphological evidences that cyclin D1/CDK4 and pRb/p107 were required for such kind of neuronal death. Addition of the PI3K inhibitor LY294002 (30 μM) or wortmannin (100 nM) significantly rescued the cultured hippocampal neurons from death caused by Pb 2+ . And that Pb 2+ -elicited phospho-AKT (Ser473) participated in the induction of cyclin D1 and partial pRb/p107 expression. These results provide evidences that cell cycle elements play a required role in the death of neurons evoked by Pb 2+ and suggest that certain signaling elements upstream of cyclin D1/CDK4 are modified and/or required for this form of neuronal death

  3. Cdk-dependent phosphorylation regulates TRF1 recruitment to PML bodies and promotes C-circle production in ALT cells.

    Science.gov (United States)

    Wilson, Florence R; Ho, Angus; Walker, John R; Zhu, Xu-Dong

    2016-07-01

    TRF1, a duplex telomeric DNA binding protein, is implicated in homologous-recombination-based alternative lengthening of telomeres, known as ALT. However, how TRF1 promotes ALT activity has yet to be fully characterized. Here we report that Cdk-dependent TRF1 phosphorylation on T371 acts as a switch to create a pool of TRF1, referred to as (pT371)TRF1, which is recruited to ALT-associated PML bodies (APBs) in S and G2 phases independently of its binding to telomeric DNA. We find that phosphorylation of T371 is essential for APB formation and C-circle production, both of which are hallmarks of ALT. We show that the interaction of (pT371)TRF1 with APBs is dependent upon ATM and homologous-recombination-promoting factors Mre11 and BRCA1. In addition, (pT371)TRF1 interaction with APBs is sensitive to transcription inhibition, which also reduces DNA damage at telomeres. Furthermore, overexpression of RNaseH1 impairs (pT371)TRF1 recruitment to APBs in the presence of campothecin, an inhibitor that prevents topoisomerase I from resolving RNA-DNA hybrids. These results suggest that transcription-associated DNA damage, perhaps arising from processing RNA-DNA hybrids at telomeres, triggers (pT371)TRF1 recruitment to APBs to facilitate ALT activity. © 2016. Published by The Company of Biologists Ltd.

  4. Synthetic inhibitors of CDKs induce different responses in androgen sensitive and androgen insensitive prostatic cancer cell lines

    Czech Academy of Sciences Publication Activity Database

    Maďarová, J.; Lukešová, M.; Hlobilková, A.; Strnad, Miroslav; Vojtěšek, B.; Lenobel, R.; Hajdúch, M.; Murray, P. G.; Perera, S.; Kolář, Z.

    2002-01-01

    Roč. 55, č. 4 (2002), s. 227-234 ISSN 1366-8714 R&D Projects: GA ČR GA301/02/0475 Institutional research plan: CEZ:AV0Z5038910 Keywords : synthetic CDK inhibitors * cell cycle * apoptosis Subject RIV: EB - Genetics ; Molecular Biology

  5. Targeted Epigenetic Remodeling of the Cdk5 Gene in Nucleus Accumbens Regulates Cocaine- and Stress-Evoked Behavior.

    Science.gov (United States)

    Heller, Elizabeth A; Hamilton, Peter J; Burek, Dominika D; Lombroso, Sonia I; Peña, Catherine J; Neve, Rachael L; Nestler, Eric J

    2016-04-27

    Recent studies have implicated epigenetic remodeling in brain reward regions following psychostimulant or stress exposure. It has only recently become possible to target a given type of epigenetic remodeling to a single gene of interest, and to probe the functional relevance of such regulation to neuropsychiatric disease. We sought to examine the role of histone modifications at the murine Cdk5 (cyclin-dependent kinase 5) locus, given growing evidence of Cdk5 expression in nucleus accumbens (NAc) influencing reward-related behaviors. Viral-mediated delivery of engineered zinc finger proteins (ZFP) targeted histone H3 lysine 9/14 acetylation (H3K9/14ac), a transcriptionally active mark, or histone H3 lysine 9 dimethylation (H3K9me2), which is associated with transcriptional repression, specifically to the Cdk5 locus in NAc in vivo We found that Cdk5-ZFP transcription factors are sufficient to bidirectionally regulate Cdk5 gene expression via enrichment of their respective histone modifications. We examined the behavioral consequences of this epigenetic remodeling and found that Cdk5-targeted H3K9/14ac increased cocaine-induced locomotor behavior, as well as resilience to social stress. Conversely, Cdk5-targeted H3K9me2 attenuated both cocaine-induced locomotor behavior and conditioned place preference, but had no effect on stress-induced social avoidance behavior. The current study provides evidence for the causal role of Cdk5 epigenetic remodeling in NAc in Cdk5 gene expression and in the control of reward and stress responses. Moreover, these data are especially compelling given that previous work demonstrated opposite behavioral phenotypes compared with those reported here upon Cdk5 overexpression or knockdown, demonstrating the importance of targeted epigenetic remodeling tools for studying more subtle molecular changes that contribute to neuropsychiatric disease. Addiction and depression are highly heritable diseases, yet it has been difficult to identify gene

  6. Class 3 Semaphorin Mediates Dendrite Growth in Adult Newborn Neurons through Cdk5/FAK Pathway

    Science.gov (United States)

    Sohn, Jae Ho; Tan, Terence; Song, Hongjun; Ming, Guo-li; Goh, Eyleen L. K.

    2013-01-01

    Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP) 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397) and serine phosphorylation (Ser 732) of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway. PMID:23762397

  7. Class 3 semaphorin mediates dendrite growth in adult newborn neurons through Cdk5/FAK pathway.

    Directory of Open Access Journals (Sweden)

    Teclise Ng

    Full Text Available Class 3 semaphorins are well-known axonal guidance cues during the embryonic development of mammalian nervous system. However, their activity on postnatally differentiated neurons in neurogenic regions of adult brains has not been characterized. We found that silencing of semaphorin receptors neuropilins (NRP 1 or 2 in neural progenitors at the adult mouse dentate gyrus resulted in newly differentiated neurons with shorter dendrites and simpler branching in vivo. Tyrosine phosphorylation (Tyr 397 and serine phosphorylation (Ser 732 of FAK were essential for these effects. Semaphorin 3A and 3F mediate serine phosphorylation of FAK through the activation of Cdk5. Silencing of either Cdk5 or FAK in newborn neurons phenocopied the defects in dendritic development seen upon silencing of NRP1 or NRP2. Furthermore, in vivo overexpression of Cdk5 or FAK rescued the dendritic phenotypes seen in NRP1 and NRP2 deficient neurons. These results point to a novel role for class 3 semaphorins in promoting dendritic growth and branching during adult hippocampal neurogenesis through the activation of Cdk5-FAK signaling pathway.

  8. Diverse models for the prediction of CDK4 inhibitory activity of ...

    Indian Academy of Sciences (India)

    Decision tree, random forest, moving average analysis (MAA), multiple linear regression (MLR), partial least square regression (PLSR) and principal component regression (PCR) were used to develop models for prediction of CDK4 inhibitory activity. The statistical significance of models was assessed through specificity, ...

  9. Inhibition of CDK7 bypasses spindle assembly checkpoint via premature cyclin B degradation during oocyte meiosis.

    Science.gov (United States)

    Wang, HaiYang; Jo, Yu-Jin; Sun, Tian-Yi; Namgoong, Suk; Cui, Xiang-Shun; Oh, Jeong Su; Kim, Nam-Hyung

    2016-12-01

    To ensure accurate chromosome segregation, the spindle assembly checkpoint (SAC) delays anaphase onset by preventing the premature activation of anaphase-promoting complex/cyclosome (APC/C) until all kinetochores are attached to the spindle. Although an escape from mitosis in the presence of unsatisfied SAC has been shown in several cancer cells, it has not been reported in oocyte meiosis. Here, we show that CDK7 activity is required to prevent a bypass of SAC during meiosis I in mouse oocytes. Inhibition of CDK7 using THZ1 accelerated the first meiosis, leading to chromosome misalignment, lag of chromosomes during chromosome segregation, and a high incidence of aneuploidy. Notably, this acceleration occurred in the presence of SAC proteins including Mad2 and Bub3 at the kinetochores. However, inhibition of APC/C-mediated cyclin B degradation blocked the THZ1-induced premature polar body extrusion. Moreover, chromosomal defects mediated by THZ1 were rescued when anaphase onset was delayed. Collectively, our results show that CDK7 activity is required to prevent premature anaphase onset by suppressing the bypass of SAC, thus ensuring chromosome alignment and proper segregation. These findings reveal new roles of CDK7 in the regulation of meiosis in mammalian oocytes. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. MEK inhibition potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells.

    Science.gov (United States)

    Zhang, Tao; Li, Yanyan; Zhu, Zhenkun; Gu, Mancang; Newman, Bryan; Sun, Duxin

    2010-10-04

    The Ras/Raf/MEK/ERK signaling has been implicated in uncontrolled cell proliferation and tumor progression in pancreatic cancer. The purpose of this study is to evaluate the antitumor activity of MEK inhibitor U0126 in combination with Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) in pancreatic cancer cells. Western blotting showed that 17-AAG caused a 2- to 3-fold transient activation of MEK/ERK signaling in pancreatic cancer cells. The activation sustained for 6 h before phospho-ERK (p-ERK) destabilization. The selective MEK inhibitor U0126 completely abolished 17-AAG induced ERK1/2 activation and resulted in more than 80% of phospho-ERK degradation after only 15 min treatment. Moreover, U0126 had complementary effect on 17-AAG regulated oncogenic and cell cycle related proteins. Although 17-AAG downregulated cyclin D1, cyclin E, CDK4 and CDK6, it led to cyclin A and CDK2 accumulation, which was reversed by the addition of U0126. Antiproliferation assay showed that combination of U0126 and 17-AAG resulted in synergistic cytotoxic effect. More importantly, 17-AAG alone only exhibited moderate inhibition of cell migration in vitro, while addition of U0126 dramatically enhanced the inhibitory effect by 2- to 5-fold. Taken together, these data demonstrate that MEK inhibitor U0126 potentiates the activity of Hsp90 inhibitor 17-AAG against pancreatic cancer cells. The combination of Hsp90 and MEK inhibition could provide a promising avenue for the treatment of pancreatic cancer.

  11. Applications of the InChI in cheminformatics with the CDK and Bioclipse.

    Science.gov (United States)

    Spjuth, Ola; Berg, Arvid; Adams, Samuel; Willighagen, Egon L

    2013-03-13

    The InChI algorithms are written in C++ and not available as Java library. Integration into software written in Java therefore requires a bridge between C and Java libraries, provided by the Java Native Interface (JNI) technology. We here describe how the InChI library is used in the Bioclipse workbench and the Chemistry Development Kit (CDK) cheminformatics library. To make this possible, a JNI bridge to the InChI library was developed, JNI-InChI, allowing Java software to access the InChI algorithms. By using this bridge, the CDK project packages the InChI binaries in a module and offers easy access from Java using the CDK API. The Bioclipse project packages and offers InChI as a dynamic OSGi bundle that can easily be used by any OSGi-compliant software, in addition to the regular Java Archive and Maven bundles. Bioclipse itself uses the InChI as a key component and calculates it on the fly when visualizing and editing chemical structures. We demonstrate the utility of InChI with various applications in CDK and Bioclipse, such as decision support for chemical liability assessment, tautomer generation, and for knowledge aggregation using a linked data approach. These results show that the InChI library can be used in a variety of Java library dependency solutions, making the functionality easily accessible by Java software, such as in the CDK. The applications show various ways the InChI has been used in Bioclipse, to enrich its functionality.

  12. MicroRNA-33 promotes the replicative senescence of mouse embryonic fibroblasts by suppressing CDK6

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Shun; Huang, Haijiao; Li, Nanhong; Zhang, Bing; Jia, Yubin; Yang, Yukun; Yuan, Yuan; Xiong, Xing-dong; Wang, Dengchuan; Zheng, Hui-ling [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China); Liu, Xinguang, E-mail: xgliu64@126.com [Institute of Aging Research, Guangdong Medical University, Dongguan (China); Guangdong Provincial Key Laboratory of Medical Molecular Diagnostics, Dongguan (China); Institute of Biochemistry & Molecular Biology, Guangdong Medical University, Zhanjiang (China)

    2016-05-13

    MicroRNAs are a large class of tiny noncoding RNAs, which have emerged as critical regulators of gene expression, and thus are involved in multiple cellular processes, including cellular senescence. MicroRNA-33 has previously been established to exert crucial effect on cell proliferation, lipid metabolism and cholesterol metabolism. Nonetheless, the association between microRNA-33 and cellular senescence and its underlying molecular mechanism are far to be elucidated. The present study has attempted to probe into the effect of microRNA-33 on MEFs senescence. Our data unveiled that microRNA-33 was dramatically down-regulated in senescent MEFs compared to the young MEFs, and ectopic expression of microRNA-33 promoted MEFs senescence, while knock-down of microRNA-33 exhibited a protective effect against senescence phenotype. Moreover, we verified CDK6 as a direct target of microRNA-33 in mouse. Silencing of CDK6 induced the premature senescence phenotype of MEFs similarly as microRNA-33, while enforced expression of CDK6 significantly reverse the senescence-induction effect of microRNA-33. Taken together, our results suggested that microRNA-33 enhanced the replicative senescence of MEFs potentially by suppressing CDK6 expression. -- Highlights: •MicroRNA-33 was dramatically down-regulated in senescent MEF cells. •Altered expression of microRNA-33 exerted a critical role in MEFs senescence. •MicroRNA-33 promoted the replicative senescence of MEFs via targeting of CDK6.

  13. Cdk5 Is Essential for Amphetamine to Increase Dendritic Spine Density in Hippocampal Pyramidal Neurons

    Directory of Open Access Journals (Sweden)

    Soledad Ferreras

    2017-11-01

    Full Text Available Psychostimulant drugs of abuse increase dendritic spine density in reward centers of the brain. However, little is known about their effects in the hippocampus, where activity-dependent changes in the density of dendritic spine are associated with learning and memory. Recent reports suggest that Cdk5 plays an important role in drug addiction, but its role in psychostimulant’s effects on dendritic spines in hippocampus remain unknown. We used in vivo and in vitro approaches to demonstrate that amphetamine increases dendritic spine density in pyramidal neurons of the hippocampus. Primary cultures and organotypic slice cultures were used for cellular, molecular, pharmacological and biochemical analyses of the role of Cdk5/p25 in amphetamine-induced dendritic spine formation. Amphetamine (two-injection protocol increased dendritic spine density in hippocampal neurons of thy1-green fluorescent protein (GFP mice, as well as in hippocampal cultured neurons and organotypic slice cultures. Either genetic or pharmacological inhibition of Cdk5 activity prevented the amphetamine–induced increase in dendritic spine density. Amphetamine also increased spine density in neurons overexpressing the strong Cdk5 activator p25. Finally, inhibition of calpain, the protease necessary for the conversion of p35 to p25, prevented amphetamine’s effect on dendritic spine density. We demonstrate, for the first time, that amphetamine increases the density of dendritic spine in hippocampal pyramidal neurons in vivo and in vitro. Moreover, we show that the Cdk5/p25 signaling and calpain activity are both necessary for the effect of amphetamine on dendritic spine density. The identification of molecular mechanisms underlying psychostimulant effects provides novel and promising therapeutic approaches for the treatment of drug addiction.

  14. Drosophila p115 is required for Cdk1 activation and G2/M cell cycle transition.

    Science.gov (United States)

    Ibar, Consuelo; Glavic, Álvaro

    2017-04-01

    Golgi complex inheritance and its relationship with the cell cycle are central in cell biology. Golgi matrix proteins, known as golgins, are one of the components that underlie the shape and functionality of this organelle. In mammalian cells, golgins are phosphorylated during mitosis to allow fragmentation of the Golgi ribbon and they also participate in spindle dynamics; both processes are required for cell cycle progression. Little is known about the function of golgins during mitosis in metazoans in vivo. This is particularly significant in Drosophila, in which the Golgi architecture is distributed in numerous units scattered throughout the cytoplasm, in contrast with mammalian cells. We examined the function of the ER/cis-Golgi golgin p115 during the proliferative phase of the Drosophila wing imaginal disc. Knockdown of p115 decreased tissue size. This phenotype was not caused by programmed cell death or cell size reductions, but by a reduction in the final cell number due to an accumulation of cells at the G2/M transition. This phenomenon frequently allows mitotic bypass and re-replication of DNA. These outcomes are similar to those observed following the partial loss of function of positive regulators of Cdk1 in Drosophila. In agreement with this, Cdk1 activation was reduced upon p115 knockdown. Interestingly, these phenotypes were fully rescued by Cdk1 overexpression and partially rescued by Myt1 depletion, but not by String (also known as Cdc25) overexpression. Additionally, we confirmed the physical interaction between p115 and Cdk1, suggesting that the formation of a complex where both proteins are present is essential for the full activation of Cdk1 and thus the correct progression of mitosis in proliferating tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Cell-cycle regulatory proteins in human wound healing

    DEFF Research Database (Denmark)

    Bartkova, Jirina; Grøn, Birgitte; Dabelsteen, Erik

    2003-01-01

    ) and A, and reduced expression of cyclins D(3) and E, the cyclin D-dependent kinase 4 (CDK4), the MCM7 component of DNA replication origin complexes and the retinoblastoma protein pRb. Among the CDK inhibitors (CKIs), p16ink4a and p21Cip1 were moderately increased and decreased, respectively, whereas...

  16. Crystal structure of the homolog of the oncoprotein gankyrin, an interactor of Rb and CDK4/6.

    Science.gov (United States)

    Padmanabhan, Balasundaram; Adachi, Naruhiko; Kataoka, Kazuhiro; Horikoshi, Masami

    2004-01-09

    The oncoprotein gankyrin plays a central role in tumorigenesis and cell proliferation. Gankyrin interacts with the retinoblastoma tumor suppressor (Rb) and cyclin-dependent kinase 4/6 (CDK4/6), increases phosphorylation at specific residues of Rb by CDK4/6 in vivo, and promotes tumorigenesis. The phosphorylation of Rb by CDK4/6 leads to the deregulation of the cell cycle during G1/S transition. Although how phosphorylation occurs on Rb has been studied extensively, the mechanism of site-specific phosphorylation of Rb remains unclear due to a lack of information on the structural arrangement of Rb and CDK4/6. Here, we have determined and refined to 2.3-A resolution the crystal structure of a gankyrin homolog, the non-ATPase subunit 6 (Nas6p) of the proteasome from yeast. The crystal structure reveals that Nas6p contains seven ankyrin repeats. The number of the repeats is different from that predicted from the primary structure. Nas6p also possesses an unusual curved structure with two acidic regions at the N- and C-terminal regions separated by one basic region, suggesting that it has at least two functional surfaces. The tertiary structure of Nas6p, together with the previous biochemical studies, indicates that the CDK4/6 and Rb binding surfaces of gankyrin are located at the N- and C-terminal regions, respectively, and face the same side of gankyrin. These observations suggest that gankyrin brings Rb and CDK4/6 together through gankyrin-Rb and gankyrin-CDK4/6 interactions and determines the relative positioning of the substrate (Rb) and the enzyme (CDK4/6). Our findings provide mechanistic insight into site-specific phosphorylation of Rb caused by CDK4/6.

  17. Crystallization and Characterization of Galdieria sulphuraria RUBISCO in Two Crystal Forms: Structural Phase Transition Observed in P21 Crystal Form

    Directory of Open Access Journals (Sweden)

    Boguslaw Stec

    2007-10-01

    Full Text Available We have isolated ribulose-1,5-bisphosphate-carboxylase/oxygenase (RUBISCOfrom the red algae Galdieria Sulphuraria. The protein crystallized in two different crystalforms, the I422 crystal form being obtained from high salt and the P21 crystal form beingobtained from lower concentration of salt and PEG. We report here the crystallization,preliminary stages of structure determination and the detection of the structural phasetransition in the P21 crystal form of G. sulphuraria RUBISCO. This red algae enzymebelongs to the hexadecameric class (L8S8 with an approximate molecular weight 0.6MDa.The phase transition in G. sulphuraria RUBISCO leads from two hexadecamers to a singlehexadecamer per asymmetric unit. The preservation of diffraction power in a phasetransition for such a large macromolecule is rare.

  18. Harvey murine sarcoma virus p21 ras protein: biological and biochemical significance of the cysteine nearest the carboxy terminus

    DEFF Research Database (Denmark)

    Willumsen, B M; Norris, K; Papageorge, A G

    1984-01-01

    localization. We have now further characterized the post-translational processing of these mutants and have also studied two C-terminal v-rasH point mutants: one encodes serine in place of cysteine-186, the other threonine for valine-187. The Thr-187 mutant was transformation-competent, and its p21 protein...... not undergo the posttranslational processing common to biologically active ras proteins: their electrophoretic migration rate did not change, they remained in the cytosol, and they failed to bind lipid. Since the cell-encoded ras proteins also contain this cysteine, we conclude that this amino acid residue......Previous studies of premature chain termination mutants and in frame deletion mutants of the p21 ras transforming protein encoded by the transforming gene of Harvey murine sarcoma virus (Ha-MuSV) have suggested that the C terminus is required for cellular transformation, lipid binding, and membrane...

  19. Sugar-sweetened beverage intake, chromosome 9p21 variants, and risk of myocardial infarction in Hispanics.

    Science.gov (United States)

    Zheng, Yan; Li, Yanping; Huang, Tao; Cheng, Han-Ling; Campos, Hannia; Qi, Lu

    2016-04-01

    Chromosome 9p21 variants are among the most robust genetic markers for coronary artery disease (CAD), and previous studies have suggested that genetic effects of this locus might be modified by dietary factors. Intake of sugar-sweetened beverages (SSBs), which are the main dietary source of added sugar, has been shown to interact with genetic factors in affecting CAD risk factors such as obesity. We aimed to test whether SSB intake modified the association between chromosome 9p21 variants and CAD risk in Hispanics living in Costa Rica. The current study included 1560 incident cases of nonfatal myocardial infarction (MI) and 1751 population-based controls. Three independent single nucleotide polymorphisms (SNPs) at the chromosome 9p21 locus were genotyped. SSB intake was assessed with the use of a food-frequency questionnaire and was defined as the frequency of intake of daily servings of sweetened beverages and fruit juice. We showed a significant interaction between SSB intake and one of the 3 variants (i.e., rs4977574) on MI risk. The per–risk allele OR (95% CI) of rs4977574 for MI was 1.44 (1.19, 1.74) in participants with higher SSB consumption (>2 servings/d), 1.21 (1.00, 1.47) in those with average consumption (1–2 servings/d), and 0.97 (0.81, 1.16) in subjects with lower consumption (intake on MI risk (P-interaction = 0.03). Our data suggest that unhealthy dietary habits such as higher intake of SSBs could exacerbate the effects of chromosome 9p21 variants on CAD.

  20. Assignment of human beta-galactosidase-A gene to 3p21.33 by fluorescence in situ hybridization.

    Science.gov (United States)

    Takano, T; Yamanouchi, Y

    1993-10-01

    GM1 gangliosidosis and Morquio syndrome type B (MPS IVB) are inherited lyosomal storage disorders associated with deficiency of beta-galactosidase-A (beta GALA) activity. A recombinant plasmid containing a biotinylated cDNA (2.4-kb insert) encoding human beta GALA was used to localize the enzyme locus by fluorescence in situ hybridization (FISH). The human beta GALA gene was assigned to 3p21.33 by FISH.

  1. Ipl1/Aurora Kinase Suppresses S-CDK-Driven Spindle Formation during Prophase I to Ensure Chromosome Integrity during Meiosis

    OpenAIRE

    Newnham, Louise; Jordan, Philip W.; Carballo, Jesus A.; Newcombe, Sonya; Hoffmann, Eva

    2013-01-01

    Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK) during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction tha...

  2. Mutational analysis of the p16 family cyclin-dependent kinase inhibitors p15INK4b and p18INK4c in tumor-derived cell lines and primary tumors.

    Science.gov (United States)

    Zariwala, M; Liu, E; Xiong, Y

    1996-01-18

    The growth suppressing activity of the retinoblastoma suspectibility gene product, pRb, is down regulated by cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) whose kinase activity is negatively regulated by CDK inhibitors of the p16 family. We have examined the genomic status of two recently isolated p16-related CDK inhibitors, p15 and p18, in 15 normal and 73 tumor-derived cell lines established from 23 different tissues, as well as 26 invasive primary breast cancers and 20 acute myelogenous leukemias. p15 was found to be homozygously deleted in 22% of the tumor derived cell lines, but no point mutations were found in either the cultured cells or the two types of primary tumors. With the exception of one breast cancer cell line, no deletions or mutations were found in the p18 gene in either cultured cell lines or primary tumors. These results indicate that mutation of the p18 gene occurs rarely in human tumors. Thus, while they share a very similar biochemical mechanism of inhibiting the kinase activity of CDK4 and CDK6, members of the p16 gene family play different roles in controlling cell proliferation and suppressing tumor growth.

  3. Phenylbutyric acid induces the cellular senescence through an Akt/p21{sup WAF1} signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hag Dong [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Jang, Chang-Young [Research Center for Cell Fate Control, College of Pharmacy, Sookmyung Women' s University, Seoul 140-742 (Korea, Republic of); Choe, Jeong Min [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of); Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Sohn, Jeongwon, E-mail: biojs@korea.ac.kr [Department of Biochemistry, Korea University College of Medicine, Seoul 136-705 (Korea, Republic of); Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705 (Korea, Republic of); Kim, Joon, E-mail: joonkim@korea.ac.kr [Laboratory of Biochemistry, School of Life Sciences and Biotechnology, and BioInstitute, Korea University, Seoul 136-701 (Korea, Republic of)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer Phenylbutyric acid induces cellular senescence. Black-Right-Pointing-Pointer Phenylbutyric acid activates Akt kinase. Black-Right-Pointing-Pointer The knockdown of PERK also can induce cellular senescence. Black-Right-Pointing-Pointer Akt/p21{sup WAF1} pathway activates in PERK knockdown induced cellular senescence. -- Abstract: It has been well known that three sentinel proteins - PERK, ATF6 and IRE1 - initiate the unfolded protein response (UPR) in the presence of misfolded or unfolded proteins in the ER. Recent studies have demonstrated that upregulation of UPR in cancer cells is required to survive and proliferate. Here, we showed that long exposure to 4-phenylbutyric acid (PBA), a chemical chaperone that can reduce retention of unfolded and misfolded proteins in ER, induced cellular senescence in cancer cells such as MCF7 and HT1080. In addition, we found that treatment with PBA activates Akt, which results in p21{sup WAF1} induction. Interestingly, the depletion of PERK but not ATF6 and IRE1 also induces cellular senescence, which was rescued by additional depletion of Akt. This suggests that Akt pathway is downstream of PERK in PBA induced cellular senescence. Taken together, these results show that PBA induces cellular senescence via activation of the Akt/p21{sup WAF1} pathway by PERK inhibition.

  4. Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

    International Nuclear Information System (INIS)

    Ragoussis, J.; Jones, T.A.; Sheer, D.; Shrimpton, A.E.; Goodfellow, P.N.; Trowsdale, J.; Ziegler, A.

    1991-01-01

    A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products

  5. Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids

    Energy Technology Data Exchange (ETDEWEB)

    Ragoussis, J.; Jones, T.A.; Sheer, D.; Shrimpton, A.E.; Goodfellow, P.N.; Trowsdale, J.; Ziegler, A. (ICRF Human Immunogenetics, London (England))

    1991-07-01

    A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.

  6. The p53-p21-DREAM-CDE/CHR pathway regulates G2/M cell cycle genes

    Science.gov (United States)

    Fischer, Martin; Quaas, Marianne; Steiner, Lydia; Engeland, Kurt

    2016-01-01

    The tumor suppressor p53 functions predominantly as a transcription factor by activating and downregulating gene expression, leading to cell cycle arrest or apoptosis. p53 was shown to indirectly repress transcription of the CCNB2, KIF23 and PLK4 cell cycle genes through the recently discovered p53-p21-DREAM-CDE/CHR pathway. However, it remained unclear whether this pathway is commonly used. Here, we identify genes regulated by p53 through this pathway in a genome-wide computational approach. The bioinformatic analysis is based on genome-wide DREAM complex binding data, p53-depedent mRNA expression data and a genome-wide definition of phylogenetically conserved CHR promoter elements. We find 210 target genes that are expected to be regulated by the p53-p21-DREAM-CDE/CHR pathway. The target gene list was verified by detailed analysis of p53-dependent repression of the cell cycle genes B-MYB (MYBL2), BUB1, CCNA2, CCNB1, CHEK2, MELK, POLD1, RAD18 and RAD54L. Most of the 210 target genes are essential regulators of G2 phase and mitosis. Thus, downregulation of these genes through the p53-p21-DREAM-CDE/CHR pathway appears to be a principal mechanism for G2/M cell cycle arrest by p53. PMID:26384566

  7. Drm/Gremlin transcriptionally activates p21(Cip1) via a novel mechanism and inhibits neoplastic transformation.

    Science.gov (United States)

    Chen, Bo; Athanasiou, Meropi; Gu, Qiuping; Blair, Donald G

    2002-08-02

    Drm/Gremlin, a member of the Dan family of BMP antagonists, is known to function in early embryonic development, but is also expressed in a tissue-specific fashion in adults and is significantly downregulated in transformed cells. In this report, we demonstrate that overexpression of Drm in the tumor-derived cell lines Daoy (primitive neuroectodermal) and Saos-2 (osteoblastic), either under ecdysone-inducible or constitutive promoters, significantly inhibits tumorigenesis. Furthermore, Drm overexpression in these cells increases the level of p21(Cip1) protein and reduces the level of phosphorylated p42/44 MAP kinase. Finally, our data indicate that Drm can induce p21(Cip1) transcriptionally via a novel pathway that is independent of p53 and the p38 and p42/44 MAP kinases. These results provide evidence that Drm can function as a novel transformation suppressor and suggest that this may occur through its affect on the levels of p21(Cip1) and phosphorylated p42/44 MAPK.

  8. Doxorubicin Activates Hepatitis B Virus Replication by Elevation of p21 (Waf1/Cip1 and C/EBPα Expression.

    Directory of Open Access Journals (Sweden)

    Yu-Fang Chen

    Full Text Available Hepatitis B virus reactivation is an important medical issue in cancer patients who undergo systemic chemotherapy. Up to half of CHB carriers receiving chemotherapy develop hepatitis and among these cases a notable proportion are associated with HBV reactivation. However, the molecular mechanism(s through which various chemotherapeutic agents induce HBV reactivation is not yet fully understood. In this study, we investigated the role of the cell cycle regulator p21 (Waf1/Cip1 in the modulation of HBV replication when a common chemotherapeutic agent, doxorubicin, is present. We showed that p21 expression was increased by doxorubicin treatment. This elevation in p21 expression enhanced the expression of CCAAT/enhancer-binding protein α (C/EBPα; such an increase is likely to promote the binding of C/EBPα to the HBV promoter, which will contribute to the activation of HBV replication. Our current study thus reveals the mechanism underlying doxorubicin modulation of HBV replication and provides an increased understanding of HBV reactivation in CHB patients who are receiving systemic chemotherapy.

  9. Synthesis and evaluation of pyrazolo[1,5-b]pyridazines as selective cyclin dependent kinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Stevens, Kirk L.; Reno, Michael J.; Alberti, Jennifer B.; Price, Daniel J.; Kane-Carson, Laurie S.; Knick, Victoria B.; Shewchuk, Lisa M.; Hassell, Anne M.; Veal, James M.; Davis, Stephen T.; Griffin, Robert J.; Peel, Michael R. (GSKNC)

    2010-10-01

    A novel series of pyrazolo[1,5-b]pyridazines have been synthesized and identified as cyclin dependant kinase inhibitors potentially useful for the treatment of solid tumors. Modification of the hinge-binding amine or the C(2)- and C(6)-substitutions on the pyrazolopyridazine core provided potent inhibitors of CDK4 and demonstrated enzyme selectivity against VEGFR-2 and GSK3{beta}.

  10. Genetic Association at the 9p21 Glaucoma Locus Contributes to Sex Bias in Normal-Tension Glaucoma.

    Science.gov (United States)

    Ng, Soo Khai; Burdon, Kathryn P; Fitzgerald, Jude T; Zhou, Tiger; Fogarty, Rhys; Souzeau, Emmanuelle; Landers, John; Mills, Richard A; Casson, Robert J; Ridge, Bronwyn; Graham, Stuart L; Hewitt, Alex W; Mackey, David A; Healey, Paul R; Wang, Jie Jin; Mitchell, Paul; MacGregor, Stuart; Craig, Jamie E

    2016-06-01

    Many genome-wide association studies have identified common single nucleotide polymorphisms (SNPs) at the 9p21 glaucoma locus (CDKN2B/CDKN2B-AS1) to be significantly associated with primary open-angle glaucoma (POAG), with association being stronger in normal tension glaucoma (NTG) and advanced glaucoma. We aimed to determine whether any observed differences in genetic association at the 9p21 locus are influenced by sex. Sex was assessed as a risk factor for POAG for 2241 glaucoma participants from the Australian and New Zealand Registry of Advanced Glaucoma, the Glaucoma Inheritance Study in Tasmania, and the Flinders Medical Centre. A total of 3176 controls were drawn from the Blue Mountains Eye Study and South Australia: 1523 advanced POAG and 718 nonadvanced POAG cases were genotyped along with 3176 controls. We selected 13 SNPs at the 9p21 locus, and association results were subanalyszd by sex for high-tension glaucoma (HTG) and NTG. Odds ratios (ORs) between sexes were compared. A sex bias was present within advanced NTG cases (57.1% female versus 42.9% male, P = 0.0026). In all POAG cases, the strongest associated SNP at 9p21 was rs1063192 (OR, 1.43; P = 4 × 10-18). This association was stronger in females (OR, 1.5; P = 5 × 10-13) than in males (OR, 1.35; P = 7 × 10-7), with a statistically significant difference in female to male OR comparison (P = 1.0 × 10-2). An NTG to HTG subanalysis yielded statistically significant results only in females (OR, 1.63; P = 1.5 × 10-4) but not in males (OR, 1.15; P = 2.8 × 10-1), with a statistically significant difference in female to male OR comparison (P = 1.4 × 10-4). This study demonstrated that female sex is a risk factor for developing advanced NTG. The stronger genetic signals at the 9p21 locus among females may contribute at least in part to the observed sex bias for NTG.

  11. Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean

    Energy Technology Data Exchange (ETDEWEB)

    Hollmann, Gabriela, E-mail: gabrielahollmann@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil); Linden, Rafael, E-mail: rlinden@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil); Giangrande, Angela, E-mail: angela.giangrande@igbmc.fr [Institut de Génétique et de Biologie Moléculaire et Cellulaire-IGBMC, INSERM, Strasbourg (France); Allodi, Silvana, E-mail: sallodi@biof.ufrj.br [Programa de Pós Graduação em Ciências Biológicas-Fisiologia, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro-UFRJ, Rio de Janeiro, RJ 21941-590 (Brazil)

    2016-04-15

    Highlights: • The paper characterizes molecular pathways of cell responses to environmental doses of UV in brain tissue of a crab species. • The UV radiation changes levels of proteins which trigger apoptotic or cell cycle arrest pathways and also it changes neurotrophins which lead to apoptosis of neural cell in the central nervous system (CNS) of the crab Ucides cordatus. • The UVB wavelengths in the solar simulator damaged the DNA, either directly or indirectly, by increasing ROS, and induced the increase of p53 and AKT, which blocked p21 and increased the expression of activated caspase-3, triggering apoptosis. The signs of death increased the expression of neurotrophins (BDNF and GDNF), which continued to stimulate the apoptosis signaling mediated by caspase-3. • In the brain of the crab U. cordatus, p53/p21 relationship in response to UV radiation is different from that of most mammals. - Abstract: Ultraviolet (UV) radiation can produce biological damage, leading the cell to apoptosis by the p53 pathway. This study evaluated some molecular markers of the apoptosis pathway induced by UVA, UVB and UVA+ UVB (Solar Simulator, SIM) in environmental doses, during five consecutive days of exposure, in the brain of the crab Ucides cordatus. We evaluated the central nervous system (CNS) by immunoblotting the content of proteins p53, p21, phosphorylated AKT, BDNF, GDNF, activated caspase-3 (C3) and phosphohistone H3 (PH3); and by immunohistochemical tests of the cells labeled for PH3 and C3. After the fifth day of exposure, UVB radiation and SIM increased the protein content of p53, increasing the content of AKT and, somehow, blocking p21, increasing the content of activated caspase-3, which led the cells to apoptosis. The signs of death affected the increase in neurotrophins, such as BDNF and GDNF, stimulating the apoptotic cascade of events. Immunohistochemical assays and immunoblotting showed that apoptosis was present in the brains of all UV groups, while

  12. New developments on the cheminformatics open workflow environment CDK-Taverna

    Directory of Open Access Journals (Sweden)

    Truszkowski Andreas

    2011-12-01

    Full Text Available Abstract Background The computational processing and analysis of small molecules is at heart of cheminformatics and structural bioinformatics and their application in e.g. metabolomics or drug discovery. Pipelining or workflow tools allow for the Lego™-like, graphical assembly of I/O modules and algorithms into a complex workflow which can be easily deployed, modified and tested without the hassle of implementing it into a monolithic application. The CDK-Taverna project aims at building a free open-source cheminformatics pipelining solution through combination of different open-source projects such as Taverna, the Chemistry Development Kit (CDK or the Waikato Environment for Knowledge Analysis (WEKA. A first integrated version 1.0 of CDK-Taverna was recently released to the public. Results The CDK-Taverna project was migrated to the most up-to-date versions of its foundational software libraries with a complete re-engineering of its worker's architecture (version 2.0. 64-bit computing and multi-core usage by paralleled threads are now supported to allow for fast in-memory processing and analysis of large sets of molecules. Earlier deficiencies like workarounds for iterative data reading are removed. The combinatorial chemistry related reaction enumeration features are considerably enhanced. Additional functionality for calculating a natural product likeness score for small molecules is implemented to identify possible drug candidates. Finally the data analysis capabilities are extended with new workers that provide access to the open-source WEKA library for clustering and machine learning as well as training and test set partitioning. The new features are outlined with usage scenarios. Conclusions CDK-Taverna 2.0 as an open-source cheminformatics workflow solution matured to become a freely available and increasingly powerful tool for the biosciences. The combination of the new CDK-Taverna worker family with the already available workflows

  13. High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27Kip Cdk-complexes

    DEFF Research Database (Denmark)

    Groth, Anja; Willumsen, Berthe Marie

    2005-01-01

    The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells...... and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent...... response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density....

  14. MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

    OpenAIRE

    Kisielewska, J.; Philipova, R.; Huang, J.-Y.; Whitaker, M.

    2009-01-01

    Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activit...

  15. A Cdk1 phosphomimic mutant of MCAK impairs microtubule end recognition

    Directory of Open Access Journals (Sweden)

    Hannah R. Belsham

    2017-12-01

    Full Text Available The microtubule depolymerising kinesin-13, MCAK, is phosphorylated at residue T537 by Cdk1. This is the only known phosphorylation site within MCAK’s motor domain. To understand the impact of phosphorylation by Cdk1 on microtubule depolymerisation activity, we have investigated the molecular mechanism of the phosphomimic mutant T537E. This mutant significantly impairs microtubule depolymerisation activity and when transfected into cells causes metaphase arrest and misaligned chromosomes. We show that the molecular mechanism underlying the reduced depolymerisation activity of this phosphomimic mutant is an inability to recognise the microtubule end. The microtubule-end residence time is reduced relative to wild-type MCAK, whereas the lattice residence time is unchanged by the phosphomimic mutation. Further, the microtubule-end specific stimulation of ADP dissociation, characteristic of MCAK, is abolished by this mutation. Our data shows that T537E is unable to distinguish between the microtubule end and the microtubule lattice.

  16. Cis-trimethoxy resveratrol induces intrinsic apoptosis via prometaphase arrest and prolonged CDK1 activation pathway in human Jurkat T cells.

    Science.gov (United States)

    Kim, Chae Eun; Jun, Do Youn; Kim, Jong-Sik; Kim, Young Ho

    2018-01-12

    Cis-trimethoxy resveratrol (cis-3M-RES) induced dose-dependent cytotoxicity and apoptotic DNA fragmentation in Jurkat T cell clones (JT/Neo); however, it induced only cytostasis in BCL-2-overexpressing cells (JT/BCL-2). Treatment with 0.25 μM cis-3M-RES induced G 2 /M arrest, BAK activation, Δψm loss, caspase-9 and caspase-3 activation, and poly (ADP-ribose) polymerase (PARP) cleavage in JT/Neo cells time-dependently but did not induce these events, except G 2 /M arrest, in JT/BCL-2 cells. Moreover, cis-3M-RES induced CDK1 activation, BCL-2 phosphorylation at Ser-70, MCL-1 phosphorylation at Ser-159/Thr-163, and BIM (BIM EL and BIM L ) phosphorylation irrespective of BCL-2 overexpression. Enforced G 1 /S arrest by using a G 1 /S blocker aphidicolin completely inhibited cis-3M-RES-induced apoptotic events. Cis-3M-RES-induced phosphorylation of BCL-2 family proteins and mitochondrial apoptotic events were suppressed by a validated CDK1 inhibitor RO3306. Immunofluorescence microscopy showed that cis-3M-RES induced mitotic spindle defects and prometaphase arrest. The rate of intracellular polymeric tubulin to monomeric tubulin decreased markedly by cis-3M-RES (0.1-1.0 μM). Wild-type Jurkat clone A3, FADD-deficient Jurkat clone I2.1, and caspase-8-deficient Jurkat clone I9.2 exhibited similar susceptibilities to the cytotoxicity of cis-3M-RES, excluding contribution of the extrinsic death receptor-dependent pathway to the apoptosis. IC 50 values of cis-3M-RES against Jurkat E6.1, U937, HL-60, and HeLa cells were 0.07-0.17 μM, whereas those against unstimulated human peripheral T cells and phytohaemagglutinin A-stimulated peripheral T cells were >10.0 and 0.23 μM, respectively. These results indicate that the antitumor activity of cis-3M-RES is mediated by microtubule damage, and subsequent prometaphase arrest and prolonged CDK1 activation that cause BAK-mediated mitochondrial apoptosis, and suggest that cis-3M-RES is a promising agent to treat leukemia.

  17. Anticancer screening of medicinal plant phytochemicals against Cyclin-Dependent Kinase-2 (CDK2: An in-silico approach

    Directory of Open Access Journals (Sweden)

    Wajahat Khan

    2017-08-01

    Full Text Available Background: Cyclin-Dependent Kinase-2 (CDK2 is a member of serine/threonine protein kinases family and plays an important role in regulation of various eukaryotic cell division events. Over-expression of CDK2 during cell cycle may lead to several cellular functional aberrations including diverse types of cancers (lung cancer, primary colorectal carcinoma, ovarian cancer, melanoma and pancreatic carcinoma in humans. Medicinal plants phytochemicals which have anticancer potential can be used as an alternative drug resource. Methods: This study was designed to find out anticancer phytochemicals from medicinal plants which could inhibit CDK2 with the help of molecular docking technique. Molecular Operating Environment (MOE v2009 software was used to dock 2300 phytochemicals in this study. Results: The outcome of this study shows that four phytochemicals Kushenol T, Remangiflavanone B, Neocalyxins A and Elenoside showed the lowest S-score (-17.83, -17.57, -17.26, -17.17 respectively and binds strongly with all eight active residues Tyr15, Lys33, Ileu52, Lys56, Leu78, phe80, Asp145 and Phe146 of CDK2 binding site. These phytochemicals could successfully inhibit the CDK2. Conclusion: These phytochemicals can be considered as potential anticancer agents and used in drug development against CDK2. We anticipate that this study would pave way for phytochemical based novel small molecules as more efficacious and selective anti-cancer therapeutic compounds.

  18. Perspective of Cyclin-dependent kinase 9 (CDK9) as a Drug Target

    Czech Academy of Sciences Publication Activity Database

    Kryštof, Vladimír; Baumli, S.; Fürst, R.

    2012-01-01

    Roč. 18, č. 20 (2012), s. 2883-2890 ISSN 1381-6128 R&D Projects: GA ČR GAP305/12/0783 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cancer * inflammation * kinase Subject RIV: ED - Physiology Impact factor: 3.311, year: 2012 http://www.benthamdirect.org/pages/article/1/3177374/perspective-of-cyclin-dependent-kinase-9-cdk9-as-a- drug -target.html

  19. Liposarcomatous differentiation in malignant phyllodes tumours is unassociated with MDM2 or CDK4 amplification.

    Science.gov (United States)

    Lyle, Pamela L; Bridge, Julia A; Simpson, Jean F; Cates, Justin M; Sanders, Melinda E

    2016-06-01

    Breast sarcomas are rare, usually occurring in the setting of malignant phyllodes tumour (MPT). Heterologous differentiation commonly resembles well-differentiated or pleomorphic liposarcoma. In extramammary sites, these subtypes have different biological behaviours and distinct genetic alterations: MDM2 and CDK4 amplification in well-differentiated liposarcoma, and polyploidy with complex structural rearrangements in pleomorphic liposarcoma. The aim of this study was to investigate foci resembling well-differentiated liposarcoma in MPT for MDM2 and CDK4 amplification. We evaluated the clinicopathological characteristics of MPTs received by the Vanderbilt Breast Consultation Service containing components resembling well-differentiated or pleomorphic liposarcoma. Cases with available tissue blocks were subjected to fluorescence in-situ hybridization with MDM2 and CDK4 probes. Thirty-eight MPTs with liposarcomatous components were available for review. The mean patient age was 49.8 years (range 26-84 years). In addition to well-differentiated liposarcoma, the following components were also present: high-grade undifferentiated sarcoma (n = 9; 23.7%), pleomorphic liposarcoma (n = 4; 10.5%), non-high-grade sarcoma not otherwise specified (n = 22; 57.9%), and malignant peripheral nerve sheath tumour-like (n = 2; 5.2%). Among 10 cases tested, none showed amplification of MDM2 or CDK4. This study examined molecular changes in the well-differentiated liposarcomatous components of MPT. Despite histological similarity to well-differentiated liposarcoma of soft tissues, liposarcomatous differentiation in MPT lacks the molecular phenotype characteristic of extramammary well-differentiated liposarcoma. © 2015 John Wiley & Sons Ltd.

  20. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    Energy Technology Data Exchange (ETDEWEB)

    Lena, A.M.; Mancini, M.; Rivetti di Val Cervo, P. [University of ' Tor Vergata' , Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133 (Italy); Istituto Dermopatico dell' Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Laboratory of Biochemistry c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); Saintigny, G.; Mahe, C. [CHANEL Parfums Beaute, 135 av. Charles de Gaulle, F 92521, Neuilly/Seine (France); Melino, G., E-mail: gerry.melino@uniroma2.it [University of ' Tor Vergata' , Department of Experimental Medicine and Biochemical Sciences, Via Montpellier 1, Rome 00133 (Italy); Istituto Dermopatico dell' Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Laboratory of Biochemistry c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); Association Cell Death and Differentiation c/o Department of Experimental Medicine and Biochemical Sciences, University of Rome ' Tor Vergata' , Rome 00133 (Italy); and others

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer miR-191 expression is upregulated in senescencent human epidermal keratinocytes. Black-Right-Pointing-Pointer miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. Black-Right-Pointing-Pointer SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. Black-Right-Pointing-Pointer SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells. -- Abstract: Keratinocyte replicative senescence has an important role in time-dependent changes of the epidermis, a tissue with high turnover. Senescence encompasses growth arrest during which cells remain metabolically active but acquire a typical enlarged, vacuolar and flattened morphology. It is also accompanied by the expression of endogenous senescence-associated-{beta}-galactosidase and specific gene expression profiles. MicroRNAs levels have been shown to be modulated during keratinocytes senescence, playing key roles in inhibiting proliferation and in the acquisition of senescent markers. Here, we identify miR-191 as an anti-proliferative and replicative senescence-associated miRNA in primary human keratinocytes. Its overexpression is sufficient per se to induce senescence, as evaluated by induction of several senescence-associated markers. We show that SATB1 and CDK6 3 Prime UTRs are two miR-191 direct targets involved in this pathway. Cdk6 and Satb1 protein levels decrease during keratinocytes replicative senescence and their silencing by siRNA is able to induce a G1 block in cell cycle, accompanied by an increase in senescence-associated markers.

  1. Structure of the sliding clamp from the fungal pathogen Aspergillus fumigatus (AfumPCNA) and interactions with Human p21.

    Science.gov (United States)

    Marshall, Andrew C; Kroker, Alice J; Murray, Lauren A M; Gronthos, Kahlia; Rajapaksha, Harinda; Wegener, Kate L; Bruning, John B

    2017-03-01

    The fungal pathogen Aspergillus fumigatus has been implicated in a drastic increase in life-threatening infections over the past decade. However, compared to other microbial pathogens, little is known about the essential molecular processes of this organism. One such fundamental process is DNA replication. The protein responsible for ensuring processive DNA replication is PCNA (proliferating cell nuclear antigen, also known as the sliding clamp), which clamps the replicative polymerase to DNA. Here we present the first crystal structure of a sliding clamp from a pathogenic fungus (A. fumigatus), at 2.6Å. Surprisingly, the structure bears more similarity to the human sliding clamp than other available fungal sliding clamps. Reflecting this, fluorescence polarization experiments demonstrated that AfumPCNA interacts with the PCNA-interacting protein (PIP-box) motif of human p21 with an affinity (K d ) of 3.1 μm. Molecular dynamics simulations were carried out to better understand how AfumPCNA interacts with human p21. These simulations revealed that the PIP-box bound to AfuPCNA forms a secondary structure similar to that observed in the human complex, with a central 3 10 helix contacting the hydrophobic surface pocket of AfumPCNA as well as a β-strand that forms an antiparallel sheet with the AfumPCNA surface. Differences in the 3 10 helix interaction with PCNA, attributed to residue Thr131 of AfumPCNA, and a less stable β-strand formation, attributed to residues Gln123 and His125 of AfumPCNA, are likely causes of the over 10-fold lower affinity of the p21 PIP-box for AfumPCNA as compared to hPCNA. The atomic coordinates and structure factors for the Aspergillus fumigatus sliding clamp can be found in the RCSB Protein Data Bank (http://www.rcsb.org) under the accession code 5TUP. © 2017 Federation of European Biochemical Societies.

  2. Structural and Functional Studies Indicate That the EPEC Effector, EspG, Directly Binds p21-Activated Kinase

    Energy Technology Data Exchange (ETDEWEB)

    Germane, Katherine L.; Spiller, Benjamin W. (Vanderbilt)

    2011-09-20

    Bacterial pathogens secrete effectors into their hosts that subvert host defenses and redirect host processes. EspG is a type three secretion effector with a disputed function that is found in enteropathogenic Escherichia coli. Here we show that EspG is structurally similar to VirA, a Shigella virulence factor; EspG has a large, conserved pocket on its surface; EspG binds directly to the amino-terminal inhibitory domain of human p21-activated kinase (PAK); and mutations to conserved residues in the surface pocket disrupt the interaction with PAK.

  3. Further genetic localization of the transforming sequences of the p21 v-ras gene of Harvey murine sarcoma virus

    DEFF Research Database (Denmark)

    Willumsen, B M; Ellis, R W; Scolnick, E M

    1984-01-01

    The sequences encoding the 21-kilodalton transforming protein (p21 ras) of Harvey murine sarcoma virus have previously been localized genetically to a 1.3-kilobase segment of the viral DNA (E. H. Chang, R. W. Ellis, E. M. Scolnick, and D. R. Lowy, Science 210:1249-1251, 1980). Within this segment...... of this open reading frame. By constructing a mutant of Harvey murine sarcoma virus DNA from which the first two ATG codons of this open reading frame have been deleted, we now show by transfection of the mutant viral DNA into NIH 3T3 cells that only the third ATG codon is necessary and sufficient...

  4. Air pollution by c-PAHs and plasma levels of p53 and p21WAF1 proteins

    Czech Academy of Sciences Publication Activity Database

    Rössner ml., Pavel; Binková, Blanka; Milcová, Alena; Solanský, I.; Židzik, J.; Lyubomirova, K.; Farmer, P. B.; Šrám, Radim

    2007-01-01

    Roč. 620, - (2007), s. 34-40 ISSN 0027-5107 R&D Projects: GA MŽP SI/340/2/00; GA MŽP SL/740/5/03 Grant - others:EU(GB) 2000 -00091 Institutional research plan: CEZ:AV0Z50390512 Source of funding: R - rámcový projekt EK Keywords : air pollution * p53 and p21WAF1 plasma levels Subject RIV: DN - Health Impact of the Environment Quality Impact factor: 4.159, year: 2007

  5. Cdk1/cyclin B-mediated phosphorylation stabilizes SREBP1 during mitosis.

    Science.gov (United States)

    Bengoechea-Alonso, Maria T; Ericsson, Johan

    2006-08-01

    Members of the sterol regulatory element-binding protein (SREBP) family of transcription factors control the biosynthesis of cholesterol and other lipids, and lipid synthesis is critical for cell growth and proliferation. We recently found that the mature forms of SREBP1a and SREBP1c are hyperphosphorylated in mitotic cells, giving rise to a phosphoepitope recognized by the mitotic protein monoclonal-2 (MPM-2) antibody. In addition, we found that mature SREBP1 was stabilized in a phosphorylation-dependent manner during mitosis. We have now mapped the major MPM-2 epitope to a serine residue, S439, in the C terminus of mature SREBP1. Using phosphorylation-specific antibodies, we demonstrate that endogenous SREBP1 is phosphorylated on S439 specifically during mitosis. Mature SREBP1 interacts with the Cdk1/cyclin B complex in mitotic cells and we demonstrate that Cdk1 phosphorylates S439, both in vitro and in vivo. Our results suggest that Cdk1-mediated phosphorylation of S439 stabilizes mature SREBP1 during mitosis, thereby preserving a critical pool of active transcription factors to support lipid synthesis. Taken together with our previous work, the current study suggests that SREBP1 may provide a link between lipid synthesis, proliferation and cell growth. This hypothesis was supported by our observation that siRNA-mediated inactivation of SREBP1 arrested cells in the G(1) phase of the cell cycle, thereby attenuating cell growth.

  6. Effective molecular targeting of CDK4/6 and IGF-1R in a rare FUS-ERG fusion CDKN2A-deletion doxorubicin-resistant Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) nude-mouse model.

    Science.gov (United States)

    Murakami, Takashi; Singh, Arun S; Kiyuna, Tasuku; Dry, Sarah M; Li, Yunfeng; James, Aaron W; Igarashi, Kentaro; Kawaguchi, Kei; DeLong, Jonathan C; Zhang, Yong; Hiroshima, Yukihiko; Russell, Tara; Eckardt, Mark A; Yanagawa, Jane; Federman, Noah; Matsuyama, Ryusei; Chishima, Takashi; Tanaka, Kuniya; Bouvet, Michael; Endo, Itaru; Eilber, Fritz C; Hoffman, Robert M

    2016-07-26

    Ewing's sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewing's sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewing's sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewing's sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer.

  7. Effective molecular targeting of CDK4/6 and IGF-1R in a rare FUS-ERG fusion CDKN2A-deletion doxorubicin-resistant Ewing's sarcoma patient-derived orthotopic xenograft (PDOX) nude-mouse model

    Science.gov (United States)

    Murakami, Takashi; Singh, Arun S.; Kiyuna, Tasuku; Dry, Sarah M.; Li, Yunfeng; James, Aaron W.; Igarashi, Kentaro; Kawaguchi, Kei; DeLong, Jonathan C.; Zhang, Yong; Hiroshima, Yukihiko; Russell, Tara; Eckardt, Mark A.; Yanagawa, Jane; Federman, Noah; Matsuyama, Ryusei; Chishima, Takashi; Tanaka, Kuniya; Bouvet, Michael; Endo, Itaru; Eilber, Fritz C.; Hoffman, Robert M.

    2016-01-01

    Ewing's sarcoma is a rare and aggressive malignancy. In the present study, tumor from a patient with a Ewing's sarcoma with cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) loss and FUS-ERG fusion was implanted in the right chest wall of nude mice to establish a patient-derived orthotopic xenograft (PDOX) model. The aim of the present study was to determine efficacy of cyclin-dependent kinase 4/6 (CDK4/6) and insulin-like growth factor-1 receptor (IGF-1R) inhibitors on the Ewing's sarcoma PDOX. The PDOX models were randomized into the following groups when tumor volume reached 50 mm3: G1, untreated control; G2, doxorubicin (DOX) (intraperitoneal (i.p.) injection, weekly, for 2 weeks); G3, CDK4/6 inhibitor (palbociclib, PD0332991, per oral (p.o.), daily, for 14 days); G4, IGF-1R inhibitor (linsitinib, OSI-906, p.o., daily, for 14 days). Tumor growth was significantly suppressed both in G3 (palbociclib) and in G4 (linsitinib) compared to G1 (untreated control) at all measured time points. In contrast, DOX did not inhibit tumor growth at any time point, which is consistent with the failure of DOX to control tumor growth in the patient. The results of the present study demonstrate the power of the PDOX model to identify effective targeted molecular therapy of a recalcitrant DOX-resistant Ewing's sarcoma with specific genetic alterations. The results of this study suggest the potential of PDOX models for individually-tailored, effective targeted therapy for recalcitrant cancer. PMID:27286459

  8. High CDK6 protects cells from fulvestrant-mediated apoptosis and is a predictor of resistance to fulvestrant in estrogen receptor-positive metastatic breast cancer

    DEFF Research Database (Denmark)

    Alves, Carla Maria Lourenco; Elias, Daniel; Lyng, Maria B

    2016-01-01

    in cell proliferation, apoptosis and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant. RESULTS: We found increased expression of CDK6 in two fulvestrant-resistant cell models vs. sensitive cells. Reduction of CDK6...... expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib re-sensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer.......511, respectively). CONCLUSIONS: Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon...

  9. Effect of 9p21 genetic variation on coronary heart disease is not modified by other risk markers. The Atherosclerosis Risk in Communities (ARIC) Study.

    Science.gov (United States)

    Folsom, Aaron R; Nambi, Vijay; Pankow, James S; Tang, Weihong; Farbakhsh, Kian; Yamagishi, Kazumasa; Boerwinkle, Eric

    2012-10-01

    To determine whether the 9p21 SNP association with coronary heart disease is modified by other classical or novel risk markers. The 9p21 SNP (rs10757274) and multiple risk markers were measured in the Atherosclerosis Risk in Communities Study, and incident coronary disease events were ascertained. Effect modification (interaction) of the 9p21 SNP with risk markers was tested in Cox proportional hazard regression models. The incidence rates of coronary heart disease per 1000 person-years were 14.4, 17.0, and 18.7 for AA, AG, and GG genotypes, yielding hazard ratios of 1.0, 1.20 (95% CI = 1.07-1.36), and 1.34 (95% CI = 1.16-1.53). There was no meaningful evidence of an interaction (all p-interaction > 0.04) between 9p21 SNP and any of 14 other risk markers for coronary heart disease. These included novel markers not previously explored for 9p21 interaction (e.g., cardiac troponin T and N-terminal pro-brain natriuretic peptide). Our study extends evidence that the 9p21 SNP association with coronary heart disease is not modified by classical or novel risk markers. Our findings therefore rule out additional plausible pathways by which 9p21 might have increased coronary heart disease risk. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  10. Receptor for advanced glycation end-products promotes premature senescence of proximal tubular epithelial cells via activation of endoplasmic reticulum stress-dependent p21 signaling.

    Science.gov (United States)

    Liu, Jun; Huang, Kun; Cai, Guang-Yan; Chen, Xiang-Mei; Yang, Ju-Rong; Lin, Li-Rong; Yang, Jie; Huo, Ben-Gang; Zhan, Jun; He, Ya-Ni

    2014-01-01

    Premature senescence is a key process in the progression of diabetic nephropathy (DN). In our study, we hypothesized that receptors for advanced glycation end-products (RAGE) mediate endoplasmic reticulum (ER) stress to induce premature senescence via p21 signaling activation in diabetic nephropathy. Here, we demonstrated that elevated expression of RAGE, ER stress marker glucose-regulated protein 78 (GRP78), and cell-cycle regulator p21 was all positively correlated with enhanced senescence-associated-β-galactosidase (SA-β-gal) activity in DN patients. In addition, the fraction of SA-β-gal or cells in the G0G1 phase were enhanced in cultured mouse proximal tubular epithelial cells (PTECs) and the expression of RAGE, GRP78 and p21 was up-regulated by advanced glycation end-products (AGEs) in a dose- and time-dependent manner. Interestingly, ER stress inducers or RAGE overexpression mimicked AGEs induced-premature senescence, and this was significantly suppressed by p21 gene silencing. However, RAGE blocking successfully attenuated AGEs-induced ER stress and p21 expression, as well as premature senescence. Moreover, ER stress inducers directly caused p21 activation, premature senescence, and also enhanced RAGE expression by positive feedback. These observations suggest that RAGE promotes premature senescence of PTECs by activation of ER stress-dependent p21 signaling. © 2013.

  11. Ribociclib (LEE011): Mechanism of Action and Clinical Impact of This Selective Cyclin-Dependent Kinase 4/6 Inhibitor in Various Solid Tumors.

    Science.gov (United States)

    Tripathy, Debu; Bardia, Aditya; Sellers, William R

    2017-07-01

    The cyclin D-cyclin-dependent kinase (CDK) 4/6-p16-retinoblastoma (Rb) pathway is commonly disrupted in cancer, leading to abnormal cell proliferation. Therapeutics targeting this pathway have demonstrated antitumor effects in preclinical and clinical studies. Ribociclib is a selective, orally bioavailable inhibitor of CDK4 and CDK6, which received FDA approval in March 2017 and is set to enter the treatment landscape alongside other CDK4/6 inhibitors, including palbociclib and abemaciclib. Here, we describe the mechanism of action of ribociclib and review preclinical and clinical data from phase I, II, and III trials of ribociclib across different tumor types, within the context of other selective CDK4/6 inhibitors. The pharmacokinetics, pharmacodynamics, safety, tolerability, and clinical responses with ribociclib as a single agent or in combination with other therapies are discussed, and an overview of the broad portfolio of ongoing clinical trials with ribociclib across a wide range of indications is presented. On the basis of the available data, ribociclib has a manageable tolerability profile and therapeutic potential for a variety of cancer types. Its high selectivity makes it an important partner drug for other targeted therapies, and it has been shown to enhance the clinical activity of existing anticancer therapies and delay the development of treatment resistance, without markedly increasing toxicity. Ongoing trials of doublet and triplet targeted therapies containing ribociclib seek to identify optimal CDK4/6-based targeted combination regimens for various tumor types and advance the field of precision therapeutics in oncology. Clin Cancer Res; 23(13); 3251-62. ©2017 AACR . ©2017 American Association for Cancer Research.

  12. A haploid genetic screen identifies the G1/S regulatory machinery as a determinant of Wee1 inhibitor sensitivity.

    Science.gov (United States)

    Heijink, Anne Margriet; Blomen, Vincent A; Bisteau, Xavier; Degener, Fabian; Matsushita, Felipe Yu; Kaldis, Philipp; Foijer, Floris; van Vugt, Marcel A T M

    2015-12-08

    The Wee1 cell cycle checkpoint kinase prevents premature mitotic entry by inhibiting cyclin-dependent kinases. Chemical inhibitors of Wee1 are currently being tested clinically as targeted anticancer drugs. Wee1 inhibition is thought to be preferentially cytotoxic in p53-defective cancer cells. However, TP53 mutant cancers do not respond consistently to Wee1 inhibitor treatment, indicating the existence of genetic determinants of Wee1 inhibitor sensitivity other than TP53 status. To optimally facilitate patient selection for Wee1 inhibition and uncover potential resistance mechanisms, identification of these currently unknown genes is necessary. The aim of this study was therefore to identify gene mutations that determine Wee1 inhibitor sensitivity. We performed a genome-wide unbiased functional genetic screen in TP53 mutant near-haploid KBM-7 cells using gene-trap insertional mutagenesis. Insertion site mapping of cells that survived long-term Wee1 inhibition revealed enrichment of G1/S regulatory genes, including SKP2, CUL1, and CDK2. Stable depletion of SKP2, CUL1, or CDK2 or chemical Cdk2 inhibition rescued the γ-H2AX induction and abrogation of G2 phase as induced by Wee1 inhibition in breast and ovarian cancer cell lines. Remarkably, live cell imaging showed that depletion of SKP2, CUL1, or CDK2 did not rescue the Wee1 inhibition-induced karyokinesis and cytokinesis defects. These data indicate that the activity of the DNA replication machinery, beyond TP53 mutation status, determines Wee1 inhibitor sensitivity, and could serve as a selection criterion for Wee1-inhibitor eligible patients. Conversely, loss of the identified S-phase genes could serve as a mechanism of acquired resistance, which goes along with development of severe genomic instability.

  13. Cdk2 silencing via a DNA/PCL electrospun scaffold suppresses proliferation and increases death of breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Clément Achille

    Full Text Available RNA interference (RNAi is a promising approach for cancer treatment. Site specific and controlled delivery of RNAi could be beneficial to the patient, while at the same time reducing undesirable off-target side effects. We utilized electrospinning to generate a biodegradable scaffold capable of incorporating and delivering a bioactive plasmid encoding for short hairpin (sh RNA against the cell cycle specific protein, Cdk2. Three electrospun scaffolds were constructed, one using polycaprolactone (PCL alone (Control and PCL with plasmid DNA encoding for either Cdk2 (Cdk2i and EGFP (EGFPi, also served as a control shRNA. Scaffold fiber diameters ranged from 1 to 20 µm (DNA containing and 0.2-3 µm (Control. While the electrospun fibers remained intact for more than two weeks in physiological buffer, degradation was visible during the third week of incubation. Approximately 20-60 ng/ml (~2.5% cumulative release of intact and bioactive plasmid DNA was released over 21 days. Further, Cdk2 mRNA expression in cells plated on the Cdk2i scaffold was decreased by ~51% and 30%, in comparison with that of cells plated on Control or EGFPi scaffold, respectively. This decrease in Cdk2 mRNA by the Cdk2i scaffold translated to a ~40% decrease in the proliferation of the breast cancer cell line, MCF-7, as well as the presence of increased number of dead cells. Taken together, these results represent the first successful demonstration of the delivery of bioactive RNAi-based plasmid DNA from an electrospun polymer scaffold, specifically, in disrupting cell cycle regulation and suppressing proliferation of cancer cells.

  14. Proteomics reveals a switch in CDK1-associated proteins upon M-phase exit during the Xenopus laevis oocyte to embryo transition.

    Science.gov (United States)

    Marteil, Gaëlle; Gagné, Jean-Philippe; Borsuk, Ewa; Richard-Parpaillon, Laurent; Poirier, Guy G; Kubiak, Jacek Z

    2012-01-01

    Cyclin-dependent kinase 1 (CDK1) is a major M-phase kinase which requires the binding to a regulatory protein, Cyclin B, to be active. CDK1/Cyclin B complex is called M-phase promoting factor (MPF) for its key role in controlling both meiotic and mitotic M-phase of the cell cycle. CDK1 inactivation is necessary for oocyte activation and initiation of embryo development. This complex process requires both Cyclin B polyubiquitination and proteosomal degradation via the ubiquitin-conjugation pathway, followed by the dephosphorylation of the monomeric CDK1 on Thr161. Previous proteomic analyses revealed a number of CDK1-associated proteins in human HeLa cells. It is, however, unknown whether specific partners are involved in CDK1 inactivation upon M-phase exit. To better understand CDK1 regulation during MII-arrest and oocyte activation, we immunoprecipitated (IPed) CDK1 together with its associated proteins from M-phase-arrested and M-phase-exiting Xenopus laevis oocytes. A mass spectrometry (MS) analysis revealed a number of new putative CDK1 partners. Most importantly, the composition of the CDK1-associated complex changed rapidly during M-phase exit. Additionally, an analysis of CDK1 complexes precipitated with beads covered with p9 protein, a fission yeast suc1 homologue well known for its high affinity for CDKs, was performed to identify the most abundant proteins associated with CDK1. The screen was auto-validated by identification of: (i) two forms of CDK1: Cdc2A and B, (ii) a set of Cyclins B with clearly diminishing number of peptides identified upon M-phase exit, (iii) a number of known CDK1 substrates (e.g. peroxiredoxine) and partners (e.g. HSPA8, a member of the HSP70 family) both in IP and in p9 precipitated pellets. In IP samples we also identified chaperones, which can modulate CDK1 three-dimensional structure, as well as calcineurin, a protein necessary for successful oocyte activation. These results shed a new light on CDK1 regulation via a dynamic

  15. Lipid Synthetic Transcription Factor SREBP-1a Activates p21WAF1/CIP1, a Universal Cyclin-Dependent Kinase Inhibitor

    OpenAIRE

    Inoue, Noriyuki; Shimano, Hitoshi; Nakakuki, Masanori; Matsuzaka, Takashi; Nakagawa, Yoshimi; Yamamoto, Takashi; Sato, Ryuichiro; Takahashi, Akimitsu; Sone, Hirohito; Yahagi, Naoya; Suzuki, Hiroaki; Toyoshima, Hideo; Yamada, Nobuhiro

    2005-01-01

    Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that regulate lipid synthetic genes. In contrast to SREBP-2, which regulates cellular cholesterol level in normal cells, SREBP-1a is highly expressed in actively growing cells and activates entire programs of genes involved in lipid synthesis such as cholesterol, fatty acids, triglycerides, and phospholipids. Previously, the physiological relevance of this potent activity of SREBP-1a has been thought ...

  16. Coordinating structural and functional synapse development: postsynaptic p21-activated kinase independently specifies glutamate receptor abundance and postsynaptic morphology.

    Science.gov (United States)

    Albin, Stephanie D; Davis, Graeme W

    2004-08-04

    Here, we show that postsynaptic p21-activated kinase (Pak) signaling diverges into two genetically separable pathways at the Drosophila neuromuscular junction. One pathway controls glutamate receptor abundance. Pak signaling within this pathway is specified by a required interaction with the adaptor protein Dreadlocks (Dock). We demonstrate that Dock is localized to the synapse via an Src homology 2-mediated protein interaction. Dock is not necessary for Pak localization but is necessary to restrict Pak signaling to control glutamate receptor abundance. A second genetically separable function of Pak kinase signaling controls muscle membrane specialization through the regulation of synaptic Discs-large. In this pathway, Dock is dispensable. We present a model in which divergent Pak signaling is able to coordinate two different features of postsynaptic maturation, receptor abundance, and muscle membrane specialization.

  17. P21 Framework Definitions

    Science.gov (United States)

    Partnership for 21st Century Skills, 2009

    2009-01-01

    To help practitioners integrate skills into the teaching of core academic subjects, the Partnership for 21st Century Skills has developed a unified, collective vision for learning known as the Framework for 21st Century Learning. This Framework describes the skills, knowledge and expertise students must master to succeed in work and life; it is a…

  18. Polyomavirus-associated Trichodysplasia spinulosa involves hyperproliferation, pRB phosphorylation and upregulation of p16 and p21.

    Directory of Open Access Journals (Sweden)

    Siamaque Kazem

    Full Text Available Trichodysplasia spinulosa (TS is a proliferative skin disease observed in severely immunocompromized patients. It is characterized by papule and trichohyalin-rich spicule formation, epidermal acanthosis and distention of dysmorphic hair follicles overpopulated by inner root sheath cells (IRS. TS probably results from active infection with the TS-associated polyomavirus (TSPyV, as indicated by high viral-load, virus protein expression and particle formation. The underlying pathogenic mechanism imposed by TSPyV infection has not been solved yet. By analogy with other polyomaviruses, such as the Merkel cell polyomavirus associated with Merkel cell carcinoma, we hypothesized that TSPyV T-antigen promotes proliferation of infected IRS cells. Therefore, we analyzed TS biopsy sections for markers of cell proliferation (Ki-67 and cell cycle regulation (p16ink4a, p21waf, pRB, phosphorylated pRB, and the putatively transforming TSPyV early large tumor (LT antigen. Intense Ki-67 staining was detected especially in the margins of TS hair follicles, which colocalized with TSPyV LT-antigen detection. In this area, staining was also noted for pRB and particularly phosphorylated pRB, as well as p16ink4a and p21waf. Healthy control hair follicles did not or hardly stained for these markers. Trichohyalin was particularly detected in the center of TS follicles that stained negative for Ki-67 and TSPyV LT-antigen. In summary, we provide evidence for clustering of TSPyV LT-antigen-expressing and proliferating cells in the follicle margins that overproduce negative cell cycle regulatory proteins. These data are compatible with a scenario of TSPyV T-antigen-mediated cell cycle progression, potentially creating a pool of proliferating cells that enable viral DNA replication and drive papule and spicule formation.

  19. p27Kip1represses the Pitx2-mediated expression of p21Cip1and regulates DNA replication during cell cycle progression.

    Science.gov (United States)

    Gallastegui, E; Biçer, A; Orlando, S; Besson, A; Pujol, M J; Bachs, O

    2017-01-19

    The tumor suppressor p21 regulates cell cycle progression and peaks at mid/late G 1 . However, the mechanisms regulating its expression during cell cycle are poorly understood. We found that embryonic fibroblasts from p27 null mice at early passages progress slowly through the cell cycle. These cells present an elevated basal expression of p21 suggesting that p27 participates to its repression. Mechanistically, we found that p27 represses the expression of Pitx2 (an activator of p21 expression) by associating with the ASE-regulatory region of this gene together with an E2F4 repressive complex. Furthermore, we found that Pitx2 binds to the p21 promoter and induces its transcription. Finally, silencing Pitx2 or p21 in proliferating cells accelerates DNA replication and cell cycle progression. Collectively, these results demonstrate an unprecedented connection between p27, Pitx2 and p21 relevant for the regulation of cell cycle progression and cancer and for understanding human pathologies associated with p27 germline mutations.

  20. Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

    Directory of Open Access Journals (Sweden)

    Huarong Guo

    2012-09-01

    Full Text Available p21CIP1/WAF1 is a p53-target gene in response to cellular DNA damage. Here we report the development of a fish cell biosensor system for high throughput genotoxicity detection of new drugs, by stably integrating two reporter plasmids of pGL3-p21-luc (human p21 promoter linked to firefly luciferase and pRL-CMV-luc (CMV promoter linked to Renilla luciferase into marine flatfish flounder gill (FG cells, referred to as p21FGLuc. Initial validation of this genotoxicity biosensor system showed that p21FGLuc cells had a wild-type p53 signaling pathway and responded positively to the challenge of both directly acting genotoxic agents (bleomycin and mitomycin C and indirectly acting genotoxic agents (cyclophosphamide with metabolic activation, but negatively to cyclophosphamide without metabolic activation and the non-genotoxic agents ethanol and D-mannitol, thus confirming a high specificity and sensitivity, fast and stable response to genotoxic agents for this easily maintained fish cell biosensor system. This system was especially useful in the genotoxicity detection of Di(2-ethylhexyl phthalate (DEHP, a rodent carcinogen, but negatively reported in most non-mammalian in vitro mutation assays, by providing a strong indication of genotoxicity for DEHP. A limitation for this biosensor system was that it might give false positive results in response to sodium butyrate and any other agents, which can trans-activate the p21 gene in a p53-independent manner.

  1. Tetrahydroanthraquinone Derivative (±)-4-Deoxyaustrocortilutein Induces Cell Cycle Arrest and Apoptosis in Melanoma Cells via Upregulation of p21 and p53 and Downregulation of NF-kappaB.

    Science.gov (United States)

    Genov, Miroslav; Kreiseder, Birgit; Nagl, Michael; Drucker, Elisabeth; Wiederstein, Martina; Muellauer, Barbara; Krebs, Julia; Grohmann, Teresa; Pretsch, Dagmar; Baumann, Karl; Bacher, Markus; Pretsch, Alexander; Wiesner, Christoph

    2016-01-01

    Malignant melanoma is an aggressive type of skin cancer with high risk for metastasis and chemoresistance. Disruption of tightly regulated processes such as cell cycle, cell adhesion, cell differentiation and cell death are predominant in melanoma development. So far, conventional treatment options have been insufficient to treat metastatic melanoma and survival rates are poor. Anthraquinone compounds have been reported to have anti-tumorigenic potential by DNA-interaction, promotion of apoptosis and suppression of proliferation in various cancer cells. In the current study, the racemic tetrahydroanthraquinone derivative (±)-4-deoxyaustrocortilutein (4-DACL) was synthesized and the cytotoxic activity against melanoma cells and melanoma spheroids determined by CellTiter-Blue viability Assay and phase contrast microscopy. Generation of reactive oxygen species (ROS) was determined with CellROX Green and Deep Red Reagent kit and microplate-based fluorometry. Luciferase reporter gene assays for nuclear factor kappa B (NF-κB) and p53 activities and western blotting analysis were carried out to detect the expression of anti-proliferative or pro-apoptotic (p53, p21, p27, MDM2, and GADD45M) and anti-apoptotic (p65, IκB-α, IKK) proteins. Cell cycle distribution and apoptosis rate were detected by flow cytometry, the morphological changes visualized by fluorescence microscopy and the activation of different caspase cascades distinguished by Caspase Glo 3/7, 8 and 9 Assays. We demonstrated that 4-DACL displayed high activity against different malignant melanoma cells and melanoma spheroids and only low toxicity to melanocytes and other primary cells. In particular, 4-DACL treatment induced mitochondrial ROS, reduced NF-κB signaling activity and increased up-regulation of the cell cycle inhibitors cyclin-dependent kinase inhibitor p21 (p21(WAF1/Cip1)) and the tumor suppressor protein p53 in a dose-dependent manner, which was accompanied by decreased cell proliferation and

  2. Cdk5 targets active Src for ubiquitin-dependent degradation by phosphorylating Src(S75)

    OpenAIRE

    Pan, Q.; Qiao, F.; Gao, C.; Norman, B.; Optican, L.; Zelenka, Peggy S.

    2011-01-01

    The non-receptor tyrosine kinase Src is a critical regulator of cytoskeletal contraction, cell adhesion, and migration. In normal cells, Src activity is stringently controlled by Csk-dependent phosphorylation of Src(Y530), and by Cullin-5-dependent ubiquitinylation, which affects active Src(pY419) exclusively, leading to its degradation by the proteosome. Previous work has shown that Src activity is also limited by Cdk5, a proline-directed kinase, which has been shown to phosphorylate Src(S75...

  3. Evidence that phosphorylation by the mitotic kinase Cdk1 promotes ICER monoubiquitination and nuclear delocalization

    Energy Technology Data Exchange (ETDEWEB)

    Memin, Elisabeth, E-mail: molinac@mail.montclair.edu [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Genzale, Megan [Department of Biochemistry and Molecular Biology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ 07103 (United States); Crow, Marni; Molina, Carlos A. [Department of Biology and Molecular Biology, Montclair State University, Montclair, NJ, 07043 (United States)

    2011-10-15

    In contrast to normal prostatic cells, the transcriptional repressor Inducible cAMP Early Repressor (ICER) is undetected in the nuclei of prostate cancer cells. The molecular mechanisms for ICER abnormal expression in prostate cancer cells remained largely unknown. In this report data is presented demonstrating that ICER is phosphorylated by the mitotic kinase cdk1. Phosphorylation of ICER on a discrete residue targeted ICER to be monoubiquitinated. Different from unphosphorylated, phosphorylated and polyubiquitinated ICER, monoubiquitinated ICER was found to be cytosolic. Taken together, these results hinted on a mechanism for the observed abnormal subcellular localization of ICER in human prostate tumors.

  4. In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

    Directory of Open Access Journals (Sweden)

    Antony M Latham

    Full Text Available Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues.We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2 and cyclin-dependent kinase 1 (CDK1. This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis.We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

  5. In silico design and biological evaluation of a dual specificity kinase inhibitor targeting cell cycle progression and angiogenesis.

    Science.gov (United States)

    Latham, Antony M; Kankanala, Jayakanth; Fearnley, Gareth W; Gage, Matthew C; Kearney, Mark T; Homer-Vanniasinkam, Shervanthi; Wheatcroft, Stephen B; Fishwick, Colin W G; Ponnambalam, Sreenivasan

    2014-01-01

    Protein kinases play a central role in tumor progression, regulating fundamental processes such as angiogenesis, proliferation and metastasis. Such enzymes are an increasingly important class of drug target with small molecule kinase inhibitors being a major focus in drug development. However, balancing drug specificity and efficacy is problematic with off-target effects and toxicity issues. We have utilized a rational in silico-based approach to demonstrate the design and study of a novel compound that acts as a dual inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) and cyclin-dependent kinase 1 (CDK1). This compound acts by simultaneously inhibiting pro-angiogenic signal transduction and cell cycle progression in primary endothelial cells. JK-31 displays potent in vitro activity against recombinant VEGFR2 and CDK1/cyclin B proteins comparable to previously characterized inhibitors. Dual inhibition of the vascular endothelial growth factor A (VEGF-A)-mediated signaling response and CDK1-mediated mitotic entry elicits anti-angiogenic activity both in an endothelial-fibroblast co-culture model and a murine ex vivo model of angiogenesis. We deduce that JK-31 reduces the growth of both human endothelial cells and human breast cancer cells in vitro. This novel synthetic molecule has broad implications for development of similar multi-kinase inhibitors with anti-angiogenic and anti-cancer properties. In silico design is an attractive and innovative method to aid such drug discovery.

  6. Sequencing Analysis of Mutant Allele $cdc$28-$srm$ of Protein Kinase CDC28 and Molecular Dynamics Study of Glycine-Rich Loop in Wild-Type and Mutant Allele G16S of CDK2 as Model

    CERN Document Server

    Koltovaya, N A; Kholmurodov, Kh T; Kretov, D A

    2005-01-01

    The central role that cyclin-dependent kinases play in the timing of cell division and the high incidence of genetic alteration of CDKs or deregulation of CDK inhibitors in a number of cancers make CDC28 of the yeast \\textit{Saccharomyces cerevisiae }very attractive model for studies of mechanisms of CDK regulation. Earlier it was found that certain gene mutations including \\textit{cdc28-srm} affect cell cycle progression, maintenance of different genetic structures and increase cell sensitivity to ionizing radiation. A~\\textit{cdc28-srm} mutation is not temperature-sensitive mutation and differs from the known \\textit{cdc28-ts }mutations because it has the evident phenotypic manifestations at 30 $^{\\circ}$C. Sequencing analysis of \\textit{cdc28-srm} revealed a single nucleotide substitution G20S. This is a third glycine in a conserved sequence GxGxxG in the G-rich loop positioned opposite the activation T-loop. Despite its demonstrated importance, the role of the G-loop has remained unclear. The crystal stru...

  7. Ipl1/Aurora kinase suppresses S-CDK-driven spindle formation during prophase I to ensure chromosome integrity during meiosis.

    Directory of Open Access Journals (Sweden)

    Louise Newnham

    Full Text Available Cells coordinate spindle formation with DNA repair and morphological modifications to chromosomes prior to their segregation to prevent cell division with damaged chromosomes. Here we uncover a novel and unexpected role for Aurora kinase in preventing the formation of spindles by Clb5-CDK (S-CDK during meiotic prophase I and when the DDR is active in budding yeast. This is critical since S-CDK is essential for replication during premeiotic S-phase as well as double-strand break induction that facilitates meiotic recombination and, ultimately, chromosome segregation. Furthermore, we find that depletion of Cdc5 polo kinase activity delays spindle formation in DDR-arrested cells and that ectopic expression of Cdc5 in prophase I enhances spindle formation, when Ipl1 is depleted. Our findings establish a new paradigm for Aurora kinase function in both negative and positive regulation of spindle dynamics.

  8. IL12A, MPHOSPH9/CDK2AP1 and RGS1 are novel multiple sclerosis susceptibility loci

    DEFF Research Database (Denmark)

    Sørensen, Per Soelberg

    2010-01-01

    and the same direction of effect observed in the discovery phase. Three loci exceeded genome-wide significance in the joint analysis: RGS1 (P value=3.55 x 10(-9)), IL12A (P=3.08 x 10(-8)) and MPHOSPH9/CDK2AP1 (P=3.96 x 10(-8)). The RGS1 risk allele is shared with celiac disease (CD), and the IL12A risk allele...... seems to be protective for celiac disease. Within the MPHOSPH9/CDK2AP1 locus, the risk allele correlates with diminished RNA expression of the cell cycle regulator CDK2AP1; this effect is seen in both lymphoblastic cell lines (P=1.18 x 10(-5)) and in peripheral blood mononuclear cells from subjects...

  9. Arecoline-induced phosphorylated p53 and p21(WAF1) protein expression is dependent on ATM/ATR and phosphatidylinositol-3-kinase in clone-9 cells.

    Science.gov (United States)

    Chou, Wen-Wen; Guh, Jinn-Yuh; Tsai, Jung-Fa; Hwang, Chi-Ching; Chiou, Shean-Jaw; Chuang, Lea-Yea

    2009-06-01

    Betel-quid use is associated with liver cancer whereas its constituent arecoline is cytotoxic, genotoxic, and induces p53-dependent p21(WAF1) protein expression in Clone-9 cells (rat hepatocytes). The ataxia telangiectasia mutated (ATM)/rad3-related (ATR)-p53-p21(WAF1) and the phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathways are involved in the DNA damage response and the pathogenesis of cancers. Thus, we studied the role of ATM/ATR and PI3K in arecoline-induced p53 and p21(WAF1) protein expression in Clone-9 cells. We found that arecoline (0.5 mM) activated the ATM/ATR kinase at 30 min. The arecoline-activated ATM/ATR substrate contained p-p53Ser15. Moreover, arecoline only increased the levels of the p-p53Ser6, p-p53Ser15, and p-p53Ser392 phosphorylated p53 isoforms among the known isoforms. ATM shRNA attenuated arecoline-induced p-p53Ser15 and p21(WAF1) at 24 h. Arecoline (0.5 mM) increased phosphorylation levels of p-AktSer473 and p-mTORSer2448 at 30-60 min. Dominant-negative PI3K plasmids attenuated arecoline-induced p21(WAF1), but not p-p53Ser15, at 24 h. Rapamycin attenuated arecoline-induced phosphrylated p-p53Ser15, but not p21(WAF1), at 24 h. ATM shRNA, but not dominant-negative PI3K plasmids, attenuated arecoline-induced p21(WAF1) gene transcription. We conclude that arecoline activates the ATM/ATR-p53-p21(WAF1) and the PI3K/Akt-mTOR-p53 pathways in Clone-9 cells. Arecoline-induced phosphorylated p-p53Ser15 expression is dependent on ATM whereas arecoline-induced p21(WAF1) protein expression is dependent on ATM and PI3K. Moreover, p21(WAF1) gene is transcriptionally induced by arecoline-activated ATM. (c) 2009 Wiley-Liss, Inc.

  10. Levels of p21WAF1/CIP1 do not affect radiation-induced cell death in human breast epithelial cells

    International Nuclear Information System (INIS)

    Kim, Harold E.; Han, Sue J.; Waid, David; Lee, Yong J.; Kim, Hyeong-Reh Choi

    1997-01-01

    Purpose/Objective: Loss of the wild-type p53 activity and/or overexpression of the proto-oncogene bcl-2 are frequently detected in breast cancer and suggested to be related to resistance to chemotherapy and radiation therapy. The long-term goals of this study are to identify the downstream signaling molecules for anti-proliferative and apoptotic activities of p53 and to investigate the interaction of bcl-2 with p53 in human breast epithelial cells. We previously showed that overexpression of bcl-2 downregulates radiation-induced expression of p21 WAF1/CIP1 , a p53 downstream molecule that functions to inhibit cyclin dependent kinases, and suppresses radiation-induced apoptosis in human breast epithelial cell line (MCF10A). In this study, we investigated the role of p21 WAF1/CIP1 in radiation-induced cell death in MCF10A cells. Materials and Methods: To determine whether downregulation of p21 WAF1/CIP1 is required for anti-apoptotic activity of bcl-2, and to investigate the roles of p21 WAF1/CIP1 in cell death following irradiation, we transfected p21 WAF1/CIP1 expression vector into bcl-2 overexpressing MCF10A cells. The effects of p21 WAF1/CIP1 overexpression on cell growth, radiation-induced apoptosis and clonogenic cell survival were analyzed. Results: Overexpression of p21 WAF1/CIP1 resulted in marked growth inhibition, but no effect on dose-dependent radiation-induced cell lethality as determined by clonogenic survival assay. Radiation-induced apoptosis was not detected in bcl-2 overexpressing MCF10A cells independent of levels of p21 WAF1/CIP1 expression. Conclusion: This study suggests that bcl-2 downregulation of p21 WAF1/CIP1 is independent of anti-apoptotic activity of bcl-2 and that levels of p21 WAF1/CIP1 do not affect radiation-induced cell death in human breast epithelial cells

  11. Cep169, a Novel Microtubule Plus-End-Tracking Centrosomal Protein, Binds to CDK5RAP2 and Regulates Microtubule Stability.

    Directory of Open Access Journals (Sweden)

    Yusuke Mori

    Full Text Available The centrosomal protein, CDK5RAP2, is a microcephaly protein that regulates centrosomal maturation by recruitment of a γ-tubulin ring complex (γ-TuRC onto centrosomes. In this report, we identified a novel human centrosomal protein, Cep169, as a binding partner of CDK5RAP2, a member of microtubule plus-end-tracking proteins (+TIPs. Cep169 interacts directly with CDK5RAP2 through CM1, an evolutionarily conserved domain, and colocalizes at the pericentriolar matrix (PCM around centrioles with CDK5RAP2. In addition, Cep169 interacts with EB1 through SxIP-motif responsible for EB1 binding, and colocalizes with CDK5RAP2 at the microtubule plus-end. EB1-binding-deficient Cep169 abolishes EB1 interaction and microtubule plus-end attachment, indicating Cep169 as a novel member of +TIPs. We further show that ectopic expression of either Cep169 or CDK5RAP2 induces microtubule bundling and acetylation in U2OS cells, and depletion of Cep169 induces microtubule depolymerization in HeLa cells, although Cep169 is not required for assembly of γ-tubulin onto centrosome by CDK5RAP2. These results show that Cep169 targets microtubule tips and regulates stability of microtubules with CDK5RAP2.

  12. A novel mutation in CDK5RAP2 gene causes primary microcephaly with speech impairment and sparse eyebrows in a consanguineous Pakistani family

    DEFF Research Database (Denmark)

    Abdullah, Uzma; Farooq, Muhammad; Mang, Yuan

    2017-01-01

    CDK5RAP2 gene encodes a centrosomal protein, highly expressed in fetal brain and essentially indispensable for its normal development, as biallelic mutations in it lead to primary microcephaly (MCPH). Despite being known as MCPH linked gene for more than a decade, the phenotypic spectrum of CDK5RAP...

  13. Melatonin Sensitizes H1975 Non-Small-Cell Lung Cancer Cells Harboring a T790M-Targeted Epidermal Growth Factor Receptor Mutation to the Tyrosine Kinase Inhibitor Gefitinib

    Directory of Open Access Journals (Sweden)

    Miyong Yun

    2014-08-01

    Full Text Available Background/Aims: The use of tyrosine kinase inhibitors (TKIs to target active epidermal growth factor receptor (EGFR-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC. However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. Methods: H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Results: Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Conclusions: Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation.

  14. Melatonin sensitizes H1975 non-small-cell lung cancer cells harboring a T790M-targeted epidermal growth factor receptor mutation to the tyrosine kinase inhibitor gefitinib.

    Science.gov (United States)

    Yun, Miyong; Kim, Eun-Ok; Lee, Duckgue; Kim, Ji-Hyun; Kim, Jaekwang; Lee, Hyemin; Lee, Jihyun; Kim, Sung-Hoon

    2014-01-01

    The use of tyrosine kinase inhibitors (TKIs) to target active epidermal growth factor receptor (EGFR)-harbouring mutations has been effective in patients with advanced non-small-cell lung cancer (NSCLC). However, the use of TKIs in NSCLS patients with somatic EGFR mutations, particularly T790M, causes drug resistance. Thus, in the present study, we investigated overcoming resistance against the TKI gefitinib by combination treatment with melatonin in H1975 NSCLC cells harbouring the T790M somatic mutation. H1975 and HCC827 cells were treated with melatonin in combination with gefitinib, and cell viability, cell cycle progression, apoptosis, and EGFR, AKT, p38, Bcl-2, Bcl-xL, caspase 3 and Bad protein levels were examined. Treatment with melatonin dose-dependently decreased the viability of H1975 cells harbouring the T790M somatic mutation compared to HCC827 cells with an EGFR active mutation. Melatonin-mediated cell death resulted in decreased phosphorylation of EGFR and Akt, leading to attenuated expression of survival proteins, such as Bcl-2, Bcl-xL and survivin, and activated caspase 3 in H1975 cells, but not in HCC827 cells. However, we did not observe a significant change in expression of cell cycle proteins, such as cyclin D, cyclin A, p21 and CDK4 in H1975 cells. Surprisingly, co-treatment of gefitinib with melatonin effectively decreased the viability of H1975 cells, but not HCC827 cells. Moreover, co-treatment of H1975 cells caused consistent down-regulation of EGFR phosphorylation and induced apoptosis compared to treatment with gefitinib or melatonin alone. Our findings demonstrate that melatonin acts as a potent chemotherapeutic agent by sensitising to gefitinib TKI-resistant H1975 cells that harbour a EGFR T790M mutation. © 2014 S. Karger AG, Basel.

  15. NuMA phosphorylation by CDK1 couples mitotic progression with cortical dynein function.

    Science.gov (United States)

    Kotak, Sachin; Busso, Coralie; Gönczy, Pierre

    2013-09-11

    Spindle positioning and spindle elongation are critical for proper cell division. In human cells, an evolutionary conserved ternary complex (NuMA/LGN/Gαi) anchors dynein at the cortex during metaphase, thus ensuring correct spindle positioning. Whether this complex contributes to anaphase spindle elongation is not known. More generally, the mechanisms coupling mitotic progression with spindle behaviour remain elusive. Here, we uncover that levels of cortical dynein markedly increase during anaphase in a NuMA-dependent manner. We demonstrate that during metaphase, CDK1-mediated phosphorylation at T2055 negatively regulates NuMA cortical localization and that this phosphorylation is counteracted by PPP2CA phosphatase activity. We establish that this tug of war is essential for proper levels of cortical dynein and thus spindle positioning during metaphase. Moreover, we find that upon CDK1 inactivation in anaphase, the rise in dephosphorylated NuMA at the cell cortex leads to cortical dynein enrichment, and thus to robust spindle elongation. Our findings uncover a mechanism whereby the status of NuMA phosphorylation coordinates mitotic progression with proper spindle function.

  16. Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury

    Energy Technology Data Exchange (ETDEWEB)

    Gu, Jun [Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu (China); Department of Orthopaedics, Xishan People' s Hospital, Wuxi, Jiangsu (China); Ni, Yingjie; Xu, Lin; Xu, Hongliang [Department of Orthopaedics, Xishan People' s Hospital, Wuxi, Jiangsu (China); Cai, Zhengdong, E-mail: caizhengdongsh@163.com [Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu (China)

    2016-01-22

    Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in the spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.

  17. Nanog interact with CDK6 to regulates astrocyte cells proliferation following spinal cord injury

    International Nuclear Information System (INIS)

    Gu, Jun; Ni, Yingjie; Xu, Lin; Xu, Hongliang; Cai, Zhengdong

    2016-01-01

    Previous research had reported transcription factors Nanog expressed in pluripotent embryonic stem cells (ESCS) that played an important role in regulating the cell proliferation. Nanog levels are frequently elevated in ESCS, but the role in the spinal cord was not clear. To examine the biological relevance of Nanog, we studied its properties in spinal cord injury model. The expression of Nanog and PCNA was gradually increased and reached a peak at 3 day by western blot analysis. The expression of Nanog was further analyzed by immunohistochemistry. Double immunofluorescent staining uncovered that Nanog can co-labeled with PCNA and GFAP in the spinal cord tissue. In vitro, Nanog can promote the proliferation of astrocyte cell by Fluorescence Activating Cell Sorter (FACS) and CCK8. Meanwhile, the cell-cycle protein CDK6 could interact with Nanog in the spinal cord tissue. Taken together, these data suggested that both Nanog may play important roles in spinal cord pathophysiology via interact with CDK6.

  18. Cellular response to antitumor cis-dichlorido platinum(II) complexes of CDK inhibitor bohemine and its analogues

    Czech Academy of Sciences Publication Activity Database

    Lišková, Barbora; Zerzánková, Lenka; Nováková, Olga; Kostrhunová, Hana; Trávníček, Z.; Brabec, Viktor

    2012-01-01

    Roč. 25, č. 2 (2012), s. 500-509 ISSN 0893-228X R&D Projects: GA ČR(CZ) GD301/09/H004; GA ČR(CZ) GAP301/10/0598 Keywords : cisplatin * DNA * bohemine Subject RIV: BO - Biophysics Impact factor: 3.667, year: 2012

  19. Conformation and recognition of DNA damaged by antitumor cis-dichlorido platinum(II) complex of CDK inhibitor bohemine

    Czech Academy of Sciences Publication Activity Database

    Nováková, Olga; Lišková, Barbora; Vystrčilová, Jana; Suchánková, Tereza; Vrána, Oldřich; Starha, P.; Trávníček, Z.; Brabec, Viktor

    2014-01-01

    Roč. 78, MAY2014 (2014), s. 54-64 ISSN 0223-5234 R&D Projects: GA ČR(CZ) GA13-08273S Institutional support: RVO:68081707 Keywords : Antitumor * Platinum * DNA Subject RIV: BO - Biophysics Impact factor: 3.447, year: 2014

  20. Association Study Between Coronary Artery Disease and rs1333049 Polymorphism at 9p21.3 Locus in Italian Population.

    Science.gov (United States)

    Pignataro, Piero; Pezone, Lucia; Di Gioia, Giuseppe; Franco, Danilo; Iaccarino, Guido; Iolascon, Achille; Ciccarelli, Michele; Capasso, Mario

    2017-12-01

    In this study, we verify the association between the rs1333049 single nucleotide polymorphism (9p21.3) within CDKN2A-CDKN2B and coronary artery disease (CAD) in an Italian population. We replicated rs1333049_G allele association with a significantly reduced risk of CAD (OR = 0.816; 95% confidence interval [0.705-0.945]; p = 0.0065) in 711 CAD patients and 755 normal healthy individuals. This effect is maintained even stratifying patients by gender and by risk factors. A significant association was found with age of CAD onset. Interestingly, we found a protective trend of association between the rs1333049_G allele and peripheral artery disease, a progressive atherosclerotic condition in which plaque builds up in the arteries that carry blood to the head, organs, and limbs (OR = 0.724; 95% CI [0.520-1.007]; p = 0.054). No genotype-phenotype association was found with more severe CAD clinical parameters. If certain genetic factors predispose individuals to adverse outcomes, the knowledge of a patient's genotype may influence clinical management.

  1. PAX8 activates a p53-p21-dependent pro-proliferative effect in high grade serous ovarian carcinoma.

    Science.gov (United States)

    Ghannam-Shahbari, Dima; Jacob, Eyal; Kakun, Reli Rachel; Wasserman, Tanya; Korsensky, Lina; Sternfeld, Ofir; Kagan, Juliana; Bublik, Debora Rosa; Aviel-Ronen, Sarit; Levanon, Keren; Sabo, Edmond; Larisch, Sarit; Oren, Moshe; Hershkovitz, Dov; Perets, Ruth

    2018-01-30

    High grade serous carcinoma (HGSC) is the most common subtype of ovarian cancer and it is now widely accepted that this disease often originates from the fallopian tube epithelium. PAX8 is a fallopian tube lineage marker with an essential role in embryonal female genital tract development. In the adult fallopian tube, PAX8 is expressed in the fallopian tube secretory epithelial cell (FTSEC) and its expression is maintained through the process of FTSEC transformation to HGSC. We now report that PAX8 has a pro-proliferative and anti-apoptotic role in HGSC. The tumor suppressor gene TP53 is mutated in close to 100% of HGSC; in the majority of cases, these are missense mutations that endow the mutant p53 protein with potential gain of function (GOF) oncogenic activities. We show that PAX8 positively regulates the expression of TP53 in HGSC and the pro-proliferative role of PAX8 is mediated by the GOF activity of mutant p53. Surprisingly, mutant p53 transcriptionally activates the expression of p21, which localizes to the cytoplasm of HGSC cells where it plays a non-canonical, pro-proliferative role. Together, our findings illustrate how TP53 mutations in HGSC subvert a normal regulatory pathway into a driver of tumor progression.

  2. A Postsynaptic AMPK→p21-Activated Kinase Pathway Drives Fasting-Induced Synaptic Plasticity in AgRP Neurons.

    Science.gov (United States)

    Kong, Dong; Dagon, Yossi; Campbell, John N; Guo, Yikun; Yang, Zongfang; Yi, Xinchi; Aryal, Pratik; Wellenstein, Kerry; Kahn, Barbara B; Sabatini, Bernardo L; Lowell, Bradford B

    2016-07-06

    AMP-activated protein kinase (AMPK) plays an important role in regulating food intake. The downstream AMPK substrates and neurobiological mechanisms responsible for this, however, are ill defined. Agouti-related peptide (AgRP)-expressing neurons in the arcuate nucleus regulate hunger. Their firing increases with fasting, and once engaged they cause feeding. AgRP neuron activity is regulated by state-dependent synaptic plasticity: fasting increases dendritic spines and excitatory synaptic activity; feeding does the opposite. The signaling mechanisms underlying this, however, are also unknown. Using neuron-specific approaches to measure and manipulate kinase activity specifically within AgRP neurons, we establish that fasting increases AMPK activity in AgRP neurons, that increased AMPK activity in AgRP neurons is both necessary and sufficient for fasting-induced spinogenesis and excitatory synaptic activity, and that the AMPK phosphorylation target mediating this plasticity is p21-activated kinase. This provides a signaling and neurobiological basis for both AMPK regulation of energy balance and AgRP neuron state-dependent plasticity. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. p21-activated kinase has substrate specificity similar to Acanthamoeba myosin I heavy chain kinase and activates Acanthamoeba myosin I.

    Science.gov (United States)

    Brzeska, H; Knaus, U G; Wang, Z Y; Bokoch, G M; Korn, E D

    1997-02-18

    Acanthamoeba class I myosins are unconventional, single-headed myosins that express actin-activated Mg2+-ATPase and in vitro motility activities only when a single serine or threonine in the heavy chain is phosphorylated by myosin I heavy chain kinase (MIHCK). Some other, but not most, class I myosins have the same consensus phosphorylation site sequence, and the two known class VI myosins have a phosphorylatable residue in the homologous position, where most myosins have an aspartate or glutamate residue. Recently, we found that the catalytic domain of Acanthamoeba MIHCK has extensive sequence similarity to the p21-activated kinase (PAK)/STE20 family of kinases from mammals and yeast, which are activated by small GTP-binding proteins. The physiological substrates of the PAK/STE20 kinases are not well characterized. In this paper we show that PAK1 has similar substrate specificity as MIHCK when assayed against synthetic substrates and that PAK1 phosphorylates the heavy chain (1 mol of P(i) per mol) and activates Acanthamoeba myosin I as MIHCK does. These results, together with the known involvement of Acanthamoeba myosin I, yeast myosin I, STE20, PAK, and small GTP-binding proteins in membrane- and cytoskeleton-associated morphogenetic transformations and activities, suggest that myosins may be physiological substrates for the PAK/STE20 family and thus mediators of these events.

  4. Hypersensitivity of primordial germ cells to compromised replication-associated DNA repair involves ATM-p53-p21 signaling.

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    Yunhai Luo

    2014-07-01

    Full Text Available Genome maintenance in germ cells is critical for fertility and the stable propagation of species. While mechanisms of meiotic DNA repair and chromosome behavior are well-characterized, the same is not true for primordial germ cells (PGCs, which arise and propagate during very early stages of mammalian development. Fanconi anemia (FA, a genomic instability syndrome that includes hypogonadism and testicular failure phenotypes, is caused by mutations in genes encoding a complex of proteins involved in repair of DNA lesions associated with DNA replication. The signaling mechanisms underlying hypogonadism and testicular failure in FA patients or mouse models are unknown. We conducted genetic studies to show that hypogonadism of Fancm mutant mice is a result of reduced proliferation, but not apoptosis, of PGCs, resulting in reduced germ cells in neonates of both sexes. Progressive loss of germ cells in adult males also occurs, overlaid with an elevated level of meiotic DNA damage. Genetic studies indicated that ATM-p53-p21 signaling is partially responsible for the germ cell deficiency.

  5. Review of renal carcinoma with t(6;11)(p21;q12) with focus on clinical and pathobiological aspects.

    Science.gov (United States)

    Kuroda, Naoto; Tanaka, Azusa; Sasaki, Naomi; Ishihara, Akira; Matsuura, Keiko; Moriyama, Masatsugu; Nagashima, Yoji; Inoue, Keiji; Petersson, Fredrik; Martignoni, Guido; Michal, Michal; Hes, Ondrej

    2013-06-01

    Recently, a new category of MiTF/TFE family translocation carcinomas of the kidney has been proposed. This category includes Xp11.2 renal cell carcinoma (RCC) and the t(6;11) RCC. These tumors share clinical, morphological, immunohistochemical and molecular genetic features. In this article, we review t(6;11) RCC. This tumor predominantly affects children and young adults. Macroscopically, the tumor generally forms a well circumscribed mass. Satellite nodules may be observed. Histologically, the tumor comprises large cells and small cells surrounded by basement membrane material. Immunohistochemically, tumor cells show nuclear immunolabeling for TFEB and usually express Cathepsin-K in the cytoplasm. Karyotyping detects the rearrangement between chromosome 6p21 and chromosome 11q12. Alpha-TFEB fusion can be detected by reverse transcriptase polymerase chain reaction (RT-PCR) or fluorescence in situ hybridization (FISH). Most cases affecting children and young adults seem to be indolent, but some adult cases have presented with metastasis or caused death. As previously reported cases remain limited to date, further examination in a large scale study will be needed in order to elucidate clinical behavior and molecular characteristics.

  6. Cloning and characterization of shk2, a gene encoding a novel p21-activated protein kinase from fission yeast.

    Science.gov (United States)

    Yang, P; Kansra, S; Pimental, R A; Gilbreth, M; Marcus, S

    1998-07-17

    We describe the characterization of a novel gene, shk2, encoding a second p21(cdc42/rac)-activated protein kinase (PAK) homolog in fission yeast. Like other known PAKs, Shk2 binds to Cdc42 in vivo and in vitro. While overexpression of either shk2 or cdc42 alone does not impair growth of wild type fission yeast cells, cooverexpression of the two genes is toxic and leads to highly aberrant cell morphology, providing evidence for functional interaction between Cdc42 and Shk2 proteins in vivo. Fission yeast shk2 null mutants are viable and exhibit no obvious phenotypic defects. Overexpression of shk2 restores viability and normal morphology but not full mating competence to fission yeast cells carrying a shk1 null mutation. Additional genetic data suggest that Shk2, like Cdc42 and Shk1, participates in Ras-dependent morphological control and mating response pathways in fission yeast. We also show that overexpression of byr2, a gene encoding a Ste11/MAPK kinase kinase homolog, suppresses the mating defect of cells partially defective for Shk1 function, providing evidence of a link between PAKs and mitogen-activated protein kinase signaling in fission yeast. Taken together, our results suggest that Shk2 is partially overlapping in function with Shk1, with Shk1 being the dominant protein in function.

  7. LncRNA-SNHG16 predicts poor prognosis and promotes tumor proliferation through epigenetically silencing p21 in bladder cancer.

    Science.gov (United States)

    Cao, Xianxiang; Xu, Jing; Yue, Dong

    2018-02-01

    More and more evidences have ensured the crucial functions of long non-coding RNAs (lncRNAs) in multiple tumors. It has been discovered that lncRNA-SNHG16 is involved in many tumors. Even so, it is still necessary to study SNHG16 comprehensively in bladder cancer. In terms of our study, the level of SNHG16 both in the tumor tissues and cell lines was measured and the relationship among SNHG16, clinicopathological traits and prognosis was explored. Interference assays were applied to determine the biological functions of SNHG16. It was discovered that the level of SNHG16 was evidently enhanced both in tissues and cell lines of bladder cancer. Patients with highly expressed SNHG16 suffered from poor overall survival. Multivariable Cox proportional hazards regression analysis implied that highly expressed SNHG16 could be used as an independent prognostic marker. It could be known from functional assays that silenced SNHG16 impaired cell proliferation, owing to the effects of SNHG16 on cell cycle and apoptosis. Finally, mechanism experiments revealed that SNHG16 could epigenetically silence the expression of p21. The facts above pointed out that lncRNA-SNHG16 might be quite vital for the diagnosis and development of bladder cancer, and could even become an important therapeutic target for bladder cancer.

  8. P21-activated kinase 7 mediates cisplatin-resistance of esophageal squamous carcinoma cells with Aurora-A overexpression.

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    Shun He

    Full Text Available Aurora-A overexpression is common in various types of cancers and has been shown to be involved in tumorigenesis through different signaling pathways, yet how the deregulation affects cancer therapeutics remains elusive. Here we showed that overexpression of Aurora-A rendered esophageal cancer cells resistance to cisplatin (CDDP by inhibiting apoptosis. By using an apoptosis array, we identified a downstream gene, p21-activated kinase 7 (PAK7. PAK7 was upregulated by Aurora-A overexpression at both mRNA and protein levels. Importantly, the expression levels of Aurora-A and PAK7 were correlated in ESCC primary samples. Chromatin immunoprecipitation (ChIP assay revealed that binding of E2F1 to the promoter of PAK7 was significantly enhanced upon Aurora-A activation, and knockdown of transcription factor E2F1 decreased PAK7 expression, suggesting that Aurora-A regulated PAK7 through E2F1. Furthermore, we demonstrated that PAK7 knockdown led to increased apoptosis, and Aurora-A-induced resistance to CDDP was reversed by downregulation of PAK7, suggesting PAK7 was a downstream player of Aurora-A that mediated chemoresistance of ESCC cells to CDDP. Our data suggest that PAK7 may serve as an attractive candidate for therapeutics in ESCC patients with Aurora-A abnormality.

  9. Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis in Mice via a Toll-Like Receptor 4/p21-Activated Kinase 1 Cascade.

    Science.gov (United States)

    Wu, Yaxin; Wu, Jiao; Chen, Ting; Li, Qing; Peng, Wei; Li, Huan; Tang, Xiaowei; Fu, Xiangsheng

    2018-03-05

    The underlying pathogenic mechanism of Fusobacterium nucleatum in the carcinogenesis of colorectal cancer has been poorly understood. Using C57BL/6-Apc Min/+ mice, we investigated gut microbial structures with F. nucleatum, antibiotics, and Toll-like receptor 4 (TLR4) antagonist TAK-242 treatment. In addition, we measured intestinal tumor formation and the expression of TLR4, p21-activated kinase 1 (PAK1), phosphorylated-PAK1 (p-PAK1), phosphorylated-β-catenin S675 (p-β-catenin S675), and cyclin D1 in mice with different treatments. Fusobacterium nucleatum and antibiotics treatment altered gut microbial structures in mice. In addition, F. nucleatum invaded into the intestinal mucosa in large amounts but were less abundant in the feces of F. nucleatum-fed mice. The average number and size of intestinal tumors in F. nucleatum groups was significantly increased compared to control groups in Apc Min/+ mice (P nucleatum groups compared to the control groups (P nucleatum groups (P nucleatum groups (P Fusobacterium nucleatum potentiates intestinal tumorigenesis in Apc Min/+ mice via a TLR4/p-PAK1/p-β-catenin S675 cascade. Fusobacterium nucleatum-induced intestinal tumorigenesis can be inhibited by TAK-242, implicating TLR4 as a potential target for the prevention and therapy of F. nucleatum-related colorectal cancer.

  10. P21 (Cdc42/Rac)-activated kinase 1 (pak1) is associated with cardiotoxicity induced by antihistamines.

    Science.gov (United States)

    Yun, Jaesuk; Kim, So Young; Yoon, Kyung Sik; Shin, Heejung; Jeong, Ho-Sang; Chung, Hyejoo; Kim, Young-Hoon; Shin, Jisoon; Cha, Hye Jin; Han, Kyoung Moon; Hyeon, Seungha; Lee, Tac-Hyung; Park, Hye-Kyung; Kim, Hyung Soo

    2016-12-01

    Astemizole, a non-sedating histamine H 1 receptor blocker, is widely known to cause cardiac arrhythmia, which prolongs the QT interval. However, the precise molecular mechanism involved in antihistamine-induced cardiovascular adverse effects other than hERG channel inhibition is still unclear. In this study, we used DNA microarray analysis to detect the mechanisms involved in life-threatening adverse effects caused by astemizole. Rat primary cardiomyocytes were treated with various concentrations of astemizole for 24 h and the corresponding cell lysates were analyzed using a DNA microarray. Astemizole altered the expression profiles of genes involved in calcium transport/signaling. Using qRT-PCR analysis, we demonstrated that, among those genes, p21 (Cdc42/Rac)-activated kinase 1 (pak1) mRNA was downregulated by treatment with terfenadine and astemizole. Astemizole also reduced pak1 protein levels in rat cardiomyocytes. In addition, astemizole decreased pak1 mRNA and protein levels in H9c2 cells and induced an increase in cell surface area (hypertrophy) and cytotoxicity. Fingolimod hydrochloride (FTY720), a pak1 activator, inhibited astemizole-induced hypertrophy and cytotoxicity in H9c2 cells. These results suggest that antihistamine-induced cardiac adverse effects are associated with pak1 expression and function.

  11. The microbe-derived short chain fatty acid butyrate targets miRNA-dependent p21 gene expression in human colon cancer.

    Directory of Open Access Journals (Sweden)

    Shien Hu

    Full Text Available Colonic microbiota ferment non-absorbed dietary fiber to produce prodigious amounts of short chain fatty acids (SCFAs that benefit the host through a myriad of metabolic, trophic, and chemopreventative effects. The chemopreventative effects of the SCFA butyrate are, in part, mediated through induction of p21 gene expression. In this study, we assessed the role of microRNA(miRNA in butyrate's induction of p21 expression. The expression profiles of miRNAs in HCT-116 cells and in human sporadic colon cancers were assessed by microarray and quantitative PCR. Regulation of p21 gene expression by miR-106b was assessed by 3' UTR luciferase reporter assays and transfection of specific miRNA mimics. Butyrate changed the expression of 44 miRNAs in HCT-116 cells, many of which were aberrantly expressed in colon cancer tissues. Members of the miR-106b family were decreased in the former and increased in the latter. Butyrate-induced p21 protein expression was dampened by treatment with a miR-106b mimic. Mutated p21 3'UTR-reporter constructs expressed in HCT-116 cells confirmed direct miR-106b targeting. Butyrate decreased HCT-116 proliferation, an effect reversed with the addition of the miR-106b mimic. We conclude that microbe-derived SCFAs regulate host gene expression involved in intestinal homeostasis as well as carcinogenesis through modulation of miRNAs.

  12. Expression Profile of p53 and p21 in Large Bowel Mucosa as Biomarkers of Inflammatory-Related Carcinogenesis in Ulcerative Colitis

    Directory of Open Access Journals (Sweden)

    Cristiana Popp

    2016-01-01

    Full Text Available Ulcerative colitis (UC is a chronic, relapsing inflammatory bowel disease that slightly increases the risk of colorectal cancer in patients with long-standing extended disease. Overexpression of p53 and p21 in colonic epithelia is usually detected in UC patients when no dysplasia is histologically seen and it is used by pathologists as a discriminator between regenerative changes and intraepithelial neoplasia, as well as a tissue biomarker useful to predict the risk of evolution toward malignancy. We present a one-year prospective observational study including a cohort of 45 patients with UC; p53 and p21 were evaluated in epithelial cells. p53 was positive in 74 samples revealed in 5% to 90% of epithelial cells, while 63 biopsies had strong positivity for p21 in 5% to 50% of epithelial cells. Architectural distortion was significantly correlated with p53 overexpression in epithelial cells. Thus, we consider that architectural distortion is a good substitute for p53 and p21 expression. We recommend use of p53 as the most valuable tissue biomarker in surveillance of UC patients, identifying the patients with higher risk for dysplasia. Association of p21 is also recommended for a better quantification of risk and for diminishing the false-negative results.

  13. A cell-based, high content screening assay reveals activators and inhibitors of cancer cell invasion

    Science.gov (United States)

    Quintavalle, Manuela; Elia, Leonardo; Price, Jeffrey H.; Heynen-Genel, Susanne; Courtneidge, Sara A.

    2012-01-01

    Acquisition of invasive cell behavior underlies tumor progression and metastasis. To define in more molecular detail the mechanisms underlying invasive behavior, we developed a high throughput screening strategy to quantitate invadopodia; actin-rich membrane protrusions of cancer cells which contribute to tissue invasion and matrix remodeling. We developed a high content, imaged-based assay, and tested the LOPAC 1280 collection of pharmacologically active agents. We found compounds that potently inhibited invadopodia formation without overt toxicity, as well as compounds that increased invadopodia number. One of the two compounds that increased both invadopodia number and invasive behavior was the chemotherapeutic agent paclitaxel, which has potential clinical implications for its use in the neoadjuvant and resistance settings. Several of the invasion inhibitors were annotated as cyclin-dependent kinase (cdk) inhibitors. Loss-of-function experiments determined that Cdk5 was the relevant target. We further determined that the mechanism by which Cdk5 promotes both invadopodia formation and cancer invasion is by phosphorylation and down regulation of the actin regulatory protein caldesmon. PMID:21791703

  14. CYCLIN-DEPENDENT KINASE INHIBITOR, PALBOCICLIB – A NEW DRUG FOR THE TREATMENT OF METASTATIC BREAST CANCER

    Directory of Open Access Journals (Sweden)

    E. N. Imyanitov

    2017-01-01

    Full Text Available The sequential use of several lines of endocrine therapy is considered the standard for the treatment of metastatic breast cancer, expressing estrogen or progesterone receptors. PALOMA-1, -2 and -3 studies showed that the combination of the inhibitor of CDK4/6, palbociclib, with endocrine therapy significantly increases the time to progression compared to the use of monotherapy with antagonists of the estrogen signaling cascade.

  15. High throughput screens yield small molecule inhibitors of Leishmania CRK3:CYC6 cyclin-dependent kinase.

    Directory of Open Access Journals (Sweden)

    Roderick G Walker

    2011-04-01

    Full Text Available Leishmania species are parasitic protozoa that have a tightly controlled cell cycle, regulated by cyclin-dependent kinases (CDKs. Cdc2-related kinase 3 (CRK3, an essential CDK in Leishmania and functional orthologue of human CDK1, can form an active protein kinase complex with Leishmania cyclins CYCA and CYC6. Here we describe the identification and synthesis of specific small molecule inhibitors of bacterially expressed Leishmania CRK3:CYC6 using a high throughput screening assay and iterative chemistry. We also describe the biological activity of the molecules against Leishmania parasites.In order to obtain an active Leishmania CRK3:CYC6 protein kinase complex, we developed a co-expression and co-purification system for Leishmania CRK3 and CYC6 proteins. This active enzyme was used in a high throughput screening (HTS platform, utilising an IMAP fluorescence polarisation assay. We carried out two chemical library screens and identified specific inhibitors of CRK3:CYC6 that were inactive against the human cyclin-dependent kinase CDK2:CycA. Subsequently, the best inhibitors were tested against 11 other mammalian protein kinases. Twelve of the most potent hits had an azapurine core with structure activity relationship (SAR analysis identifying the functional groups on the 2 and 9 positions as essential for CRK3:CYC6 inhibition and specificity against CDK2:CycA. Iterative chemistry allowed synthesis of a number of azapurine derivatives with one, compound 17, demonstrating anti-parasitic activity against both promastigote and amastigote forms of L. major. Following the second HTS, 11 compounds with a thiazole core (active towards CRK3:CYC6 and inactive against CDK2:CycA were tested. Ten of these hits demonstrated anti-parasitic activity against promastigote L. major.The pharmacophores identified from the high throughput screens, and the derivatives synthesised, selectively target the parasite enzyme and represent compounds for future hit

  16. Two Blood Monocytic Biomarkers (CCL15 and p21 Combined with the Mini-Mental State Examination Discriminate Alzheimer’s Disease Patients from Healthy Subjects

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    Tanja Hochstrasser

    2011-10-01

    Full Text Available Background: Alzheimer’s disease (AD is a progressive neurodegenerative disorder. In AD, monocytes migrate across the blood-brain barrier and differentiate into microglia, are linked to inflammatory responses and display age-dependent decreases in telomere lengths. Methods: Six monocyte-specific chemokines and the (telomere-associated tumor suppressor proteins p53 and p21 were determined by multiplex immunoassay in plasma and monocyte extracts of patients with AD or mild cognitive impairment, and levels were compared between patients and controls (without cognitive impairment. Results: CCL15 (macrophage inflammatory protein-1δ, CXCL9 (monokine-induced by interferon-γ and p21 levels were decreased in monocytes of AD patients compared with controls. Conclusion: The combination of monocytic CCL15 and p21 together with the Mini-Mental State Examination enables to differentiate AD patients from controls with high specificity and sensitivity.

  17. Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G0/G1 phase and differentiation into monocyte.

    Science.gov (United States)

    Lee, Kyung-Won; Kim, Hyoung Ja; Lee, Yong Sup; Park, Hee-Jun; Choi, Jong-Won; Ha, Joohun; Lee, Kyung-Tae

    2007-09-01

    We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC50, approximately 30 microM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G1 phase in HL-60 human promyelocytic leukemia cells. Among the G1 phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21(CIP1/WAF1) and p27(KIP1), were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21(CIP1/WAF1) and p27(KIP1) to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G1 arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21(CIP1/WAF1) and p27(KIP1) levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.

  18. MicroRNA-490-3P targets CDK1 and inhibits ovarian epithelial carcinoma tumorigenesis and progression.

    Science.gov (United States)

    Chen, Shuo; Chen, Xi; Xiu, Yin-Ling; Sun, Kai-Xuan; Zhao, Yang

    2015-06-28

    The expression of microRNA-490-3P has been reported to regulate hepatocellular carcinoma cell proliferation, migration and invasion, and its overexpression significantly inhibits A549 lung cancer cell proliferation. Here, we demonstrated for the first time that miR-490 mRNA expression was significantly lower in ovarian carcinoma and borderline tumors compared to benign tumors, and lower in metastatic ovarian carcinoma (omentum) than primary ovarian carcinoma, and was negatively associated with differentiation and International Federation of Gynecology and Obstetrics (FIGO) staging. MiR-490-3P overexpression promoted G1/S or G2/M arrest and apoptosis; reduced cell proliferation, migration and invasion; reduced CDK1, Bcl-xL, MMP2/9, CCND1, SMARCD1 mRNA or protein expression; and induced P53 expression. Dual-luciferase reporter assay indicated miR-490-3P directly targeted CDK1. In vivo studies showed that miR-490-3P transfection suppressed tumor development and CDK1, Bcl-xL, MMP2/9 expression while inducing P53 expression. These findings indicate that miR-490-3P may target CDK1 and inhibit ovarian epithelial carcinoma tumorigenesis and progression. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. Phosphorylation of CRMP2 by Cdk5 Regulates Dendritic Spine Development of Cortical Neuron in the Mouse Hippocampus

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    Xiaohua Jin

    2016-01-01

    Full Text Available Proper density and morphology of dendritic spines are important for higher brain functions such as learning and memory. However, our knowledge about molecular mechanisms that regulate the development and maintenance of dendritic spines is limited. We recently reported that cyclin-dependent kinase 5 (Cdk5 is required for the development and maintenance of dendritic spines of cortical neurons in the mouse brain. Previous in vitro studies have suggested the involvement of Cdk5 substrates in the formation of dendritic spines; however, their role in spine development has not been tested in vivo. Here, we demonstrate that Cdk5 phosphorylates collapsin response mediator protein 2 (CRMP2 in the dendritic spines of cultured hippocampal neurons and in vivo in the mouse brain. When we eliminated CRMP2 phosphorylation in CRMP2KI/KI mice, the densities of dendritic spines significantly decreased in hippocampal CA1 pyramidal neurons in the mouse brain. These results indicate that phosphorylation of CRMP2 by Cdk5 is important for dendritic spine development in cortical neurons in the mouse hippocampus.

  20. Therapeutic rationale to target highly expressed CDK7 conferring poor outcomes in triple-negative breast cancer

    NARCIS (Netherlands)

    Li, Bo; Chonghaile, Triona Ni; Fan, Yue; Madden, Stephen F.; Klinger, Rut; O'Connor, Aisling E.; Walsh, Louise; O'Hurley, Gillian; Udupi, Girish Mallya; Joseph, Jesuchristopher; Tarrant, Finbarr; Conroy, Emer; Gaber, Alexander; Chin, Suet-Feung; Bardwell, Helen A; Provenzano, Elena; Crown, John; Dubois, Thierry; Linn, Sabine; Jirstrom, Karin; Caldas, Carlos; O'Connor, Darran P; Gallagher, William M

    2017-01-01

    Triple-negative breast cancer (TNBC) patients commonly exhibit poor prognosis and high relapse after treatment, but there remains a lack of biomarkers and effective targeted therapies for this disease. Here, we report evidence highlighting the cell-cycle–related kinase CDK7 as a driver and candidate

  1. Tumors initiated by constitutive Cdk2 activation exhibit transforming growth factor beta resistance and acquire paracrine mitogenic stimulation during progression

    DEFF Research Database (Denmark)

    Corsino, P.; Davis, B.; Law, M.

    2007-01-01

    Cyclin D1/cyclin-dependent kinase 2 (Cdk2) complexes are present at high frequency in human breast cancer cell lines, but the significance of this observation is unknown. This report shows that expression of a cyclin D1-Cdk2 fusion protein under the control of the mouse mammary tumor virus (MMITV......) promoter results in mammary gland hyperplasia and fibrosis, and mammary tumors. Cell lines isolated from MMTV-cyclin D1-Cdk2 (MMTV-D1K2) tumors exhibit Rb and p130 hyperphosphorylation and up-regulation of the protein products of E2F-dependent genes. These results suggest that cyclin D1/Cdk2 complexes may...... mediate some of the transforming effects that result from cyclin D1 overexpression in human breast cancers. MMTV-DIK2 cancer cells express the hepatocyte growth factor (HGF) receptor, c-Met. MMTV-D1K2 cancer cells also secrete transforming growth factor beta (TGF beta), but are relatively resistant to TGF...

  2. The Chemistry Development Kit (CDK) v2.0: atom typing, depiction, molecular formulas, and substructure searching.

    Science.gov (United States)

    Willighagen, Egon L; Mayfield, John W; Alvarsson, Jonathan; Berg, Arvid; Carlsson, Lars; Jeliazkova, Nina; Kuhn, Stefan; Pluskal, Tomáš; Rojas-Chertó, Miquel; Spjuth, Ola; Torrance, Gilleain; Evelo, Chris T; Guha, Rajarshi; Steinbeck, Christoph

    2017-06-06

    The Chemistry Development Kit (CDK) is a widely used open source cheminformatics toolkit, providing data structures to represent chemical concepts along with methods to manipulate such structures and perform computations on them. The library implements a wide variety of cheminformatics algorithms ranging from chemical structure canonicalization to molecular descriptor calculations and pharmacophore perception. It is used in drug discovery, metabolomics, and toxicology. Over the last 10 years, the code base has grown significantly, however, resulting in many complex interdependencies among components and poor performance of many algorithms. We report improvements to the CDK v2.0 since the v1.2 release series, specifically addressing the increased functional complexity and poor performance. We first summarize the addition of new functionality, such atom typing and molecular formula handling, and improvement to existing functionality that has led to significantly better performance for substructure searching, molecular fingerprints, and rendering of molecules. Second, we outline how the CDK has evolved with respect to quality control and the approaches we have adopted to ensure stability, including a code review mechanism. This paper highlights our continued efforts to provide a community driven, open source cheminformatics library, and shows that such collaborative projects can thrive over extended periods of time, resulting in a high-quality and performant library. By taking advantage of community support and contributions, we show that an open source cheminformatics project can act as a peer reviewed publishing platform for scientific computing software. Graphical abstract CDK 2.0 provides new features and improved performance.

  3. Suppression of chondrosarcoma cells by 15-deoxy-Δ12,14-prostaglandin J2 is associated with altered expression of Bax/Bcl-xL and p21

    International Nuclear Information System (INIS)

    Shen, Zheng-Nan; Nishida, Keiichiro; Doi, Hideyuki; Oohashi, Toshitaka; Hirohata, Satoshi; Ozaki, Toshifumi; Yoshida, Aki; Ninomiya, Yoshifumi; Inoue, Hajime

    2005-01-01

    We previously reported that 15-deoxy-Δ 12,14 -prostaglandin J 2 (15d-PGJ 2 ), the most potent agonist for peroxisome proliferator-activated receptor γ (PPARγ), induces apoptosis of human chondrosarcoma cell line OUMS-27. The current study aimed to explore the mechanism of 15d-PGJ 2 -induced apoptosis and inhibition of cell proliferation in OUMS-27 cells. The preliminary results of cDNA microarray analysis showed the down-regulation of anti-apoptotic Bcl-xL and up-regulation of pro-apoptotic Bax in the process of 15d-PGJ 2 -induced apoptosis. These changes were further confirmed at mRNA and protein levels by RT-PCR and Western blot analysis, respectively. Among cyclin-dependent kinase inhibitors, p21 was induced and up-regulated by 15d-PGJ 2 , but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ 2 was partly, but not completely, blocked by PPARγ antagonist (GW9662) suggesting the 15d-PGJ 2 exerted its effect by PPARγ-dependent and -independent pathways. Interestingly, immunohistochemical study on human chondrosarcoma samples revealed that Bcl-xL is frequently expressed by tumor cells. The results of the current study suggest that the potential ability of 15d-PGJ 2 in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma

  4. Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation.

    Science.gov (United States)

    Sørensen, Belinda Halling; Nielsen, Dorthe; Thorsteinsdottir, Unnur Arna; Hoffmann, Else Kay; Lambert, Ian Henry

    2016-06-01

    The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of