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Sample records for cdk inhibitor p21

  1. The functions of the cdk-cyclin kinase inhibitor p21WAF1

    International Nuclear Information System (INIS)

    p21WAF1 plays a critical role in regulating cell growth and the cell response to DNA damage. The primary targets of p21WAF1 (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 δ. 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)

  2. CDK inhibitors, p21Cip1 and p27Kip1, participate in cell cycle exit of mammalian cardiomyocytes

    International Nuclear Information System (INIS)

    Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21Cip1 and p27Kip1 but not p57Kip2 showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21Cip1 and p27Kip1 bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21Cip1 and p27Kip1 knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21Cip1 and p27Kip play important roles in the cell cycle exit of postnatal cardiomyocytes

  3. CDK inhibitors, p21{sup Cip1} and p27{sup Kip1}, participate in cell cycle exit of mammalian cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Tane, Shoji; Ikenishi, Aiko; Okayama, Hitomi; Iwamoto, Noriko [School of Life Sciences, Faculty of Medicine, Tottori University, Yonago 683-8503 (Japan); Nakayama, Keiichi I. [Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582 (Japan); Takeuchi, Takashi, E-mail: takeuchi@med.tottori-u.ac.jp [School of Life Sciences, Faculty of Medicine, Tottori University, Yonago 683-8503 (Japan)

    2014-01-17

    Highlights: •Expression of p21 and p27 in the hearts showed a peak during postnatal stages. •p21 and p27 bound to cyclin E, cyclin A and CDK2 in the hearts at postnatal stages. •Cardiomyocytes in both KO mice showed failure in the cell cycle exit at G1-phase. •These data show the first apparent phenotypes in the hearts of Cip/Kip KO mice. -- Abstract: Mammalian cardiomyocytes actively proliferate during embryonic stages, following which cardiomyocytes exit their cell cycle after birth. The irreversible cell cycle exit inhibits cardiac regeneration by the proliferation of pre-existing cardiomyocytes. Exactly how the cell cycle exit occurs remains largely unknown. Previously, we showed that cyclin E- and cyclin A-CDK activities are inhibited before the CDKs levels decrease in postnatal stages. This result suggests that factors such as CDK inhibitors (CKIs) inhibit CDK activities, and contribute to the cell cycle exit. In the present study, we focused on a Cip/Kip family, which can inhibit cyclin E- and cyclin A-CDK activities. Expression of p21{sup Cip1} and p27{sup Kip1} but not p57{sup Kip2} showed a peak around postnatal day 5, when cyclin E- and cyclin A-CDK activities start to decrease. p21{sup Cip1} and p27{sup Kip1} bound to cyclin E, cyclin A and CDK2 at postnatal stages. Cell cycle distribution patterns of postnatal cardiomyocytes in p21{sup Cip1} and p27{sup Kip1} knockout mice showed failure in the cell cycle exit at G1-phase, and endoreplication. These results indicate that p21{sup Cip1} and p27{sup Kip} play important roles in the cell cycle exit of postnatal cardiomyocytes.

  4. Complete inhibition of Cdk/cyclin by one molecule of p21Cip1

    OpenAIRE

    Hengst, L; Göpfert, U.; Lashuel, H. A.; Reed, S I

    1998-01-01

    Cell-cycle phase transitions are controlled by cyclin-dependent kinases (Cdks). Key to the regulation of these kinase activities are Cdk inhibitors, proteins that are induced in response to various antiproliferative signals but that can also oscillate during cell-cycle progression, leading to Cdk inactivation. A current dogma is that kinase complexes containing the prototype Cdk inhibitor p21 transit between active and inactive states, in that Cdk complexes associated with one p21 molecule re...

  5. Molecular interplay between cdk4 and p21 dictates G0/G1 cell cycle arrest in prostate cancer cells

    OpenAIRE

    Gulappa, Thippeswamy; Reddy, Ramadevi Subramani; Suman, Suman; Nyakeriga, Alice M; Damodaran, Chendil

    2013-01-01

    This study examined the effect of 3, 9-dihydroxy-2-prenylcoumestan (pso), a furanocoumarin, on PC-3 and C4-2B castration-resistant prostate cancer (CRPC) cell lines. Pso caused significant G0/G1 cell cycle arrest and inhibition of cell growth. Molecular analysis of cyclin (D1, D2, D3, and E), cyclin-dependent kinase (cdk) (cdks 2, 4, and 6), and cdk inhibitor (p21 and p27) expression suggested transcriptional regulation of the cdk inhibitors and more significant downregulation of cdk4 than of...

  6. Resistance to Apoptosis Conferred by Cdk Inhibitors During Myocyte Differentiation

    OpenAIRE

    Jian WANG; Walsh, Kenneth

    1996-01-01

    Proliferating murine C2C12 myoblasts can undergo either terminal differentiation or programmed cell death under conditions of mitogen deprivation. Unlike myoblasts, differentiated myotubes were resistant to apoptosis. During myogenesis the appearance of the apoptosis-resistant phenotype was correlated with the induction of the cyclin-dependent kinase (Cdk) inhibitor p21CIP1 but not with the appearance of myogenin, a marker expressed earlier in differentiation. Forced expression of the Cdk inh...

  7. Development of mice without Cip/Kip CDK inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    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

  8. Development of mice without Cip/Kip CDK inhibitors

    International Nuclear Information System (INIS)

    Highlights: ► Mice lacking Cip/Kip CKIs (p21, p27, and p57) survive until embryonic day 13.5. ► Proliferation of MEFs lacking all three Cip/Kip CKIs appears unexpectedly normal. ► 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 G0 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 normal development (although it is thought to be a key player in the response to

  9. Inhibition of X-ray and doxorubicin-induced apoptosis by butyrolactone I, a CDK-specific inhibitor, in human tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Lu Yanjun [Shanghai Celstar Bio-Pharmaceutical Co. Ltd. (China). Cancer Research Center; Takebe, Hiraku; Yagi, Takashi

    2000-12-01

    Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21 {sup wafl/Cipl/Sdil} prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21 +/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21 -/-) and DLD1 (p21 +/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDK-inhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis. (author)

  10. Inhibition of X-ray and doxorubicin-induced apoptosis by butyrolactone I, a CDK-specific inhibitor, in human tumor cells

    International Nuclear Information System (INIS)

    Cell-cycle progression is coordinately regulated by cyclin-dependent kinases (CDKs). The inhibition of CDKs by p21 wafl/Cipl/Sdil prevents the apoptosis of cells treated with DNA-damaging agents. In this study, we found that butyrolactone I, a specific inhibitor of CDC2 family kinases, blocks the X-ray- or doxorubicin-induced apoptosis of DLD1 (p21 +/+) human colorectal carcinoma cells in a dose-dependent manner. We also found that butyrolactone I inhibits the CDK2 activity and enhances cell survival after an X-ray irradiation or doxorubicin treatment in both DLD1 (p21 -/-) and DLD1 (p21 +/+) cells. These findings suggest that butyrolactone I prevents apoptosis by the direct inhibition of CDK and also, possibly, by CDK-inhibition through p53-independent p21-induction. Our findings indicate that CDK activity is required for DNA-damaging agent-induced apoptosis. (author)

  11. HPV16 E6、P21WAF1、CDK2、Livin在宫颈癌中的表达及其意义%Expression and significance of HPV16 E6, P21WAF1, CDK2 and livin in cervical cancer

    Institute of Scientific and Technical Information of China (English)

    孙峰; 邢传平; 刘斌; 哈小琴; 王芳; 柳红; 曹晓哲

    2009-01-01

    目的 探讨人乳头瘤病毒(HPV)16 E6、P21WAF1、CDK2、livin在宫颈癌中的表达及P21WAF1、CDK2、livin与E6的关系.方法 采用免疫组化SP法检测HPV16 E6、P21 P21WAF1、CDK2、livin在20例宫颈癌、40例宫颈上皮内瘤变(CIN)及20例正常对照组中的表达.应用图像分析软件分别对此四个因子的表达进行平均光密度分析.结果 E6、P21WAF1、CDK2、livin蛋白于宫颈癌组高于CIN1-2组及正常组(P0.05).CIN1-2组与正常组之间差异无统计学意义(P>0.05).E6分别与P21WAF1、CDK2、livin成正相关(相关系数r分别为0.706、0.713、0.711,P<0.05).结论 E6、CDK2与宫颈癌的发生、发展密切相关,E6、CDK2异常高表达可能是宫颈疾病恶变早期事件.livin可能成为宫颈癌潜在的治疗分子靶向.

  12. Expression of Cdk2 and p21 in gastric cancer and its relationship with clinicopathological features and prognosis%胃癌中Cdk2和p21的表达及其与临床病理和预后的关系

    Institute of Scientific and Technical Information of China (English)

    刘雷; 陈国昌; 宋振云; 郜恒骏; 陈卫昌

    2014-01-01

    目的 研究Cdk2和p21在胃癌中的表达及其与临床病理和预后的关系.方法 将130例胃癌组织和248例癌旁胃组织标本石蜡块制成组织芯片,采用免疫组化法检测Cdk2及p21的表达.结果 胃癌组织组Cdk2及p21阳性表达率分别为63.8%(83/130)和70.0%(91/130),明显高于癌旁组织的18.1%(45/248)和37.1%(92/248)(P<0.05).p21的阳性表达与胃癌的分化程度有关,Cdk2的阳性表达与胃癌的分化程度、淋巴结转移、TNM分期有关(P<0.05).p21阳性表达组5年生存率低于阴性表达组.多因素分析显示,Cdk2和p21的表达可作为胃癌独立的预后因素.结论 Cdk2及p21在胃癌的发生、发展中可能起重要作用,可作为判断胃癌预后的指标.

  13. Versatile templates for the development of novel kinase inhibitors: Discovery of novel CDK inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Dwyer, Michael P.; Paruch, Kamil; Alvarez, Carmen; Doll, Ronald J.; Keertikar, Kerry; Duca, Jose; Fischmann, Thierry O.; Hruza, Alan; Madison, Vincent; Lees, Emma; Parry, David; Seghezzi, Wolfgang; Sgambellone, Nicole; Shanahan, Frances; Wiswell, Derek; Guzi, Timothy J. (SPRI)

    2008-06-30

    A series of four bicyclic cores were prepared and evaluated as cyclin-dependent kinase-2 (CDK2) inhibitors. From the in-vitro and cell-based analysis, the pyrazolo[1,5-a]pyrimidine core (represented by 9) emerged as the superior core for further elaboration in the identification of novel CDK2 inhibitors.

  14. 鲍恩样丘疹病中p21CDK2的表达与人乳头瘤病毒感染的关系%Relationship between the expressions of p21 and CDK2 and infection of human papillomavirus in Bowenoid papulosis

    Institute of Scientific and Technical Information of China (English)

    杨永生; 梁俊; 杜娟; 林尽染; 金晶; 朱小华; 徐金华

    2010-01-01

    目的: 评价鲍恩样丘疹病(Bowenoid populosis, BP)皮损中p21CDK2的表达与HPV感染的相关性.方法: 石蜡包埋标本免疫组化Envision法和IMS细胞图像分析系统分析p21CDK2蛋白表达.冰冻标本采用煮沸法提取DNA,用导流杂交基因芯片技术作HPV分型检测.结果: 在28例BP中p21CDK2阳性细胞数百分比分别为(31.6±10.5)%和(49.9±13.8)%,显著高于包皮对照组的(22.5±5.4)%和(17.3±8.5)%,P<0.05.HPV阳性组p21CDK2阳性细胞百分比分别为(30.56±11.78)%和(54.42±13.69)%;HPV阴性组p21CDK2阳性细胞百分比为(33.3±8.36)%和(43.15±11.55)%.p21两组差别无统计学意义(P>0.05),而CDK2差别有统计学意义(P<0.05).p21CDK2直线相关分析无相关性(P>0.05).结论: BP中存在细胞周期相关蛋白的高表达,HPV可能通过作用于细胞周期相关蛋白使细胞周期紊乱,继而导致非典型增生.

  15. Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization.

    Science.gov (United States)

    Mahale, S; Bharate, S B; Manda, S; Joshi, P; Jenkins, P R; Vishwakarma, R A; Chaudhuri, B

    2015-01-01

    The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G0/G1 phase of the cell cycle, the block occurs primarily at the G2/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC50anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G0/G1. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development. PMID:25950473

  16. Inhibition of human immunodeficiency virus type-1 by cdk inhibitors

    Directory of Open Access Journals (Sweden)

    Kehn-Hall Kylene

    2010-03-01

    Full Text Available Abstract Current therapy for human immunodeficiency virus (HIV-1 infection relies primarily on the administration of anti-retroviral nucleoside analogues, either alone or in combination with HIV-protease inhibitors. Although these drugs have a clinical benefit, continuous therapy with the drugs leads to drug-resistant strains of the virus. Recently, significant progress has been made towards the development of natural and synthetic agents that can directly inhibit HIV-1 replication or its essential enzymes. We previously reported on the pharmacological cyclin-dependent kinase inhibitor (PCI r-roscovitine as a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. Potent activity on viral replication combined with proliferation inhibition without the emergence of resistant viruses, which are normally observed in HAART patients; make PCIs ideal candidates for HIV-1 inhibition. To this end we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro. Screening of these compounds identified alsterpaullone as the most potent inhibitor of HIV-1 with activity at 150 nM. We found that alsterpaullone effectively inhibits cdk2 activity in HIV-1 infected cells with a low IC50 compared to control uninfected cells. The effects of alsterpaullone were associated with suppression of cdk2 and cyclin expression. Combining both alsterpaullone and r-roscovitine (cyc202 in treatment exhibited even stronger inhibitory activities in HIV-1 infected PBMCs.

  17. Enhanced susceptibility of cyclin kinase inhibitor p21 knockout mice to high fat diet induced atherosclerosis

    OpenAIRE

    Khanna Ashwani K

    2009-01-01

    Abstract Cyclin kinase inhibitor p21 is one of the most potent inhibitors of aortic smooth muscle cell proliferation, a key mediator of atherosclerosis. This study tests if p2l deficiency will result in severe atherosclerosis in a mouse model. p21-/- and strain matched wild type mice were fed with high fat diet for 21 weeks. Analysis for biochemical parameters (cholesterol, triglycerides) in serum and mRNA expression of CD36, HO-1, TGF-β, IFN-γ, TNF-α, PPAR-γ and NADPH oxidase components (p22...

  18. Enhanced susceptibility of cyclin kinase inhibitor p21 knockout mice to high fat diet induced atherosclerosis

    Directory of Open Access Journals (Sweden)

    Khanna Ashwani K

    2009-07-01

    Full Text Available Abstract Cyclin kinase inhibitor p21 is one of the most potent inhibitors of aortic smooth muscle cell proliferation, a key mediator of atherosclerosis. This study tests if p2l deficiency will result in severe atherosclerosis in a mouse model. p21-/- and strain matched wild type mice were fed with high fat diet for 21 weeks. Analysis for biochemical parameters (cholesterol, triglycerides in serum and mRNA expression of CD36, HO-1, TGF-β, IFN-γ, TNF-α, PPAR-γ and NADPH oxidase components (p22phox, NOX-1 and Rac-1 was performed in aortic tissues by Real Time PCR. p21-/- mice gained significantly (p -/- compared to wild type mice fed with high fat diet. High fat diet resulted in significantly decreased TGF-β (p -/- mice compared to animal fed with regular diet. IFN-γ mRNA expression (235 ± 11 folds increased significantly in high fat diet fed p21-/- mice and a multifold modulation of PPAR-γ(136 ± 7, p22phox, NOX-1 and Rac-1 (15–35-folds mRNA in aortic tissues from p21-/- mice compared to the wild type mice. Severity of atherosclerotic lesions was significantly higher in p21-/- compared to wild type mice. The results demonstrate that the deficiency of p21 leads to altered expression of pro-atherogenic genes, and severe atherosclerosis in mice fed with high fat diet. This opens the possibility of p21 protein as a therapeutic tool to control progression of atherosclerosis.

  19. The gene expressions of cyclin E, CDK2 and p21WAF1 and their significant in esophageal carsinogenesis%Cyclin E、CDK2和p21WAF1在食管上皮癌变过程中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    李丽; 齐凤英; 左连富; 李萍; 王辉

    2005-01-01

    目的探讨食管上皮癌变过程中细胞周期调控因子cyclin E、CDK2和p21WAF1的表达状况及其意义.方法应用免疫组化SP法和原位杂交方法分别检测48例食管癌组织、31例非典型增生组织和17例正常食管粘膜中cyclin E、CDK2和p21WAF1蛋白及mRNA表达.应用半定量RT-PCR和Western blot检测22例新鲜食管癌及相应癌旁组织的mRNA和蛋白表达.结果从食管正常粘膜、非典型增生组织到癌组织,cyclin E和CDK2蛋白和mRNA阳性表达率逐渐上升,差异具有统计学意义(P<0.01或P<0.05).食管癌组织中cyclin E、CDK2和p21WAF1蛋白及mRNA高表达,与癌旁组织或切缘正常食管粘膜有显著性差异(P<0.01).cyclin E、CDK2和p21WAF1基因表达显著正相关(P<0.01或P<0.05).结论食管上皮癌变过程中,细胞周期相关基因cyclin E和CDK2表达逐渐增强.cyclin E基因表达异常是食管癌变过程中的早期事件.p21WAF1基因在食管癌中高表达,可能与细胞周期调控的反馈机制有关.

  20. Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells

    Directory of Open Access Journals (Sweden)

    Pal-Bhadra Manika

    2012-05-01

    Full Text Available Abstract Background Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. Methods Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27. The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. Results Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. Conclusion Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (−692 to −684 region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating

  1. Plant HDAC inhibitor chrysin arrest cell growth and induce p21WAF1 by altering chromatin of STAT response element in A375 cells

    International Nuclear Information System (INIS)

    Chrysin and its analogues, belongs to flavonoid family and possess potential anti-tumour activity. The aim of this study is to determine the molecular mechanism by which chrysin controls cell growth and induce apoptosis in A375 cells. Effect of chrysin and its analogues on cell viability and cell cycle analysis was determined by MTT assay and flowcytometry. A series of Western blots was performed to determine the effect of chrysin on important cell cycle regulatory proteins (Cdk2, cyclin D1, p53, p21, p27). The fluorimetry and calorimetry based assays was conducted for characterization of chrysin as HDAC inhibitor. The changes in histone tail modification such as acetylation and methylation was studied after chrysin treatment was estimated by immuno-fluorescence and western blot analysis. The expression of Bcl-xL, survivin and caspase-3 was estimated in chrysin treated cells. The effect of chrysin on p21 promoter activity was studied by luciferase and ChIP assays. Chrysin cause G1 cell cycle arrest and found to inhibit HDAC-2 and HDAC-8. Chrysin treated cells have shown increase in the levels of H3acK14, H4acK12, H4acK16 and decrease in H3me2K9 methylation. The p21 induction by chrysin treatment was found to be independent of p53 status. The chromatin remodelling at p21WAF1 promoter induces p21 activity, increased STAT-1 expression and epigenetic modifications that are responsible for ultimate cell cycle arrest and apoptosis. Chrysin shows in vitro anti-cancer activity that is correlated with induction of histone hyperacetylation and possible recruitment of STAT-1, 3, 5 proteins at STAT (−692 to −684) region of p21 promoter. Our results also support an unexpected action of chrysin on the chromatin organization of p21WAF1 promoter through histone methylation and hyper-acetylation. It proposes previously unknown sequence specific chromatin modulations in the STAT responsive elements for regulating cell cycle progression negatively via the induction of the CDK

  2. 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.

  3. DACH1 regulates cell cycle progression of myeloid cells through the control of cyclin D, Cdk 4/6 and p21Cip1

    International Nuclear Information System (INIS)

    Highlights: ► DACH1 increases cyclin D, F and Cdk 1, 4, 6 in mouse myeloid progenitor cells. ► The knockdown of DACH1 blocked the cell cycle progression of HL-60 cells. ► 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 p27Kip1 and repressed p21Cip1, 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 p21Cip1, 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.

  4. Development of a Potent, Specific CDK8 Kinase Inhibitor Which Phenocopies CDK8/19 Knockout Cells.

    Science.gov (United States)

    Koehler, Michael F T; Bergeron, Philippe; Blackwood, Elizabeth M; Bowman, Krista; Clark, Kevin R; Firestein, Ron; Kiefer, James R; Maskos, Klaus; McCleland, Mark L; Orren, Linda; Salphati, Laurent; Schmidt, Steve; Schneider, Elisabeth V; Wu, Jiansheng; Beresini, Maureen H

    2016-03-10

    Beginning with promiscuous COT inhibitors, which were found to inhibit CDK8, a series of 6-aza-benzothiophene containing compounds were developed into potent, selective CDK8 inhibitors. When cocrystallized with CDK8 and cyclin C, these compounds exhibit an unusual binding mode, making a single hydrogen bond to the hinge residue A100, a second to K252, and a key cation-π interaction with R356. Structure-based drug design resulted in tool compounds 13 and 32, which are highly potent, kinase selective, permeable compounds with a free fraction >2% and no measurable efflux. Despite these attractive properties, these compounds exhibit weak antiproliferative activity in the HCT-116 colon cancer cell line. Further examination of the activity of 32 in this cell line revealed that the compound reduced phosphorylation of the known CDK8 substrate STAT1 in a manner identical to a CDK8 knockout clone, illustrating the complex effects of inhibition of CDK8 kinase activity in proliferation in these cells. PMID:26985305

  5. Expression and signification of cell cycle regulation protein Cyclin D1-CDK4-p21 in scar cancer%细胞周期调控系统相关因子 Cyclin D1-CDK4-p21在瘢痕癌中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    林宇静; 郭瑞珍; 王海青

    2014-01-01

    Objective Dysfunction of cell cycle regulation is one of the key factors for cellular carcinogenesis .This paper aimed to study the expression and significance of cell cycle regulation protein Cyclin D 1-CDK4-p21 in scar cancer . Methods The expressions of Cyclin D1, CDK4 and p21 protains were detected in scar cancer group , pathological scar group and normal skin group respectively by using immunohistochemical staining (SP).The mRNA expression levels of Cyclin D1, CDK4 and p21 were detected by the use of nucleic acid-mediated in-situ hybridization .Correlation analysis was made on the indexes , and the average optical density and positive area were analyzed using image analysis . Results The expressions of Cyclin D1, CDK4 and p21 protains and the mRNA ex-pression levels of cyclin D1, CDK4 and p21 were high in scar cancer group, low in pathological scar group , and negative in normal skin group.The mean optical density and positive area in scar cancer group were significantly different from pathological scar group and normal skin group (P0.05).In terms of correlation analysis , the expressions of Cyclin D 1 and CDK4 as well as p21 and CDK4 in scar cancer tissue were both in posi-tive correlations. Conclusion The occurrence of scar cancer is related to the abnormal expression of Cyclin D 1 and CDK4.The complex formed by Cyclin D1 and CDK4 may promote the G1/S transition, proliferation and tumorigenesis of scar cancer .In scar canc-er, the inhibition of Cyclin D1-CDK4 complex might be caused by other members of CKI family or even inbibitors of other families apart from CDK family.%目的:细胞周期调控机制失调是细胞增生肿瘤发生的重要因素。文中探讨细胞周期调控系统相关因子Cyclin D1-CDK4-p21在瘢痕癌中的表达及意义。方法选取遵义医学院病理教研室和中山大学附属第五医院病理科2005-2011年石蜡包埋标本,分为瘢痕癌组、病理性瘢痕组和正常皮肤组。应

  6. Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

    International Nuclear Information System (INIS)

    The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation of the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML)

  7. Variation in transcriptional regulation of cyclin dependent kinase inhibitor p21waf1/cip1 among human bronchogenic carcinomas

    Directory of Open Access Journals (Sweden)

    Reed Cheryl AM

    2005-07-01

    Full Text Available Abstract Background Cell proliferation control depends in part on the carefully ordered regulation of transcription factors. The p53 homolog p73, contributes to this control by directly upregulating the cyclin dependent kinase inhibitor, p21waf1/cip1. E2F1, an inducer of cell proliferation, directly upregulates p73 and in some systems upregulates p21 directly. Because of its central role in controlling cell proliferation, upregulation of p21 has been explored as a modality for treating bronchogenic carcinoma (BC. Improved understanding of p21 transcriptional regulation will facilitate identification of BC tissues that are responsive to p21-directed therapies. Toward this goal, we investigated the role that E2F1 and p73 each play in the transcriptional regulation of p21. Results Among BC samples (N = 21 p21 transcript abundance (TA levels varied over two orders of magnitude with values ranging from 400 to 120,000 (in units of molecules/106 molecules β-actin. The p21 values in many BC were high compared to those observed in normal bronchial epithelial cells (BEC (N = 18. Among all BC samples, there was no correlation between E2F1 and p21 TA but there was positive correlation between E2F1 and p73α (p Conclusion p21 TA levels vary considerably among BC patients which may be attributable to 1 genetic alterations in Rb and p53 and 2 variation in TA levels of upstream transcription factors E2F1 and p73. Here we provide evidence that p73 upregulates p21 TA in BC tissues and upregulated p21 TA may result from E2F1 upregulation of p73 but not from E2F1 directly.

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

    International Nuclear Information System (INIS)

    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

  9. 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.

  10. Benzamide capped peptidomimetics as non-ATP competitive inhibitors of CDK2 using the REPLACE strategy.

    Science.gov (United States)

    Premnath, Padmavathy Nandha; Craig, Sandra N; Liu, Shu; McInnes, Campbell

    2016-08-01

    Inhibition of cyclin dependent kinase 2 (CDK2) in complex with cyclin A in G1/S phase of the cell cycle has been shown to promote selective apoptosis of cancer cells through the E2F1 pathway. An alternative approach to catalytic inhibition is to target the substrate recruitment site also known as the cyclin binding groove (CBG) to generate selective non-ATP competitive inhibitors. The REPLACE strategy has been applied to identify fragment alternatives and substituted benzoic acid derivatives were evaluated as a promising scaffold to present appropriate functionality to mimic key peptide determinants. Fragment Ligated Inhibitory Peptides (FLIPs) are described which potently inhibit both CDK2/cyclin A and CDK4/cyclin D1 and have preliminary anti-tumor activity. A structural rationale for binding was obtained through molecular modeling further demonstrating their potential for further development as next generation non ATP competitive CDK inhibitors. PMID:27297568

  11. Iterative Conversion of Cyclin Binding Groove Peptides into Druglike CDK Inhibitors with Antitumor Activity

    OpenAIRE

    Premnath, Padmavathy Nandha; Craig, Sandra N.; Liu, Shu; Anderson, Erin L.; Grigoroudis, Asterios I.; Kontopidis, George; Perkins, Tracy L.; Wyatt, Michael D.; Pittman, Douglas L.; McInnes, Campbell

    2014-01-01

    The cyclin groove is an important recognition site for substrates of the cell cycle cyclin dependent kinases and provides an opportunity for highly selective inhibition of kinase activity through a non-ATP competitive mechanism. The key peptide residues of the cyclin binding motif have been studied in order to precisely define the structure–activity relationship for CDK kinase inhibition. Through this information, new insights into the interactions of peptide CDK inhibitors with key subsites ...

  12. Synthesis and Biological Evaluation of Scutellaria Flavone Cyclaneaminol Mannich Base Derivatives as Novel CDK1 Inhibitors.

    Science.gov (United States)

    Ha, Lisha; Qian, Yuan; Zhang, Shixuan; Ju, Xiulan; Sun, Shiyou; Guo, Hongmin; Wang, Qianru; Li, Kangjian; Fan, Qingyu; Zheng, Yang; Li, Hailiang

    2016-01-01

    Cyclin-dependent kinase 1 (CDK1) is the only necessary CDK in the cell proliferation process and a new target in the research and development of anti-cancer drugs. Natural flavones are selective CDK1 inhibitors which can suppress the proliferation of cancer cells. However, their bioavailability is poor. To solve these problems, 6 Scutellaria flavones were isolated from hydrolyzed products of Scutellaria baicalensis and used as lead compounds, 18 Scutellaria flavones cyclane-aminol Mannich base derivatives were semi-synthesized and their biological activity as novel CDK1 inhibitors was evaluated. Results indicated that the biological activity of 8-Hydroxypiperidinemethyl-baicalein (BA-j) is the highest among these compounds. BA-j is a selective CDK1 inhibitor, and has broad-spectrum anti-proliferative activity in human cancer cells (IC50 12.3μM). BA-j can capture oxygen free radicals (.O2(-)) and selectively increase intracellular H2O2 level in cancer cells and activated lymphocytes, thus inducing their apoptosis rather than in normal cells. These findings suggest that BA-j selectively induces apoptosis in cancer and activated lymphocyte by controlling intracellular H2O2 level, and can be developed into a novel anti-proliferative agent for the treatment of cancer, AIDS, and some immune diseases. PMID:26411959

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

    International Nuclear Information System (INIS)

    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

  14. 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.

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

    International Nuclear Information System (INIS)

    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

  16. Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors

    OpenAIRE

    Premnath, Padmavathy Nandha; Liu, Shu; Perkins, Tracy; Abbott, Jennifer; Anderson, Erin; McInnes, Campbell

    2013-01-01

    In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating b...

  17. Prognostic value of expression of cyclin dependent kinase inhibitor p21 and lung resistance protein as predicting factors in breast cancer

    International Nuclear Information System (INIS)

    The purpose of the study was to investigate whether cyclin dependent kinase inhibitor p21 (p21) and lung resistance protein (LRP) could be predicting factors for recurrence after surgery for breast cancer. Immunostainings of p53, p21 and LRP were carried out to breast tissues which had been curatively resected. Prognostic factors were searched by means of multivariate analysis. In a Cox proportional hazard model, p21 and LRP were factors affecting the recurrence. Patients with p21 (-) and LRP (+) developed postoperative recurrence in a high frequency. Of patients administered anticancer drugs, the disease-free survical rate in those with p21 (-) was significantly low and all patients with p21 (-) and LRP (+) developed recurrence. Of patients having received postoperative irradiation, a significant difference was seen in the disease-free survical rate between groups with p21 (+) and p21 (-). In conclusion, expression of p21 (+) and LRP can each be an independent prognostic factor. And a combination of p21 (+) and LRP (-) predict the best prognosis along with favourable response to chemoradiotherapy following surgery. On the century a combination of p21 (-) and LRP (+) meet disappointing response to chemoradiotherapy. (author)

  18. CDK2 Is Required for the DNA Damage Response During Porcine Early Embryonic Development.

    Science.gov (United States)

    Wang, HaiYang; Kim, Nam-Hyung

    2016-08-01

    Cyclin-dependent kinase (CDK) 2 inhibition plays a central role in DNA damage-induced cell cycle arrest and DNA repair. However, whether CDK2 also influences early porcine embryo development is unknown. In this study, we examined whether CDK2 is involved in the regulation of oocyte meiosis and early embryonic development of porcine embryos. We found that disrupting CDK2 activity with RNAi or an inhibitor did not affect meiotic resumption or meiosis II arrest. However, CDK2 inhibitor-treated embryos showed delayed cleavage and ceased development before the blastocyst stage. Disrupting CDK2 activity is able to induce sustained DNA damage, as demonstrated by the formation of distinct gammaH2AX foci in nuclei of Day-3 and Day-5 embryos. Inhibiting CDK2 triggers a DNA damage checkpoint by activation of the ataxia telangiectasia mutated (ATM)-P53-P21 pathway. However, the mRNA expression of genes involved in nonhomologous end joining or homologous recombination pathways for double-strand break repair were reduced after administering CDK2 inhibitor to 5-day-old embryos. Furthermore, CDK2 inhibition caused apoptosis in Day-7 blastocysts. Thus, our results indicate that an ATM-P53-P21 DNA damage checkpoint is intact in the absence of CDK2; however, CDK2 is important for proper repair of the damaged DNA by either directly or indirectly influencing DNA repair-related gene expression. PMID:27307074

  19. Momilactone B induces apoptosis and G1 arrest of the cell cycle in human monocytic leukemia U937 cells through downregulation of pRB phosphorylation and induction of the cyclin-dependent kinase inhibitor p21Waf1/Cip1.

    Science.gov (United States)

    Park, Cheol; Jeong, Na Young; Kim, Gi-Young; Han, Min Ho; Chung, Ill-Min; Kim, Wun-Jae; Yoo, Young Hyun; Choi, Yung Hyun

    2014-04-01

    Momilactone B, a terpenoid phytoalexin present in rice bran, has been shown to exhibit several biological activities. The present study was conducted using cultured human leukemia U937 cells to elucidate the possible mechanisms by which momilactone B exerts its anticancer activity, which to date has remained poorly understood. Momilactone B treatment of U937 cells resulted in a dose-dependent inhibition of cell growth and induced apoptotic cell death as detected by chromatin condensation, DNA fragmentation, the cleavage of poly(ADP-ribose) polymerase and Annexin V-FITC staining. Flow cytometric analysis revealed that momilactone B resulted in G1 arrest in cell cycle progression, which was associated with the dephosphorylation of retinoblastoma protein (pRB) and enhanced binding of pRB with the E2F transcription factor family proteins. Treatment with momilactone B also increased the expression of cyclin-dependent kinase (Cdk) inhibitor p21Waf1/Cip1 in a p53-independent manner, without any noticeable changes in G1 cyclins and cyclin-dependent kinases (Cdks), except a slight decrease in cyclin E. Moreover, in vitro kinase assay indicated that momilactone B significantly decreased Cdk4- and Cdk6-associated kinase activities through a notably increased binding of p21 to Cdk4 and Cdk6. Our results demonstrated that momilactone B caused G1 cell cycle arrest and apoptosis in U937 cells through the induction of p21 expression, inhibition of Cdk/cyclin-associated kinase activities, and reduced phosphorylation of pRB, which may be related to anticancer activity. PMID:24503697

  20. Cyclin-dependent Kinase Inhibitor, p21WAF1/CIP1, Is Involved in Adipocyte Differentiation and Hypertrophy, Linking to Obesity, and Insulin Resistance*S⃞

    OpenAIRE

    Inoue, Noriyuki; Yahagi, Naoya; Yamamoto, Takashi; Ishikawa, Mayumi; Watanabe, Kazuhisa; Matsuzaka, Takashi; Nakagawa, Yoshimi; Takeuchi, Yoshinori; Kobayashi, Kazuto; Takahashi, Akimitsu; Suzuki, Hiroaki; Hasty, Alyssa H.; Toyoshima, Hideo; Yamada, Nobuhiro; Shimano, Hitoshi

    2008-01-01

    Both adipocyte hyperplasia and hypertrophy are determinant factors for adipocyte differentiation during the development of obesity. p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, is induced during adipocyte differentiation; however, its precise contribution to this process is unknown. Using both in vitro and in vivo systems, we show that p21 is crucial for maintaining adipocyte hypertrophy and obesity-induced insulin resistance. The absence of p21 in 3T3-L1 fibroblasts ...

  1. Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly

    OpenAIRE

    Liu, Shu; Premnath, Padmavathy Nandha; Bolger, Joshua K.; Perkins, Tracy; Kirkland, Lindsay O.; Kontopidis, George; McInnes, Campbell

    2013-01-01

    A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a ...

  2. Maintenance of Leukemia-Initiating Cells Is Regulated by the CDK Inhibitor Inca1

    OpenAIRE

    Bäumer, Nicole; Bäumer, Sebastian; Berkenfeld, Frank; Stehling, Martin; Köhler, Gabriele; Berdel, Wolfgang E.; Müller-Tidow, Carsten; Tschanter, Petra

    2014-01-01

    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...

  3. Targeting Transcriptional Addictions In Small Cell Lung Cancer With a Covalent CDK7 Inhibitor

    OpenAIRE

    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

    2014-01-01

    Small cell lung cancer (SCLC) is an aggressive disease with high mortality. 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, and in particular to THZ1, a recent identified covalent inhibitor of cyclin-dependent kinase 7 (CDK7). We find that expression of super-enhancer associated transcription fact...

  4. 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 in...

  5. Acetylation of p53 at Lysine 373/382 by the Histone Deacetylase Inhibitor Depsipeptide Induces Expression of p21Waf1/Cip1

    OpenAIRE

    Zhao, Ying; Lu, Shaoli; Wu, Lipeng; Chai, Guolin; Wang, Haiying; Chen, Yingqi; Sun, Jia; Yu, Yu; Zhou, Wen; Zheng, Quanhui; Wu, Mian; Otterson, Gregory A.; Zhu, Wei-Guo

    2006-01-01

    Generally, histone deacetylase (HDAC) inhibitor-induced p21Waf1/Cip1 expression is thought to be p53 independent. Here we found that an inhibitor of HDAC, depsipeptide (FR901228), but not trichostatin A (TSA), induces p21Waf1/Cip1 expression through both p53 and Sp1/Sp3 pathways in A549 cells (which retain wild-type p53). This is demonstrated by measuring relative luciferase activities of p21 promoter constructs with p53 or Sp1 binding site mutagenesis and was further confirmed by transfectio...

  6. Antidepressants stimulate hippocampal neurogenesis by inhibiting p21 expression in the subgranular zone of the hipppocampus.

    Directory of Open Access Journals (Sweden)

    Robert N Pechnick

    Full Text Available The relationships among hippocampal neurogenesis, depression and the mechanism of action of antidepressant drugs have generated a considerable amount of controversy. The cyclin-dependent kinase (Cdk inhibitor p21(Cip1 (p21 plays a crucial role in restraining cellular proliferation and maintaining cellular quiescence. Using in vivo and in vitro approaches the present study shows that p21 is expressed in the subgranular zone of the dentate gyrus of the hippocampus in early neuronal progenitors and in immature neurons, but not in mature neurons or astroglia. In vitro, proliferation is higher in neuronal progenitor cells derived from p21-/- mice compared to cells derived from wild-type mice. Proliferation is increased in neuronal progenitor cells after suppression of p21 using lentivirus expressing short hairpin RNA against p21. In vivo, chronic treatment with the non-selective antidepressant imipramine as well as the norepinephrine-selective reuptake inhibitor desipramine or the serotonin-selective reuptake inhibitor fluoxetine all decrease p21 expression, and this was associated with increased neurogenesis. Chronic antidepressant treatment did not affect the expression of other Cdk inhibitors. Untreated p21-/- mice exhibit a higher degree of baseline neurogenesis and decreased immobility in the forced swim test. Although chronic imipramine treatment increased neurogenesis and reduced immobility in the forced swim test in wild-type mice, it reduced neurogenesis and increased immobility in p21-/- mice. These results demonstrate the unique role of p21 in the control of neurogenesis, and support the hypothesis that different classes of reuptake inhibitor-type antidepressant drugs all stimulate hippocampal neurogenesis by inhibiting p21 expression.

  7. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

    OpenAIRE

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

    2014-01-01

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2...

  8. 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.

  9. Fragment based discovery of Arginine isosteres through REPLACE: towards non-ATP competitive CDK inhibitors

    Science.gov (United States)

    Premnath, Padmavathy Nandha; Liu, Shu; Perkins, Tracy; Abbott, Jennifer; Anderson, Erin; McInnes, Campbell

    2013-01-01

    In order to develop non-ATP competitive CDK2/cyclin A inhibitors, the REPLACE strategy has been applied to generate fragment alternatives for the N-terminal tetrapeptide of the cyclin binding motif (HAKRRLIF) involved in substrate recruitment prior to phosphotransfer. The docking approach used for the prediction of small molecule mimics for peptide determinants was validated through reproduction of experimental binding modes of known inhibitors and provides useful information for evaluating binding to protein-protein interaction sites. Further to this, potential arginine isosteres predicted using the validated LigandFit docking method were ligated to the truncated C-terminal peptide, RLIF using solid phase synthesis and evaluated in a competitive binding assay. After testing, identified fragments were shown to represent not only appropriate mimics for a critical arginine residue but also to interact effectively with a minor hydrophobic pocket present in the binding groove. Further evaluation of binding modes was undertaken to optimize the potency of these compounds. Through further application of the REPLACE strategy in this study, peptide-small molecule hybrid CDK2 inhibitors were identified that are more drug-like and suitable for further optimization as anti-tumor therapeutics. PMID:24286762

  10. 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...

  11. An opposite effect of the CDK inhibitor, p18(INK4c on embryonic stem cells compared with tumor and adult stem cells.

    Directory of Open Access Journals (Sweden)

    Yanxin Li

    Full Text Available Self-renewal is a feature common to both adult and embryonic stem (ES cells, as well as tumor stem cells (TSCs. The cyclin-dependent kinase inhibitor, p18(INK4c, is a known tumor suppressor that can inhibit self-renewal of tumor cells or adult stem cells. Here, we demonstrate an opposite effect of p18 on ES cells in comparison with teratoma cells. Our results unexpectedly showed that overexpression of p18 accelerated the growth of mouse ES cells and embryonic bodies (EB; on the contrary, inhibited the growth of late stage teratoma. Up-regulation of ES cell markers (i.e., Oct4, Nanog, Sox2, and Rex1 were detected in both ES and EB cells, while concomitant down-regulation of various differentiation markers was observed in EB cells. These results demonstrate that p18 has an opposite effect on ES cells as compared with tumor cells and adult stem cells. Mechanistically, expression of CDK4 was significantly increased with overexpression of p18 in ES cells, likely leading to a release of CDK2 from the inhibition by p21 and p27. As a result, self-renewal of ES cells was enhanced. Our current study suggests that targeting p18 in different cell types may yield different outcomes, thereby having implications for therapeutic manipulations of cell cycle machinery in stem cells.

  12. Cyclin E1 and RTK/RAS signaling drive CDK inhibitor resistance via activation of E2F and ETS

    Science.gov (United States)

    Taylor-Harding, Barbie; Aspuria, Paul-Joseph; Agadjanian, Hasmik; Cheon, Dong-Joo; Mizuno, Takako; Greenberg, Danielle; Allen, Jenieke R.; Spurka, Lindsay; Funari, Vincent; Spiteri, Elizabeth; Wang, Qiang; Orsulic, Sandra; Walsh, Christine; Karlan, Beth Y.; Wiedemeyer, W. Ruprecht

    2015-01-01

    High-grade serous ovarian cancers (HGSOC) are genomically complex, heterogeneous cancers with a high mortality rate, due to acquired chemoresistance and lack of targeted therapy options. Cyclin-dependent kinase inhibitors (CDKi) target the retinoblastoma (RB) signaling network, and have been successfully incorporated into treatment regimens for breast and other cancers. Here, we have compared mechanisms of response and resistance to three CDKi that target either CDK4/6 or CDK2 and abrogate E2F target gene expression. We identify CCNE1 gain and RB1 loss as mechanisms of resistance to CDK4/6 inhibition, whereas receptor tyrosine kinase (RTK) and RAS signaling is associated with CDK2 inhibitor resistance. Mechanistically, we show that ETS factors are mediators of RTK/RAS signaling that cooperate with E2F in cell cycle progression. Consequently, CDK2 inhibition sensitizes cyclin E1-driven but not RAS-driven ovarian cancer cells to platinum-based chemotherapy. In summary, this study outlines a rational approach for incorporating CDKi into treatment regimens for HGSOC. PMID:25557169

  13. Preclinical Metabolism and Pharmacokinetics of SB1317 (TG02), a Potent CDK/JAK2/FLT3 Inhibitor

    NARCIS (Netherlands)

    Pasha, Mohammed Khalid; Jayaraman, Ramesh; Reddy, Venkatesh Pilla; Yeo, Pauline; Goh, Evelyn; Williams, Anthony; Goh, Kee Chuan; Kantharaj, Ethirajulu

    2012-01-01

    SB1317 (TG02) is a novel small molecule potent CDK/JAK2/FLT3 inhibitor. To evaluate full potential of this development candidate, we conducted drug metabolism and pharmacokinetic studies of this novel anti-cancer agent. SB1317 was soluble, highly permeable in Caco-2 cells, and showed >99% binding to

  14. Optimization of non-ATP competitive CDK/cyclin groove Inhibitors through REPLACE mediated Fragment Assembly

    Science.gov (United States)

    Liu, Shu; Premnath, Padmavathy Nandha; Bolger, Joshua K.; Perkins, Tracy; Kirkland, Lindsay O.; Kontopidis, George; McInnes, Campbell

    2013-01-01

    A major challenge in drug discovery is to develop and improve methods for targeting protein-protein interactions. Further exemplification of the REPLACE strategy for generating inhibitors of protein-protein interactions demonstrated that it can be used to optimize fragment alternatives of key determinants, to combine these in an effective way and was achieved for compounds targeting the CDK2 substrate recruitment site on the cyclin regulatory subunit. Phenylheterocyclic isosteres replacing a critical charge-charge interaction provided new structural insights for binding to the cyclin groove. In particular, these results shed light onto the key contributions of a H-bond observed in crystal structures of N-terminally capped peptides. Furthermore the structure-activity relationship of a bisarylether C-terminal capping group mimicking dipeptide interactions, was probed through ring substitutions, allowing increased complementarity with the primary hydrophobic pocket. This study further validates REPLACE as an effective strategy for converting peptidic compounds to more pharmaceutically relevant compounds. PMID:23323521

  15. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase

    Energy Technology Data Exchange (ETDEWEB)

    Filgueira de Azevedo, W. Jr.; Mueller-Dieckmann, H.J.; Schulze-Gahmen, U. [Lawrence Berkeley National Lab., CA (United States)] [and others

    1996-04-02

    The central role of cyclin-dependent kinases (CDKs) in cell cycle regulation makes them a promising target for studying inhibitory molecules that can modify the degree of cell proliferation. The discovery of specific inhibitors of CDKs such as polyhydroxylated flavones has opened the way to investigation and design of antimitotic compounds. A novel flavone, (-)-cis-5,7-dihydroxyphenyl-8-[4-(3-hydroxy-1-methyl)piperidinyl]-4H-1-benzopyran-4-one hydrochloride hemihydrate (L868276), is a potent inhibitor of CDKs. A chlorinated form, flavopiridol, is currently in phase I clinical trials as a drug against breast tumors. We determined the crystal structure of a complex between CDK2 and L868276 at 2.33-{Angstrom} resolution and refined to an R{sub factor} of 20.3%. The aromatic portion of the inhibitor binds to the adenine-binding pocket of CDK2, and the position of the phenyl group of the inhibitor enables the inhibitor to make contacts with the enzyme not observed in the ATP complex structure. The analysis of the position of this phenyl ring not only explains the great differences of kinase inhibition among the flavonoid inhibitors but also explains the specificity of L868276 to inhibit CDK2 and CDC2. 36 refs., 4 figs., 2 tabs.

  16. p16-Cdk4-Rb axis controls sensitivity to a cyclin-dependent kinase inhibitor PD0332991 in glioblastoma xenograft cells

    OpenAIRE

    Cen, Ling; Carlson, Brett L.; Schroeder, Mark A.; Ostrem, Jamie L.; Kitange, Gaspar J.; Mladek, Ann C.; Fink, Stephanie R.; Decker, Paul A.; Wu, Wenting; Kim, Jung-Sik; Waldman, Todd; Jenkins, Robert B.; Sarkaria, Jann N.

    2012-01-01

    Deregulation of the p16INK4a-Cdk4/6-Rb pathway is commonly detected in patients with glioblastoma multiforme (GBM) and is a rational therapeutic target. Here, we characterized the p16INK4a-Cdk4/6-Rb pathway in the Mayo panel of GBM xenografts, established from primary tissue samples from patients with GBM, and evaluated their response to PD0332991, a specific inhibitor of Cdk4/6. All GBM xenograft lines evaluated in this study had disruptions in the p16INK4a-Cdk4/6-Rb pathway. In vitro evalua...

  17. Inhibitor of Cyclin-dependent Kinase (CDK) Interacting with Cyclin A1 (INCA1) Regulates Proliferation and Is Repressed by Oncogenic Signaling*

    OpenAIRE

    Bäumer, Nicole; Tickenbrock, Lara; Tschanter, Petra; Lohmeyer, Lisa; Diederichs, Sven; Bäumer, Sebastian; Skryabin, Boris V.; Zhang, Feng; Agrawal-Singh, Shuchi; Köhler, Gabriele; Berdel, Wolfgang E.; Serve, Hubert; Koschmieder, Steffen; Müller-Tidow, Carsten

    2011-01-01

    The cell cycle is driven by the kinase activity of cyclin·cyclin-dependent kinase (CDK) complexes, which is negatively regulated by CDK inhibitor proteins. Recently, we identified INCA1 as an interaction partner and a 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 inhibitory effects depended on the cyclin-interacting domain. Mitogenic and oncogenic sig...

  18. Effects of activin and TGFβ on p21 in colon cancer.

    Directory of Open Access Journals (Sweden)

    Jessica Bauer

    Full Text Available Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21(cip1/waf1. Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.

  19. Protein kinase C delta (PKCδ affects proliferation of insulin-secreting cells by promoting nuclear extrusion of the cell cycle inhibitor p21Cip1/WAF1.

    Directory of Open Access Journals (Sweden)

    Felicia Ranta

    Full Text Available BACKGROUND: High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS: Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1. This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1 at Ser146. In kinase dead PKCδ (PKCδKN overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1 was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1 with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE: These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1, which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.

  20. 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.

  1. Molecular modelling on small molecular CDK2 inhibitors: an integrated approach using a combination of molecular docking, 3D-QSAR and pharmacophore modelling.

    Science.gov (United States)

    Yuan, H; Liu, H; Tai, W; Wang, F; Zhang, Y; Yao, S; Ran, T; Lu, S; Ke, Z; Xiong, X; Xu, J; Chen, Y; Lu, T

    2013-10-01

    Cyclin-dependent kinase 2 (CDK2) has been identified as an important target for developing novel anticancer agents. Molecular docking, three-dimensional quantitative structure-activity relationship (3D-QSAR) and pharmacophore modelling were combined with the ultimate goal of studying the structure-activity relationship of CDK2 inhibitors. The comparative molecular similarity indices analysis (CoMSIA) model constructed based on a set of 3-aminopyrazole derivatives as CDK2 inhibitors gave statistically significant results (q (2) = 0.700; r (2) = 0.982). A HypoGen pharmacophore model, constructed using diverse CDK2 inhibitors, also showed significant statistics ([Formula: see text]Cost = 61.483; RMSD = 0.53; Correlation coefficient = 0.98). The small residues and error values between the estimated and experimental activities of the training and test set compounds proved their strong capability of activity prediction. The structural insights obtained from these two models were consistent with each other. The pharmacophore model summarized the important pharmacophoric features required for protein-ligand binding. The 3D contour maps in combination with the comprehensive pharmacophoric features helped to better interpret the structure-activity relationship. The results will be beneficial for the discovery and design of novel CDK2 inhibitors. The simplicity of this approach provides expansion to its applicability in optimizing other classes of small molecular CDK2 inhibitors. PMID:23941641

  2. Specific Antileukemic Activity of PD0332991, a CDK4/6 Inhibitor, against Philadelphia Chromosome-Positive Lymphoid Leukemia.

    Science.gov (United States)

    Nemoto, Atsushi; Saida, Satoshi; Kato, Itaru; Kikuchi, Jiro; Furukawa, Yusuke; Maeda, Yasuhiro; Akahane, Koshi; Honna-Oshiro, Hiroko; Goi, Kumiko; Kagami, Keiko; Kimura, Shinya; Sato, Yuko; Okabe, Seiichi; Niwa, Akira; Watanabe, Kenichiro; Nakahata, Tatsutoshi; Heike, Toshio; Sugita, Kanji; Inukai, Takeshi

    2016-01-01

    S-phase progression of the cell cycle is accelerated in tumors through various genetic abnormalities, and, thus, pharmacologic inhibition of altered cell-cycle progression would be an effective strategy to control tumors. In the current study, we analyzed the antileukemic activity of three available small molecules targeting CDK4/CDK6 against lymphoid crisis of chronic myeloid leukemia (CML-LC) and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL), and found that all three molecules showed specific activities against leukemic cell lines derived from CML-LC and Ph(+) ALL. In particular, PD0332991 exhibited extremely high antileukemic activity against CML-LC and Ph(+) ALL cell lines in the nanomolar range by the induction of G0-G1 arrest and partially cell death through dephosphorylation of pRb and downregulation of the genes that are involved in S-phase transition. As an underlying mechanism for favorable sensitivity to the small molecules targeting CDK4/CDK6, cell-cycle progression of Ph(+) lymphoid leukemia cells was regulated by transcriptional and posttranscriptional modulation of CDK4 as well as Cyclin D2 gene expression under the control of BCR-ABL probably through the PI3K pathway. Consistently, the gene expression level of Cyclin D2 in Ph(+) lymphoid leukemia cells was significantly higher than that in Ph(-) lymphoid leukemia cells. Of note, three Ph(+) ALL cell lines having the T315I mutation also showed sensitivity to PD0332991. In a xenograft model, PD0332991, but not imatinib, suppressed dissemination of Ph(+) ALL having the T315I mutation and prolonged survival, demonstrating that this reagent would be a new therapeutic modality for relapsed CML-LC and Ph(+) ALL patients after treatment with tyrosine kinase inhibitors. PMID:26637365

  3. Combination treatment with proteasome inhibitors and antiestrogens has a synergistic effect mediated by p21WAF1 in estrogen receptor-positive breast cancer.

    Science.gov (United States)

    Maynadier, Marie; Basile, Ilaria; Gallud, Audrey; Gary-Bobo, Magali; Garcia, Marcel

    2016-08-01

    Although antiestrogens significantly improve the survival of patients with ER-positive breast cancer, therapeutic resistance remains a major limitation. The combinatorial use of antiestrogen with other therapies was proposed to increase their efficiency and more importantly, to prevent or delay the resistance phenomenon. In the present study, we addressed their combined effects with proteasome inhibitors (PIs). The effects of antiestrogens (hydroxyl-tamoxifen, raloxifen and fulvestrant) currently used in endocrine therapy were tested in combination with PIs, bortezomib or MG132, on the growth of three ER-positive breast cancer cell lines and in two cellular models of acquired antiestrogen resistance. When compared to single treatments, these combined treatments were significantly more effective in preventing the growth of the cell lines. The regulation of key cell cycle proteins, the cyclin-dependent kinase inhibitors, p21WAF1 and p27KIP1, were also studied. Bortezomib and MG132 drastically increased p21WAF1 expression through elevation of its mRNA concentration. Notably, p27KIP1 regulation was quite different from that of p21WAF1. Furthermore, the effect of bortezomib in combination with antiestrogen was evaluated on antiestrogen-resistant cell lines. The growth of two antiestrogen-resistant cell lines appeared responsive to proteasome inhibition and was strongly decreased by a combined therapy with an antiestrogen. Collectively, these findings provide new perspectives for the use of PIs in combination with endocrine therapies for breast cancer and possibly to overcome acquired hormonal resistance. PMID:27373750

  4. Free-energy-based methods for binding profile determination in a congeneric series of CDK2 inhibitors.

    Science.gov (United States)

    Fidelak, Jérémy; Juraszek, Jarek; Branduardi, Davide; Bianciotto, Marc; Gervasio, Francesco Luigi

    2010-07-29

    Free-energy pathway methods show great promise in computing the mode of action and the free energy profile associated with the binding of small molecules with proteins, but are generally very computationally demanding. Here we apply a novel approach based on metadynamics and path collective variables. We show that this combination is able to find an optimal reaction coordinate and the free energy profile of binding with explicit solvent and full flexibility, while minimizing human intervention and computational costs. We apply it to predict the binding affinity of a congeneric series of 5 CDK2 inhibitors. The predicted binding free energy profiles are in accordance with experiment. PMID:20593892

  5. Free Energy Analysis of CDK2-Inhibitor Interaction%CDK2-抑制剂结合自由能计算

    Institute of Scientific and Technical Information of China (English)

    蒋勇军; 曾敏; 周先波; 邹建卫; 俞庆森

    2004-01-01

    细胞周期蛋白依赖性激酶Ⅱ(cyclin-dependent kinase 2,CDK2)是一种重要的治疗癌症的靶标.本文中采用分子动力学取样,运用MM-PBSA/GBSA两种方法计算了CDK2-NU6102复合物的绝对结合自由能.通过能量分解的方法考察了CDK2大分子主要残基与配体NU6102之间的相互作用和识别.

  6. Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation

    Directory of Open Access Journals (Sweden)

    Dubrovsky Larisa

    2008-03-01

    Full Text Available Abstract It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.

  7. X-rays Induce Dose-dependent and Cell Cycle-independent Accumulation of p21sdi1/WAF1

    OpenAIRE

    Tsuyama, Naohiro; Ide, Toshinori; Noda, Asao; Iwamoto, Keisuke S.; Mizuno, Terumi; Kyoizumi, Seishi; Seyama, Toshio

    2001-01-01

    Cell cycle arrest at the G1 checkpoint is governed by a function ofwild-typep53. We assessed the behavior of the sdi1 gene, which codes for a 21kDa 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 p21sdi1/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 p21sdi1/WAF1 was...

  8. Effect of mimetic CDK9 inhibitors on HIV-1 activated transcription

    OpenAIRE

    Van Duyne, Rachel; Guendel, Irene; Jaworski, Elizabeth; Sampey, Gavin; Klase, Zachary; Chen, Hao; Zeng, Chen; Kovalskyy, Dmytro; el Kouni, Mahmoud H.; Lepene, Benjamin; Patanarut, Alexis; Nekhai, Sergei; Price, David H; Kashanchi, Fatah

    2012-01-01

    Potent antiretroviral therapy (ART) has transformed HIV-1 infection into a chronic manageable disease; however drug resistance remains a common problem that limits the effectiveness and clinical benefits of this type of treatment. The discovery of viral reservoirs in the body, in which HIV-1 may persist, has helped to explain why therapeutic eradication of HIV-1 has proved so difficult. In the current study we utilized a combination of structure based analysis of Cyclin/CDK complexes with our...

  9. 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.

  10. Apoptosis, cell proliferation and modulation of cyclin-dependent kinase inhibitor p21(cip1) in vascular remodelling during vein arterialization in the rat.

    Science.gov (United States)

    Borin, Thaiz Ferraz; Miyakawa, Ayumi Aurea; Cardoso, Leandro; de Figueiredo Borges, Luciano; Gonçalves, Giovana Aparecida; Krieger, Jose Eduardo

    2009-06-01

    Neo-intima development and atherosclerosis limit long-term vein graft use for revascularization of ischaemic tissues. Using a rat model, which is technically less challenging than smaller rodents, we provide evidence that the temporal morphological, cellular, and key molecular events during vein arterialization resemble the human vein graft adaptation. Right jugular vein was surgically connected to carotid artery and observed up to 90 days. Morphometry demonstrated gradual thickening of the medial layer and important formation of neo-intima with deposition of smooth muscle cells (SMC) in the subendothelial layer from day 7 onwards. Transmission electron microscopy showed that SMCs switch from the contractile to synthetic phenotype on day 3 and new elastic lamellae formation occurs from day 7 onwards. Apoptosis markedly increased on day 1, while alpha-actin immunostaining for SMC almost disappeared by day 3. On day 7, cell proliferation reached the highest level and cellular density gradually increased until day 90. The relative magnitude of cellular changes was higher in the intima vs. the media layer (100 vs. 2 times respectively). Cyclin-dependent kinase inhibitors (CDKIs) p27(Kip1) and p16(INKA) remained unchanged, whereas p21(Cip1) was gradually downregulated, reaching the lowest levels by day 7 until day 90. Taken together, these data indicate for the first time that p21(Cip1) is the main CDKI protein modulated during the arterialization process the rat model of vein arterialization that may be useful to identify and validate new targets and interventions to improve the long-term patency of vein grafts. PMID:19563615

  11. Induction of p21(Waf1/Cip1) by garcinol via downregulation of p38-MAPK signaling in p53-independent H1299 lung cancer.

    Science.gov (United States)

    Yu, Sheng-Yung; Liao, Chiung-Ho; Chien, Ming-Hsien; Tsai, Tsung-Yu; Lin, Jen-Kun; Weng, Meng-Shih

    2014-03-01

    Garcinol, a polyisoprenylated benzophenone, from Garcinia indica fruit rind has possessed anti-inflammatory, antioxidant, antiproliferation, and anticancer activities. However, the anticancer mechanisms of garcinol in lung cancer were still unclear. Therefore, we examine the effects of garcinol on antiproliferation in human lung cancer cells. Treatments with garcinol for 24 h exhibited morphological changes and inhibited the proliferation of H460 (p53-wild type) and H1299 (p53-null) cells in dose- and time-dependent manners. Furthermore, a significant G1 cell cycle arrest was observed in a dose-dependent treatment after H1299 cells were exposed in garcinol, whereas garcinol induced apoptosis rather than cell cycle arrest in H460 cells. Moreover, cyclin-dependent kinase 2 (CDK2), cyclin-dependent kinase 4 (CDK4), cyclin D1, and cyclin D3 were decreased, although cyclin E and cyclin-dependent kinase 6 (CDK6) were increased in garcinol-treated H1299 cells. Meanwhile, the protein levels of CDK inhibitors p21(Waf1/Cip1) and p27(KIP1) also exhibited upregulation after garcinol treatments. The enhanced protein-associated level between p21(Waf1/Cip1) and CDK4/2 rather than p27(KIP1) and CDK4/2 was demonstrated in garcinol-treated cells. Additionally, knock-down p21(Waf1/Cip1) by specific siRNA competently prevented garcinol-induced G1 arrest. Besides, garcinol also inhibited ERK and p38-MAPK activations in time-dependent mode. The pretreatment with p38-MAPK inhibitor but not ERK inhibitor raised garcinol-induced G1 population cells. Co-treatment with p38-MAPK inhibitor and garcinol synergistically elevated cyclin E, p21(Waf1/Cip1), and p27(Kip1) expressions. Meanwhile, overexpression dominant negative p38-MAPK also enhanced garcinol-induced p21(Waf1/Cip1) expression in H1299 cells. Accordingly, our data suggested that garcinol induced G1 cell cycle arrest and apoptosis in lung cancer cells under different p53 statuses. The p53-independent G1 cell cycle arrest induced by

  12. Structural requirements of pyrido[2,3-d]pyrimidin-7-one as CDK4/D inhibitors: 2D autocorrelation, CoMFA and CoMSIA analyses.

    Science.gov (United States)

    Caballero, Julio; Fernández, Michael; González-Nilo, Fernando D

    2008-06-01

    2D autocorrelation, comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were undertaken for a series of pyrido[2,3-d]pyrimidin-7-ones to correlate cyclin-dependent kinase (CDK) cyclin D/CDK4 inhibition with 2D and 3D structural properties of 60 known compounds. QSAR models with considerable internal as well as external predictive ability were obtained. The relevant 2D autocorrelation descriptors for modeling CDK4/D inhibitory activity were selected by linear and nonlinear genetic algorithms (GAs) using multiple linear regression (MLR) and Bayesian-regularized genetic neural network (BRGNN) approaches, respectively. Both models showed good predictive statistics; but BRGNN model enables better external predictions. A weight-based input ranking scheme and Kohonen self-organized maps (SOMs) were carried out to interpret the final net weights. The 2D autocorrelation space brings different descriptors for CDK4/D inhibition, and suggests the atomic properties relevant for the inhibitors to interact with CDK4/D active site. CoMFA and CoMSIA analyses were developed with a focus on interpretative ability using coefficient contour maps. CoMSIA produced significantly better results. The results indicate a strong correlation between the inhibitory activity of the modeled compounds and the electrostatic and hydrophobic fields around them. PMID:18468903

  13. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    International Nuclear Information System (INIS)

    Highlights: → p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. → p21 colocalized with the DSB marker γ-H2AX and the DSB sensor Ku80. → Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. → Accumulation activity of p21 was conserved among human and animal cells. → p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker γ-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful tool as a detection marker of DNA

  14. Accumulation of p21 proteins at DNA damage sites independent of p53 and core NHEJ factors following irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Koike, Manabu, E-mail: m_koike@nirs.go.jp [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Yutoku, Yasutomo [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan); Graduate School of Science, Chiba University, Chiba 263-8522 (Japan); Koike, Aki [DNA Repair Gene Res., National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555 (Japan)

    2011-08-19

    Highlights: {yields} p21 accumulated rapidly at laser-irradiated sites via its C-terminal region. {yields} p21 colocalized with the DSB marker {gamma}-H2AX and the DSB sensor Ku80. {yields} Accumulation of p21 is dependent on PCNA, but not p53 and the NHEJ core factors. {yields} Accumulation activity of p21 was conserved among human and animal cells. {yields} p21 is a useful tool as a detection marker of DNA damaged sites. -- Abstract: The cyclin-dependent kinase (CDK) inhibitor p21 plays key roles in p53-dependent DNA-damage responses, i.e., cell cycle checkpoints, senescence, or apoptosis. p21 might also play a role in DNA repair. p21 foci arise at heavy-ion-irradiated DNA-double-strand break (DSB) sites, which are mainly repaired by nonhomologous DNA-end-joining (NHEJ). However, no mechanisms of p21 accumulation at double-strand break (DSB) sites have been clarified in detail. Recent works indicate that Ku70 and Ku80 are essential for the accumulation of other NHEJ core factors, e.g., DNA-PKcs, XRCC4 and XLF, and other DNA damage response factors, e.g., BRCA1. Here, we show that p21 foci arise at laser-irradiated sites in cells from various tissues from various species. The accumulation of EGFP-p21 was detected in not only normal cells, but also transformed or cancer cells. Our results also showed that EGFP-p21 accumulated rapidly at irradiated sites, and colocalized with the DSB marker {gamma}-H2AX and with the DSB sensor protein Ku80. On the other hand, the accumulation occurred in Ku70-, Ku80-, or DNA-PKcs-deficient cell lines and in human papillomavirus 18-positive cells, whereas the p21 mutant without the PCNA-binding region (EGFP-p21(1-146)) failed to accumulate at the irradiated sites. These findings suggest that the accumulation of p21, but not functional p53 and the NHEJ core factors, is dependent on PCNA. These findings also suggest that the accumulation activity of p21 at DNA damaged sites is conserved among human and animal cells, and p21 is a useful

  15. G{sub 1} arrest and down-regulation of cyclin E/cyclin-dependent kinase 2 by the protein kinase inhibitor staurosporine are dependent on the retinoblastoma protein in the bladder carcinoma cell line 5637

    Energy Technology Data Exchange (ETDEWEB)

    Schnier, J.B.; Nishi, K. [Univ. of California, Davis, CA (United States); Goodrich, D.W. [Univ. of Texas, Houston, TX (United States)] [and others

    1996-06-11

    The protein kinase inhibitor staurosporine has been shown to induce G{sub 1} phase arrest in normal cells but not in most transformed cells. Staurosporine did not induce G{sub 1} phase arrest in the bladder carcinoma cell line 5637 that lacks a functional retinoblastoma protein (pRB{sup {minus}}). However, when infected with a pRB-expressing retrovirus, these cells, now pRB{sup +} and pRB{sup {minus}} cells, cyclin D1-associated kinase activities were reduced on staurosporine treatment. In contrast, cylin-dependent kinase (CDK) 2 and cyclin E/CDK2 activities were inhibited only in pRB{sup +} cells. Staurosporine treatment did not cause reductions in the protein levels of CDK4, cyclin D1, CDK2, or cyclin E. The CDK inhibitor proteins p21{sup (Wafl/Cipl)} and p27{sup (Kipl}) levels increased in staurosporine-treated cells. Immunoprecipitation of CDK2, cyclin E, and p21 form staurosporine-treated pRB{sup +} cells revealed a 2.5- to 3-fold higher ratio of p21 bound to CDK2 compared with staurosporine-treated pRB cells. In pRB{sup +} cells, p21 was preferentially associated with Thr160 phosphorylated active CDK2. In pRB{sup {minus}} cells, however, p21 was bound preferentially to the unphosphorylated, inactive form of CDK2 even though the phosphorylated form was abundant. This is the first evidence suggesting that G{sub 1} arrest by 4 nM staurosporine is dependent on a functional pRB protein. Cell cycle arrest at the pRB-dependent checkpoint may prevent activation of cyclin E/CDK2 by stabilizing its interaction with inhibitor proteins p21 and p27. 47 refs.

  16. The Establishment of a Hyperactive Structure Allows the Tumour Suppressor Protein p53 to Function through P-TEFb during Limited CDK9 Kinase Inhibition.

    Directory of Open Access Journals (Sweden)

    Thomas K Albert

    Full Text Available CDK9 is the catalytic subunit of positive elongation factor b (P-TEFb that controls the transition of RNA polymerase II (RNAPII into elongation. CDK9 inhibitors block mRNA synthesis and trigger activation of the stress-sensitive p53 protein. This in turn induces transcription of CDKN1A (p21 and other cell cycle control genes. It is presently unclear if and how p53 circumvents a general P-TEFb-requirement when it activates its target genes. Our investigations using a panel of specific inhibitors reason for a critical role of CDK9 also in the case of direct inhibition of the kinase. At the prototypic p21 gene, the activator p53 initially accumulates at the pre-bound upstream enhancer followed-with significant delay-by de novo binding to a secondary enhancer site within the first intron of p21. This is accompanied by recruitment of the RNAPII initiation machinery to both elements. ChIP and functional analyses reason for a prominent role of CDK9 itself and elongation factor complexes PAF1c and SEC involved in pause and elongation control. It appears that the strong activation potential of p53 facilitates gene activation in the situation of global repression of RNAPII transcription. The data further underline the fundamental importance of CDK9 for class II gene transcription.

  17. 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. PMID:27212103

  18. 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: 8.431, year: 2014

  19. 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.

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

    International Nuclear Information System (INIS)

    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 G2-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.

  1. FLT3 and CDK4/6 inhibitors: signaling mechanisms and tumor burden in subcutaneous and orthotopic mouse models of acute myeloid leukemia.

    Science.gov (United States)

    Zhang, Yaping; Hsu, Cheng-Pang; Lu, Jian-Feng; Kuchimanchi, Mita; Sun, Yu-Nien; Ma, Ji; Xu, Guifen; Zhang, Yilong; Xu, Yang; Weidner, Margaret; Huard, Justin; D'Argenio, David Z

    2014-12-01

    FLT3(ITD) subtype acute myeloid leukemia (AML) has a poor prognosis with currently available therapies. A number of small molecule inhibitors of FLT3 and/or CDK4/6 are currently under development. A more complete and quantitative understanding of the mechanisms of action of FLT3 and CDK4/6 inhibitors may better inform the development of current and future compounds that act on one or both of the molecular targets, and thus may lead to improved treatments for AML. In this study, we investigated in both subcutaneous and orthotopic AML mouse models, the mechanisms of action of three FLT3 and/or CDK4/6 inhibitors: AMG925 (Amgen), sorafenib (Bayer and Onyx), and quizartinib (Ambit Biosciences). A composite model was developed to integrate the plasma pharmacokinetics of these three compounds on their respective molecular targets, the coupling between the target pathways, as well as the resulting effects on tumor burden reduction in the subcutaneous xenograft model. A sequential modeling approach was used, wherein model structures and estimated parameters from upstream processes (e.g. PK, cellular signaling) were fixed for modeling subsequent downstream processes (cellular signaling, tumor burden). Pooled data analysis was employed for the plasma PK and cellular signaling modeling, while population modeling was applied to the tumor burden modeling. The resulting model allows the decomposition of the relative contributions of FLT3(ITD) and CDK4/6 inhibition on downstream signaling and tumor burden. In addition, the action of AMG925 on cellular signaling and tumor burden was further studied in an orthotopic tumor mouse model more closely representing the physiologically relevant environment for AML. PMID:25326874

  2. Impact of roscovitine, a selective CDK inhibitor, on cancer cells: bi-functionality increases its therapeutic potential.

    Science.gov (United States)

    Wesierska-Gadek, Józefa; Borza, Andreea; Komina, Oxana; Maurer, Margarita

    2009-01-01

    Increased expression and activity of proteins driving cell cycle progression as well as inactivation of endogenous inhibitors of cyclin-dependent kinases (CDKs) enhance the proliferative potential of cells. Escape of cells during malignant transformation from the proper cell cycle control rendering them independent from growth factors provides rationale for therapeutic targeting of CDKs. Exposure of rapidly growing human MCF-7 breast cancer and HeLa cervix cancer cells to roscovitine (ROSC), a selective inhibitor of CDKs, inhibits their proliferation by induction of cell cycle arrest and/or apoptosis. The outcome strongly depends on the intrinsic traits of the tumor cells, on their cell cycle status prior to the onset of treatment and also on ROSC concentration. At lower dose ROSC primarily inhibits the cell cycle-related CDKs resulting in a strong cell cycle arrest. Interestingly, ROSC arrests asynchronously growing cells at the G(2)/M transition irrespective of the status of their restriction checkpoint. However, the exposure of cancer cells synchronized after serum starvation in the late G(1) phase results in a transient G(1) arrest only in cells displaying the intact G(1)/S checkpoint. At higher dosage ROSC triggers apoptosis. In HeLa cells inhibition of the activity of CDK7 and, in consequence, that of RNA polymerase II is a major event that facilitates the initiation of caspase-dependent apoptosis. In contrast, in the caspase-3-deficient MCF-7 breast cancer cells ROSC induces apoptosis by a p53-dependent pathway. HIPK2-mediated activation of the p53 transcription factor by phosphorylation at Ser46 results in upregulation of p53AIP1 protein. This protein after de novo synthesis and translocation into the mitochondria promotes depolarization of the mitochondrial membrane. PMID:19724778

  3. Selective CDK inhibitors:promising candidates for future clinical traumatic brain injury trials

    Institute of Scientific and Technical Information of China (English)

    Shruti V.Kabadi; Alan I.Faden

    2014-01-01

    Traumatic brain injury induces secondary injury that contributes to neuroinlfammation, neuronal loss, and neurological dysfunction. One important injury mechanism is cell cycle activation which causes neuronal apoptosis and glial activation. The neuroprotective effects of both non-selective (Flavopiridol) and selective (Roscovitine and CR-8) cyclin-dependent kinase inhibitors have been shown across multiple experimental traumatic brain injury models and species. Cyclin-depen-dent kinaseinhibitors, administered as a single systemic dose up to 24 hours after traumatic brain injury, provide strong neuroprotection-reducing neuronal cell death, neuroinflammation and neurological dysfunction. Given their effectiveness and long therapeutic window, cyclin-depen-dent kinase inhibitors appear to be promising candidates for clinical traumatic brain injury trials.

  4. 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

    2014-12-01

    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 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. PMID:25490451

  5. 2,6,8,9-Tetrasubstituted Purines as New CDK1 Inhibitors

    Czech Academy of Sciences Publication Activity Database

    Moravec, Jiří; Kryštof, Vladimír; Hanuš, Jan; Havlíček, Libor; Moravcová, Daniela; Fuksová, K.; Kuzma, Marek; Lenobel, René; Otyepka, M.; Strnad, Miroslav

    2003-01-01

    Roč. 13, č. 18 (2003), s. 2993ů2996. ISSN 0960-894X R&D Projects: GA ČR GA301/02/0475; GA AV ČR KJB6137301 Institutional research plan: CEZ:AV0Z5020903; CEZ:AV0Z5038910 Keywords : Purine inhibitors * cyclin-dependent kinases * roscovitine Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.182, year: 2003

  6. 123I-labeled HIV-1 tat peptide radioimmunoconjugates are imported into the nucleus of human breast cancer cells and functionally interact in vitro and in vivo with the cyclin-dependent kinase inhibitor, p21WAF-1/Cip-1

    International Nuclear Information System (INIS)

    To evaluate the internalization and nuclear translocation of 123I-tat-peptide radioimmunoconjugates in MDA-MB-468 breast cancer cells and their ability to interact with the cyclin-dependent kinase inhibitor, p21WAF-1/Cip-1. Peptides [GRKKRRQRRRPPQGYGC] harboring the nuclear-localizing sequence from HIV tat domain were conjugated to anti-p21WAF-1/Cip-1 antibodies. Immunoreactivity was assessed by Western blot using lysate from MDA-MB-468 cells exposed to EGF to induce p21WAF-1/Cip-1. Internalization and nuclear translocation were measured. The ability of tat-anti-p21WAF-1/Cip-1 to block G1-S phase arrest in MDA-MB-468 cells caused by EGF-induced p21WAF-1/Cip-1 was evaluated. Tumor and normal tissue uptake were determined at 48 h p.i. in athymic mice implanted s.c. with MDA-MB-468 xenografts injected intratumorally with EGF. There was 13.4±0.2% of radioactivity internalized by MDA-MB-468 cells incubated with 123I-tat-anti-p21WAF-1/Cip-1 and 34.6±3.1% imported into the nucleus. Tat-anti-p21WAF-1/Cip-1(8 μM) decreased the proportion of EGF-treated cells in G1 phase from 81.9±0.7% to 46.1±0.7% (p1 phase fraction to that of unexposed cells (25.8±0.2%). Non-specific tat-mouse IgG did not block EGF-induced G1-S phase arrest. Tumor uptake of radioactivity was higher in mice injected with EGF to induce p21WAF-1/Cip-1 than in mice not receiving EGF (3.1±0.4% versus 1.8±0.2% ID/g; p=0.04). Western blot analysis of tumors revealed a threefold increase in the p21WAF-1/Cip-1/β-actin ratio. We conclude that intracellular and nuclear epitopes in cancer cells can be functionally targeted with tat-radioimmunoconjugates to exploit many more epitopes for imaging and radiotherapeutic applications than have previously been accessible. (orig.)

  7. A Novel High-Throughput 3D Screening System for EMT Inhibitors: A Pilot Screening Discovered the EMT Inhibitory Activity of CDK2 Inhibitor SU9516.

    Science.gov (United States)

    Arai, Kazuya; Eguchi, Takanori; Rahman, M Mamunur; Sakamoto, Ruriko; Masuda, Norio; Nakatsura, Tetsuya; Calderwood, Stuart K; Kozaki, Ken-Ichi; Itoh, Manabu

    2016-01-01

    Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-β promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-βreceptor I (TGFβR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-β-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFβR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor

  8. CDK1 structures reveal conserved and unique features of the essential cell cycle CDK

    Science.gov (United States)

    Brown, Nicholas R.; Korolchuk, Svitlana; Martin, Mathew P.; Stanley, Will A.; Moukhametzianov, Rouslan; Noble, Martin E. M.; Endicott, Jane A.

    2015-04-01

    CDK1 is the only essential cell cycle CDK in human cells and is required for successful completion of M-phase. It is the founding member of the CDK family and is conserved across all eukaryotes. Here we report the crystal structures of complexes of CDK1-Cks1 and CDK1-cyclin B-Cks2. These structures confirm the conserved nature of the inactive monomeric CDK fold and its ability to be remodelled by cyclin binding. Relative to CDK2-cyclin A, CDK1-cyclin B is less thermally stable, has a smaller interfacial surface, is more susceptible to activation segment dephosphorylation and shows differences in the substrate sequence features that determine activity. Both CDK1 and CDK2 are potential cancer targets for which selective compounds are required. We also describe the first structure of CDK1 bound to a potent ATP-competitive inhibitor and identify aspects of CDK1 structure and plasticity that might be exploited to develop CDK1-selective inhibitors.

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

    Directory of Open Access Journals (Sweden)

    Hemant Varma

    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.

  10. Epstein-Barr virus nuclear antigen 3A promotes cellular proliferation by repression of the cyclin-dependent kinase inhibitor p21WAF1/CIP1.

    Directory of Open Access Journals (Sweden)

    Melissa L Tursiella

    2014-10-01

    Full Text Available Latent infection by Epstein-Barr virus (EBV is highly associated with the endemic form of Burkitt lymphoma (eBL, which typically limits expression of EBV proteins to EBNA-1 (Latency I. Interestingly, a subset of eBLs maintain a variant program of EBV latency - Wp-restricted latency (Wp-R - that includes expression of the EBNA-3 proteins (3A, 3B and 3C, in addition to EBNA-1. In xenograft assays, Wp-R BL cell lines were notably more tumorigenic than their counterparts that maintain Latency I, suggesting that the additional latency-associated proteins expressed in Wp-R influence cell proliferation and/or survival. Here, we evaluated the contribution of EBNA-3A. Consistent with the enhanced tumorigenic potential of Wp-R BLs, knockdown of EBNA-3A expression resulted in abrupt cell-cycle arrest in G0/G1 that was concomitant with conversion of retinoblastoma protein (Rb to its hypophosphorylated state, followed by a loss of Rb protein. Comparable results were seen in EBV-immortalized B lymphoblastoid cell lines (LCLs, consistent with the previous observation that EBNA-3A is essential for sustained growth of these cells. In agreement with the known ability of EBNA-3A and EBNA-3C to cooperatively repress p14(ARF and p16(INK4a expression, knockdown of EBNA-3A in LCLs resulted in rapid elevation of p14(ARF and p16I(NK4a. By contrast, p16(INK4a was not detectably expressed in Wp-R BL and the low-level expression of p14(ARF was unchanged by EBNA-3A knockdown. Amongst other G1/S regulatory proteins, only p21(WAF1/CIP1, a potent inducer of G1 arrest, was upregulated following knockdown of EBNA-3A in Wp-R BL Sal cells and LCLs, coincident with hypophosphorylation and destabilization of Rb and growth arrest. Furthermore, knockdown of p21(WAF1/CIP1 expression in Wp-R BL correlated with an increase in cellular proliferation. This novel function of EBNA-3A is distinct from the functions previously described that are shared with EBNA-3C, and likely contributes to

  11. 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 ostatní: 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

  12. 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

  13. 1α,25 dihydroxi-vitamin D3 modulates CDK4 and CDK6 expression and localization

    International Nuclear Information System (INIS)

    We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)2-vitamin D3 [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) p21Waf1/Cip1 and p27Kip1 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-localizates with VDR after 1,25D

  14. 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.

  15. Virtual screening of and in vitro activity study on allosteric small-molecule CDK2 inhibitors%CDK2别构小分子抑制剂的虚拟筛选和体外活性研究

    Institute of Scientific and Technical Information of China (English)

    邵媛媛; 张璐; 沈瑛; 张健

    2016-01-01

    目的 根据CDK2的晶体结构(PDB ID:3PXF),在已验证的别构口袋处,拟筛选出CDK2新型别构小分子抑制剂.方法 通过计算机辅助药物设计方法,基于CDK2蛋白晶体别构位点进行虚拟筛选,综合分析化合物与CDK2的作用模式;构建CDK2体外激酶活性检测体系,对化合物进行初步的体外生物活性研究.结果 虚拟筛选得到打分前1 000名的化合物,最终挑选并购买10个候选化合物.其中,化合物S2和S5表现出较好的抑制效果,在100 μmol/L的浓度下对CDK2活性的抑制率分别为57.59%和41.64%.结论 综合利用虚拟筛选、结构分析以及生物活性测试,筛选出具有明显的CDK2抑制活性的先导化合物S2和S5,为设计开发新型的CDK2别构小分子抑制剂奠定了基础.

  16. Loss of CDKN2A expression is a frequent event in primary invasive melanoma and correlates with sensitivity to the CDK4/6 inhibitor PD0332991 in melanoma cell lines.

    Science.gov (United States)

    Young, Richard J; Waldeck, Kelly; Martin, Claire; Foo, Jung H; Cameron, Donald P; Kirby, Laura; Do, Hongdo; Mitchell, Catherine; Cullinane, Carleen; Liu, Wendy; Fox, Stephen B; Dutton-Regester, Ken; Hayward, Nicholas K; Jene, Nicholas; Dobrovic, Alexander; Pearson, Richard B; Christensen, James G; Randolph, Sophia; McArthur, Grant A; Sheppard, Karen E

    2014-07-01

    We have investigated the potential for the p16-cyclin D-CDK4/6-retinoblastoma protein pathway to be exploited as a therapeutic target in melanoma. In a cohort of 143 patients with primary invasive melanoma, we used fluorescence in situ hybridization to detect gene copy number variations (CNVs) in CDK4, CCND1, and CDKN2A and immunohistochemistry to determine protein expression. CNVs were common in melanoma, with gain of CDK4 or CCND1 in 37 and 18% of cases, respectively, and hemizygous or homozygous loss of CDKN2A in 56%. Three-quarters of all patients demonstrated a CNV in at least one of the three genes. The combination of CCND1 gain with either a gain of CDK4 and/or loss of CDKN2A was associated with poorer melanoma-specific survival. In 47 melanoma cell lines homozygous loss, methylation or mutation of CDKN2A gene or loss of protein (p16(INK) (4A) ) predicted sensitivity to the CDK4/6 inhibitor PD0332991, while RB1 loss predicted resistance. PMID:24495407

  17. Binding of the potential antitumour agent indirubin-5-sulphonate at the inhibitor site of rabbit muscle glycogen phosphorylase b. Comparison with ligand binding to pCDK2-cyclin A complex.

    Science.gov (United States)

    Kosmopoulou, Magda N; Leonidas, Demetres D; Chrysina, Evangelia D; Bischler, Nicolas; Eisenbrand, Gerhard; Sakarellos, Constantinos E; Pauptit, Richard; Oikonomakos, Nikos G

    2004-06-01

    The binding of indirubin-5-sulphonate (E226), a potential anti-tumour agent and a potent inhibitor (IC(50) = 35 nm) of cyclin-dependent kinase 2 (CDK2) and glycogen phosphorylase (GP) has been studied by kinetic and crystallographic methods. Kinetic analysis revealed that E226 is a moderate inhibitor of GPb (K(i) = 13.8 +/- 0.2 micro m) and GPa (K(i) = 57.8 +/- 7.1 micro m) and acts synergistically with glucose. To explore the molecular basis of E226 binding we have determined the crystal structure of the GPb/E226 complex at 2.3 A resolution. Structure analysis shows clearly that E226 binds at the purine inhibitor site, where caffeine and flavopiridol also bind [Oikonomakos, N.G., Schnier, J.B., Zographos, S.E., Skamnaki, V.T., Tsitsanou, K.E. & Johnson, L.N. (2000) J. Biol. Chem.275, 34566-34573], by intercalating between the two aromatic rings of Phe285 and Tyr613. The mode of binding of E226 to GPb is similar, but not identical, to that of caffeine and flavopiridol. Comparative structural analyses of the GPb-E226, GPb-caffeine and GPb-flavopiridol complex structures reveal the structural basis of the differences in the potencies of the three inhibitors and indicate binding residues in the inhibitor site that can be exploited to obtain more potent inhibitors. Structural comparison of the GPb-E226 complex structure with the active pCDK2-cyclin A-E226 complex structure clearly shows the different binding modes of the ligand to GPb and CDK2; the more extensive interactions of E226 with the active site of CDK2 may explain its higher affinity towards the latter enzyme. PMID:15153119

  18. Dual Targeting of CDK4 and ARK5 Using a Novel Kinase Inhibitor ON123300 Exerts Potent Anticancer Activity against Multiple Myeloma.

    Science.gov (United States)

    Perumal, Deepak; Kuo, Pei-Yu; Leshchenko, Violetta V; Jiang, Zewei; Divakar, Sai Krishna Athaluri; Cho, Hearn Jay; Chari, Ajai; Brody, Joshua; Reddy, M V Ramana; Zhang, Weijia; Reddy, E Premkumar; Jagannath, Sundar; Parekh, Samir

    2016-03-01

    Multiple myeloma is a fatal plasma cell neoplasm accounting for over 10,000 deaths in the United States each year. Despite new therapies, multiple myeloma remains incurable, and patients ultimately develop drug resistance and succumb to the disease. The response to selective CDK4/6 inhibitors has been modest in multiple myeloma, potentially because of incomplete targeting of other critical myeloma oncogenic kinases. As a substantial number of multiple myeloma cell lines and primary samples were found to express AMPK-related protein kinase 5(ARK5), a member of the AMPK family associated with tumor growth and invasion, we examined whether dual inhibition of CDK4 and ARK5 kinases using ON123300 results in a better therapeutic outcome. Treatment of multiple myeloma cell lines and primary samples with ON123300 in vitro resulted in rapid induction of cell-cycle arrest followed by apoptosis. ON123300-mediated ARK5 inhibition or ARK5-specific siRNAs resulted in the inhibition of the mTOR/S6K pathway and upregulation of the AMPK kinase cascade. AMPK upregulation resulted in increased SIRT1 levels and destabilization of steady-state MYC protein. Furthermore, ON123300 was very effective in inhibiting tumor growth in mouse xenograft assays. In addition, multiple myeloma cells sensitive to ON123300 were found to have a unique genomic signature that can guide the clinical development of ON123300. Our study provides preclinical evidence that ON123300 is unique in simultaneously inhibiting key oncogenic pathways in multiple myeloma and supports further development of ARK5 inhibition as a therapeutic approach in multiple myeloma. PMID:26873845

  19. The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

    Directory of Open Access Journals (Sweden)

    Pumfery Anne

    2004-04-01

    Full Text Available Abstract Background The human T-cell leukemia virus type 1 (HTLV-1 Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells. Results To further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4. Conclusion These data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.

  20. Preclinical Characterization of G1T28: A Novel CDK4/6 Inhibitor for Reduction of Chemotherapy-Induced Myelosuppression.

    Science.gov (United States)

    Bisi, John E; Sorrentino, Jessica A; Roberts, Patrick J; Tavares, Francis X; Strum, Jay C

    2016-05-01

    Chemotherapy-induced myelosuppression continues to represent the major dose-limiting toxicity of cytotoxic chemotherapy, which can be manifested as neutropenia, lymphopenia, anemia, and thrombocytopenia. As such, myelosuppression is the source of many of the adverse side effects of cancer treatment including infection, sepsis, bleeding, and fatigue, thus resulting in the need for hospitalizations, hematopoietic growth factor support, and transfusions (red blood cells and/or platelets). Moreover, clinical concerns raised by myelosuppression commonly lead to chemotherapy dose reductions, therefore limiting therapeutic dose intensity, and reducing the antitumor effectiveness of the treatment. Currently, the only course of treatment for myelosuppression is growth factor support which is suboptimal. These treatments are lineage specific, do not protect the bone marrow from the chemotherapy-inducing cytotoxic effects, and the safety and toxicity of each agent is extremely specific. Here, we describe the preclinical development of G1T28, a novel potent and selective CDK4/6 inhibitor that transiently and reversibly regulates the proliferation of murine and canine bone marrow hematopoietic stem and progenitor cells and provides multilineage protection from the hematologic toxicity of chemotherapy. Furthermore, G1T28 does not decrease the efficacy of cytotoxic chemotherapy on RB1-deficient tumors. G1T28 is currently in clinical development for the reduction of chemotherapy-induced myelosuppression in first- and second-line treatment of small-cell lung cancer. Mol Cancer Ther; 15(5); 783-93. ©2016 AACR. PMID:26826116

  1. 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

  2. p21与周期蛋白依赖性激酶2在Bowen病皮损中的表达%Expression of p21 and Cyclin-dependent Kinase 2 in Bowen's Disease

    Institute of Scientific and Technical Information of China (English)

    尤德渊; 蔡丽敏; 尤海燕

    2003-01-01

    目的探讨细胞周期调节蛋白p21及周期蛋白依赖性激酶2(CDK2)在Bowen病中的表达和意义.方法采用链霉亲和素-过氧化物酶法检测28例Bowen病和10例正常人皮肤中p21CDK2的表达和分布.结果p21CDK2在Bowen病中均为高表达,而10例正常人皮肤标本中表皮均未见表达.28例Bowen病标本,其中22例(78.6%)呈p21阳性染色,26例(92.9%)呈CDK2阳性染色,两者表达水平呈正相关(r=0.84,P<0.001).结论Bowen病中肿瘤细胞的增殖能力高于正常细胞,可表达高水平促增殖因子CDK2.p21在Bowen病中的高表达可能与肿瘤细胞的分化有关.

  3. Frequent amplification of CENPF, GMNN and CDK13 genes in hepatocellular carcinomas.

    Directory of Open Access Journals (Sweden)

    Hye-Eun Kim

    Full Text Available Genomic changes frequently occur in cancer cells during tumorigenesis from normal cells. Using the Illumina Human NS-12 single-nucleotide polymorphism (SNP chip to screen for gene copy number changes in primary hepatocellular carcinomas (HCCs, we initially detected amplification of 35 genes from four genomic regions (1q21-41, 6p21.2-24.1, 7p13 and 8q13-23. By integrated screening of these genes for both DNA copy number and gene expression in HCC and colorectal cancer, we selected CENPF (centromere protein F/mitosin, GMNN (geminin, DNA replication inhibitor, CDK13 (cyclin-dependent kinase 13, and FAM82B (family with sequence similarity 82, member B as common cancer genes. Each gene exhibited an amplification frequency of ~30% (range, 20-50% in primary HCC (n = 57 and colorectal cancer (n = 12, as well as in a panel of human cancer cell lines (n = 70. Clonogenic and invasion assays of NIH3T3 cells transfected with each of the four amplified genes showed that CENPF, GMNN, and CDK13 were highly oncogenic whereas FAM82B was not. Interestingly, the oncogenic activity of these genes (excluding FAM82B was highly correlated with gene-copy numbers in tumor samples (correlation coefficient, r>0.423, indicating that amplifications of CENPF, GMNN, and CDK13 genes are tightly linked and coincident in tumors. Furthermore, we confirmed that CDK13 gene copy number was significantly associated with clinical onset age in patients with HCC (P = 0.0037. Taken together, our results suggest that coincidently amplified CDK13, GMNN, and CENPF genes can play a role as common cancer-driver genes in human cancers.

  4. Molecular Modeling Studies of 4,5-Dihydro-1H-pyrazolo[4,3-h] quinazoline Derivatives as Potent CDK2/Cyclin A Inhibitors Using 3D-QSAR and Docking

    Directory of Open Access Journals (Sweden)

    Fa-Jun Song

    2010-09-01

    Full Text Available CDK2/cyclin A has appeared as an attractive drug targets over the years with diverse therapeutic potentials. A computational strategy based on comparative molecular fields analysis (CoMFA and comparative molecular similarity indices analysis (CoMSIA followed by molecular docking studies were performed on a series of 4,5-dihydro-1H-pyrazolo[4,3-h]quinazoline derivatives as potent CDK2/cyclin A inhibitors. The CoMFA and CoMSIA models, using 38 molecules in the training set, gave r2cv values of 0.747 and 0.518 and r2 values of 0.970 and 0.934, respectively. 3D contour maps generated by the CoMFA and CoMSIA models were used to identify the key structural requirements responsible for the biological activity. Molecular docking was applied to explore the binding mode between the ligands and the receptor. The information obtained from molecular modeling studies may be helpful to design novel inhibitors of CDK2/cyclin A with desired activity.

  5. 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

  6. 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)

    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

  7. Genetic Evidence for Functional Dependency of p18Ink4c on Cdk4

    OpenAIRE

    Pei, Xin-Hai; Bai, Feng; Tsutsui, Tateki; Kiyokawa, Hiroaki; Xiong, Yue

    2004-01-01

    The INK4 family of cyclin-dependent kinase (CDK) inhibitors negatively regulates cyclin D-dependent CDK4 and CDK6 and induces the growth-suppressive function of Rb family proteins. Mutations in the Cdk4 gene conferring INK4 resistance are associated with familial and sporadic melanoma in humans and result in a wide spectrum of tumors in mice, suggesting that INK4 is a major regulator of CDK4. Mice lacking the Cdk4 gene exhibit various defects in many organs associated with hypocellularity, wh...

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

    International Nuclear Information System (INIS)

    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

  9. Synergic effects of the cyclin-dependent kinase (CDK) inhibitor olomoucine and androgen-antagonist bicalutamide on prostatic cancer cell lines

    Czech Academy of Sciences Publication Activity Database

    Knillová, J.; Bouchal, J.; Hlobilková, A.; Strnad, Miroslav; Kolář, Z.

    2004-01-01

    Roč. 51, č. 5 (2004), s. 358-367. ISSN 0028-2685 R&D Projects: GA ČR(CZ) GA301/02/0475 Institutional research plan: CEZ:AV0Z5038910 Keywords : olomoucine * bikalutamid * prostate cancer * CDK Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.822, year: 2004

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

    International Nuclear Information System (INIS)

    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

  11. Identification of Candidate Cyclin-dependent kinase 1 (Cdk1) Substrates in Mitosis by Quantitative Phosphoproteomics.

    Science.gov (United States)

    Petrone, Adam; Adamo, Mark E; Cheng, Chao; Kettenbach, Arminja N

    2016-07-01

    Cyclin-dependent kinase 1 (Cdk1) is an essential regulator of many mitotic processes including the reorganization of the cytoskeleton, chromosome segregation, and formation and separation of daughter cells. Deregulation of Cdk1 activity results in severe defects in these processes. Although the role of Cdk1 in mitosis is well established, only a limited number of Cdk1 substrates have been identified in mammalian cells. To increase our understanding of Cdk1-dependent phosphorylation pathways in mitosis, we conducted a quantitative phosphoproteomics analysis in mitotic HeLa cells using two small molecule inhibitors of Cdk1, Flavopiridol and RO-3306. In these analyses, we identified a total of 24,840 phosphopeptides on 4,273 proteins, of which 1,215 phosphopeptides on 551 proteins were significantly reduced by 2.5-fold or more upon Cdk1 inhibitor addition. Comparison of phosphopeptide quantification upon either inhibitor treatment revealed a high degree of correlation (R(2) value of 0.87) between the different datasets. Motif enrichment analysis of significantly regulated phosphopeptides revealed enrichment of canonical Cdk1 kinase motifs. Interestingly, the majority of proteins identified in this analysis contained two or more Cdk1 inhibitor-sensitive phosphorylation sites, were highly connected with other candidate Cdk1 substrates, were enriched at specific subcellular structures, or were part of protein complexes as identified by the CORUM database. Furthermore, candidate Cdk1 substrates were enriched in G2 and M phase-specific genes. Finally, we validated a subset of candidate Cdk1 substrates by in vitro kinase assays. Our findings provide a valuable resource for the cell signaling and mitosis research communities and greatly increase our knowledge of Cdk1 substrates and Cdk1-dependent signaling pathways. PMID:27134283

  12. Germ Line Transmission of the Cdk4R24C Mutation Facilitates Tumorigenesis and Escape from Cellular Senescence

    OpenAIRE

    Rane, Sushil G; Cosenza, Stephen C.; Mettus, Richard V.; Reddy, E. Premkumar

    2002-01-01

    Mutations in CDK4 and its key kinase inhibitor p16INK4a have been implicated in the genesis and progression of familial human melanoma. The importance of the CDK4 locus in human cancer first became evident following the identification of a germ line CDK4-Arg24Cys (R24C) mutation, which abolishes the ability of CDK4 to bind to p16INK4a. To determine the role of the Cdk4R24C germ line mutation in the genesis of other cancer types, we introduced the R24C mutation in the Cdk4 locus of mice by usi...

  13. Euphol arrests breast cancer cells at the G1 phase through the modulation of cyclin D1, p21 and p27 expression.

    Science.gov (United States)

    Wang, Lin; Wang, Guiying; Yang, Dandan; Guo, Xudong; Xu, Yanxin; Feng, Bo; Kang, Jiuhong

    2013-10-01

    Euphorbia tirucalli is a long‑established treatment for a wide variety of cancers. However, the mechanism of its anticancer effect is yet to be elucidated. In the present study, we examined the anticancer effect of euphol, a tetracyclic triterpene alcohol isolated from the sap of Euphorbia tirucalli, in T47D human breast cancer cells. Following the treatment of cells with different doses of euphol for 24, 48 and 72 h, the cell proliferation, cell cycle, and mRNA and protein levels of cell cycle regulatory molecules were analyzed, respectively. Treatment of the cells with euphol resulted in decreased cell viability, which was accompanied by an accumulation of cells in the G1 phase. Further studies demonstrated that euphol treatment downregulated cyclin D1 expression and the hypophosphorylation of Rb. Furthermore, this effect was correlated with the downregulation of cyclin‑dependent kinase 2 (CDK2) expression and the upregulation of the CDK inhibitors p21 and p27. Reduced expression levels of cyclin A and B1 were also observed, corresponding to the decreased distribution of cells in the S and G2/M phases, respectively. These findings indicated that euphol is an active agent in Euphorbia tirucalli that exerts anticancer activity by arresting the cell cycle of cancer cells. PMID:23969579

  14. The Role of Cdk5 in Alzheimer's Disease.

    Science.gov (United States)

    Liu, Shu-Lei; Wang, Chong; Jiang, Teng; Tan, Lan; Xing, Ang; Yu, Jin-Tai

    2016-09-01

    Alzheimer's disease (AD) is known as the most fatal chronic neurodegenerative disease in adults along with progressive loss of memory and other cognitive function disorders. Cyclin-dependent kinase 5 (Cdk5), a unique member of the cyclin-dependent kinases (Cdks), is reported to intimately associate with the process of the pathogenesis of AD. Cdk5 is of vital importance in the development of CNS and neuron movements such as neuronal migration and differentiation, synaptic functions, and memory consolidation. However, when neurons suffer from pathological stimuli, Cdk5 activity becomes hyperactive and causes aberrant hyperphosphorylation of various substrates of Cdk5 like amyloid precursor protein (APP), tau and neurofilament, resulting in neurodegenerative diseases like AD. Deregulation of Cdk5 contributes to an array of pathological events in AD, ranging from formation of senile plaques and neurofibrillary tangles, synaptic damage, mitochondrial dysfunction to cell cycle reactivation as well as neuronal cell apoptosis. More importantly, an inhibition of Cdk5 activity with inhibitors such as RNA inference (RNAi) could protect from memory decline and neuronal cell loss through suppressing β-amyloid (Aβ)-induced neurotoxicity and tauopathies. This review will briefly describe the above-mentioned possible roles of Cdk5 in the physiological and pathological mechanisms of AD, further discussing recent advances and challenges in Cdk5 as a therapeutic target. PMID:26227906

  15. 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.

  16. INHIBITION STUDIES OF TERPENE BASED NATURAL PRODUCTS WITH CYCLIN-DEPENDENT KINASE 4 (CDK4 MIMIC CDK2

    Directory of Open Access Journals (Sweden)

    Dr. Sunil H. Ganatra et al

    2012-09-01

    Full Text Available Cyclin dependent kinases (CDKs are known as cell cycle regulators in eukaryotic cell cycle. Different CDKs (CDK2, CDK4 etc. are having structure homology among them. Using computer based molecular modeling tools, interactions between naturally occurring terpene based compounds with crystal structure of CDK4 mimic CDK2 enzyme having PDB ID : 1GII. Using In-silico techniques, the binding energies between terpene based compounds and receptor enzymes are calculated in the form of ΔG in kcal/mol. The reported binding energies for series of molecules are ranging from –5.35 to –13.20 kcal/mol. The negative docking energies and a few hydrogen bonds between selected ligands and receptor enzyme support the affinity of Terpene based compounds with CDK4 mimic CDK2 enzymes. It is also found out that those compounds having carbon atoms 30-31 interacts better with enzyme, whereas larger size compounds having carbon atoms higher than 40 show weak interactions. It is concluded that Tri-terpene class of compounds are the best CDK4 mimic CDK2 inhibitors.

  17. 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.

  18. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activation via loss of mitochondrial membrane potential.

    Science.gov (United States)

    Meeran, Syed M; Katiyar, Santosh K

    2007-05-01

    Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers. PMID:17437483

  19. Tissue Inhibitor of Matrix Metalloproteinases-1 Knockdown Suppresses the Proliferation of Human Adipose-Derived Stem Cells

    Science.gov (United States)

    Zhang, Peihua; Li, Jin; Qi, Yawei; Tang, Xudong; Duan, Jianfeng; Liu, Li; Wu, Zeyong; Liang, Jie; Li, Jiangfeng; Wang, Xian; Zeng, Guofang; Liu, Hongwei

    2016-01-01

    Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional matrix metalloproteinase, and it is involved in the regulation of cell proliferation and apoptosis in various cell types. However, little is known about the effect of TIMP-1 expression on the proliferation of adipose-derived stem cells (ADSCs). Therefore, TIMP-1 expression in the ADSCs was firstly detected by western blotting, and TIMP-1 gene was knocked down by lentivirus-mediated shRNA. Cell proliferation was then evaluated by MTT assay and Ki67 staining, respectively. Cell cycle progression was determined by flow cytometry. The changes of p51, p21, cyclin E, cyclin-dependent kinase 2 (CDK2), and P-CDK2 caused by TIMP-1 knockdown were detected by western blotting. The results indicated that ADSCs highly expressed TIMP-1 protein, and the knockdown of TIMP-1 inhibited cell proliferation and arrested cell cycle progression at G1 phase in the ADSCs possibly through the upregulation of p53, p21, and P-CDK2 protein levels and concurrent downregulation of cyclin E and CDK2 protein levels. These findings suggest that TIMP-1 works as a positive regulator of cell proliferation in ADSCs. PMID:27239203

  20. 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

  1. 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: 3.447, year: 2014

  2. Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities

    OpenAIRE

    Jorge Franco; Uthra Balaji; Elizaveta Freinkman; Agnieszka K. Witkiewicz; Erik S. Knudsen

    2016-01-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models, CDK4/6 inhibition had a variable effect on cell cycle but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was ass...

  3. Effect of CDK9 Inhibitor F200 on the Apoptosis of Human Breast Cancer MCF7 Cells%CDK9抑制剂F200对乳腺癌细胞MCF7凋亡的影响

    Institute of Scientific and Technical Information of China (English)

    侯贝; 罗楹; 陈文峻

    2014-01-01

    Objective This study aimed to investigate the effects of cell cycle dependent kinase inhibitor F200 on ap-optosis of human breast cancer cell MCF7. Methods MCF7 cells were cultured in RPMI-1640 medium with 0.01 mg·mL-1 human recombinant insulin and 10%fetal bovine serum. Cells were subcultured at exponential growth phase. 24 h later, cells were adherent to the plate and treatments were given according to groups setup, negative control (0.5%DMSO), posi-tive control (R-Roscovitine 5.66μM), F200 groups (F200 0.1μM, 0.71μM). At 48 h after treatment, cell morphological change of apoptosis was measured by TUNEL methods and the apoptosis rate was detected by flow cytometry. The expres-sion of PRAP was measured by Western Bolt. Results The TUNEL results showed that as the F200 concentration increased, the cell apoptosis features like cell pyknosis and dense granule in nucleus were significantly clearer. More cells were stained with DAB after treated with F200. The flow cytometry results showed 0.71μM F200 could induce 32.6%cell apoptosis. Moreover, the Western Blot results showed cleaved PARP was increased along with the increase of F200 concentration. Conclusion CDK inhibitor F200 could inhibit MCF7 cell growth and induce the cell apoptosis.%目的:研究细胞周期依赖性激酶(cell cycle dependent kinase,CDK)CDK9抑制剂F200对乳腺癌细胞MCF7凋亡的影响。方法 MCF7细胞培养于含0.01 mg·mL-1人重组胰岛素及10%胎牛血清的RPMI 1640培养液中,待对数生长期时接种细胞进行实验,24 h贴壁后给药:分为阴性对照组(0.5% DMSO)、阳性对照组(R-Roscovitine 5.66μM)及药物组(F2000.1μM,0.71μM),给药48 h后利用TUNEL法染色观察细胞凋亡DNA断裂情况及细胞凋亡形态学变化、流式细胞技术检测细胞的凋亡比率、免疫印迹法检测凋亡标志蛋白PARP的表达情况。结果 TUNEL结果显示,随着F200浓度增加,细胞出现明显的固缩变圆,细

  4. Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

    OpenAIRE

    Polier, G; Ding, J.; Konkimalla, B V; Eick, D; Ribeiro, N.; Köhler, R.; Giaisi, M; Efferth, T.; Desaubry, L; Krammer, P.H.; Li-Weber, M

    2011-01-01

    The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent ...

  5. 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

    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...... relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we...... suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2....

  6. 含氮查尔酮类细胞周期蛋白依赖性激酶抑制剂的合成及抗癌活性研究Ⅳ%Synthesis and anticancer activity study of nitrogen-containing chalcones as CDK inhibitors IV

    Institute of Scientific and Technical Information of China (English)

    李艳玲; 方浩; 徐文方

    2011-01-01

    The key role of CDKs in tumorigenesis have raised great interest for the development of CDK inhibitors as potential anticancer agents. Flavopiridol,a synthetic flavone,is the fist CDK inhibitors that entered clinical trial. And chalcones,as a branch of flavone,having important effects on cancer cell growth and proliferation. Herein, based on the structure of flavopiridol, eight novel chalcones analogs were synthesized by aldol condensation of a substituted acetophenone with various benzaldehyde, acetophenone was obtained by Hoesch reaction and methylation,chalcones was then converted target compounds by Mannich reaction with morpholine. The structures of target compounds were confirmed by IR,'H-NMR and ESI-MS,and their CDK1 inhibition as well as cytotoxicity activity against HCT116 were determined with flavopiridol as a positive control. The results showed that compounds 5a,5b,5c,5d,5f,5g and 5h exhibited higher CDK1 inhibition than flavopiridol,with the IC50 values of 63. 83,46.45,59. 70,48. 97,51. 40,52. 84,45. 70 nrnol·L-1 respectively, while flavopiridol was 64. 05 nmol·L-1 ,and compounds 5g and 5h showed higher cytotoxicity against HCT116 with the Icj,, values 2. 17,2. 80 μmol·L-1 respectively. The results showed that piperidine ring of flavopiridol can be replaced by Mannich base with morpholine ring.%目的 寻找活性更好的类黄酮细胞周期蛋白依赖激酶(CDKs)抑制剂.方法 利用Mannich反应制得8个查尔酮类黄酮.结果与结论 目标化合物的结构经1R、1 H-NMR、质谱确证,并测定了化合物对CDK1的抑制活性以及对HCT116肿瘤细胞的体外抗肿瘤活性,其中有7个化合物对CDK1抑制活性高于阳性对照flavopiridol,所有化合物对HCT116肿瘤细胞均显示出较强的抑制活性.

  7. The nitric oxide-sensitive p21Ras-ERK pathway mediates S-nitrosoglutathione-induced apoptosis

    International Nuclear Information System (INIS)

    p21Ras protein plays a critical role in cellular signaling that induces either cell cycle progression or apoptosis. Nitric oxide (NO) has been consistently reported to activate p21Ras through the redox sensitive cysteine residue (118). In this study, we demonstrated that the p21Ras-ERK pathway regulates THP-1 monocyte/macrophage apoptosis induced by S-nitrosoglutathione (SNOG). This was apparent from studies in THP-1 cells expressing NO-insensitive p21Ras (p21RasC118S) where the pro-apoptotic action of SNOG was almost abrogated. Three major MAP kinase pathways (ERK, JNK, and p38) that are downstream to p21Ras were investigated. It was observed that only the activation of ERK1/2 MAP kinases by SNOG in THP-1 cells was attributable to p21Ras. The inhibition of the ERK pathway by PD98059 markedly attenuated apoptosis in SNOG-treated THP-1 cells, but had a marginal effect on SNOG-treated THP-1 cells expressing NO-insensitive p21Ras. The inhibition of the JNK and p38 pathways by selective inhibitors had no marked effects on the percentage of apoptosis. The induction of p21Waf1 expression by SNOG was observed in THP-1 cells harboring mutant and wild-type p21Ras, however in cells expressing mutant Ras, the expression of p21Waf1 was significantly attenuated. The treatment of THP-1 cells expressing wild-type p21Ras with PD98059 resulted in significant attenuation of p21Waf1 expression. These results indicate that the redox sensitive p21Ras-ERK pathway plays a critical role in sensing and delivering the pro-apoptotic signaling mediated by SNOG

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

    International Nuclear Information System (INIS)

    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 (p21RasC118S). 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

  9. G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21Waf1 signaling.

    Science.gov (United States)

    Suvorova, Irina I; Grigorash, Bogdan B; Chuykin, Ilya A; Pospelova, Tatiana V; Pospelov, Valery A

    2016-01-01

    Mouse embryonic stem cells (mESCs) lack of G1 checkpoint despite that irradiation (IR) activates ATM/ATR-mediated DDR signaling pathway. The IR-induced p53 localizes in the nuclei and up-regulates p21/Waf1 transcription but that does not lead to accumulation of p21/Waf1 protein. The negative control of the p21Waf1 expression appears to occur at 2 levels of regulation. First, both p21/Waf1 gene transcription and the p21/Waf1 protein content increase in mESCs treated with histone-deacetylase inhibitors, implying its epigenetic regulation. Second, proteasome inhibitors cause the p21/Waf1 accumulation, indicating that the protein is a subject of proteasome-dependent degradation in ESСs. Then, the dynamics of IR-induced p21Waf1 protein show its accumulation at long-term time points (3 and 5 days) that coincides with an increase in the proportion of G1-phase cells, down-regulation of Oct4 and Nanog pluripotent gene transcription and activation of endoderm-specific genes sox17 and afp. In addition, nutlin-dependent stabilization of p53 in mESC was also accompanied by the accumulation of p21/Waf1 as well as restoration of G1 checkpoint and an onset of differentiation. Thus, the lack of functional p21/Waf1 is indispensable for maintaining self-renewal and pluripotency of mESCs. PMID:26636245

  10. Quinazolines as cyclin dependent kinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sielecki, Thais M.; Johnson, Tricia L.; Liu, Jie; Muckelbauer, Jodi K.; Grafstrom, Robert H.; Cox, Sarah; Boylan, John; Burton, Catherine R.; Chen, Haiying; Smallwood, Angela; Chang, Chong-Hwan; Boisclair, Michael; Benfield, Pamela A.; Trainor, George L.; Seitza, Steven P. (Dupont)

    2010-03-08

    Quinazolines have been identified as inhibitors of CDK4/D1 and CDK2/E. Aspects of the SAR were investigated using solution-phase, parallel synthesis. An X-ray crystal structure was obtained of quinazoline 51 bound in CDK2 and key interactions within the ATP binding pocket are defined.

  11. 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...... and inhibits DNA replication. Here, we show that p21WAF1/CIP1 binds to the regulatory beta-subunit of protein kinase CK2 but not to the catalytic alpha-subunit. Binding of p21WAF1/CIP1 down regulates the kinase activity of CK2 with respect to the phosphorylation of the beta-subunit of CK2, casein and...... the C-terminus of p53. This study demonstrates a new binding partner for the regulatory beta-subunit of protein kinase CK2 which regulates the activity of the holoenzyme....

  12. Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula

    2005-01-10

    Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with anti-mitotic and/or anti-neurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinity and specificity.

  13. Metabolic re-programming of pancreatic cancer mediated by CDK4/6 inhibition elicits unique vulnerabilities

    OpenAIRE

    Franco, Jorge; Balaji, Uthra; Freinkman, Elizaveta; Witkiewicz, Agnieszka K.; Knudsen, Erik S.

    2016-01-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models CDK4/6 inhibition had variable effect on cell cycle, but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was assoc...

  14. p21 suppresses inflammation and tumorigenesis on pRB-deficient stratified epithelia

    Science.gov (United States)

    Garín, Marina; Ruiz, Sergio; Santos, Mirentxu; Lorz, Corina; García-Escudero, Ramón; Martínez-Fernández, Mónica; Bravo, Ana; Fernández-Capetillo, Oscar; Segrelles, Carmen; Paramio, Jesús M

    2016-01-01

    The retinoblastoma gene product (pRb) controls proliferation and differentiation processes in stratified epithelia. Importantly, an in contrast to other tissues, Rb deficiency does not lead to spontaneous skin tumor formation. As the cyclin dependent kinase inhibitor p21 regulates proliferation and differentiation in the absence of pRb, we analyzed the consequences of deleting p21 in pRb-ablated stratified epithelia (hereafter pRbΔEpi;p21-/-). These mice display an enhancement of the phenotypic abnormalities observed in pRbΔEpi animals, indicating that p21 partially compensates pRb absence. Remarkably, pRbΔEpi;p21-/- mice show an acute skin inflammatory phenotype and develop spontaneous epithelial tumors, particularly affecting tongue and oral tissues. Biochemical analyses and transcriptome studies reveal changes affecting multiple pathways, including DNA damage and p53-dependent signaling responses. Comparative metagenomic analyses, together with the histopathological profiles, indicate that these mice constitute a faithful model for human head and neck squamous cell carcinomas. Collectively, our findings demonstrate that p21, in conjunction with pRb, plays a central role in regulating multiple epithelial processes and orchestrating specific tumor suppressor functions. PMID:24121270

  15. EIF3D silencing suppresses renal cell carcinoma tumorigenesis via inducing G2/M arrest through downregulation of Cyclin B1/CDK1 signaling.

    Science.gov (United States)

    Pan, Xiu-Wu; Chen, Lu; Hong, Yi; Xu, Dan-Feng; Liu, Xi; Li, Lin; Huang, Yi; Cui, Li-Ming; Gan, Si-Shun; Yang, Qi-Wei; Huang, Hai; Qu, Fa-Jun; Ye, Jian-Qing; Wang, Lin-Hui; Cui, Xin-Gang

    2016-06-01

    There are no effective therapies for advanced renal cell carcinoma (RCC), except for VEGFR inhibitors with only ~50% response rate. To identify novel targets and biomarkers for RCC is of great importance in treating RCC. In this study, we observed that eukaryotic initiation factor 3d (EIF3D) expression was significantly increased in RCC compared with paracarcinoma tissue using immunohistochemistry staining and western blot analysis. Furthermore, bioinformatics meta-analysis using ONCOMINE microarray datasets showed that EIF3D mRNA expressions in CCRCC tissue specimens were significantly higher than that in normal tissue specimens. In addition, RCC tissue microarray demonstrated that elevated EIF3D expression was positively correlated with TNM stage and tumor size. EIF3D silencing in human 786-O and ACHN CCRCC cell lines by RNA interference demonstrated that EIF3D knockdown obviously inhibited cell proliferation and colony formation, caused G2/M arrest through downregulation of Cyclin B1 and Cdk1 and upregulation of p21, and induced apoptosis shown by sub-G1 accumulation and RARP cleavage. Moreover, correlation analysis using ONCOMINE microarray datasets indicated that increased EIF3D mRNA expression was positively correlated to PCNA, Cyclin B1 and CDK1 mRNA expression in RCC. Collectively, these results provide reasonable evidences that EIF3D may function as a potential proto-oncogene that participates in the occurrence and progression of RCC. PMID:27035563

  16. Inhibition of CDK9 as a therapeutic strategy for inflammatory arthritis.

    Science.gov (United States)

    Hellvard, Annelie; Zeitlmann, Lutz; Heiser, Ulrich; Kehlen, Astrid; Niestroj, André; Demuth, Hans-Ulrich; Koziel, Joanna; Delaleu, Nicolas; Jan Potempa; Mydel, Piotr

    2016-01-01

    Rheumatoid arthritis is characterised by synovial inflammation and proliferation of fibroblast-like synoviocytes. The induction of apoptosis has long been proposed as a target for proliferative autoimmune diseases, and has further been shown to act as a successful treatment of experimental models of arthritis, such as collagen-induced arthritis. Here we examined the effects of specific oral small-molecule inhibitors of the transcription regulating cyclin-dependent kinase 9 on the development and progression of collagen-induced arthritis. DBA/1 mice were immunised with bovine collagen type II and treated orally with specific CDK9 inhibitors. The effects of CDK9 inhibition on RNA levels and protein expression, apoptosis induction, caspase activation and lymphocyte phenotype were further analysed. Mice showed a significant delay in disease onset and a reduction in disease severity following treatment with CDK9 inhibitors. Inhibiting CDK9 activity in peripheral blood mononuclear cells resulted in the loss of Mcl-1 expression at both the protein and RNA levels, along with a subsequent increase in apoptosis. CDK9 specific inhibitors may be a potential alternative treatment not only of cancer, but also for autoimmune- and inflammatory diseases. Taken together, these results show that transient inhibition of CDK9 induces apoptosis in leukocyte subsets and modulates the immune response. PMID:27511630

  17. 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

    2014-07-01

    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. PMID:24764564

  18. Atypical role of sprouty in p21 dependent inhibition of cell proliferation in colorectal cancer.

    Science.gov (United States)

    Zhang, Qiong; Shim, Katherine; Wright, Kevin; Jurkevich, Alexander; Khare, Sharad

    2016-09-01

    Sprouty (SPRY) appears to act as a tumor suppressor in cancer, whereas we reported that SPRY2 functions as a putative oncogene in colorectal cancer (CRC) [Oncogene, 2010, 29: 5241-5253]. In general, various studies established inhibition of cell proliferation by SPRY in cancer. The mechanisms by which SPRY regulates cell proliferation in CRC are investigated. We demonstrate, for the first time, suppression of SPRY2 augmented EGF-dependent oncogenic signaling, however, surprisingly decreased cell proliferation in colon cancer cells. Our data suggest that cell cycle inhibitor p21(WAF1/CIP1) transcriptional activity being regulated by SPRY2. Indeed, suppression of SPRY2 significantly increased p21(WAF1/CIP1) mRNA and protein expression as well as p21(WAF1/CIP1) promoter activity. Conversely, overexpressing SPRY2 triggered a decrease in p21(WAF1/CIP1) promoter activity. Concurrent down-regulation of both SPRY1 and SPRY2 also increased p21(WAF1/CIP1) expression in colon cancer cells. Increased nuclear localization of p21(WAF1/CIP1) in SPRY2 downregulated colon cancer cells may explain the inhibition of cell proliferation in colon cancer cells. Underscoring the biological relevance of these findings in SPRY1 and SPRY2 mutant mouse, recombination of floxed SPRY1 and SPRY2 alleles in mouse embryonic fibroblasts (MEFs) resulted in increased expression and nuclear localization of p21(WAF1/CIP1) and decreased cell proliferation. In CRC, the relationship of SPRY with p21 may provide unique strategies for cancer prevention and treatment. © 2015 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc. PMID:26293890

  19. Metabolic Reprogramming of Pancreatic Cancer Mediated by CDK4/6 Inhibition Elicits Unique Vulnerabilities.

    Science.gov (United States)

    Franco, Jorge; Balaji, Uthra; Freinkman, Elizaveta; Witkiewicz, Agnieszka K; Knudsen, Erik S

    2016-02-01

    Due to loss of p16ink4a in pancreatic ductal adenocarcinoma (PDA), pharmacological suppression of CDK4/6 could represent a potent target for treatment. In PDA models, CDK4/6 inhibition had a variable effect on cell cycle but yielded accumulation of ATP and mitochondria. Pharmacological CDK4/6 inhibitors induce cyclin D1 protein levels; however, RB activation was required and sufficient for mitochondrial accumulation. CDK4/6 inhibition stimulated glycolytic and oxidative metabolism and was associated with an increase in mTORC1 activity. MTOR and MEK inhibitors potently cooperate with CDK4/6 inhibition in eliciting cell-cycle exit. However, MTOR inhibition fully suppressed metabolism and yielded apoptosis and suppression of tumor growth in xenograft models. The metabolic state mediated by CDK4/6 inhibition increases mitochondrial number and reactive oxygen species (ROS). Concordantly, the suppression of ROS scavenging or BCL2 antagonists cooperated with CDK4/6 inhibition. Together, these data define the impact of therapeutics on PDA metabolism and provide strategies for converting cytostatic response to tumor cell killing. PMID:26804906

  20. Role of CDK5/cyclin complexes in ischemia-induced death and survival of renal tubular cells

    Science.gov (United States)

    Guevara, Tatiana; Sancho, Mónica; Pérez-Payá, Enrique; Orzáez, Mar

    2014-01-01

    Ischemia reperfusion processes induce damage in renal tubules and compromise the viability of kidney transplants. Understanding the molecular events responsible for tubule damage and recovery would help to develop new strategies for organ preservation. CDK5 has been traditionally considered a neuronal kinase with dual roles in cell death and survival. Here, we demonstrate that CDK5 and their regulators p35/p25 and cyclin I are also expressed in renal tubular cells. We show that treatment with CDK inhibitors promotes the formation of pro-survival CDK5/cyclin I complexes and enhances cell survival upon an ischemia reperfusion pro-apoptotic insult. These findings support the benefit of treating with CDK inhibitors for renal preservation, assisting renal tubule protection. PMID:24675881

  1. p21CDKN1A Regulates the Binding of Poly(ADP-Ribose) Polymerase-1 to DNA Repair Intermediates

    OpenAIRE

    Dutto, Ilaria; Sukhanova, Maria; Tillhon, Micol; Cazzalini, Ornella; Stivala, Lucia A.; Scovassi, A. Ivana; Lavrik, Olga; Prosperi, Ennio

    2016-01-01

    The cell cycle inhibitor p21CDKN1A was previously found to interact directly with DNA nick-sensor poly(ADP-ribose) polymerase-1 (PARP-1) and to promote base excision repair (BER). However, the molecular mechanism responsible for this BER-related association of p21 with PARP-1 remains to be clarified. In this study we investigate the capability of p21 to influence PARP-1 binding to DNA repair intermediates in a reconstituted BER system in vitro. Using model photoreactive BER substrates contain...

  2. The HTLV-1 Tax protein binding domain of cyclin-dependent kinase 4 (CDK4 includes the regulatory PSTAIRE helix

    Directory of Open Access Journals (Sweden)

    Grassmann Ralph

    2005-09-01

    Full Text Available Abstract Background The Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1 is leukemogenic in transgenic mice and induces permanent T-cell growth in vitro. It is found in active CDK holoenzyme complexes from adult T-cell leukemia-derived cultures and stimulates the G1- to-S phase transition by activating the cyclin-dependent kinase (CDK CDK4. The Tax protein directly and specifically interacts with CDK4 and cyclin D2 and binding is required for enhanced CDK4 kinase activity. The protein-protein contact between Tax and the components of the cyclin D/CDK complexes increases the association of CDK4 and its positive regulatory subunit cyclin D and renders the complex resistant to p21CIP inhibition. Tax mutants affecting the N-terminus cannot bind cyclin D and CDK4. Results To analyze, whether the N-terminus of Tax is capable of CDK4-binding, in vitro binding -, pull down -, and mammalian two-hybrid analyses were performed. These experiments revealed that a segment of 40 amino acids is sufficient to interact with CDK4 and cyclin D2. To define a Tax-binding domain and analyze how Tax influences the kinase activity, a series of CDK4 deletion mutants was tested. Different assays revealed two regions which upon deletion consistently result in reduced binding activity. These were isolated and subjected to mammalian two-hybrid analysis to test their potential to interact with the Tax N-terminus. These experiments concurrently revealed binding at the N- and C-terminus of CDK4. The N-terminal segment contains the PSTAIRE helix, which is known to control the access of substrate to the active cleft of CDK4 and thus the kinase activity. Conclusion Since the N- and C-terminus of CDK4 are neighboring in the predicted three-dimensional protein structure, it is conceivable that they comprise a single binding domain, which interacts with the Tax N-terminus.

  3. Recombined adenovirus mediated delivery of p21 inhibits oxygen-induced retinal neovascularization in mice%重组腺病毒介导p21对小鼠视网膜新生血管生成的抑制作用

    Institute of Scientific and Technical Information of China (English)

    韩金栋; 袁志刚; 郑华宾; 颜华

    2012-01-01

    Objective To observe the the inhibitory effect of recombined adenovirus mediated delivery of p21 (rAd-p21) on oxygen-induced retinal neovascularization in mice.Methods A total of 56 C57BL/6 mice at the age of seven days were divided into control group,phosphate buffer solution (PBS) group,rAdp21 group and rAd-no purpose gene control (rAd-NC) group,14 mice in each group.The retinal neovascularization of PBS,rAd-p21and rAd-NC group were induced by oxygen,and received an intravitreal injection 1 μl PBS,rAd-p21 and rAd-NC at postnatal day 11,respectively.The rats of control group were not intervened.At postnatal day 17,RNV was determined by retinal flat mounts and retinal section; nonperfusion areas of retina were analyzed by Image-Pro plus 6.0 software; reverse transcription-polymerase chain reaction (RT-PCR) and Western blot was used to measure the mRNA and protein expression of p21 and CDK2.Results Compared with PBS and rAd-NC groups,the retinal non-perfusion areas,neovascularization and the numbers of endothelial cell nuclei breaking through the internal limiting membrane in rAd-p21 group were reduced significantly.Non-perfusion areas of retina in rAd-p21 group was less than that in PBS and rAd-NC groups,the difference among these three groups was significantly (F= 101.634,P<0.05).Compared with the other three groups,the level of p21 mRNA and protein in rAd-p21 group increased significantly (F=839.664,509.817; P<0.05) ; the level of CDK2 mRNA and protein in rAd-p21 group decreased significantly (F=301.858,592.882; P<0.05).Conclusion rAd-p21can inhibit oxygen-induced retinal neovascularization,up-regulated p21 expression and down-regulated CDK2 expression may be the mechanism.%目的 观察重组腺病毒-p21 (rAd-p21)对氧诱导小鼠视网膜新生血管(RNV)的抑制作用.方法 将56只健康7日龄C57BL/6J小鼠随机分为对照组、磷酸盐缓冲液(PBS)组、rAd-p21组及rAd-无目的基因对照(rAd-NC)组,每组14只.PBS组、rAd-p21组及rAd

  4. 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.

  5. Cristacarpin promotes ER stress-mediated ROS generation leading to premature senescence by activation of p21(waf-1).

    Science.gov (United States)

    Chakraborty, Souneek; Rasool, Reyaz Ur; Kumar, Sunil; Nayak, Debasis; Rah, Bilal; Katoch, Archana; Amin, Hina; Ali, Asif; Goswami, Anindya

    2016-06-01

    Stress-induced premature senescence (SIPS) is quite similar to replicative senescence that is committed by cells exposed to various stress conditions viz. ultraviolet radiation (DNA damage), hydrogen peroxide (oxidative stress), chemotherapeutic agents (cytotoxic threat), etc. Here, we report that cristacarpin, a natural product obtained from the stem bark of Erythrina suberosa, promotes endoplasmic reticulum (ER) stress, leading to sub-lethal reactive oxygen species (ROS) generation and which eventually terminates by triggering senescence in pancreatic and breast cancer cells through blocking the cell cycle in the G1 phase. The majority of cristacarpin-treated cells responded to conventional SA-β-gal stains; showed characteristic p21(waf1) upregulation along with enlarged and flattened morphology; and increased volume, granularity, and formation of heterochromatin foci-all of these features are the hallmarks of senescence. Inhibition of ROS generation by N-acetyl-L-cysteine (NAC) significantly reduced the expression of p21(waf1), confirming that the modulation in p21(waf1) by anti-proliferative cristacarpin was ROS dependent. Further, the elevation in p21(waf1) expression in PANC-1 and MCF-7 cells was consistent with the decrease in the expression of Cdk-2 and cyclinD1. Here, we provide evidence that cristacarpin promotes senescence in a p53-independent manner. Moreover, cristacarpin treatment induced p38MAPK, indicating the ROS-dependent activation of the MAP kinase pathway, and thus abrogates the tumor growth in mouse allograft tumor model. PMID:27246693

  6. Knockdown of cyclin-dependent kinase 10 (cdk10) gene impairs neural progenitor survival via modulation of raf1a gene expression.

    Science.gov (United States)

    Yeh, Chi-Wei; Kao, Shoa-Hsuan; Cheng, Yi-Chuan; Hsu, Li-Sung

    2013-09-27

    In this study, we used zebrafish as an animal model to elucidate the developmental function of cdk10 in vertebrates. In situ hybridization analyses demonstrated that cdk10 is expressed throughout development with a relative enrichment in the brain in the late stages. Similar to its mammalian ortholog, cdk10 can interact with the transcription factor ETS2 and exhibit kinase activity by phosphorylating histone H1. Morpholino-based loss of cdk10 expression caused apoptosis in sox2-positive cells and decreased the expression of subsequent neuronal markers. Acetylated tubulin staining revealed a significant reduction in the number of Rohon-Beard sensory neurons in cdk10 morphants. This result is similar to that demonstrated by decreased islet2 expression in the dorsal regions. Moreover, cdk10 morphants exhibited a marked loss of huC-positive neurons in the telencephalon and throughout the spinal cord axis. The population of retinal ganglion cells was also diminished in cdk10 morphants. These phenotypes were rescued by co-injection of cdk10 mRNA. Interestingly, the knockdown of cdk10 significantly elevated raf1a mRNA expression. Meanwhile, an MEK inhibitor (U0126) recovered sox2 and ngn1 transcript levels in cdk10 morphants. Our findings provide the first functional characterization of cdk10 in vertebrate development and reveal its critical function in neurogenesis by modulation of raf1a expression. PMID:23902762

  7. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  8. Role of p21 RAS in p210 bcr-abl transformation of murine myeloid cells.

    Science.gov (United States)

    Mandanas, R A; Leibowitz, D S; Gharehbaghi, K; Tauchi, T; Burgess, G S; Miyazawa, K; Jayaram, H N; Boswell, H S

    1993-09-15

    The p21 RAS product has been implicated as part of the downstream signaling of certain nonreceptor tyrosine kinase oncogenes and several growth factor receptor-ligand interactions. We have reported that the chronic myelogenous leukemia oncogene p210 bcr-abl transforms a growth-factor-dependent myeloid cell line NFS/N1.H7 to interleukin-3 (IL-3) independence. In these p210 bcr-abl-transformed cells (H7 bcr-abl.A54) and in two other murine myeloid cell lines transformed to IL-3 independence by p210 bcr-abl, endogenous p21 RAS is activated as determined by an elevated ratio of associated guanosine triphosphate (GTP)/guanosine diphosphate (GDP), assayed by thin-layer chromatography of the nucleotides eluted from p21 RAS after immunoprecipitation with the Y13-259 antibody. Treatment of p210 bcr-abl-transformed cells with a specific tyrosine kinase inhibitor herbimycin A resulted in diminished tyrosine phosphorylation of p210 bcr-abl and associated proteins, without major reduction in expression of the p210 bcr-abl protein itself. Inhibition of p210 bcr-abl-dependent tyrosine phosphorylation resulted in a reduction of active p21RAS-GTP complexes in the transformed cells, in diminished expression of the nuclear early response genes c-jun and c-fos, and in lower cellular proliferation rate. To further implicate p21 RAS in these functional events downstream of p210 bcr-abl tyrosine phosphorylation, we targeted G-protein function directly by limiting the availability of GTP with the inosine monophosphate dehydrogenase inhibitor, tiazofurin (TR). In p210 bcr-abl-transformed cells treated for 4 hours with TR, in which the levels of GTP were reduced by 50%, but GDP, guanosine monophosphate, and adenosine triphosphate (ATP) were unaffected, p210 bcr-abl tyrosine phosphorylation was at control levels. However, expression of c-fos and c-jun nuclear proto-oncogenes were strongly inhibited and p21 RAS activity was downregulated. These findings show that p210 bcr-abl transduces

  9. Anti-p21 autoantibodies detected in colorectal cancer patients: A proof of concept study

    OpenAIRE

    Bishehsari, Faraz; Gach, Johannes S.; Akagi, Naomi; Webber, Molly K; Bauer, Jessica; Jung, Barbara H.

    2014-01-01

    Whereas the presence of autoantibodies in cancer patients has been acknowledged, their diagnostic or therapeutic significance has yet to be established. This is due, at least in part, to the lack of robust screening techniques to detect and characterize such antibodies for further assessment. In this study, we screened colorectal cancer (CRC) patient sera for antibodies specifically targeting the key cell cycle inhibitory factor p21 encoded by the cyclin-dependent kinase inhibitor 1A (CDKN1A)...

  10. ING5 is phosphorylated by CDK2 and controls cell proliferation independently of p53.

    Directory of Open Access Journals (Sweden)

    Ulrike Linzen

    Full Text Available Inhibitor of growth (ING proteins have multiple functions in the control of cell proliferation, mainly by regulating processes associated with chromatin regulation and gene expression. ING5 has been described to regulate aspects of gene transcription and replication. Moreover deregulation of ING5 is observed in different tumors, potentially functioning as a tumor suppressor. Gene transcription in late G1 and in S phase and replication is regulated by cyclin-dependent kinase 2 (CDK2 in complex with cyclin E or cyclin A. CDK2 complexes phosphorylate and regulate several substrate proteins relevant for overcoming the restriction point and promoting S phase. We have identified ING5 as a novel CDK2 substrate. ING5 is phosphorylated at a single site, threonine 152, by cyclin E/CDK2 and cyclin A/CDK2 in vitro. This site is also phosphorylated in cells in a cell cycle dependent manner, consistent with it being a CDK2 substrate. Furthermore overexpression of cyclin E/CDK2 stimulates while the CDK2 inhibitor p27KIP1 represses phosphorylation at threonine 152. This site is located in a bipartite nuclear localization sequence but its phosphorylation was not sufficient to deregulate the subcellular localization of ING5. Although ING5 interacts with the tumor suppressor p53, we could not establish p53-dependent regulation of cell proliferation by ING5 and by phospho-site mutants. Instead we observed that the knockdown of ING5 resulted in a strong reduction of proliferation in different tumor cell lines, irrespective of the p53 status. This inhibition of proliferation was at least in part due to the induction of apoptosis. In summary we identified a phosphorylation site at threonine 152 of ING5 that is cell cycle regulated and we observed that ING5 is necessary for tumor cell proliferation, without any apparent dependency on the tumor suppressor p53.

  11. Pharmacologic inhibition of cdk4/6 arrests the growth of glioblastoma multiforme intracranial xenografts

    OpenAIRE

    Michaud, Karine; Solomon, David A.; Oermann, Eric; Kim, Jung-Sik; Zhong, Wei-Zhu; Prados, Michael D.; Ozawa, Tomoko; James, C. David; Waldman, Todd

    2010-01-01

    Activation of cyclin-dependent kinases 4 and 6 (cdk4/6) occurs in the majority of glioblastoma multiforme (GBM) tumors, and represents a promising molecular target for the development of small molecule inhibitors. In the current study we investigated the molecular determinants and in vivo response of diverse GBM cell lines and xenografts to PD-0332991, a cdk4/6 specific inhibitor. In vitro testing of PD-0332991 against a panel of GBM cell lines revealed a potent G1 cell cycle arrest and induc...

  12. Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

    Directory of Open Access Journals (Sweden)

    Ade Kallas

    2014-01-01

    Full Text Available As cyclin-dependent kinases (CDKs regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction on human embryonic stem (hES cells and embryonal carcinoma-derived (hEC cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

  13. 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.

  14. Expression,Purification and Spectral Characterization of p21Waf1/Cip1

    Institute of Scientific and Technical Information of China (English)

    SHI Qiao-yun; ZHENG Yong-chen; REN Jin-song; QU Xiao-gang

    2008-01-01

    p21Wafl/Cip1 ,best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes,can interact with various target proteins,and this ability relies on its structural plasticity.Therefore,studies on the structural properties of p21 are very important to understand its structure-function relationship.However,detailed studies on its secondary strcture and biophysical properties have been comparatively sparse.A human p21 gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109.Recombinant protein was expressed as a non-fusion protein and purified by gel filtration and anion exchange chromatography.The purified protein was verified by Western blot and the functional activity was recognized by pull-down assay.Furthermore,circular dichroisrn,fluorescence spectroscopy,and fluorescence quenching methods were used to characterize the conformational properties of the purified protein.The results indicate that it was largely unstructured under the native solution conditions,and its tryptophan residues were exposed and located in a positively charged microenvironment.This study lays a good foundation for further study of p21 binding to its different partners.

  15. 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.

  16. High glucose increases Cdk5 activity in podocytes via transforming growth factor-β1 signaling pathway

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Yue [Department of Diagnostics, Hebei Medical University, Shijiazhuang 050017 (China); Li, Hongbo; Hao, Jun [Department of Pathology, Hebei Medical University, Shijiazhuang 050017 (China); Zhou, Yi [Department of Neurology, the Second Hospital of Hebei Medical University, Shijiazhuang 050000 (China); Liu, Wei, E-mail: lwei929@126.com [Department of Pathology, Hebei Medical University, Shijiazhuang 050017 (China)

    2014-08-15

    Podocytes are highly specialized and terminally differentiated glomerular cells that play a vital role in the development and progression of diabetic nephropathy (DN). Cyclin-dependent kinase 5 (Cdk5), who is an atypical but essential member of the Cdk family of proline-directed serine/threonine kinases, has been shown as a key regulator of podocyte differentiation, proliferation and morphology. Our previous studies demonstrated that the expression of Cdk5 was significantly increased in podocytes of diabetic rats, and was closely related with podocyte injury of DN. However, the mechanisms of how expression and activity of Cdk5 are regulated under the high glucose environment have not yet been fully elucidated. In this study, we showed that high glucose up-regulated the expression of Cdk5 and its co-activator p35 with a concomitant increase in Cdk5 kinase activity in conditionally immortalized mouse podocytes in vitro. When exposed to 30 mM glucose, transforming growth factor-β1 (TGF-β1) was activated. Most importantly, we found that SB431542, the Tgfbr1 inhibitor, significantly decreased the expression of Cdk5 and p35 and Cdk5 kinase activity in high glucose-treated podocytes. Moreover, high glucose increased the expression of early growth response-1 (Egr-1) via TGF-β1-ERK1/2 pathway in podocytes and inhibition of Egr-1 by siRNA decreased p35 expression and Cdk5 kinase activity. Furthermore, inhibition of Cdk5 kinase activity effectively alleviated podocyte apoptosis induced by high glucose or TGF-β1. Thus, the TGF-β1-ERK1/2-Egr-1 signaling pathway may regulate the p35 expression and Cdk5 kinase activity in high glucose-treated podocytes, which contributes to podocyte injury of DN. - Highlights: • HG up-regulated the expression of Cdk5 and p35, and Cdk5 activity in podocytes. • HG activated TGF-β1 pathway and SB431542 inhibited Cdk5 expression and activity. • HG increased the expression of Egr-1 via TGF-β1-ERK1/2 pathway. • Inhibition of Egr-1

  17. Identification of TSG101 functional domains and p21 loci required for TSG101-mediated p21 gene regulation.

    Directory of Open Access Journals (Sweden)

    Yu-Shiuan Lin

    Full Text Available TSG101 (tumor susceptibility gene 101 is a multi-domain protein known to act in the cell nucleus, cytoplasm, and periplasmic membrane. Remarkably, TSG101, whose location within cells varies with the stage of the cell cycle, affects biological events as diverse as cell growth and proliferation, gene expression, cytokinesis, and endosomal trafficking. The functions of TSG101 additionally are recruited for viral and microvesicle budding and for intracellular survival of invading bacteria. Here we report that the TSG101 protein also interacts with and down-regulates the promoter of the p21 (CIP1/WAF1 tumor suppressor gene, and identify a p21 locus and TSG101 domains that mediate this interaction. TSG101 deficiency in Saos-2 human osteosarcoma cells was accompanied by an increased abundance of p21 mRNA and protein and the retardation of cell proliferation. A cis-acting element in the p21 promoter that interacts with TSG101 and is required for promoter repression was located using chromatin immunoprecipitation (ChIP analysis and p21-driven luciferase reporter gene expression, respectively. Additional analysis of TSG101 deletion mutants lacking specific domains established the role of the central TSG101 domains in binding to the p21 promoter and demonstrated the additional essentiality of the TSG101 C-terminal steadiness box (SB in the repression of p21 promoter activity. Neither binding of TSG101 to the p21 promoter nor repression of this promoter required the TSG101 N-terminal UEV domain, which mediates the ubiquitin-recognition functions of TSG101 and its actions as a member of ESCRT endocytic trafficking complexes, indicating that regulation of the p21 promoter by TSG101 is independent of its role in such trafficking.

  18. p21(CIP1/WAF1)-dependent inhibition of cardiac hypertrophy in response to Angiotensin II involves Akt/Myc and pRb signaling.

    Science.gov (United States)

    Hauck, Ludger; Grothe, Daniela; Billia, Filio

    2016-09-01

    The cyclin-dependent kinase inhibitor p21(CIP1/WAF1) (p21) is highly expressed in the adult heart. However, in response to stress, its expression is downregulated. Therefore, we investigated the role of p21 in the regulation of cardiac hypertrophic growth. At 2 months of age, p21 knockout mice (p21KO) lack an overt cardiac phenotype. In contrast, by 10 months of age, p21KO developed age-dependent cardiac hypertrophy and heart failure. After 3 weeks of trans-aortic banding (TAB), the heart/body weight ratio in 11 week old p21KO mice increased by 57%, as compared to 42% in wild type mice indicating that p21KO have a higher susceptibility to pressure overload-induced cardiac hypertrophy. We then chronically infused 8 week old wild type mice with Angiotensin II (2.0mg/kg/min) or saline subcutaneously by osmotic pumps for 14 days. Recombinant TAT conjugated p21 protein variants (10mg/kg body weight) or saline were intraperitoneally injected once daily for 14 days into Angiotensin II and saline-infused animals. Angiotensin II treated mice developed pathological cardiac hypertrophy with an average increase of 38% in heart/body weight ratios, as compared to saline-treated controls. Reconstitution of p21 function by TAT.p21 protein transduction prevented Angiotensin II-dependent development of cardiac hypertrophy and failure. Taken together, our genetic and biochemical data show an important function of p21 in the regulation of growth-related processes in the heart. PMID:27486069

  19. Inhibitors

    Science.gov (United States)

    ... wrong place in the body. Immune Tolerance Induction (ITI) Therapy: The goal of ITI therapy is to stop the inhibitor reaction from ... body to accept clotting factor concentrate treatments. With ITI therapy, people receive large amounts of clotting factor ...

  20. CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1-induced T-cell acute lymphoblastic leukemia.

    Science.gov (United States)

    Jena, N; Sheng, J; Hu, J K; Li, W; Zhou, W; Lee, G; Tsichlis, N; Pathak, A; Brown, N; Deshpande, A; Luo, C; Hu, G F; Hinds, P W; Van Etten, R A; Hu, M G

    2016-05-01

    T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new therapeutic approaches are needed. We showed previously that cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy. PMID:26707936

  1. The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1.

    Science.gov (United States)

    Voets, Erik; Marsman, Judith; Demmers, Jeroen; Beijersbergen, Roderick; Wolthuis, Rob

    2015-01-01

    Cyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cdk1 inhibitors, is also detrimental for proliferating cells, but has been associated with defects in mitotic progression, and the formation of aneuploid daughter cells. Here, we used a large-scale RNAi screen to identify the human genes that critically determine the cellular toxicity of Cdk1 inhibition. We show that Cdk1 inhibition leads to fatal sister chromatid alignment errors and mitotic arrest in the spindle checkpoint. These problems start early in mitosis and are alleviated by depletion of isoform 1 of PRC1 (PRC1-1), by gene ablation of its binding partner KIF4, or by abrogation of KIF4 motor activity. Our results show that, normally, Cdk1 activity must rise above the level required for mitotic entry. This prevents KIF4-dependent PRC1-1 translocation to astral microtubule tips and safeguards proper chromosome congression. We conclude that cell death in response to Cdk1 inhibitors directly relates to chromosome alignment defects generated by insufficient repression of PRC1-1 and KIF4 during prometaphase. PMID:26423135

  2. Synthesis and CDK2 kinase inhibitory activity of 7/7′-azaindirubin derivatives

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    A series of novel 7'-azaindirubin(1a-g) and 7-azaindirubin(2a,2c,2e and 2f) derivatives were designed and synthesized.Their structures were characterized by ~1H NMR and MS spectroscopy as well as by elemental analysis.Their inhibitory properties against CDK2/cylinA were evaluated in vitro.In contrast to indirubin,some of the described azaindirubins emerged as potent inhibitors of CDK2/cylinA and compound 2b had more potent activity.Biological tests also showed that nitrogen atom at 7-position of azaindir...

  3. Securin and not CDK1/cyclin B1 regulates sister chromatid disjunction during meiosis II in mouse eggs.

    Science.gov (United States)

    Nabti, Ibtissem; Reis, Alexandra; Levasseur, Mark; Stemmann, Olaf; Jones, Keith T

    2008-09-15

    Mammalian eggs remain arrested at metaphase of the second meiotic division (metII) for an indeterminate time before fertilization. During this period, which can last several hours, the continued attachment of sister chromatids is thought to be achieved by inhibition of the protease separase. Separase is known to be inhibited by binding either securin or Maturation (M-Phase)-Promoting Factor, a heterodimer of CDK1/cyclin B1. However, the relative contribution of securin and CDK/cyclin B1 to sister chromatid attachment during metII arrest has not been assessed. Although there are conditions in which either CDK1/cyclinB1 activity or securin can prevent sister chromatid disjunction, principally by overexpression of non-degradable cyclin B1 or securin, we find here that separase activity is primarily regulated by securin and not CDK1/cyclin B1. Thus the CDK1 inhibitor roscovitine and an antibody we designed to block the interaction of CDK1/cyclin B1 with separase, both failed to induce sister disjunction. In contrast, securin morpholino knockdown specifically induced loss of sister attachment, that could be restored by securin cRNA rescue. During metII arrest separase appears primarily regulated by securin binding, not CDK1/cyclin B1. PMID:18639540

  4. Ovarian Cancers Harboring Inactivating Mutations in CDK12 Display a Distinct Genomic Instability Pattern Characterized by Large Tandem Duplications.

    Science.gov (United States)

    Popova, Tatiana; Manié, Elodie; Boeva, Valentina; Battistella, Aude; Goundiam, Oumou; Smith, Nicholas K; Mueller, Christopher R; Raynal, Virginie; Mariani, Odette; Sastre-Garau, Xavier; Stern, Marc-Henri

    2016-04-01

    CDK12 is a recurrently mutated gene in serous ovarian carcinoma, whose downregulation is associated with impaired expression of DNA damage repair genes and subsequent hypersensitivity to DNA-damaging agents and PARP1/2 inhibitors. In this study, we investigated the genomic landscape associated with CDK12 inactivation in patients with serous ovarian carcinoma. We show that CDK12 loss was consistently associated with a particular genomic instability pattern characterized by hundreds of tandem duplications of up to 10 megabases (Mb) in size. Tandem duplications were characterized by a bimodal (∼0.3 and ∼3 Mb) size distribution and overlapping microhomology at the breakpoints. This genomic instability, denoted as the CDK12 TD-plus phenotype, is remarkably distinct from other alteration patterns described in breast and ovarian cancers. The CDK12 TD-plus phenotype was associated with a greater than 10% gain in genomic content and occurred at a 3% to 4% rate in The Cancer Genome Atlas-derived and in-house cohorts of patients with serous ovarian carcinoma. Moreover, CDK12-inactivating mutations together with the TD-plus phenotype were also observed in prostate cancers. Our finding provides new insight toward deciphering the function of CDK12 in genome maintenance and oncogenesis. Cancer Res; 76(7); 1882-91. ©2016 AACR. PMID:26787835

  5. Characterization of p21Ras-mediated apoptosis induced by protein kinase C inhibition and application to human tumor cell lines.

    Science.gov (United States)

    Liou, James S; Chen, James S; Faller, Douglas V

    2004-02-01

    Suppression of PKC activity can selectively induce apoptosis in cells expressing a constitutively activated p21Ras protein. We demonstrate that continued expression of p21Ras activity is required in PKC-mediated apoptosis because farnesyltransferase inhibitors abrogated the loss of viability in p21Ras-transformed cells occurring following PKC inhibition. Studies utilizing gene transfer or viral vectors demonstrate that transient expression of oncogenic p21Ras activity is sufficient for induction of apoptosis by PKC inhibition, whereas physiologic activation of p21Ras by growth factor is not sufficient to induce apoptosis. Mechanistically, the p21Ras-mediated apoptosis induced by PKC inhibition is dependent upon mitochondrial dysregulation, with a concurrent loss of mitochondrial membrane potential (psim). Cyclosporine A, which prevented the loss of psim, also inhibited HMG-induced DNA fragmentation in cells expressing an activated p21Ras. Induction of apoptosis by PKC inhibition in human tumors with oncogenic p21Ras mutations was demonstrated. Inhibition of PKC caused increased apoptosis in MIA-PaCa-2, a human pancreatic tumor line containing a mutated Ki-ras allele, when compared to HS766T, a human pancreatic tumor line with normal Ki-ras alleles. Furthermore, PKC inhibition induced apoptosis in HCT116, a human colorectal tumor line containing an oncogenic Ki-ras allele but not in a subline (Hke3) in which the mutated Ki-ras allele had been disrupted. The PKC inhibitor 1-O-hexadecyl-2-O-methyl-rac-glycerol (HMG), significantly reduced p21Ras-mediated tumor growth in vivo in a nude mouse MIA-PaCa-2 xenograft model. Collectively these studies suggest the therapeutic feasibility of targeting PKC activity in tumors expressing an activated p21Ras oncoprotein. PMID:14603530

  6. AP4 directly downregulates p16 and p21 to suppress senescence and mediate transformation

    OpenAIRE

    Jackstadt, R; Jung, P.; Hermeking, H

    2013-01-01

    Here we analyzed the function of the c-MYC-inducible basic helix–loop–helix leucine-zipper transcription factor AP4 in AP4-deficient mouse embryo fibroblasts (MEFs). Loss of AP4 resulted in premature senescence and resistance towards immortalization. Senescence was accompanied by induction of the cyclin-dependent kinase inhibitor-encoding genes p16, a known tumor suppressor, and p21, a previously described target for repression by AP4. Notably, AP4 directly repressed p16 expression via conser...

  7. Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies.

    Science.gov (United States)

    Prével, Camille; Pellerano, Morgan; González-Vera, Juan A; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May C

    2016-11-15

    Melanoma constitutes the most aggressive form of skin cancer, which further metastasizes into a deadly form of cancer. The p16(INK4a)-Cyclin D-CDK4/6-pRb pathway is dysregulated in 90% of melanomas. CDK4/Cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of Cyclin D or loss of p16(INK4a) leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker in melanoma and an emerging pharmacological target, however there are no tools enabling direct detection or quantification of its activity. Here we report on the design and application of a fluorescent peptide biosensor to quantify CDK4 activity in melanoma cell extracts, skin biopsies and melanoma xenografts. This biosensor provides sensitive means of comparing CDK4 activity between different melanoma cell lines and further responds to CDK4 downregulation by siRNA or small-molecule inhibitors. By affording means of monitoring CDK4 hyperactivity consequent to cancer-associated molecular alterations in upstream signaling pathways that converge upon this kinase, this biosensor offers an alternative to immunological identification of melanoma-specific biomarkers, thereby constituting an attractive tool for diagnostic purposes, providing complementary functional information to histological analysis, of particular utility for detection of melanoma onset in precancerous lesions. This is indeed the first fluorescent peptide biosensor which has been successfully implemented to monitor kinase activity in skin samples and melanoma tumour xenografts. Moreover by enabling to monitor response to CDK4 inhibitors, this biosensor constitutes an attractive companion assay to identify compounds of therapeutic relevance for melanoma. PMID:27203461

  8. 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.

  9. CDK2-AP1基因过表达对乳腺癌MCF-7细胞增殖及周期的影响%Effect of CDK2-AP1 gene over-expression on proliferation and cell cycle regulation of breast cancer cell line MCF-7

    Institute of Scientific and Technical Information of China (English)

    关晓燕; 周卫兵; 黄隽; 王龙云; 廖遇平

    2012-01-01

    Objective: To over-express cyclin-dependent kinase 2-associated protein 1 (CDK2-AP1) gene, and investigate its effect on the proliferation and cell cycle regulation in breast cancer cell line MCF-7. Methods: CDK2-AP1 gene coding region was cloned into lentivirus vector. Lentivirus particles were infected into MCF-7 cells to upregulate the expression of CDK2-AP1 gene. The expression level of CDK2-AP1 was detected at both mRNA and protein levels by real-time PCR and Western blot. MTT assay, colony formatting assay, and flow cytometry were performed to detect the change of proliferation and cell cycle in MCF-7 cells. We examined the expression of cell cycle associated genes (CDK2, CDK4, P16Ink4A, and P2lCiP1/Wafl) followed by CDK2-AP1 over-expression by Western blot.Results: CDK2-AP1 gene was up-regulated significantly at both mRNA (6.94 folds) and protein level. MTT based growth curve, colony formatting assay and flow cytometry showed that CDK2- API over-expression lentivirus inhibited the proliferation of MCF-7 cells with statistical difference (P<0.05). In addition, with CDK2-AP1 over-expression, MCF-7 cells were arrested in G1 phase accompanied by apoptosis. Western blot showed that the expression level of P21Clpl/wafl and P16Int4A was upregulated, while the expression level of CDK2 and CDK4, members of the CDK family, was downregulated.Conclusion: CDK2-AP1 gene plays a cancer suppressor role in breast cancer. Its function includes inhibiting the proliferation of MCF-7 cells and arresting the cell cycle in G, phase.%目的:通过过表达手段上调细胞周期调节蛋白依赖性激酶2-关联蛋白1(CDK2-AP1)基因在乳腺癌细胞MCF-7中的表达,并观察其对MCF-7细胞生长和细胞周期调控的作用.方法:将CDK2-AP1基因的编码框构建于慢病毒表达载体,导入MCF-7细胞,应用实时定量PCR和Western印迹验证CDK2-AP1基因mRNA和蛋白的表达效率.利用MTT法绘制生长曲线、克隆形成实验观察CDK2-AP1

  10. Established and New Mouse Models Reveal E2f1 and Cdk2 Dependency of Retinoblastoma and Expose Strategies to Block Tumor Initiation

    Science.gov (United States)

    Sangwan, Monika; McCurdy, Sean R.; Livne-bar, Izzy; Ahmad, Mohammad; Wrana, Jeffery L.; Chen, Danian; Bremner, Rod

    2016-01-01

    RB +/− individuals develop retinoblastoma and, subsequently, many other tumors. The Rb relatives p107 and p130 protect the tumor-resistant Rb−/− mouse retina. Determining the mechanism underlying this tumor suppressor function may expose novel strategies to block Rb-pathway cancers. p107/p130 are best known as E2f inhibitors, but here we implicate E2f-independent Cdk2 inhibition as the critical p107 tumor suppressor function in vivo. Like p107 loss, deleting p27 or inactivating its Cdk inhibitor (CKI) function (p27CK−) cooperated with Rb loss to induce retinoblastoma. Genetically, p107 behaved like a CKI because inactivating Rb and one allele each of p27 and p107 was tumorigenic. While Rb loss induced canonical E2f targets, unexpectedly p107 loss did not further induce these genes but instead caused post-transcriptional Skp2-induction and Cdk2 activation. Strikingly, Cdk2 activity correlated with tumor penetrance across all the retinoblastoma models. Therefore, Rb restrains E2f, but p107 inhibits cross-talk to Cdk. While removing either E2f2 or E2f3 genes had little effect, removing only one E2f1 allele blocked tumorigenesis. More importantly, exposing retinoblastoma-prone fetuses to small molecule E2f or Cdk inhibitors for merely one week dramatically inhibited subsequent tumorigenesis in adult mice. Protection was achieved without disrupting normal proliferation. Thus, exquisite sensitivity of the cell-of-origin to E2f and Cdk activity can be exploited to prevent Rb pathway-induced cancer in vivo without perturbing normal cell division. These data suggest that E2f inhibitors, never before tested in vivo, or Cdk inhibitors, largely disappointing as therapeutics, may be effective preventive agents. PMID:22286767

  11. A dual role of Cdk2 in DNA damage response

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2009-05-01

    Full Text Available Abstract Once it was believed that Cdk2 was the master regulator of S phase entry. Gene knockout mouse studies of cell cycle regulators revealed that Cdk2 is dispensable for S phase initiation and progression whereby Cdk1 can compensate for the loss of Cdk2. Nevertheless, recent evidence indicates that Cdk2 is involved in cell cycle independent functions such as DNA damage repair. Whether these properties are unique to Cdk2 or also being compensated by other Cdks in the absence of Cdk2 is under extensive investigation. Here we review the emerging new role of Cdk2 in DNA damage repair and also discuss how the loss of Cdk2 impacts the G1/S phase DNA damage checkpoint.

  12. The CDK Subunit CKS2 Counteracts CKS1 to Control Cyclin A/CDK2 Activity in Maintaining Replicative Fidelity and Neurodevelopment

    Science.gov (United States)

    Frontini, Mattia; Kukalev, Alexander; Leo, Elisabetta; Ng, Yiu-Ming; Cervantes, Marcella; Cheng, Chi-Wai; Holic, Roman; Dormann, Dirk; Tse, Eric; Pommier, Yves; Yu, Veronica

    2012-01-01

    Summary CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCFSKP2 to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2−/− knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2−/− cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In vivo, Cks2−/− cortical progenitor cells are limited in their capacity to differentiate into mature neurons, a phenotype akin to animals lacking p27. We propose that the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation. PMID:22898779

  13. 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.

  14. Upregulation of miR-572 transcriptionally suppresses SOCS1 and p21 and contributes to human ovarian cancer progression

    OpenAIRE

    Zhang, Xin; Liu, Junling; Zang, Dan; Wu, Shu; Liu, Aibin; Zhu, Jinrong; Wu, Geyan; Li, Jun; Jiang, Lili

    2015-01-01

    Ovarian cancer is a gynecological malignancy with high mortality rates worldwide and novel diagnostic and prognostic markers and therapeutic targets are urgently required. The suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibitor 1A (p21KIP) are known to regulate tumor cell proliferation. However, the mechanisms that regulate these genes have not yet been completely elucidated. In the present study, analysis of a published microarray-based high-throughput assessment...

  15. Molecular basis for viral selective replication in cancer cells: activation of CDK2 by adenovirus-induced cyclin E.

    Directory of Open Access Journals (Sweden)

    Pei-Hsin Cheng

    Full Text Available Adenoviruses (Ads with deletion of E1b55K preferentially replicate in cancer cells and have been used in cancer therapies. We have previously shown that Ad E1B55K protein is involved in induction of cyclin E for Ad replication, but this E1B55K function is not required in cancer cells in which deregulation of cyclin E is frequently observed. In this study, we investigated the interaction of cyclin E and CDK2 in Ad-infected cells. Ad infection significantly increased the large form of cyclin E (cyclin EL, promoted cyclin E/CDK2 complex formation and increased CDK2 phosphorylation at the T160 site. Activated CDK2 caused pRb phosphorylation at the S612 site. Repression of CDK2 activity with the chemical inhibitor roscovitine or with specific small interfering RNAs significantly decreased pRb phosphorylation, with concomitant repression of viral replication. Our results suggest that Ad-induced cyclin E activates CDK2 that targets the transcriptional repressor pRb to generate a cellular environment for viral productive replication. This study reveals a new molecular basis for oncolytic replication of E1b-deleted Ads and will aid in the development of new strategies for Ad oncolytic virotherapies.

  16. Cdk5 Is Required for Memory Function and Hippocampal Plasticity via the cAMP Signaling Pathway

    Science.gov (United States)

    Gao, Jun; Joseph, Nadine; Xie, Zhigang; Zhou, Ying; Durak, Omer; Zhang, Lei; Zhu, J. Julius; Clauser, Karl R.; Carr, Steven A.; Tsai, Li-Huei

    2011-01-01

    Memory formation is modulated by pre- and post-synaptic signaling events in neurons. The neuronal protein kinase Cyclin-Dependent Kinase 5 (Cdk5) phosphorylates a variety of synaptic substrates and is implicated in memory formation. It has also been shown to play a role in homeostatic regulation of synaptic plasticity in cultured neurons. Surprisingly, we found that Cdk5 loss of function in hippocampal circuits results in severe impairments in memory formation and retrieval. Moreover, Cdk5 loss of function in the hippocampus disrupts cAMP signaling due to an aberrant increase in phosphodiesterase (PDE) proteins. Dysregulation of cAMP is associated with defective CREB phosphorylation and disrupted composition of synaptic proteins in Cdk5-deficient mice. Rolipram, a PDE4 inhibitor that prevents cAMP depletion, restores synaptic plasticity and memory formation in Cdk5-deficient mice. Collectively, our results demonstrate a critical role for Cdk5 in the regulation of cAMP-mediated hippocampal functions essential for synaptic plasticity and memory formation. PMID:21984943

  17. Neutralizing monoclonal antibody against ras oncogene product p21 which impairs guanine nucleotide exchange.

    OpenAIRE

    Hattori, S; Clanton, D J; Satoh, T.; Nakamura, S.; Kaziro, Y; Kawakita, M; Shih, T Y

    1987-01-01

    The neutralizing monoclonal antibody Y13-259 severely hampers the nucleotide exchange reaction between p21-bound and exogenous guanine nucleotides but does not interfere with the association of GDP to p21. These results suggest that the nucleotide exchange reaction is critical for p21 function. Interestingly, the v-ras p21 has a much faster dissociation rate than the p21 of the c-ras proto-oncogene.

  18. Clinicopathologic and prognostic significance of p21 (Cip1/Waf1) expression in bladder cancer

    OpenAIRE

    Tang, Kun; Wang, Chenghe; Chen, Zhong; Xu, Hua; Ye, Zhangqun

    2015-01-01

    Recent studies have shown that altered expression p21 is shown to associate with tumorigenesis and tumor progression. To investigate the clinicopathological significance and prognostic value of p21 in bladder cancer (BCa). A total of 48 patients with BCa were included in this study. The correlation between p21 expression and clinicopathologic features and survival was studied. Also, a meta-analysis was performed to investigate the relationship between the p21 and BCa survival. Low p21 express...

  19. Analysis list: Cdk7 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Cdk7 Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cd...k7.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk7.5.tsv http://dbarchive.bioscienced...bc.jp/kyushu-u/mm9/target/Cdk7.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk7.Pluripotent_s

  20. Analysis list: Cdk8 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Cdk8 Pluripotent stem cell + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cd...k8.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Cdk8.5.tsv http://dbarchive.bioscienced...bc.jp/kyushu-u/mm9/target/Cdk8.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Cdk8.Pluripotent_s

  1. Biochemical characterization of Cdk2-Speedy/Ringo A2

    Directory of Open Access Journals (Sweden)

    Kaldis Philipp

    2005-09-01

    Full Text Available Abstract Background Normal cell cycle progression requires the precise activation and inactivation of cyclin-dependent protein kinases (CDKs, which consist of a CDK and a cyclin subunit. A novel cell cycle regulator called Speedy/Ringo shows no sequence similarity to cyclins, yet can directly bind to and activate CDKs. Speedy/Ringo proteins, which bind to and activate Cdc2 and Cdk2 in vitro, are required for the G2 to M transition during Xenopus oocyte maturation and for normal S-phase entry in cultured human cells. Results We have characterized the substrate specificity and enzymatic activity of human Cdk2-Speedy/Ringo A2 in order to gain insights into the possible functions of this complex. In contrast to Cdk2-cyclin A, which has a well-defined consensus target site ((S/TPX(K/R that strongly favors substrates containing a lysine at the +3 position of substrates, Cdk2-Speedy/Ringo A2 displayed a broad substrate specificity at this position. Consequently, Cdk2-Ringo/Speedy A2 phosphorylated optimal Cdk2 substrates such as histone H1 and a KSPRK peptide poorly, only ~0.08% as well as Cdk2-cyclin A, but non-canonical Cdk2 substrates such as a KSPRY peptide relatively well, with an efficiency of ~80% compared to Cdk2-cyclin A. Cdk2-Speedy/Ringo A2 also phosphorylated authentic Cdk2 substrates, such as Cdc25 proteins, which contain non-canonical CDK phosphorylation sites, nearly as well as Cdk2-cyclin A. Phosphopeptide mapping indicated that Cdk2-Speedy/Ringo A2 and Cdk2-cyclin A phosphorylate distinct subsets of sites on Cdc25 proteins. Thus, the low activity that Cdk2-Speedy/Ringo A2 displays when assayed on conventional Cdk2 substrates may significantly underestimate the potential physiological importance of Cdk2-Speedy/Ringo A2 in phosphorylating key subsets of Cdk2 substrates. Unlike Cdk2-cyclin A, whose activity depends strongly on activating phosphorylation of Cdk2 on Thr-160, neither the overall catalytic activity nor the substrate

  2. INHIBITION STUDIES OF TERPENE BASED NATURAL PRODUCTS WITH CYCLIN-DEPENDENT KINASE 4 (CDK4 MIMIC CDK2)

    OpenAIRE

    Dr. Sunil H. Ganatra et al

    2012-01-01

    Cyclin dependent kinases (CDKs) are known as cell cycle regulators in eukaryotic cell cycle. Different CDKs (CDK2, CDK4 etc.) are having structure homology among them. Using computer based molecular modeling tools, interactions between naturally occurring terpene based compounds with crystal structure of CDK4 mimic CDK2 enzyme having PDB ID : 1GII. Using In-silico techniques, the binding energies between terpene based compounds and receptor enzymes are calculated in the form of ΔG in kcal/mol...

  3. THE EXPRESSION OF P53 PROTEIN AND P21WAFl/cipl/sdil IN GASTRIC CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective To study the relation along p53, p21 protein, p21 gene and their clinical significances in 40 gastric comparing with normal gastric tissues.Methods In this study, the p53 and p21 protein were investigated in 40 gastric carcinomas using IHC(Immunohistochemistry). At the same time, the possible presence of p21 gene mutation was also analyzed by silver staining PCR-SSCP method.Results The abnormal expression of p53 and p21 protein occurs only in gastric carcinoma; The expression of p53 protein and p21 is not related to the clinico pathological features. There was relationship between the expression of p53 protein and p21 protein. In 40 cases of gastric carcinoma, single strand conformational polymorphism of PCR product for p21 gene in tumor tissue shows no altered band or mobility shifting.Conclusion The abnormal expression of p53 and p21 protein occurs only in gastric carcinoma and is not related to the clinicopathological features. The expression of p21 protein is related to that of p53 protein. The mutation of p21 gene was not found in all of 40 tumor specimens. This suggests that p21 alteration in gastric carcinoma is caused through the inactivation of p53 protein rather than through intragenic mutation of the p21 gene itself.Using drugs which can stimulate p21 gene is a new method to cure gastric cancer with mutation-p53 protein.

  4. 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.

  5. TPPII, MYBBP1A and CDK2 form a protein-protein interaction network.

    Science.gov (United States)

    Nahálková, Jarmila; Tomkinson, Birgitta

    2014-12-15

    Tripeptidyl-peptidase II (TPPII) is an aminopeptidase with suggested regulatory effects on cell cycle, apoptosis and senescence. A protein-protein interaction study revealed that TPPII physically interacts with the tumor suppressor MYBBP1A and the cell cycle regulator protein CDK2. Mutual protein-protein interaction was detected between MYBBP1A and CDK2 as well. In situ Proximity Ligation Assay (PLA) using HEK293 cells overexpressing TPPII forming highly enzymatically active oligomeric complexes showed that the cytoplasmic interaction frequency of TPPII with MYBBP1A increased with the protein expression of TPPII and using serum-free cell growth conditions. A specific reversible inhibitor of TPPII, butabindide, suppressed the cytoplasmic interactions of TPPII and MYBBP1A both in control HEK293 and the cells overexpressing murine TPPII. The interaction of MYBBP1A with CDK2 was confirmed by in situ PLA in two different mammalian cell lines. Functional link between TPPII and MYBBP1A has been verified by gene expression study during anoikis, where overexpression of TPP II decreased mRNA expression level of MYBBP1A at the cell detachment conditions. All three interacting proteins TPPII, MYBBP1A and CDK2 have been previously implicated in the research for development of tumor-suppressing agents. This is the first report presenting mutual protein-protein interaction network of these proteins. PMID:25303791

  6. Variants at the 9p21 locus and melanoma risk

    International Nuclear Information System (INIS)

    The influence of variants at the 9p21 locus on melanoma risk has been reported through investigation of CDKN2A variants through candidate gene approach as well as by genome wide association studies (GWAS). In the present study we genotyped, 25 SNPs that tag 273 variants on chromosome 9p21 in 837 melanoma cases and 1154 controls from Spain. Ten SNPs were selected based on previous associations, reported in GWAS, with either melanocytic nevi or melanoma risk or both. The other 15 SNPs were selected to fine map the CDKN2A gene region. All the 10 variants selected from the GWAS showed statistically significant association with melanoma risk. Statistically significant association with melanoma risk was also observed for the carriers of the variant T-allele of rs3088440 (540 C>T) at the 3’ UTR of CDKN2A gene with an OR 1.52 (95% CI 1.14-2.04). Interaction analysis between risk associated polymorphisms and previously genotyped MC1R variants, in the present study, did not show any statistically significant association. Statistical significant association was observed for the interaction between phototypes and the rs10811629 (located in intron 5 of MTAP). The strongest association was observed between the homozygous carrier of the A–allele and phototype II with an OR of 15.93 (95% CI 5.34-47.54). Our data confirmed the association of different variants at chromosome 9p21 with melanoma risk and we also found an association of a variant with skin phototypes

  7. Cytoplasmic p21WAF1/CIP1 expression is correlated with HER-2/ neu in breast cancer and is an independent predictor of prognosis

    International Nuclear Information System (INIS)

    HER-2 (c-erbB2/Neu) predicts the prognosis of and may influence treatment responses in breast cancer. HER-2 activity induces the cytoplasmic location of p21WAFI/CIPI in cell culture, accompanied by resistance to apoptosis. p21WAFI/CIPI is a cyclin-dependent kinase inhibitor activated by p53 to produce cell cycle arrest in association with nuclear localisation of p21WAFI/CIPI. We previously showed that higher levels of cytoplasmic p21WAFI/CIPI in breast cancers predicted reduced survival at 5 years. The present study examined HER-2 and p21WAFI/CIPI expression in a series of breast cancers with up to 9 years of follow-up, to evaluate whether in vitro findings were related to clinical data and the effect on outcome. The CB11 anti-HER2 monoclonal antibody and the DAKO Envision Plus system were used to evaluate HER-2 expression in 73 patients. p21WAFI/CIPI staining was performed as described previously using the mouse monoclonal antibody Ab-1 (Calbiochem, Cambridge, MA, USA). HER-2 was evaluable in 67 patients and was expressed in 19% of cases, predicting reduced overall survival (P = 0.02) and reduced relapse-free survival (P = 0.004; Cox regression model). HER-2-positive tumours showed proportionately higher cytoplasmic p21WAFI/CIPI staining using an intensity distribution score (median, 95) compared with HER-2-negative cancers (median, 47) (P = 0.005). There was a much weaker association between nuclear p21WAFI/CIPI and HER-2 expression (P = 0.05), suggesting an inverse relationship between nuclear p21WAF1/CIP1 and HER-2. This study highlights a new pathway by which HER-2 may modify cancer behaviour. HER-2 as a predictor of poor prognosis may partly relate to its ability to influence the relocalisation of p21WAFI/CIPI from the nucleus to the cytoplasm, resulting in a loss of p21WAFI/CIPItumour suppressor functions. Cytoplasmic p21WAFI/CIPI may be a surrogate marker of functional HER-2 in vivo

  8. Prediction of paclitaxel sensitivity by CDK1 and CDK2 activity in human breast cancer cells

    OpenAIRE

    Nakayama, Satoshi; Torikoshi, Yasuhiro; Takahashi, Takeshi; Yoshida, Tomokazu; Sudo, Tamotsu; Matsushima, Tomoko; Kawasaki, Yuko; Katayama, Aya; Gohda, Keigo; Hortobagyi, Gabriel N.; Noguchi, Shinzaburo; Sakai, Toshiyuki; Ishihara, Hideki; Ueno, Naoto T.

    2009-01-01

    Introduction Paclitaxel is used widely in the treatment of breast cancer. Not all tumors respond to this drug, however, and the characteristics that distinguish resistant tumors from sensitive tumors are not well defined. Activation of the spindle assembly checkpoint is required for paclitaxel-induced cell death. We hypothesized that cyclin-dependent kinase (CDK) 1 activity and CDK2 activity in cancer cells, which reflect the activation state of the spindle assembly checkpoint and the growth ...

  9. Upregulation of miR-572 transcriptionally suppresses SOCS1 and p21 and contributes to human ovarian cancer progression.

    Science.gov (United States)

    Zhang, Xin; Liu, Junling; Zang, Dan; Wu, Shu; Liu, Aibin; Zhu, Jinrong; Wu, Geyan; Li, Jun; Jiang, Lili

    2015-06-20

    Ovarian cancer is a gynecological malignancy with high mortality rates worldwide and novel diagnostic and prognostic markers and therapeutic targets are urgently required. The suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibitor 1A (p21(KIP)) are known to regulate tumor cell proliferation. However, the mechanisms that regulate these genes have not yet been completely elucidated. In the present study, analysis of a published microarray-based high-throughput assessment (NCBI/E-MTAB-1067) and real-time PCR demonstrated that miR-572 was upregulated in human ovarian cancer tissues and cell lines. Kaplan-Meir analysis indicated that high level expression of miR-572 was associated with poorer overall survival. Ectopic miR-572 promoted ovarian cancer cell proliferation and cell cycle progression in vitro and tumorigenicity in vivo. SOCS1 and p21 were identified as direct targets of miR-572 and suppression of SOCS1 or p21 reversed the inhibiting-function of miR-572-silenced cell on proliferation and tumorigenicity in ovarian cancer cells. Additionally, the expression of miR-572 correlated inversely with the protein expression levels of SOCS1, p21 and positively with Cyclin D1 in ovarian carcinoma specimens. This study demonstrates that miR-572 post-transcriptionally regulates SOCS1 and p21 and may play an important role in ovarian cancer progression; miR-572 may represent a potential therapeutic target for ovarian cancer therapy. PMID:25893382

  10. IL-1-induced ERK1/2 activation up-regulates p21Waf1/Cip1 protein by inhibition of degradation via ubiquitin-independent pathway in human melanoma cells A375

    International Nuclear Information System (INIS)

    IL-1 inhibits the proliferation of human melanoma cells A375 by arresting the cell cycle at G0/G1 phase, which accompanies the increase of p21Waf1/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.

  11. Study of expression of CDK2 and CDK4 in Hamster Buckle Pouch Carcinogesis%CDK2、CDK4在金黄地鼠颊囊癌变过程中表达的研究

    Institute of Scientific and Technical Information of China (English)

    孙淑芬; 高文信; 罗兰; 顾彦成

    2006-01-01

    目的探讨CDK2 、CDK4在金黄地鼠颊囊黏膜从正常黏膜到单纯增生、异常增生及鳞状细胞癌的表达变化及相关性.方法采用DMBA诱导48只金黄地鼠颊囊癌变动物模型,SABC免疫组化法检测CDK2 、CDK4蛋白的表达.结果 CDK2 、CDK4均在异常增生上皮及鳞状细胞癌的表达与正常和单纯增生组相比明显提高(P<0.05),阳性染色等级随病理等级改变提高(P<0.05).CDK2与CDK4呈高度正相关.结论 CDK2 、CDK4参与了口腔黏膜癌前病变和鳞状细胞癌的发生与发展.

  12. Functional specialization of chordate CDK1 paralogs during oogenic meiosis.

    Science.gov (United States)

    Øvrebø, Jan Inge; Campsteijn, Coen; Kourtesis, Ioannis; Hausen, Harald; Raasholm, Martina; Thompson, Eric M

    2015-01-01

    Cyclin-dependent kinases (CDKs) are central regulators of eukaryotic cell cycle progression. In contrast to interphase CDKs, the mitotic phase CDK1 is the only CDK capable of driving the entire cell cycle and it can do so from yeast to mammals. Interestingly, plants and the marine chordate, Oikopleura dioica, possess paralogs of the highly conserved CDK1 regulator. However, whereas in plants the 2 CDK1 paralogs replace interphase CDK functions, O. dioica has a full complement of interphase CDKs in addition to its 5 odCDK1 paralogs. Here we show specific sub-functionalization of odCDK1 paralogs during oogenesis. Differential spatiotemporal dynamics of the odCDK1a, d and e paralogs and the meiotic polo-like kinase 1 (Plk1) and aurora kinase determine the subset of meiotic nuclei in prophase I arrest that will seed growing oocytes and complete meiosis. Whereas we find odCDK1e to be non-essential, knockdown of the odCDK1a paralog resulted in the spawning of non-viable oocytes of reduced size. Knockdown of odCDK1d also resulted in the spawning of non-viable oocytes. In this case, the oocytes were of normal size, but were unable to extrude polar bodies upon exposure to sperm, because they were unable to resume meiosis from prophase I arrest, a classical function of the sole CDK1 during meiosis in other organisms. Thus, we reveal specific sub-functionalization of CDK1 paralogs, during the meiotic oogenic program. PMID:25714331

  13. p21WAF1/CIP1 gene DNA sequencing and its expression in human osteosarcoma

    Institute of Scientific and Technical Information of China (English)

    廖威明; 张春林; 李佛保; 曾炳芳; 曾益新

    2004-01-01

    Background Mutation and expression change of p21WAF1/CIP1 may play a role in the growth of osteosarcoma. This study was to investigate the expression of the p21WAF1/CIP1 gene in human osteosarcoma, p21WAF1/CIP1 gene DNA sequence change and their relationships with the phenotype and clinical prognosis.Methods p21WAF1/CIP1 gene in 10 normal people and the tumours of 45 osteosarcoma patients were examined using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) with silver staining. The PCR product with an abnormal strand was sequenced directly. The p21WAF1/CIP1 gene mRNA and P21 protein of 45 cases of osteosarcoma were investigated by using in situ hybridization and immunohistochemistry, respectively. Results The occurrence of P21 protein in osteosarcoma was 17.78% (8/45), and p21WAF1/CIP1 mRNA expression in osteosarcoma was 42.22% (19/45). The p21WAF1/CIP1 gene DNA sequencing of amplified production showed that in p21WAF1/CIP1 gene exon 3 of 36 cases of human osteosarcoma, there were 17 cases (47.22%) with C→T at position 609; 10 normal blood samples' DNA sequence analysis yielded 8 cases (80.00%) with C→T at the same position. Conclusions Along with the increase of malignancy, the expression of p21WAF1/CIP1mRNA and P21 protein in osteosarcoma tends to decrease. It is uncommon for the p21WAF1/CIP1 gene mutation to occur in human osteosarcoma. As a result, the possible existence of tumour subtypes of p21WAF1/CIP1 gene mutation should be investigated. Our research leads to the location of p21WAF1/CIP1 gene polymorphism of Chinese osteosarcoma patients, which can provide a basis for further research.

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

    International Nuclear Information System (INIS)

    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 (pKIpl (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

  15. Berry anthocyanins reduce proliferation of human colorectal carcinoma cells by inducing caspase-3 activation and p21 upregulation.

    Science.gov (United States)

    Anwar, Sirajudheen; Fratantonio, Deborah; Ferrari, Daniela; Saija, Antonella; Cimino, Francesco; Speciale, Antonio

    2016-08-01

    Colorectal cancer is the fourth most common type of cancer worldwide, and adenocarcinoma cells that form the majority of colorectal tumors are markedly resistant to antineoplastic agents. Epidemiological studies have demonstrated that consumption of fruits and vegetables that are rich in polyphenols, is linked to reduced risk of colorectal cancer. In the present study, the effect of a standardized anthocyanin (ACN)‑rich extract on proliferation, apoptosis and cell cycle in the Caco-2 human colorectal cancer cell line was evaluated by trypan blue and clonogenic assays and western blot analysis of cleaved caspase‑3 and p21Waf/Cif1. The results of the current study demonstrated that the ACN extract markedly decreased Caco‑2 cell proliferation, induced apoptosis by activating caspase‑3 cleavage, and upregulated cyclin‑dependent kinase inhibitor 1 (p21Waf/Cif1) expression in a dose dependent manner. Furthermore, ACN extract was able to produce a dose‑dependent increase of intracellular reactive oxygen species (ROS) in Caco‑2 cells, together with a light increase of the cell total antioxidant status. In conclusion, the present study demonstrated that a standardized berry anthocyanin rich extract inhibited proliferation of Caco‑2 cells by promoting ROS accumulation, inducing caspase‑3 activation, and upregulating the expression of p21Waf/Cif1. PMID:27314273

  16. Expressions of CDK2 and CDK4 in intimal cell proliferation in autologous vein grafts%CDK2、CDK4基因与自体移植静脉内膜增殖的关系

    Institute of Scientific and Technical Information of China (English)

    亓明; 王新文; 罗英伟; 秦岭峰; 马文锋; 张强; 辛世杰; 段志泉

    2012-01-01

    [ Objecttive ] To observe CDK2, CDK4 expressions of proliferative intima in autologous grafted vein of rat. [Methods] Rat autologous vein graft model was established. Fifty rats were divided into five groups randomly, and the graft veins were respectively collectived at the 1st, 2nd, 3rd, 7th, and 14th day after the operation. Then the protein expressions of CDK2 and CDK4 were detected by immunohistochemistry and the expressions of their mRNA were detected by RT - PCR in intima of the graft vein. Normal veins were used for control. [ Results] At 7d after operation, the intima proliferation of autologous grafted vein reached nearly to the high point, and it was more obvious than those of control group and groups at 1 d, 2 d and 3 d after operation (P < 0.05) . The numbers of CDK2 and CDK4 positive cells in graft vein increased from 2 d after operation and reached to the peak at 7 d after operation. The expressions of CDK2, CDK4 mRNA reached to the peak during 7 d to 14 d after operation. [ Conclusions ] The expressions of CDK2 and CDK4 increased from early phrase after vein graft, and reached the peak during 7 d to 14 d after operation. The CDK2 and CDK4 played a role in intima proliferation of autologous graft vein.%[目的]了解CDK2、CDK4在大鼠移植血管的表达及对平滑肌细胞增殖的影响.[方法]Wistar大鼠50只,随机分为5组,建立自体静脉移植模型,分别于术后1、2、3、7及14 d取组织形态学观察,并用免疫组织化学和RT-PCR方法检测血管移植后不同时期CDK2、CDK4的表达情况,取正常静脉为对照组.[结果]移植后7d,内膜厚度与管壁厚度接近高峰,与对照组及移植后1、2、3d比较,差异有显著性意义(P<0.05).免疫组织化学显示,移植静脉CDK2、CDK4阳性细胞在移植后2d明显增加,7d达到高峰,与移植后1d比较,差异有显著性意义(P<0.05).RT-PCR检测结果显示,CDK2、CDK4基因mRNA表达产量7~14 d达到高峰,与移植后1、2、3d比

  17. 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/Cd...k9.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

  18. 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://dba...rchive.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://dba...rchive.biosciencedbc.jp/kyushu-u/hg19/colo/CDK9.Liver.tsv http://dbarchive.bios...ciencedbc.jp/kyushu-u/hg19/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Liver.gml ...

  19. 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.

  20. P21-activated kinase 1 and breast cancer

    Institute of Scientific and Technical Information of China (English)

    Jun-Xiang Zhang; Da-Qiang Li; Rakesh Kumar

    2010-01-01

    @@ The p21 activated kinase 1 (PAK1) belongs to PAKs family, a group of highly evolutionarily conserved protein family of serine/threonine kinases, which acts as a downstream effector of the small GTPases Cdc42 and Rac1, firstly reported in 1994[1]. As a serine/threonine kinase, PAK1 plays an important role in many cellular functions including cell morphogenesis, motility, survival, mitosis, angiogenesis, and tumorigenesis. More than 40 proteins have been reported to be phosphorylated by PAK1[2]. Accumulating experimental data in multiple experimental systems provide compelling evidence that PAK1 plays an important role in breast cancer promotion and progression. PAK1 is overexpressed and/or hyperactivated in more than 50% of breast cancers[3]. On the other hand, PAK1 overexpression in estrogen receptor alpha (ER α) positive breast cancer is also closely associated with a reduced responsiveness to tamoxifen therapy[4]. Since PAK1 plays such a vital role in breast cancer, PAK1 targeted therapeutic approaches are likely to be useful in breast cancer treatment as well as in other human cancers with PAK1 upregulation and/or hyperactivation[5].

  1. Dux4 induces cell cycle arrest at G1 phase through upregulation of p21 expression

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hongliang; Wang, Zhaoxia; Jin, Suqin; Hao, Hongjun [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Zheng, Lemin [The Institute of Cardiovascular Sciences, Peking University Health Science Center, Key Laboratory of Molecular Cardiovascular Sciences of Education Ministry, Key Laboratory of Cardiovascular Molecular Biology and Regulatory Peptides of Health Ministry, Beijing 100191 (China); Zhou, Boda [The Department of Cardiology, Peking University Third Hospital, Beijing 100191 (China); Zhang, Wei; Lv, He [Department of Neurology, Peking University First Hospital, Beijing 100034 (China); Yuan, Yun, E-mail: yuanyun2002@sohu.com [Department of Neurology, Peking University First Hospital, Beijing 100034 (China)

    2014-03-28

    Highlights: • Dux4 induced TE671 cell proliferation defect and G1 phase arrest. • Dux4 upregulated p21 expression without activating p53. • Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. • Sp1 binding site was required for Dux4-induced p21 promoter activation. - Abstract: It has been implicated that Dux4 plays crucial roles in development of facioscapulohumeral dystrophy. But the underlying myopathic mechanisms and related down-stream events of this retrogene were far from clear. Here, we reported that overexpression of Dux4 in a cell model TE671 reduced cell proliferation rate, and increased G1 phase accumulation. We also determined the impact of Dux4 on p53/p21 signal pathway, which controls the checkpoint in cell cycle progression. Overexpression of Dux4 increased p21 mRNA and protein level, while expression of p53, phospho-p53 remained unchanged. Silencing p21 rescued Dux4 mediated proliferation defect and cell cycle arrest. Furthermore, we demonstrated that enhanced Dux4 expression increased p21 promoter activity and elevated expression of Sp1 transcription factor. Mutation of Sp1 binding site decreased dux4 induced p21 promoter activation. Chromatin immunoprecipitation (ChIP) assays confirmed the Dux4-induced binding of Sp1 to p21 promoter in vivo. These results suggest that Dux4 might induce proliferation inhibition and G1 phase arrest through upregulation of p21.

  2. p21WAF1/Cip1/Sdi1 knockout mice respond to doxorubicin with reduced cardiotoxicity

    International Nuclear Information System (INIS)

    Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21WAF1/Cip1/Sdi1 (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFNγ and TNFα in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: ► Doxorubicin induces p21 elevation in the myocardium. ► Doxorubicin causes dilated cardiomyopathy in wild type mice. ► p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. ► Lack of inflammatory response correlates with the resistance in p21 knockout mice.

  3. Involvement of calpain/p35-p25/Cdk5/NMDAR signaling pathway in glutamate-induced neurotoxicity in cultured rat retinal neurons.

    Directory of Open Access Journals (Sweden)

    Yanying Miao

    Full Text Available We investigated possible involvement of a calpain/p35-p25/cyclin-dependent kinase 5 (Cdk5 signaling pathway in modifying NMDA receptors (NMDARs in glutamate-induced injury of cultured rat retinal neurons. Glutamate treatment decreased cell viability and induced cell apoptosis, which was accompanied by an increase in Cdk5 and p-Cdk5(T15 protein levels. The Cdk5 inhibitor roscovitine rescued the cell viability and inhibited the cell apoptosis. In addition, the protein levels of both calpain 2 and calpain-specific alpha-spectrin breakdown products (SBDPs, which are both Ca(2+-dependent, were elevated in glutamate-induced cell injury. The protein levels of Cdk5, p-Cdk5(T15, calpain 2 and SBDPs tended to decline with glutamate treatments of more than 9 h. Furthermore, the elevation of SBDPs was attenuated by either D-APV, a NMDAR antagonist, or CNQX, a non-NMDAR antagonist, but was hardly changed by the inhibitors of intracellular calcium stores dantrolene and xestospongin. Moreover, the Cdk5 co-activator p35 was significantly up-regulated, whereas its cleaved product p25 expression showed a transient increase. Glutamate treatment for less than 9 h also considerably enhanced the ratio of the Cdk5-phosphorylated NMDAR subunit NR2A at Ser1232 site (p-NR2A(S1232 and NR2A (p-NR2A(S1232/NR2A, and caused a translocation of p-NR2A(S1232 from the cytosol to the plasma membrane. The enhanced p-NR2A(S1232 was inhibited by roscovitine, but augmented by over-expression of Cdk5. Calcium imaging experiments further showed that intracellular Ca(2+ concentrations ([Ca(2+](i of retinal cells were steadily increased following glutamate treatments of 2 h, 6 h and 9 h. All these results suggest that the activation of the calpain/p35-p25/Cdk5 signaling pathway may contribute to glutamate neurotoxicity in the retina by up-regulating p-NR2A(S1232 expression.

  4. 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.

  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. 皮肤瘢痕癌中CDK4、CDK6蛋白的表达及意义%The expression and its significance of CDK4 and CDK6 in skin scar cancer

    Institute of Scientific and Technical Information of China (English)

    林宇静; 郭瑞珍

    2013-01-01

    目的 探讨细胞周期素依赖激酶CDK4、CDK6蛋白在皮肤瘢痕癌组织中的表达及意义.方法 采用免疫组织化学S-P法检测正常皮肤表皮、皮肤病理性瘢痕被覆上皮和瘢痕癌组织中CDK4、CDK6蛋白的表达.结果 CDK4、CDK6蛋白在皮肤瘢痕癌组中呈阳性或强阳性表达,在皮肤病理性瘢痕组中呈弱阳性表达,在正常皮肤组中呈阴性或弱阳性表达.瘢痕癌组分别与正常皮肤组和皮肤病理性瘢痕组比较,差别有统计学意义(P<0.01).结论 CDK4、CDK6蛋白的过表达可能与瘢痕癌的发生具有相关性.

  7. Up-regulation of p21 and TNF-α is mediated in lycorine-induced death of HL-60 cells

    Directory of Open Access Journals (Sweden)

    He Yan

    2010-08-01

    Full Text Available Abstract Background Leukemia is one of the most life-threatening cancers today, and acute promyelogenous leukemia (APL is a common type of leukemia. Many natural compounds have already been found to exhibit significant anti-tumor effects. Lycorine, a natural alkaloid extracted from Amaryllidaceae, exhibited anti-leukemia effects in vitro and in vivo. The survival rate of HL-60 cells exposed to lycorine was decreased, cell growth was slowed down, and cell regeneration potential was inhibited. HL-60 cells exhibited typical apoptotic characteristic. Lycorine can suppress leukemia growth and reduce cell survival and inducing apoptosis of tumor cells. The purpose of this work is to elucidate the mechanism by which lycorine induces APL cells. Results When HL-60 cells were treated with different concentration of lycorine, the expression of p21 and TNF-α was up-regulated in a concentration-dependent manner as shown by real-time quantitative reverse transcriptase-polymerase chain reaction and Western blotting. Lycorine also down-regulated p21-related gene expression, including Cdc2, Cyclin B, Cdk2 and Cyclin E, promoted Bid truncation, decreased IκB phosphorylation and blocked NF-κB nuclear import. Cytochrome c was released from mitochondria as observed with confocal laser microscopy. Conclusions The TNF-α signal transduction pathway and p21-mediated cell-cycle inhibition were involved in the apoptosis of HL-60 cells induced by lycorine. These results contribute to the development of new lycorine-based anti-leukemia drugs.

  8. 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)

  9. p21WAF1/CIP1 deficiency induces mitochondrial dysfunction in HCT116 colon cancer cells

    International Nuclear Information System (INIS)

    Highlights: ► p21−/− HCT116 cells exhibited an increase in mitochondrial mass. ► The expression levels of PGC-1α and AMPK were upregulated in p21−/− HCT116 cells. ► The proliferation of p21−/− HCT116 cells in galactose medium was significantly impaired. ► p21 may play a role in maintaining proper mitochondrial mass and respiratory function. -- Abstract: p21WAF1/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−/− HCT116 cells, as measured by 10-N-nonyl-acridine orange staining, as well as an increase in the mitochondrial DNA content. In contrast, p53−/− cells had a mitochondrial mass comparable to that of wild-type HCT116 cells. In addition, the expression levels of the mitochondrial biogenesis regulators PGC-1α and TFAM and AMPK activity were also elevated in p21−/− cells, indicating that p21 deficiency induces the rate of mitochondrial biogenesis through the AMPK-PGC-1α axis. However, the increase in mitochondrial biogenesis in p21−/− cells did not accompany an increase in the cellular steady-state level of ATP. Furthermore, p21−/− 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.

  10. 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

    OpenAIRE

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

    2014-01-01

    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 NRASQ61K (constitutively active RAS) or mutant activated CDK4R24C/R24C (prevents binding of CDK4 by kinase inhibitor p16INK4A) with an epidermis-specific knockout...

  11. Mechanism of p53 downstream effectors p21 and Gadd45 in DNA damage surveillance

    Institute of Scientific and Technical Information of China (English)

    孟祥兵; 董燕; 孙志贤

    1999-01-01

    Both p21 (WAF1/CIP1) and Gadd45 were activated in a p53-dependent manner in MCF-7 cells after being exposed to ionizing radiation. In order to investigate their roles in DNA damage surveillance, p21as/MCF-7 cells stably transfected by p21 antisense expression plasmid pC-WAF1-AS and Gadd45as/MCF-7 stably transfected by Gadd45 antisense expression plasmid pCMVas45 were established. It was observed that G1 arrest induced by radiation was significantly reduced in Gadd45as/MCF-7 cells as well as in p21as/MCF-7 cells. Repair of radiation damaged report gene greatly reduced in Gadd45as/MCF-7 and p21as/MCF-7 cells. Apoptosis significantly increased in p21as/MCF-7 after exposure to radiation. These results suggest that both p21 and Gadd45 support cellular survival by taking roles in G1 arrest and DNA repair, furthermore, p21 protects cells from death by inhibiting apoptosis after exposure to ionizing radiation.

  12. KSHV G protein-coupled receptor inhibits lytic gene transcription in primary-effusion lymphoma cells via p21-mediated inhibition of Cdk2

    OpenAIRE

    Cannon, M; Cesarman, E; Boshoff, C

    2006-01-01

    Kaposi sarcoma (KS) remains the most common AIDS-associated malignancy worldwide. In sub-Saharan Africa especially, this aggressive endothelial-cell tumor is a cause of widespread morbidity and mortality. Infection with Kaposi sarcoma-associated herpesvirus (KSHV) is now known to be an etiologic force behind KS and primary-effusion lymphoma (PEL). Over time, KSHV has pirated many human genes whose products regulate angiogenesis, inflammation, and the cell cycle. One of these, the KSHV vGPCR, ...

  13. CDK4 is an essential insulin effector in adipocytes

    Science.gov (United States)

    Lagarrigue, Sylviane; Lopez-Mejia, Isabel C.; Denechaud, Pierre-Damien; Escoté, Xavier; Castillo-Armengol, Judit; Jimenez, Veronica; Chavey, Carine; Giralt, Albert; Lai, Qiuwen; Zhang, Lianjun; Martinez-Carreres, Laia; Delacuisine, Brigitte; Annicotte, Jean-Sébastien; Blanchet, Emilie; Huré, Sébastien; Abella, Anna; Tinahones, Francisco J.; Vendrell, Joan; Dubus, Pierre; Bosch, Fatima; Kahn, C. Ronald; Fajas, Lluis

    2015-01-01

    Insulin resistance is a fundamental pathogenic factor that characterizes various metabolic disorders, including obesity and type 2 diabetes. Adipose tissue contributes to the development of obesity-related insulin resistance through increased release of fatty acids, altered adipokine secretion, and/or macrophage infiltration and cytokine release. Here, we aimed to analyze the participation of the cyclin-dependent kinase 4 (CDK4) in adipose tissue biology. We determined that white adipose tissue (WAT) from CDK4-deficient mice exhibits impaired lipogenesis and increased lipolysis. Conversely, lipolysis was decreased and lipogenesis was increased in mice expressing a mutant hyperactive form of CDK4 (CDK4R24C). A global kinome analysis of CDK4-deficient mice following insulin stimulation revealed that insulin signaling is impaired in these animals. We determined that insulin activates the CCND3-CDK4 complex, which in turn phosphorylates insulin receptor substrate 2 (IRS2) at serine 388, thereby creating a positive feedback loop that maintains adipocyte insulin signaling. Furthermore, we found that CCND3 expression and IRS2 serine 388 phosphorylation are increased in human obese subjects. Together, our results demonstrate that CDK4 is a major regulator of insulin signaling in WAT. PMID:26657864

  14. Structure-based library approach to kinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Norman, T.C.; Gray, N.S.; Koh, J.T.; Schultz, P.G. [Univ. of California, Berkeley, CA (United States)

    1996-08-07

    While purine analogs were being screened for inhibition of various protein kinases, a relatively selective inhibitor, olomoucine, was identified that competitively inhibits CDK2/cyclin A with an IC{sub 50} of 7 {mu}M. A comparison of the CDK2 crystal structures containing bound ATP and bound olomoucine confirms that olomoucine binds in the adenine binding pocket of CDK2, but its purine nucleus adopts an entirely different orientation than that observed for ATP. In spite of the good shape complementarity shown by the olomoucine-CDK2 complex, structural variations at C-6, C-2, and N-9 might be expected to lead to enhanced affinity and selectivity for CDK2. The coupling of this structural information with combinatorial methods is an obvious strategy for optimizing olomoucine`s potency. Herein we apply this approach to the solid-phase synthesis and screening of combinatorial libraries based on the purine scaffold found in olomoucine. The iteration of library synthesis with structural analysis of the optimized leads should provide an effective strategy for the development of more potent and selective inhibitors of CDK2. In addition, libraries containing purine derivatives may prove useful in the search for inhibitors of a large number of cellular processes. 24 refs., 1 fig.

  15. p21 controls patterning but not homologous recombination in RPE development.

    Science.gov (United States)

    Bishop, A J R; Kosaras, B; Hollander, M C; Fornace, A; Sidman, R L; Schiestl, R H

    2006-01-01

    p21/WAF1/CIP1/MDA6 is a key cell cycle regulator. Cell cycle regulation is an important part of development, differentiation, DNA repair and apoptosis. Following DNA damage, p53 dependent expression of p21 results in a rapid cell cycle arrest. p21 also appears to be important for the development of melanocytes, promoting their differentiation and melanogenesis. Here, we examine the effect of p21 deficiency on the development of another pigmented tissue, the retinal pigment epithelium. The murine mutation pink-eyed unstable (p(un)) spontaneously reverts to a wild-type allele by homologous recombination. In a retinal pigment epithelium cell this results in pigmentation, which can be observed in the adult eye. The clonal expansion of such cells during development has provided insight into the pattern of retinal pigment epithelium development. In contrast to previous results with Atm, p53 and Gadd45, p(un) reversion events in p21 deficient mice did not show any significant change. These results suggest that p21 does not play any role in maintaining overall genomic stability by regulating homologous recombination frequencies during development. However, the absence of p21 caused a distinct change in the positions of the reversion events within the retinal pigment epithelium. Those events that would normally arrest to produce single cell events continued to proliferate uncovering a cell cycle dysregulation phenotype. It is likely that p21 is involved in controlling the developmental pattern of the retinal pigment. We also found a C57BL/6J specific p21 dependent ocular defect in retinal folding, similar to those reported in the absence of p53. PMID:16202662

  16. Mutagenesis in vivo in T cells of p21-deficient mice

    OpenAIRE

    Shao, Changshun; Liang, Li; Zhao, Xin; Chen, Yanping; Zheng, Betty; Chen, Jianmin; Luo, Minjie; Tischfield, Jay A

    2009-01-01

    Mice that are deficient in p53 exhibit an early onset of multiple types of tumors, especially thymic lymphoma. However, it remains unclear to what extent each of the p53-regulated pathways exerts its tumor suppressor activity. p21Cip1/Waf1, acting down stream of p53, is a major G1/S checkpoint protein that restricts cell cycle progression into S phase in the presence of DNA damage. While at old ages p21−/− mice have a higher incidence of many types of tumors than p21+/+ mice, they are more re...

  17. Cdk Activity Couples Epigenetic Centromere Inheritance to Cell Cycle Progression

    OpenAIRE

    Silva, Mariana C.C.; Bodor, Dani L.; Stellfox, Madison E.; Martins, Nuno M.C.; Hochegger, Helfrid; Foltz, Daniel R.; Jansen, Lars E.T.

    2012-01-01

    Centromeres form the site of chromosome attachment to microtubules during mitosis. Identity of these loci is maintained epigenetically by nucleosomes containing the histone H3 variant CENP-A. Propagation of CENP-A chromatin is uncoupled from DNA replication initiating only during mitotic exit. We now demonstrate that inhibition of Cdk1 and Cdk2 activities is sufficient to trigger CENP-A assembly throughout the cell cycle in a manner dependent on the canonical CENP-A assembly machinery. We fur...

  18. Structural and functional analysis of cyclin D1 reveals p27 and substrate inhibitor binding requirements

    Science.gov (United States)

    Liu, Shu; Bolger, Joshua K.; Kirkland, Lindsay O.; Premnath, Padmavathy N.; McInnes, Campbell

    2012-01-01

    An alternative strategy for inhibition of the cyclin dependent kinases in anti-tumor drug discovery is afforded through the substrate recruitment site on the cyclin positive regulatory subunit. Critical CDK substrates such as the Rb and E2F families must undergo cyclin groove binding before phosphorylation and hence inhibitors of this interaction also block substrate specific kinase activity. This approach offers the potential of generating highly selective and cell cycle specific CDK inhibitors and to reduce the inhibition of transcription mediated through CDK7 and 9, commonly observed with ATP competitive compounds. While highly potent peptide and small molecule inhibitors of CDK2/cyclin A, E substrate recruitment have been reported, little information has been generated on the determinants of inhibitor binding to the cyclin groove of the CDK4/cyclin D1 complex. CDK4/cyclin D is a validated anti-cancer drug target and continues to be widely pursued in the development of new therapeutics based on cell cycle blockade. We have therefore investigated the structural basis for peptide binding to its cyclin groove and have examined the features contributing to potency and selectivity of inhibitors. Peptidic inhibitors of CDK4/cyclin D of pRb phosphorylation have been synthesized, and their complexes with CDK4/cyclin D1 crystal structures have been generated. Based on available structural information, comparisons of the cyclin grooves of cyclin A2 and D1 are presented and provide insights into the determinants for peptide binding and the basis for differential binding and inhibition. In addition, a complex structure has been generated in order to model the interactions of the CDKI, p27KIP1, with cyclin D1. This information has been used shed light onto the endogenous inhibition of CDK4 and also to identify unique aspects of cyclin D1 and which can be exploited in the design of cyclin groove based CDK inhibitors. Peptidic and non-peptidic compounds have been synthesized

  19. Trisomy for 8p21→pter owing to a familial translocation

    OpenAIRE

    Allen, Elizabeth F.; Hodgkin, William E

    1983-01-01

    A girl with developmental delay and physical abnormalities was trisomic for the segment 8p21→pter owing to a familial translocation t(8;11). The child's father and paternal grandmother carry the same translocation.

  20. EXPRESSION AND SIGNIFICANCE OF SMAD4 AND p21WAF1 IN ENDOMETRIAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    葛秀君; 李英勇

    2003-01-01

    Objective: To investigate the expression of Smad4 and p21WAF1 in endometrial carcinoma and its clinical significance. Methods: Immunohistochemical method was used to detect Smad4 and p21WAF1 expression in 56 cases of endometrial carcinoma. Results: The positive rate of Smad4 was 80.36% in endometrial carcinoma. The Samd4 expression was significantly correlated with histological grade (P0.05). Conclusion: Smad4 may play an important role in the tumorigenesis, differentiation and progression of endometrial carcinoma. The expression of p21WAF1 was associated with the tumorigenesis of endometrial carcinoma, but the association between p21WAF1 and differentiation and progression of endometrial carcinomas needs to be further investigated.

  1. Dysregulation of CDK8 and Cyclin C in tumorigenesis %Dysregulation of CDK8 and Cyclin C in tumorigenesis

    Institute of Scientific and Technical Information of China (English)

    Wu Xu; Jun-Yuan Ji

    2011-01-01

    Appropriately controlled gene expression is fundamental for normal growth and survival of all living organisms.In eukaryotes,the transcription of protein-coding mRNAs is dependent on RNA polymerase Ⅱ (Pol Ⅱ).The multi-subunit transcription cofactor Mediator complex is proposed to regulate most,if not all,of the Pol Ⅱ-dependent transcription.Here we focus our discussion on two subunits of the Mediator complex,cyclin-dependent kinase 8 (CDK8) and its regulatory partner Cyclin C (CycC),because they are either mutated or amplified in a variety of human cancers.CDK8 functions as an oncoprotein in melanoma and colorectal cancers,thus there are considerable interests in developing drugs specifically targeting the CDK8 kinase activity.However,to evaluate the feasibility of targeting CDK8 for cancer therapy and to understand how their dysregulation contributes to tumorigenesis,it is essential to elucidate the in vivo function and regulation of CDK8-CycC,which are still poorly understood in multi-cellular organisms.We summarize the evidence linking their dysregulation to various cancers and present our bioinformatics and computational analyses on the structure and evolution of CDK8.We also discuss the implications of these observations in tumorigenesis.Because most of the Mediator subunits,including CDK8 and CycC,are highly conserved during eukaryotic evolution,we expect that investigations using model organisms such as Drosophila will provide important insights into the function and regulation of CDK8 and CycC in different cellular and developmental contexts.

  2. Androgen via p21 Inhibits Tumor Necrosis Factor α-induced JNK Activation and Apoptosis*

    OpenAIRE

    Tang, Fangming; Kokontis, John; Lin, Yuting; Liao, Shutsung; Lin, Anning; Xiang, Jialing

    2009-01-01

    The male hormone androgen is a growth/survival factor for its target tissues or organs. Yet, the underlying mechanism is incompletely understood. Here, we report that androgen via p21 inhibits tumor necrosis factor α-induced JNK activation and apoptosis. Inhibition by androgen requires the transcription activity of androgen receptor (AR) and de novo protein synthesis. Androgen·AR induces expression of p21 that in turn inhibits tumor necrosis factor α-induced JNK and apoptosis. Furthermore, ge...

  3. Association of p21 SNPs and risk of cervical cancer among Chinese women

    International Nuclear Information System (INIS)

    The p21 codon 31 single nucleotide polymorphism (SNP), rs1801270, has been linked to cervical cancer but with controversial results. The aims of this study were to investigate the role of p21 SNP-rs1801270 and other untested p21 SNPs in the risk of cervical cancer in a Chinese population. We genotyped five p21 SNPs (rs762623, rs2395655, rs1801270, rs3176352, and rs1059234) using peripheral blood DNA from 393 cervical cancer patients and 434 controls. The frequency of the rs1801270 A allele in patients (0.421) was significantly lower than that in controls (0.494, p = 0.003). The frequency of the rs3176352 C allele in cases (0.319) was significantly lower than that in controls (0.417, p < 0.001).The allele frequency of other three p21 SNPs showed not statistically significantly different between patients and controls. The rs1801270 AA genotype was associated with a decreased risk for the development of cervical cancer (OR = 0.583, 95%CI: 0.399 - 0.853, P = 0.005). We observed that the three p21 SNPs (rs1801270, rs3176352, and rs1059234) was in linkage disequilibrium (LD) and thus haplotype analysis was performed. The AGT haplotype (which includes the rs1801270A allele) was the most frequent haplotype among all subjects, and both homozygosity and heterozygosity for the AGT haplotype provided a protective effect from development of cervical cancer. We show an association between the p21 SNP rs1801270A allele and a decreased risk for cervical cancer in a population of Chinese women. The AGT haplotype formed by three p21 SNPs in LD (rs1801270, rs3176352 and rs1059234) also provided a protective effect in development of cervical cancer in this population

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

    OpenAIRE

    Zhang, Yanhong; Yan, Wensheng; 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 multipl...

  5. Influence of CDK1 and CDK2 siRNA interference on tumor cell cycle and cell apoptosis%CDK1、CDK2 siRNA干扰对肿瘤细胞凋亡和细胞周期的影响

    Institute of Scientific and Technical Information of China (English)

    Hui Xiao; Wanjun Gong; Jingpeng Cao; Xiaolan Li; Deding Tao; Junbo Hu; Jianping Gong

    2009-01-01

    Objective: We investigated the influence of CDK1 and CDK2 expression inhibited by cotransfection of CDK1 and CDK2 siRNA on cell cycle and apoptosis, explored the exact role of cell cycle master regulator in tumor cell apoptosis process. Methods: The siRNA targeting the CDK1 and CDK2 genes were synthesized and simultaneously cotransfected into Hela cells by lipofectamine 2000.48 or 60 h after the cotransfection, CDK1 and CDK2 protein expressions were examined by Western blot. Cell cycle distribution was analyzed by flow cytometry. Cell apoptosis was detected by the Annexin V/PI method. The changes of the transfected cell morphological under a microscope after Wright-Giemsa Staining were studied. Results: CDK1 and CDK2 protein expression was decreased at 48 or 60 h after cotransfection. The accumulation of the G2/M and S phase population in cell cycle of the cotrensfected cells at 48 or 60 h after transfection was enhanced obviously compared with control. The ratio of apoptotic cell of cotransfected cells at 48 or 60 h after transfection was increased significantly compared with control. More binucleate or multinucleate cells among cotransfected cells were observed under the microscope. Conclu- sion: The decreased expression of CDK1 and CDK2 by cotransfection of CDK1 and CDK2 siRNA not only leads to tumor cell cycle arrest in S phase and G2/M phase, but also induces tumor cell apoptosis.

  6. Flow cytometric analysis of p21 protein expression on irradiated human lymphocytes

    International Nuclear Information System (INIS)

    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. 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

  8. Novel structural features of CDK inhibition revealed by an ab initio computational method combined with dynamic simulations

    CERN Document Server

    Heady, Lucy; Mancera, Ricardo L; Joyce, Sian; Venkitaraman, Ashok R; Artacho, Emilio; Skylaris, Chris-Kriton; Ciacchi, Lucio Colombi; Payne, Mike C

    2008-01-01

    The rational development of specific inhibitors for the ~500 protein kinases encoded in the human genome is impeded by a poor understanding of the structural basis for the activity and selectivity of small molecules that compete for ATP binding. Combining classical dynamic simulations with a novel ab initio computational approach linear-scalable to molecular interactions involving thousands of atoms, we have investigated the binding of five distinct inhibitors to the cyclin-dependent kinase CDK2. We report here that polarization and dynamic hydrogen bonding effects, so far undetected by crystallography, affect both their activity and selectivity. The effects arise from the specific solvation patterns of water molecules in the ATP binding pocket or the intermittent formation of hydrogen bonds during the dynamics of CDK/inhibitor interactions and explain the unexpectedly high potency of certain inhibitors such as 3-(3H-imidazol-4-ylmethylene)-5-methoxy-1,3-dihydro-indol-2-one (SU9516). The Lys89 residue in the ...

  9. 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.

  10. Sequencing analysis of mutant allele cdc28-srm of protein kinase CDC28 and molecular dynamics study of glycine-rich loop in wild-type and mutant allele G16S of CDK2 as model

    International Nuclear Information System (INIS)

    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 Saccharomyces cerevisiae a very attractive model for studies of mechanisms of CDK regulation. Earlier it was found that certain gene mutations including cdc28-srm affect cell cycle progression, maintenance of different genetic structures and increase cell sensitivity to ionizing radiation. A cdc28-srm mutation is not a temperature-sensitive mutation and differs from the known cdc28-ts mutations because it has the evident phenotypic manifestations at 30 deg C. Sequencing analysis of 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 structure of the human CDK2 has served as a model for the catalytic core of other CDKs, including CDC28. Nanoseconds long molecular dynamics (MD) trajectories of the CDK2/ATP complex were analyzed. The MD simulations of CDK2-G16S (CDC28-G20S) substitution show conformational changes of CDK2 structure resulting in the moving of the G-loop away from ATP and a new rearrangement of amino acids in the T-loop

  11. Arsenic trioxide phosphorylates c-Fos to transactivate p21WAF1/CIP1 expression

    International Nuclear Information System (INIS)

    An infamous poison, arsenic also has been used as a drug for nearly 2400 years; in recently years, arsenic has been effective in the treatment of acute promyelocytic leukemia. Increasing evidence suggests that opposite effects of arsenic trioxide (ATO) on tumors depend on its concentrations. For this reason, the mechanisms of action of the drug should be elucidated, and it should be used therapeutically only with extreme caution. Previously, we demonstrated the opposing effects of ERK1/2 and JNK on p21WAF1/CIP1 (p21) expression in response to ATO in A431 cells. In addition, JNK phosphorylates c-Jun (Ser63/73) to recruit TGIF/HDAC1 to suppress p21 gene expression. Presently, we demonstrated that a high concentration of ATO sustains ERK1/2 phosphorylation, and increases c-Fos biosynthesis and stability, which enhances p21 gene expression. Using site-directed mutagenesis, a DNA affinity precipitation assay, and functional assays, we demonstrated that phosphorylation of the C-terminus of c-Fos (Thr232, Thr325, Thr331, and Ser374) plays an important role in its binding to the p21 promoter, and in conjunction with N-terminus phosphorylation of c-Fos (Ser70) to transactivate p21 promoter expression. In conclusion, a high concentration of ATO can sustain ERK1/2 activation to enhance c-Fos expression, then dimerize with dephosphorylated c-Jun (Ser63/73) and recruit p300/CBP to the Sp1 sites (- 84/- 64) to activate p21 gene expression in A431 cells

  12. 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.

  13. Lipotoxic effect of p21 on free fatty acid-induced steatosis in L02 cells.

    Directory of Open Access Journals (Sweden)

    Jie-wei Wang

    Full Text Available Nonalcoholic fatty liver disease (NAFLD is increasingly regarded as a hepatic manifestation of metabolic syndrome. Though with high prevalence, the mechanism is poorly understood. This study aimed to investigate the effects of p21 on free fatty acid (FFA-induced steatosis in L02 cells. We therefore analyzed the L02 cells with MG132 and siRNA treatment for different expression of p21 related to lipid accumulation and lipotoxicity. Cellular total lipid was stained by Oil Red O, while triglyceride content, cytotoxicity assays, lipid peroxidation markers and anti-oxidation levels were measured by enzymatic kits. Treatment with 1 mM FFA for 48 hr induced magnificent intracellular lipid accumulation and increased oxidative stress in p21 overload L02 cells compared to that in p21 knockdown L02 cells. By increasing oxidative stress and peroxidation, p21 accelerates FFA-induced lipotoxic effect in L02 cells and might provide information about potentially new targets for drug development and treatments of NAFLD.

  14. Trisubstituted pyrazolopyrimidines as novel angiogenesis inhibitors.

    Directory of Open Access Journals (Sweden)

    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.

  15. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

    OpenAIRE

    Matsui, Y.; Kikuchi, A; Araki, S; Hata, Y; Kondo, J; Teranishi, Y; Takai, Y.

    1990-01-01

    We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe ...

  16. Oridonin Up-regulates Expression of P21 and Induces Autophagy and Apoptosis in Human Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Xiang Li, Xiang Li, Jiaxiong Wang, Zaiyuan Ye, Ji-Cheng Li

    2012-01-01

    Full Text Available Background: Oridonin (ORI could inhibit the proliferation and induce apoptosis in various cancer cell lines. However, the mechanism is not fully understood.Methods: Human prostate cancer (HPC cells were cultured in vitro and cell viability was detected by the CCK-8 assay. The ultrastructure changes were observed under transmission electron microscope (TEM. Chemical staining with acridine orange (AO, MDC or DAPI was used to detect acidic vesicular organelles (AVOs and alternation of DNA. Expression of LC3 and P21 was detected by Western Blot. Apoptotic rates and cell cycle arrest were detected by FACS.Results: Our study demonstrated that after ORI treatment, the proliferations of human prostate cancer (HPC cell lines PC-3 and LNCaP were inhibited in a concentration and time-dependent manner. ORI induced cell cycle arrest at the G2/M phase. A large number of autophagosomes with double-membrane structure and acidic vesicular organelles (AVOs were detected in the cytoplasm of HPC cells treated with ORI for 24 hours. ORI resulted in the conversion of LC3-I to LC3-II and recruitment of LC3-II to the autophagosomal membranes. Autophagy inhibitor 3-methyladenine (3-MA reduced AVOs formation and inhibited LC3-I to LC3-II conversion. At 48 h, DNA fragmentation, chromatin condensation and disappearance of surface microvilli were detected in ORI-treated cells. ORI induced a significant increase in the number of apoptotic cells (PC-3: 5.4% to 27.0%, LNCaP: 5.3% to 31.0%. Promoting autophagy by nutrient starvation increased cell viability, while inhibition of autophagy by 3-MA promoted cell death. The expression of P21 was increased by ORI, which could be completely reversed by the inhibition of autophagy.Conclusions: Our findings indicated that autophagy occurred before the onset of apoptosis and protected cancer cells in ORI-treated HPC cells. P21 was involved in ORI-induced autophagy and apoptosis. Our results provide an experimental basis for understand

  17. 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.

  18. EZH2 regulates neuroepithelium structure and neuroblast proliferation by repressing p21

    Science.gov (United States)

    Akizu, Naiara; García, María Alejandra; Estarás, Conchi; Fueyo, Raquel; Badosa, Carmen; de la Cruz, Xavier

    2016-01-01

    The function of EZH2 as a transcription repressor is well characterized. However, its role during vertebrate development is still poorly understood, particularly in neurogenesis. Here, we uncover the role of EZH2 in controlling the integrity of the neural tube and allowing proper progenitor proliferation. We demonstrate that knocking down the EZH2 in chick embryo neural tubes unexpectedly disrupts the neuroepithelium (NE) structure, correlating with alteration of the Rho pathway, and reduces neural progenitor proliferation. Moreover, we use transcriptional profiling and functional assays to show that EZH2-mediated repression of p21WAF1/CIP1 contributes to both processes. Accordingly, overexpression of cytoplasmic p21WAF1/CIP1 induces NE structural alterations and p21WAF1/CIP1 suppression rescues proliferation defects and partially compensates for the structural alterations and the Rho activity. Overall, our findings describe a new role of EZH2 in controlling the NE integrity in the neural tube to allow proper progenitor proliferation.

  19. 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.

  20. An emerging role for p21-activated kinases (Paks) in viral infections

    DEFF Research Database (Denmark)

    Van den Broeke, Celine; Radu, Maria; Chernoff, Jonathan;

    2010-01-01

    p21-activated protein kinases (Paks) are cytosolic serine/threonine protein kinases that act as effectors for small (p21) GTPases of the Cdc42 and Rac families. It has long been established that Paks play a major role in a host of vital cellular functions such as proliferation, survival and...... motility, and abnormal Pak function is associated with a number of human diseases. Here, we discuss emerging evidence that these enzymes also play a major role in the entry, replication and spread of many important pathogenic human viruses, including HIV. Careful assessment of the potential role of Paks in...

  1. INK4/ARF Transcript Expression Is Associated with Chromosome 9p21 Variants Linked to Atherosclerosis

    OpenAIRE

    Liu, Yan; Sanoff, Hanna K.; Cho, Hyunsoon; Burd, Christin E.; Torrice, Chad; Mohlke, Karen L.; Ibrahim, Joseph G.; Thomas, Nancy E.; Sharpless, Norman E.

    2009-01-01

    Background Genome-wide association studies (GWAS) have linked common single nucleotide polymorphisms (SNPs) on chromosome 9p21 near the INK4/ARF (CDKN2A/B) tumor suppressor locus with risk of atherosclerotic diseases and type 2 diabetes mellitus. To explore the mechanism of this association, we investigated whether expression of proximate transcripts (p16INK4a , p15INK4b , ARF, ANRIL and MTAP) correlate with genotype of representative 9p21 SNPs. Methodology/Principal Findings We analyzed expr...

  2. Mutations in CDK5RAP2 cause Seckel syndrome

    OpenAIRE

    Karabey Kayserili, Hülya; Yiğit, G.; Brown, KE.; Pohl, E.; Caliebe, A.; Zahnleiter, D.; Rosser, E.; Bögershausen, N.; Uyguner, ZO.; Altunoğlu, U.; Nürnberg, G.; Nürnberg, P.; Rauch, A.; Li, Y.; Thiel, CT.; Wollnik, B.

    2015-01-01

    Seckel syndrome is a heterogeneous, autosomal recessive disorder marked by prenatal proportionate short stature, severe microcephaly, intellectual disability, and characteristic facial features. Here, we describe the novel homozygous splice-site mutations c.383+1G>C and c.4005-9A>G in CDK5RAP2 in two consanguineous families with Seckel syndrome. CDK5RAP2 (CEP215) encodes a centrosomal protein which is known to be essential for centrosomal cohesion and proper spindle formation and has been sho...

  3. 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.

  4. File list: Oth.EmF.05.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.05.Cdk9.AllCell mm9 TFs and others Cdk9 Embryonic fibroblast SRX620288,SRX6...20289,SRX255482,SRX620286,SRX620287 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.05.Cdk9.AllCell.bed ...

  5. File list: Oth.Bld.20.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.20.Cdk9.AllCell mm9 TFs and others Cdk9 Blood SRX277329,SRX020973,SRX020972...,SRX020974,SRX020971 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.20.Cdk9.AllCell.bed ...

  6. File list: Oth.PSC.05.Cdk8.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.05.Cdk8.AllCell mm9 TFs and others Cdk8 Pluripotent stem cell SRX236482,SRX...668247 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Cdk8.AllCell.bed ...

  7. File list: Oth.Bld.05.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.Bld.05.Cdk9.AllCell mm9 TFs and others Cdk9 Blood SRX277329,SRX020971,SRX020972...,SRX020973,SRX020974 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Bld.05.Cdk9.AllCell.bed ...

  8. File list: Oth.PSC.10.Cdk8.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.10.Cdk8.AllCell mm9 TFs and others Cdk8 Pluripotent stem cell SRX668247,SRX...236482 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.10.Cdk8.AllCell.bed ...

  9. File list: Oth.PSC.50.Cdk8.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.PSC.50.Cdk8.AllCell mm9 TFs and others Cdk8 Pluripotent stem cell SRX236482,SRX...668247 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.50.Cdk8.AllCell.bed ...

  10. File list: Oth.EmF.50.Cdk9.AllCell [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Oth.EmF.50.Cdk9.AllCell mm9 TFs and others Cdk9 Embryonic fibroblast SRX620288,SRX6...20289,SRX620287,SRX620286,SRX255482 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Cdk9.AllCell.bed ...

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

    Science.gov (United States)

    Modi, Khushbu K; Roy, Avik; Brahmachari, Saurabh; Rangasamy, Suresh B; Pahan, Kalipada

    2015-01-01

    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. PMID:26102198

  12. 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.

  13. Identification of new targets of human glioma CDK2 siRNA%人脑胶质瘤CDK2干扰RNA新靶点的检验

    Institute of Scientific and Technical Information of China (English)

    呼格吉乐; 张军力; 段美庆; 王俊瑞; 高乃康

    2012-01-01

    Objective To construct four new eukaryotic expression vectors of small interference RNA(siRNA) specific for CDK2 and confirm the interferential efficiency of siRNA on the expression of CDK2. Methods (l)Four new eukaryotic expression vectors of siRNA specific for CDK2 were constructed and identified by double enzymic digestion. (2)SHG44 cell line of human brain gliocytoma was transiently transfected with the four new vectors via oligofectamine. (3) Vector, with the strongest interferential efficiency, was confirmed by detecting the expression level of CDK2 Mrna using reverse transcription-polymerase chain reactionCRT-PCR). Results (l)Four eukaryotic expression vectors of siRNA specific for new targets of CDK2 was constructed and denominated as Pgpu6/GFP/Neo-CDK2-l,Pgpu6/GFP/Neo-CDK2-2,Pgpu6/GFP/Neo-CDK2-3 and Pgpu6/GFP/Neo-CDK2-4. (2)The expression of CDK2 Mrna was obviously suppressed and the vector with the strongest interferential efficiency was obtained. Conclusion The eukaryotic expression vectors of siRNA,specific for new target of CDK2 and with the strongest interferential efficiency, was successfully constructed and indentified,which could obviously suppress the expression of CDK2 Mrna in SHG44 cell line.%目的 构建4个新靶点CDK2干扰RNA真核表达载体,转染人脑胶质细胞瘤SHG44细胞,经逆转录-聚合酶链反应(RT-PCR)检测mRNA表达,获得干扰效果最好的真核表达载体,为CDK2成为人脑肿瘤标志物提供有价值的资料.方法 (1)构建4个新靶点CDK2干扰RNA真核表达载体并用双酶切和测序鉴定;(2)用脂质体法瞬时转染上述4个载体到SHG44细胞株;(3)通过 RT-PCR 比较转染后CDK2 mRNA表达量,选出干扰效果最好的一个载体.结果 (1)成功构建了4个新靶点CDK2干扰RNA真核表达载体即pGPU6/GFP/Neo-CDK2-1、pGPU6/GFP/Neo-CDK2-2、pGPU6/GFP/Neo-CDK2-3、pGPU6/GFP/Neo-CDK2-4;(2)CDK2 mRNA表达明显受抑制,并获得效果最好的CDK2干扰RNA真核表达载体.结论 成

  14. 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.

  15. p21{sup WAF1/Cip1/Sdi1} knockout mice respond to doxorubicin with reduced cardiotoxicity

    Energy Technology Data Exchange (ETDEWEB)

    Terrand, Jerome; Xu, Beibei; Morrissy, Steve; Dinh, Thai Nho [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Williams, Stuart [Biomedical Engineering Program, College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States); Chen, Qin M., E-mail: qchen@email.arizona.edu [Department of Pharmacology,College of Medicine, University of Arizona, 1501 N. Campbell Ave, Tucson, AZ 85724 (United States)

    2011-11-15

    Doxorubicin (Dox) is an antineoplastic agent that can cause cardiomyopathy in humans and experimental animals. As an inducer of reactive oxygen species and a DNA damaging agent, Dox causes elevated expression of p21{sup WAF1/Cip1/Sdi1} (p21) gene. Elevated levels of p21 mRNA and p21 protein have been detected in the myocardium of mice following Dox treatment. With chronic treatment of Dox, wild type (WT) animals develop cardiomyopathy evidenced by elongated nuclei, mitochondrial swelling, myofilamental disarray, reduced cardiac output, reduced ejection fraction, reduced left ventricular contractility, and elevated expression of ANF gene. In contrast, p21 knockout (p21KO) mice did not show significant changes in the same parameters in response to Dox treatment. In an effort to understand the mechanism of the resistance against Dox induced cardiomyopathy, we measured levels of antioxidant enzymes and found that p21KO mice did not contain elevated basal or inducible levels of glutathione peroxidase and catalase. Measurements of 6 circulating cytokines indicated elevation of IL-6, IL-12, IFN{gamma} and TNF{alpha} in Dox treated WT mice but not p21KO mice. Dox induced elevation of IL-6 mRNA was detected in the myocardium of WT mice but not p21KO mice. While the mechanism of the resistance against Dox induced cardiomyopathy remains unclear, lack of inflammatory response may contribute to the observed cardiac protection in p21KO mice. -- Highlights: Black-Right-Pointing-Pointer Doxorubicin induces p21 elevation in the myocardium. Black-Right-Pointing-Pointer Doxorubicin causes dilated cardiomyopathy in wild type mice. Black-Right-Pointing-Pointer p21 Knockout mice are resistant against doxorubicin induced cardiomyopathy. Black-Right-Pointing-Pointer Lack of inflammatory response correlates with the resistance in p21 knockout mice.

  16. 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

  17. Variants in 9p21 Predicts Severity of Coronary Artery Disease in a Chinese Han Population.

    Science.gov (United States)

    Jing, Jinjin; Su, Li; Zeng, Ying; Tang, Xiaojun; Wei, Jie; Wang, Long; Zhou, Li

    2016-09-01

    Recent genome-wide association studies identified the common genetic variants in 9p21 were associated with the coronary artery disease (CAD). However, whether this locus could predict the severity of CAD in Chinese Han population is unclear. 499 CAD patients who underwent coronary angiography (CAG) have been enrolled for this study. The single-nucleotide polymorphisms rs2383207 and rs2383206 in 9p21 were genotyped in 499 CAG cases and 1519 controls in Chinese Han population. The gene dosage of 9p21 was stratified by the degree of vascular lesions and tested for association with the severity of CAD. Rs2383207 and rs2383206 demonstrated significant associations with 2-vessel and 3-vessel disease (P = 2.0×10(-3) and 1.9×10(-4) , respectively). GG genotypes of rs2383206 occurred higher proportion of left main trunk (LM) disease (P = 6.0×10(-3) ). GG genotypes of rs2383207 occurred higher proportion of left anterior descending artery disease (LAD) and right CAD (RCA) (P = 2.7×10(-6) and 1.6×10(-4) , respectively). The risk allele G of rs2383207 was associated with severity of CAD estimated by the Gensini score (P = 3.6×10(-5) ). Rs2383207 may strongly influence the development of CAD in Chinese Han population. The gene dosage in 9p21 could predict the severity of CAD. PMID:27461153

  18. Familial chromosome translocation t(3;18)(p21;p11).

    OpenAIRE

    Buchinger, G; Wettstein, A; Metze, H

    1981-01-01

    A familial translocation t(3;18)(p21;p11) was observed in a newborn male. He had multiple malformations resulting from partial trisomy 3 and partial monosomy 18. The mother, maternal uncle, and maternal grandmother were found to be balanced translocation carriers. A daughter of the maternal uncle with similar malformations probably had the same unbalanced karyotype as the proband.

  19. The p21 ras C-terminus is required for transformation and membrane association

    DEFF Research Database (Denmark)

    Willumsen, B M; Christensen, A; Hubbert, N L;

    1984-01-01

    , undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these...

  20. INK4/ARF transcript expression is associated with chromosome 9p21 variants linked to atherosclerosis.

    Directory of Open Access Journals (Sweden)

    Yan Liu

    Full Text Available Genome-wide association studies (GWAS have linked common single nucleotide polymorphisms (SNPs on chromosome 9p21 near the INK4/ARF (CDKN2A/B tumor suppressor locus with risk of atherosclerotic diseases and type 2 diabetes mellitus. To explore the mechanism of this association, we investigated whether expression of proximate transcripts (p16(INK4a, p15(INK4b, ARF, ANRIL and MTAP correlate with genotype of representative 9p21 SNPs.We analyzed expression of 9p21 transcripts in purified peripheral blood T-cells (PBTL from 170 healthy donors. Samples were genotyped for six selected disease-related SNPs spanning the INK4/ARF locus. Correlations among these variables were determined by univariate and multivariate analysis. Significantly reduced expression of all INK4/ARF transcripts (p15(INK4b, p16(INK4a, ARF and ANRIL was found in PBTL of individuals harboring a common SNP (rs10757278 associated with increased risk of coronary artery disease, stroke and aortic aneurysm. Expression of MTAP was not influenced by rs10757278 genotype. No association of any these transcripts was noted with five other tested 9p21 SNPs.Genotypes of rs10757278 linked to increased risk of atherosclerotic diseases are also associated with decreased expression in PBTL of the INK4/ARF locus, which encodes three related anti-proliferative transcripts of known importance in tumor suppression and aging.

  1. Proteomics of CDK inhibition in cancer cells

    Czech Academy of Sciences Publication Activity Database

    Kovářová, Hana; Skalníková, Helena; Halada, Petr; Strnad, M.; Hajdúch, M.

    Olomouc: -, 2007, s. 1-1. [Symposium and Workshop on Molecular Pathology /3./. Olomouc (CZ), 04.05.2007-05.05.2007] R&D Projects: GA ČR GA301/05/0418; GA MŠk LC07017 Institutional research plan: CEZ:AV0Z50450515; CEZ:AV0Z50200510 Keywords : cyclin-dependent kinase inhibitors * cancer * proteomics Subject RIV: EB - Genetics ; Molecular Biology

  2. Effect of CDK1/CDK2 interference on cell cycle by lentivirus vector in cancer cells%慢病毒介导 CDK1/CDK2干扰对肿瘤细胞周期的影响❉

    Institute of Scientific and Technical Information of China (English)

    江文娇; 李慧萍; 齐庆远

    2015-01-01

    In order to investigate the influence of CDK1 and CDK2 interference on cell cycle in CBRH-7919 cell, the CDK1, CDK2 specific shRNA lentiviral expression vectors were structured, then three plasmids were contransfected into 293 FT cells to produce viral particles, which infected the CBRH-7919 cells after collecting and concentrating the virals.The morphological changes of cells were observed by fluorescence microscope, Real-time PCR and Western Blotting demonstrated the level changes of CDK1 , CDK2 mRNA and protein ex-pression in CBRH-7919 cells.It was analyzed the changes of cell proliferation and cycle effect by MTT and flow cytometry.The results showed that it was successful to construct the CDK1 and CDK2 specific shRNA lentiviral expression vector;silencing of CDK1 led to arrest of cells in G2/M phase, cell proliferation rate de-creased obviously, and increased cell debris, while silencing CDK2 cells remained growth as normal.%为了检测细胞周期性蛋白激酶CDK1与CDK2干扰对CBRH-7919细胞周期的影响,构建了CDK1和CDK2特异性shRNA慢病毒沉默表达载体,三质粒共转染293 FT细胞产生病毒颗粒,收集浓缩后感染CBRH-7919细胞,荧光显微镜下观察了细胞形态,实时定量荧光PCR和聚丙烯酰胺凝胶电泳检测了细胞中CDK1和CDK2 mRNA和蛋白质表达水平的变化,MTT法和流式细胞仪分别检测了细胞增殖和细胞周期的变化情况。结果表明:成功构建了CDK1与CDK2特异性shRNA慢病毒表达载体,干扰CDK1导致细胞G2/M期的阻滞,细胞增殖明显降低,细胞碎片增多;而干扰CDK2后细胞仍正常生长。

  3. Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

    OpenAIRE

    Kaibuchi, K; Mizuno, T; Fujioka, H.; Yamamoto, T; Kishi, K; Y. Fukumoto; Hori, Y.(University of Tokyo, Tokyo, Japan); Takai, Y.

    1991-01-01

    We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleoti...

  4. 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 dynami...

  5. p21(WAF1) (/Cip1) limits senescence and acinar-to-ductal metaplasia formation during pancreatitis.

    Science.gov (United States)

    Grabliauskaite, Kamile; Hehl, Adrian B; Seleznik, Gitta M; Saponara, Enrica; Schlesinger, Kathryn; Zuellig, Richard A; Dittmann, Anja; Bain, Martha; Reding, Theresia; Sonda, Sabrina; Graf, Rolf

    2015-02-01

    Trans-differentiation of pancreatic acinar cells into ductal-like lesions, a process defined as acinar-to-ductal metaplasia (ADM), is observed in the course of organ regeneration following pancreatitis. In addition, ADM is found in association with pre-malignant PanIN lesions and correlates with an increased risk of pancreatic adenocarcinoma (PDAC). Human PDAC samples show down-regulation of p21(WAF1) (/Cip1) , a key regulator of cell cycle and cell differentiation. Here we investigated whether p21 down-regulation is implicated in controlling the early events of acinar cell trans-differentiation and ADM formation. p21-mediated regulation of ADM formation and regression was analysed in vivo during the course of cerulein-induced pancreatitis, using wild-type (WT) and p21-deficient (p21(-/-) ) mice. Biochemical and immunohistochemical methods were used to evaluate disease progression over 2 weeks of the disease and during a recovery phase. We found that p21 was strongly up-regulated in WT acinar cells during pancreatitis, while it was absent in ADM areas, suggesting that p21 down-regulation is associated with ADM formation. In support of this hypothesis, p21(-/-) mice showed a significant increase in number and size of metaplasia. In addition, p21 over-expression in acinar cells reduced ADM formation in vitro, suggesting that the protein regulates the metaplastic transition in a cell-autonomous manner. p21(-/-) mice displayed increased expression and relocalization of β-catenin both during pancreatitis and in the subsequent recovery phase. Finally, loss of p21 was accompanied by increased DNA damage and development of senescence. Our findings are consistent with a gate-keeper role of p21 in acinar cells to limit senescence activation and ADM formation during pancreatic regeneration. PMID:25212177

  6. Preventing DNA over-replication: a Cdk perspective

    Directory of Open Access Journals (Sweden)

    Porter Andrew CG

    2008-01-01

    Full Text Available Abstract The cell cycle is tightly controlled to ensure that replication origins fire only once per cycle and that consecutive S-phases are separated by mitosis. When controls fail, DNA over-replication ensues: individual origins fire more than once per S-phase (re-replication or consecutive S-phases occur without intervening mitoses (endoreduplication. In yeast the cell cycle is controlled by a single cyclin dependent kinase (Cdk that prevents origin licensing at times when it promotes origin firing, and that is inactivated, via proteolysis of its partner cyclin, as cells undergo mitosis. A quantitative model describes three levels of Cdk activity: low activity allows licensing, intermediate activity allows firing but prevents licensing, and high activity promotes mitosis. In higher eukaryotes the situation is complicated by the existence of additional proteins (geminin, Cul4-Ddb1Cdt2, and Emi1 that control licensing. A current challenge is to understand how these various control mechanisms are co-ordinated and why the degree of redundancy between them is so variable. Here the experimental induction of DNA over-replication is reviewed in the context of the quantitative model of Cdk action. Endoreduplication is viewed as a consequence of procedures that cause Cdk activity to fall below the threshold required to prevent licensing, and re-replication as the result of procedures that increase that threshold value. This may help to explain why over-replication does not necessarily require reduced Cdk activity and how different mechanisms conspire to prevent over-replication. Further work is nevertheless required to determine exactly how losing just one licensing control mechanism often causes over-replication, and why this varies between cell systems.

  7. 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)

    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

  8. 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

  9. Expressions of P53 and P21 protein in retinoblastoma%P53蛋白及P21蛋白在视网膜母细胞瘤表达的研究

    Institute of Scientific and Technical Information of China (English)

    罗红; 范寒桂; 邓平

    2001-01-01

    目的:探讨P53和P21在视网膜母细胞瘤发生中的作用,及其与预后的关系.方法:应用抗人P53、P21抗体,采用链霉素亲生物素蛋白-过氧化霉(SP)免疫组化方法,对30例RB常规石蜡标本进行P53和P21蛋白的测定.结果:P53和P21在RB中的阳性表达率分别为60%和80%.P53的表达与临床分期有关(P<0.05).P53和P21的表达均与生存时间有关,P53阳性组生存时间低于P53阴性组(P<0.05),P21阳性组生存时间高于P21阴性组(P<0.01); P53阳性组的2年生存率低于P53阴性组,而P21阳性组的2年生存率高于P21的阴性组.结论:在RB的发生中P53和P21起着重要的作用.P53和P21的表达可以作为临床预后的一个参考指标.

  10. 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

  11. 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

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: These results suggest that Pak functions in novel signaling pathways in NF2, and may serve as a useful therapeutic target in this disease.

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

    International Nuclear Information System (INIS)

    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 p27kip1. The elevated p27kip1 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 p27kip1. • Isoangustone A inhibits CDK2 and mTOR activity. • Isoangustone A directly binds with CDK2 and mTOR complex in prostate cancer cells

  13. 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.

  14. AC1MMYR2 impairs high dose paclitaxel-induced tumor metastasis by targeting miR-21/CDK5 axis.

    Science.gov (United States)

    Ren, Yu; Zhou, Xuan; Yang, Juan-Juan; Liu, Xia; Zhao, Xiao-hui; Wang, Qi-xue; Han, Lei; Song, Xin; Zhu, Zhi-yan; Tian, Wei-ping; Zhang, Lun; Mei, Mei; Kang, Chun-sheng

    2015-07-01

    Paclitaxel (taxol) is a widely used chemo-drug for many solid tumors, while continual taxol treatment is revealed to stimulate tumor dissemination. We previously found that a small molecule inhibitor of miR-21, termed AC1MMYR2, had the potential to impair tumorigenesis and metastasis. The aim of this study was to investigate whether combining AC1MMYR2 with taxol could be explored as a means to limit tumor metastasis. Here we showed that abnormal activation of miR-21/CDK5 axis was associated with breast cancer lymph node metastasis, which was also contribute to high dose taxol-induced invasion and epithelial mesenchymal transition (EMT) in both breast cancer cell line MDA-MB-231 and glioblastoma cell line U87VIII. AC1MMYR2 attenuated CDK5 activity by functional targeting CDK5RAP1, CDK5 activator p39 and target p-FAK(ser732). A series of in vitro assays indicated that treatment of AC1MMYR2 combined with taxol suppressed tumor migration and invasion ability in both MDA-MB-231 and U87VIII cell. More importantly, combination therapy impaired high-dose taxol induced invadopodia, and EMT markers including β-catenin, E-cadherin and vimentin. Strikingly, a significant reduction of lung metastasis in mice was observed in the AC1MMYR2 plus taxol treatment. Taken together, our work demonstrated that AC1MMYR2 appeared to be a promising strategy in combating taxol induced cancer metastasis by targeting miR-21/CDK5 axis, which highlighted the potential for development of therapeutic modalities for better clinic taxol application. PMID:25827073

  15. Phenyl-1-Pyridin-2yl-Ethanone-Based Iron Chelators Increase IκB-α Expression, Modulate CDK2 and CDK9 Activities, and Inhibit HIV-1 Transcription

    Science.gov (United States)

    Kumari, Namita; Iordanskiy, Sergey; Kovalskyy, Dmytro; Breuer, Denitra; Niu, Xiaomei; Lin, Xionghao; Xu, Min; Gavrilenko, Konstantin; Kashanchi, Fatah; Dhawan, Subhash

    2014-01-01

    HIV-1 transcription is activated by the Tat protein, which recruits CDK9/cyclin T1 to the HIV-1 promoter. CDK9 is phosphorylated by CDK2, which facilitates formation of the high-molecular-weight positive transcription elongation factor b (P-TEFb) complex. We previously showed that chelation of intracellular iron inhibits CDK2 and CDK9 activities and suppresses HIV-1 transcription, but the mechanism of the inhibition was not understood. In the present study, we tested a set of novel iron chelators for the ability to inhibit HIV-1 transcription and elucidated their mechanism of action. Novel phenyl-1-pyridin-2yl-ethanone (PPY)-based iron chelators were synthesized and examined for their effects on cellular iron, HIV-1 inhibition, and cytotoxicity. Activities of CDK2 and CDK9, expression of CDK9-dependent and CDK2-inhibitory mRNAs, NF-κB expression, and HIV-1- and NF-κB-dependent transcription were determined. PPY-based iron chelators significantly inhibited HIV-1, with minimal cytotoxicity, in cultured and primary cells chronically or acutely infected with HIV-1 subtype B, but they had less of an effect on HIV-1 subtype C. Iron chelators upregulated the expression of IκB-α, with increased accumulation of cytoplasmic NF-κB. The iron chelators inhibited CDK2 activity and reduced the amount of CDK9/cyclin T1 in the large P-TEFb complex. Iron chelators reduced HIV-1 Gag and Env mRNA synthesis but had no effect on HIV-1 reverse transcription. In addition, iron chelators moderately inhibited basal HIV-1 transcription, equally affecting HIV-1 and Sp1- or NF-κB-driven transcription. By virtue of their involvement in targeting several key steps in HIV-1 transcription, these novel iron chelators have the potential for the development of new therapeutics for the treatment of HIV-1 infection. PMID:25155598

  16. Combined p21WAF1/CIP1 and p53 overexpression predict improved survival in muscle-invasive bladder cancer treated by radical radiotherapy

    International Nuclear Information System (INIS)

    Purpose: The prognostic value of p21 and p53 expression was evaluated for patients with muscle-invasive bladder cancer treated by radical radiotherapy. Methods and Materials: Sixty-eight paraffin-embedded sections from surgically resected tumors taken prior to irradiation were immunostained for p21 and p53. Results: Nuclear staining for p21 and p53 was demonstrated in 32/68 (47%) and 46/68 (68%) tumors, respectively. There was no correlation between p21 and p53 immunopositivity in this group (r=0.067, p=0.56). Patients were stratified into four distinct groups depending on staining for p21 and p53: p21+p53+, p21+p53-, p21-p53+, and p21-p53-. Patients with p21+p53+ tumors had the best prognosis with a 3-year survival of 82% compared to 12% for p21-p53+ tumors (p=0.0031), 29% for p21+p53- tumors (p=0.0108); and 45% for p21-p53- tumors (p=0.0375). The p21+p53+ group also demonstrated significantly improved survival when a combined analysis was performed of p21-p53+, p21-p53-, and p21+p53- tumors (3-year survival = 30%, p=0.0062). In a multivariate model, p21+p53+ tumors (p=0.0108, relative risk [RR] 5.18) and complete/partial response (p=0.0019, RR=3.76) were the only independent predictors of improved survival. Conclusions: With muscle-invasive bladder tumors treated by radical radiotherapy, stratification for p21 and p53 identifies distinct prognostic groups, with p21+p53+ tumors being associated with the best survival and p21-p53+ the worst

  17. Alteration of p53and p21 during hepatocarcinogenesis in shrews

    Institute of Scientific and Technical Information of China (English)

    Jian-Jia Su; Rui-Qi Yang; Ke-Chen Ban; Yuan Li; Liu-Liang Qin; Hui-Yun Wang; Chun Yang; Chao Ou; Xiao-Xian Duan; Young-Lk Lee

    2004-01-01

    AIM: To investigate p53 mutation and p21 expression in hepatocarcinogenesis induced by hepatitis B virus (HBV)and aflatoxin B1 (AFB1) in tree shrews, and to reveal the role of these genes in hepatocarcinogenesis.METHODS: Tree shrews were divided into four groups:group A, those infected with HBV and fed with AFB1 (n = 39);group B, those infected with HBV alone (n = 28); group C,those fed with AFB1 alone (n = 29); and group D, normal controls (n = 20). The tree shrews underwent liver biopsies once every 15 wk. Expression of p53 and p21 proteins and genes in the biopsies and tumor tissues of the experimental tree shrews was detected, respectively, by immunohistochemistry,and by Southern blottdng and reverse transcription-polymerase chain reaction and sequencing.RESULTS: The incidence of hepatocellular carcinomas (HCC) was higher in group A (66.7%) than that in group B (3.57%) and C (30%). The time of HCC occurrence was also earlier in group A than that in group C (120.0±16.6 wk vs 153.3±5.8 wk, respectively, P<0.01). p53 protein was not detected by immunohistochemistry in all groups before the 75th wk of the experiment. At the 105th wk, the positive rates fo p53 were 78.6%, 60% and 71.4% in groups A, B and C, respectively, which were significantly higher than that in group D (10%) (all P<0.05). An abnormal band of p53gene was observed in groups A and C. The mutation points of p53gene in tree shrews with HCC were at codons 275, 78 and 13. The nucleotide sequence and amino acid sequence of tree shrew's wild-type p53 showed 91.7%and 93.4% homologies with those of human p53,respectively. The immunopositivity for p21 was found before HCC development. The incidence of HCC was significantly higher in tree shrews that were positive for p21 than those negative for p21 (80.0% vs 11.0%, P<0.001).The incidence of HCC in p21 positive animals in group A was significantly higher than those positive for p21 in group C (P<0.05).CONCLUSION: A remarkable synergistic effect on HCC

  18. Targeting p35/Cdk5 Signalling via CIP-Peptide Promotes Angiogenesis in Hypoxia

    Science.gov (United States)

    Bosutti, Alessandra; Qi, Jie; Pennucci, Roberta; Bolton, David; Matou, Sabine; Ali, Kamela; Tsai, Li-Huei; Krupinski, Jerzy; Petcu, Eugene B.; Montaner, Joan; Al Baradie, Raid; Caccuri, Francesca; Caruso, Arnaldo; Alessandri, Giulio; Kumar, Shant; Rodriguez, Cristina; Martinez-Gonzalez, Jose; Slevin, Mark

    2013-01-01

    Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition. PMID:24098701

  19. Targeting p35/Cdk5 signalling via CIP-peptide promotes angiogenesis in hypoxia.

    Directory of Open Access Journals (Sweden)

    Alessandra Bosutti

    Full Text Available Cyclin-dependent kinase-5 (Cdk5 is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.

  20. Cdk2-Null Mice Are Resistant to ErbB-2-Induced Mammary Tumorigenesis

    Directory of Open Access Journals (Sweden)

    Dipankar Ray

    2011-05-01

    Full Text Available The concept of targeting G1 cyclin-dependent kinases (CDKs in breast cancer treatments is supported by the fact that the genetic ablation of Cdk4 had minimal impacts on normal cell proliferation in majority of cell types, resulting in near-normal mouse development, whereas such loss of Cdk4 completely abrogated ErbB-2/neu-induced mammary tumorigenesis in mice. In most human breast cancer tissues, another G1-regulatory CDK, CDK2, is also hyperactivated by various mechanisms and is believed to be an important therapeutic target. In this report, we provide genetic evidence that CDK2 is essential for proliferation and oncogenesis of murine mammary epithelial cells. We observed that 87% of Cdk2-null mice were protected from ErbB-2-induced mammary tumorigenesis. Mouse embryonic fibroblasts isolated from Cdk2-null mouse showed resistance to various oncogene-induced transformation. Previously, we have reported that hemizygous loss of Cdc25A, the major activator of CDK2, can also protect mice from ErbB-2-induced mammary tumorigenesis [Cancer Res (2007 67(14: 6605–11]. Thus, we propose that CDC25A-CDK2 pathway is critical for the oncogenic action of ErbB-2 in mammary epithelial cells, in a manner similar to Cyclin D1/CDK4 pathway.

  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. The proline-histidine-rich CDK2/CDK4 interaction region of C/EBPalpha is dispensable for C/EBPalpha-mediated growth regulation in vivo

    DEFF Research Database (Denmark)

    Porse, Bo Torben; Pedersen, Thomas Askov; Hasemann, Marie Sigurd;

    2006-01-01

    a short, centrally located, 15-amino-acid proline-histidine-rich region (PHR) of C/EBPalpha is responsible for the growth-inhibitory function of the protein through its ability to interact with CDK2 and CDK4, thereby inhibiting their activities. Homozygous Cebpa(DeltaPHR/DeltaPHR) (DeltaPHR) mice...

  3. Structure-guided discovery of cyclin-dependent kinase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Fischmann, Thierry O.; Hruza, Alan; Duca, Jose S.; Ramanathan, Lata; Mayhood, Todd; Windsor, William T.; Le, Hung V.; Guzi, Timothy J.; Dwyer, Michael P.; Paruch, Kamil; Doll, Ronald J.; Lees, Emma; Parry, David; Seghezzi, Wolfgang; Madison, Vincent (SPRI)

    2008-10-02

    CDK2 inhibitors containing the related bicyclic heterocycles pyrazolopyrimidines and imidazopyrazines were discovered through high-throughput screening. Crystal structures of inhibitors with these bicyclic cores and two more related ones show that all but one have a common binding mode featuring two hydrogen bonds (H-bonds) to the backbone of the kinase hinge region. Even though ab initio computations indicated that the imidazopyrazine core would bind more tightly to the hinge, pyrazolopyrimidines gain an advantage in potency through participation of N4 in an H-bond network involving two catalytic residues and bridging water molecules. Further insight into inhibitor/CDK2 interactions was gained from analysis of additional crystal structures. Significant gains in potency were obtained by optimizing the fit of hydrophobic substituents to the gatekeeper region of the ATP binding site. The most potent inhibitors have good selectivity.

  4. Postnatal telomere dysfunction induces cardiomyocyte cell-cycle arrest through p21 activation.

    Science.gov (United States)

    Aix, Esther; Gutiérrez-Gutiérrez, Óscar; Sánchez-Ferrer, Carlota; Aguado, Tania; Flores, Ignacio

    2016-06-01

    The molecular mechanisms that drive mammalian cardiomyocytes out of the cell cycle soon after birth remain largely unknown. Here, we identify telomere dysfunction as a critical physiological signal for cardiomyocyte cell-cycle arrest. We show that telomerase activity and cardiomyocyte telomere length decrease sharply in wild-type mouse hearts after birth, resulting in cardiomyocytes with dysfunctional telomeres and anaphase bridges and positive for the cell-cycle arrest protein p21. We further show that premature telomere dysfunction pushes cardiomyocytes out of the cell cycle. Cardiomyocytes from telomerase-deficient mice with dysfunctional telomeres (G3 Terc(-/-)) show precocious development of anaphase-bridge formation, p21 up-regulation, and binucleation. In line with these findings, the cardiomyocyte proliferative response after cardiac injury was lost in G3 Terc(-/-) newborns but rescued in G3 Terc(-/-)/p21(-/-) mice. These results reveal telomere dysfunction as a crucial signal for cardiomyocyte cell-cycle arrest after birth and suggest interventions to augment the regeneration capacity of mammalian hearts. PMID:27241915

  5. Phenylbutyric acid induces the cellular senescence through an Akt/p21WAF1 signaling pathway

    International Nuclear Information System (INIS)

    Highlights: ► Phenylbutyric acid induces cellular senescence. ► Phenylbutyric acid activates Akt kinase. ► The knockdown of PERK also can induce cellular senescence. ► Akt/p21WAF1 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 p21WAF1 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/p21WAF1 pathway by PERK inhibition.

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

    International Nuclear Information System (INIS)

    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

  7. Low p21(Waf1/Cip1) protein level sensitizes testicular germ cell tumor cells to Fas-mediated apoptosis

    NARCIS (Netherlands)

    Spierings, DCJ; de Vries, EGE; Stel, AJ; Rietstap, NT; Vellenga, E; de Jong, S

    2004-01-01

    In the present study, we investigated the relation between p21 expression and the sensitivity of testicular germ cell tumor (TGCT) cells to apoptotic stimuli. Despite similar cisplatin-induced wild-type p53 accumulation, the TGCT cell lines Tera and Scha expressed low p21 protein and mRNA levels in

  8. 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

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

    International Nuclear Information System (INIS)

    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

  10. CDK2 accelerates early erythroid differentiation of K562 cells%CDK2促进K562细胞早期红系分化

    Institute of Scientific and Technical Information of China (English)

    李均; 岳瑞华; 沈钧乐; 肖俊

    2011-01-01

    目的 探讨细胞周期调节蛋白CDK2对K562细胞红系分化的影响.方法 分别用CDK2表达质粒和干扰RNA分子转染K562细胞,用Western blot法检测过表达或干扰效率,使用real-time PCR和联苯胺染色法检测K562细胞分化.结果 CDK2在K562细胞红系分化早期呈现表达上升趋势;在K562细胞中过表达CDK2可促进hemin诱导的红系分化;反之,干扰K562内源的CDK2表达会对K562红系分化产生抑制作用.结论 CDK2在K562细胞早期红系分化过程中发挥促进作用.%Objective To study the roles of a cell cycle regulator cyclin-dependent kinase 2 (CDK2) in erythroid differentiation of K562 cells. Methods K562 cells were transfected with the construct expressing CDK2 and siRNAs specifically targeting at CDK2. The effects of over-expression or knocking-down of CDK2 were examined by Western blot. Quantitative RT-PCR was performed to detect the level of γ-globin mRNA expression. The benzidine staining assay was used to identify the differentiation state of K562 cells. Results CDK2 was up-regulated at the early stage of K562 erythroid differentiation. Over-expression of CDK2 in K562 cells accelerated erythroid differentiation. Inhibition of CDK2 attenuates globin accumulation in K562 cells. Conclusion CDK2 is necessary for early erythroid differentiation of K562 cells.

  11. Cyclin-Dependent Kinase Inhibitor P27Kip1 Is Required for Mouse Mammary Gland Morphogenesis and Function

    OpenAIRE

    Muraoka, Rebecca S.; Lenferink, Anne E.G.; Simpson, Jean; Brantley, Dana M.; Roebuck, L. Renee; Yakes, F. Michael; Arteaga, Carlos L

    2001-01-01

    We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1–Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1–deficient animals. These hypotheses were investigated in postna...

  12. Induction of apoptosis in cancer cells by tumor necrosis factor and butyrolactone, an inhibitor of cyclin-dependent kinases

    OpenAIRE

    Belizário, J E; S. Sherwood; Beçak, W.

    1999-01-01

    Induction of apoptosis by tumor necrosis factor (TNF) is modulated by changes in the expression and activity of several cell cycle regulatory proteins. We examined the effects of TNF (1-100 ng/ml) and butyrolactone I (100 µM), a specific inhibitor of cyclin-dependent kinases (CDK) with high selectivity for CDK-1 and CDK-2, on three different cancer cell lines: WEHI, L929 and HeLa S3. Both compounds blocked cell growth, but only TNF induced the common events of apoptosis, i.e., chromatin conde...

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

    International Nuclear Information System (INIS)

    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 p21WAF/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

  14. THE EXPRESSION AND CLINICAL SIGNIFICANCE OF P21 (WAF1/CIP1)AND CYCLIN D1 PROTEIN IN COLORECTAL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To study the effect of P21 (WAF1/CIP1) and cyclin D1 and their relationship in colorec- tal carcinoma. Methods The expression of P21 and cyclin D1 was studied in 40 colorectal carcinoma and 10 normal tissues using S-P immunohistochemical technique. Results Decreased expression of P12 and overexpression of cyclin D1 were revealed in colorectal carcinoma. Decreased expression of P21 was related to lymph node metastasis. No cor- relation was found between cyclin D1 and clinicopathological parameters. Conclusion Decreased expression of P21 and overexpression of cyclin D1 may be involved in colorectal tumorigenesis,and were associated with poor prognosis. No correlation was found between P21 and cyclin D1 in colorectai carcinoma.

  15. Cell cycle sibling rivalry: Cdc2 vs. Cdk2.

    Science.gov (United States)

    Kaldis, Philipp; Aleem, Eiman

    2005-11-01

    It has been long believed that the cyclin-dependent kinase 2 (Cdk2) binds to cyclin E or cyclin A and exclusively promotes the G1/S phase transition and that Cdc2/cyclin B complexes play a major role in mitosis. We now provide evidence that Cdc2 binds to cyclin E (in addition to cyclin A and B) and is able to promote the G1/S transition. This new concept indicates that both Cdk2 and/or Cdc2 can drive cells through G1/S phase in parallel. In this review we discuss the classic cell cycle model and how results from knockout mice provide new evidence that refute this model. We focus on the roles of Cdc2 and p27 in regulating the mammalian cell cycle and propose a new model for cell cycle regulation that accommodates these novel findings. PMID:16258277

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

    Directory of Open Access Journals (Sweden)

    Catherine Oikonomou

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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

  17. Cyclin-dependent kinase CDK1/CDC28 and checkpoints

    International Nuclear Information System (INIS)

    The genetic instability induced by defects in the cell cycle progression contributes to different human diseases, particularly neoplastic transformation. The control mechanisms of correct cell cycle progression are the most studied in the yeast Saccharomyces cerevisiae, in which checkpoint was first discovered. Many components of these processes have been identified by now. Here, the role of the central kinase of cell cycle CDK1/CDC28 is considered in checkpoint in different phases.

  18. 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.

  19. Cyclin-dependent protein kinase inhibitors including palbociclib as anticancer drugs.

    Science.gov (United States)

    Roskoski, Robert

    2016-05-01

    Cyclins and cyclin-dependent protein kinases (CDKs) are important regulatory components that are required for cell cycle progression. The levels of the cell cycle CDKs are generally constant and their activities are controlled by cyclins, proteins whose levels oscillate during each cell cycle. Additional CDK family members were subsequently discovered that play significant roles in a wide range of activities including the control of gene transcription, metabolism, and neuronal function. In response to mitogenic stimuli, cells in the G1 phase of the cell cycle produce cyclins of the D type that activate CDK4/6. These activated enzymes catalyze the monophosphorylation of the retinoblastoma protein. Then CDK2-cyclin E catalyzes the hyperphosphorylation of Rb that promotes the release and activation of the E2F transcription factors, which in turn lead to the generation of several proteins required for cell cycle progression. As a result, cells pass through the G1-restriction point and are committed to complete cell division. CDK2-cyclin A, CDK1-cyclin A, and CDK1-cyclin B are required for S, G2, and M-phase progression. Increased cyclin or CDK expression or decreased levels of endogenous CDK inhibitors such as INK4 or CIP/KIP have been observed in various cancers. In contrast to the mutational activation of EGFR, Kit, or B-Raf in the pathogenesis of malignancies, mutations in the CDKs that cause cancers are rare. Owing to their role in cell proliferation, CDKs represent natural targets for anticancer therapies. Abemaciclib (LY2835219), ribociclib (Lee011), and palbociclib (Ibrance(®) or PD0332991) target CDK4/6 with IC50 values in the low nanomolar range. Palbociclib and other CDK inhibitors bind in the cleft between the small and large lobes of the CDKs and inhibit the binding of ATP. Like ATP, palbociclib forms hydrogen bonds with residues in the hinge segment of the cleft. Like the adenine base of ATP, palbociclib interacts with catalytic spine residues CS6 and CS7

  20. Cell-cycle protein expression in a population-based study of ovarian and endometrial cancers

    OpenAIRE

    Felix, Ashley S.; Sherman, Mark E.; Hewitt, Stephen M.; Munira eGunja; Yang, Hannah P.; Renata eCora; Vicky eBoudreau; Kris eYlaya; Jolanta eLissowska; Brinton, Louise A.; Nicolas eWentzensen

    2015-01-01

    Aberrant expression of cyclin-dependent kinase (CDK) inhibitors is implicated in the carcinogenesis of many cancers, including ovarian and endometrial cancers. We examined associations between CDK inhibitor expression, cancer risk factors, tumor characteristics, and survival outcomes among ovarian and endometrial cancer patients enrolled in a population-based case control study. Expression (negative vs. positive) of three CDK inhibitors (p16, p21, p27) and ki67 was examined with immunohistoch...

  1. Cell-Cycle Protein Expression in a Population-Based Study of Ovarian and Endometrial Cancers

    OpenAIRE

    Felix, Ashley S.; Sherman, Mark E.; Hewitt, Stephen M.; Gunja, Munira Z.; Yang, Hannah P.; Cora, Renata L.; Boudreau, Vicky; Ylaya, Kris; Lissowska, Jolanta; Brinton, Louise A.; Wentzensen, Nicolas

    2015-01-01

    Aberrant expression of cyclin-dependent kinase (CDK) inhibitors is implicated in the carcinogenesis of many cancers, including ovarian and endometrial cancers. We examined associations between CDK inhibitor expression, cancer risk factors, tumor characteristics, and survival outcomes among ovarian and endometrial cancer patients enrolled in a population-based case-control study. Expression (negative vs. positive) of three CDK inhibitors (p16, p21, and p27) and ki67 was examined with immunohis...

  2. Roles of the CDK Phosphorylation Sites of Yeast Cdc6 in Chromatin Binding and Rereplication

    OpenAIRE

    Honey, Sangeet; Futcher, Bruce

    2007-01-01

    The Saccharomyces cerevisiae Cdc6 protein is crucial for DNA replication. In the absence of cyclin-dependent kinase (CDK) activity, Cdc6 binds to replication origins, and loads Mcm proteins. In the presence of CDK activity, Cdc6 does not bind to origins, and this helps prevent rereplication. CDK activity affects Cdc6 function by multiple mechanisms: CDK activity affects transcription of CDC6, degradation of Cdc6, nuclear import of Cdc6, and binding of Cdc6 to Clb2. Here we examine some of the...

  3. Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    HE Kan; CHEN Yue; LI Jing-hua; ZHAN Zhuo; WU Yong-ge; KONG Wei; JIN Ying-hua

    2007-01-01

    Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli. To determine a specific substrate of Cyclin A-Cdk2 for apoptosis, in this study, we carried out anin vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source, and recombinant protein GST-Bad as a substrate. Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro. To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo, we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn, a dominant negative version of Cdk2, in Hela cells and determined the phosphorylation status of protein Bad. The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent. Moreover, etoposide also caused the phosphorylation of endogenetic Bad. In conclusion, here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.

  4. Cdk12 is essential for embryonic development and the maintenance of genomic stability.

    Science.gov (United States)

    Juan, H-C; Lin, Y; Chen, H-R; Fann, M-J

    2016-06-01

    The maintenance of genomic integrity during early embryonic development is important in order to ensure the proper development of the embryo. Studies from cultured cells have demonstrated that cyclin-dependent kinase 12 (Cdk12) is a multifunctional protein that maintains genomic stability and the pluripotency of embryonic stem cells. Perturbation of its functions is also known to be associated with pathogenesis and drug resistance in human cancers. However, the biological significance of Cdk12 in vivo is unclear. Here we bred mice that are deficient in Cdk12 and demonstrated that Cdk12 depletion leads to embryonic lethality shortly after implantation. We also used an in vitro culture system of blastocysts to examine the molecular mechanisms associated with the embryonic lethality of Cdk12-deficient embryos. Cdk12(-/-) blastocysts fail to undergo outgrowth of the inner cell mass because of an increase in the apoptosis of these cells. Spontaneous DNA damage was revealed by an increase in 53BP1 foci among cells cultured from Cdk12(-/-) embryos. Furthermore, the expression levels of various DNA damage response genes, namely Atr, Brca1, Fanci and Fancd2, are reduced in Cdk12(-/-) embryos. These findings indicate that Cdk12 is important for the correct expression of some DNA damage response genes and indirectly has an influence on the efficiency of DNA repair. Our report also highlights that DNA breaks occurring during DNA replication are frequent in mouse embryonic cells and repair of such damage is critical to the successful development of mouse embryos. PMID:26658019

  5. Waves of Cdk1 Activity in S Phase Synchronize the Cell Cycle in Drosophila Embryos.

    Science.gov (United States)

    Deneke, Victoria E; Melbinger, Anna; Vergassola, Massimo; Di Talia, Stefano

    2016-08-22

    Embryos of most metazoans undergo rapid and synchronous cell cycles following fertilization. While diffusion is too slow for synchronization of mitosis across large spatial scales, waves of Cdk1 activity represent a possible process of synchronization. However, the mechanisms regulating Cdk1 waves during embryonic development remain poorly understood. Using biosensors of Cdk1 and Chk1 activities, we dissect the regulation of Cdk1 waves in the Drosophila syncytial blastoderm. We show that Cdk1 waves are not controlled by the mitotic switch but by a double-negative feedback between Cdk1 and Chk1. Using mathematical modeling and surgical ligations, we demonstrate a fundamental distinction between S phase Cdk1 waves, which propagate as active trigger waves in an excitable medium, and mitotic Cdk1 waves, which propagate as passive phase waves. Our findings show that in Drosophila embryos, Cdk1 positive feedback serves primarily to ensure the rapid onset of mitosis, while wave propagation is regulated by S phase events. PMID:27554859

  6. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus.

    Science.gov (United States)

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-04-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging "allele-specific" functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

  7. Different molecular mechanisms causing 9p21 deletions in acute lymphoblastic leukemia of childhood.

    Science.gov (United States)

    Novara, Francesca; Beri, Silvana; Bernardo, Maria Ester; Bellazzi, Riccardo; Malovini, Alberto; Ciccone, Roberto; Cometa, Angela Maria; Locatelli, Franco; Giorda, Roberto; Zuffardi, Orsetta

    2009-10-01

    Deletion of chromosome 9p21 is a crucial event for the development of several cancers including acute lymphoblastic leukemia (ALL). Double strand breaks (DSBs) triggering 9p21 deletions in ALL have been reported to occur at a few defined sites by illegitimate action of the V(D)J recombination activating protein complex. We have cloned 23 breakpoint junctions for a total of 46 breakpoints in 17 childhood ALL (9 B- and 8 T-lineages) showing different size deletions at one or both homologous chromosomes 9 to investigate which particular sequences make the region susceptible to interstitial deletion. We found that half of 9p21 deletion breakpoints were mediated by ectopic V(D)J recombination mechanisms whereas the remaining half were associated to repeated sequences, including some with potential for non-B DNA structure formation. Other mechanisms, such as microhomology-mediated repair, that are common in other cancers, play only a very minor role in ALL. Nucleotide insertions at breakpoint junctions and microinversions flanking the breakpoints have been detected at 20/23 and 2/23 breakpoint junctions, respectively, both in the presence of recombination signal sequence (RSS)-like sequences and of other unspecific sequences. The majority of breakpoints were unique except for two cases, both T-ALL, showing identical deletions. Four of the 46 breakpoints coincide with those reported in other cases, thus confirming the presence of recurrent deletion hotspots. Among the six cases with heterozygous 9p deletions, we found that the remaining CDKN2A and CDKN2B alleles were hypermethylated at CpG islands. PMID:19484265

  8. 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.

  9. Allelic Imbalance in Regulation of ANRIL through Chromatin Interaction at 9p21 Endometriosis Risk Locus

    Science.gov (United States)

    Nakaoka, Hirofumi; Gurumurthy, Aishwarya; Hayano, Takahide; Ahmadloo, Somayeh; Omer, Waleed H; Yoshihara, Kosuke; Yamamoto, Akihito; Kurose, Keisuke; Enomoto, Takayuki; Akira, Shigeo; Hosomichi, Kazuyoshi; Inoue, Ituro

    2016-01-01

    Genome-wide association studies (GWASs) have discovered numerous single nucleotide polymorphisms (SNPs) associated with human complex disorders. However, functional characterization of the disease-associated SNPs remains a formidable challenge. Here we explored regulatory mechanism of a SNP on chromosome 9p21 associated with endometriosis by leveraging “allele-specific” functional genomic approaches. By re-sequencing 1.29 Mb of 9p21 region and scrutinizing DNase-seq data from the ENCODE project, we prioritized rs17761446 as a candidate functional variant that was in perfect linkage disequilibrium with the original GWAS SNP (rs10965235) and located on DNase I hypersensitive site. Chromosome conformation capture followed by high-throughput sequencing revealed that the protective G allele of rs17761446 exerted stronger chromatin interaction with ANRIL promoter. We demonstrated that the protective allele exhibited preferential binding affinities to TCF7L2 and EP300 by bioinformatics and chromatin immunoprecipitation (ChIP) analyses. ChIP assays for histone H3 lysine 27 acetylation and RNA polymerase II reinforced the enhancer activity of the SNP site. The allele specific expression analysis for eutopic endometrial tissues and endometrial carcinoma cell lines showed that rs17761446 was a cis-regulatory variant where G allele was associated with increased ANRIL expression. Our work illuminates the allelic imbalances in a series of transcriptional regulation from factor binding to gene expression mediated by chromatin interaction underlie the molecular mechanism of 9p21 endometriosis risk locus. Functional genomics on common disease will unlock functional aspect of genotype-phenotype correlations in the post-GWAS stage. PMID:27055116

  10. Recombinant human decorin suppresses liver HepG2 carcinoma cells by p21 upregulation

    Directory of Open Access Journals (Sweden)

    Zhang Y

    2012-08-01

    Full Text Available Yucheng Zhang, Yali Wang,* Zhenwu Du, Qian Wang, Mei Wu, Xiaofeng Wang, Lingling Wang, Linlin Cao, Abdu Selim Hamid, Guizhen Zhang*Central Laboratory, China-Japan Union Hospital, Jilin University, Changchun, People's Republic of China *These authors contributed equally to this workBackground: Decorin is a multifunctional molecule of the extracellular matrix and impedes different kinds of tumor cell growth, but the role and molecular mechanism by which decorin inhibits HepG2 cells is not fully understood. Our objective was to construct recombinant human decorin (pcDNA3.1-DCN and to explore the mechanism by which it inhibits HepG2 cells.Methods: This experiment was divided into three groups, ie, a control group, an empty vector group, and a pcDNA3.1-DCN group. pcDNA3.1-DCN was constructed using recombinant DNA technology, and the vector for pcDNA3.1-DCN and pcDNA3.1 was then transfected into HepG2 cells using Lipofectamine 2000.Results: Compared with cells in the control group and in the empty vector group, growth of cells in the pcDNA3.1-DCN group was significantly suppressed, the ratios of cells in the G0/G1 phases and proportion of early apoptotic cells were significantly increased, and the level of p21WAF1/CIP1 (p21 protein was markedly upregulated (P < 0.05. However, there was no significant difference among the three groups in p53 protein expression (P > 0.05.Conclusion: The pcDNA3.1-DCN vector was successfully constructed and transfected into HepG2 cells, and decorin overexpression suppressed the growth of HepG2 cells by upregulation of p21 via a p53-independent pathway.Keywords: decorin, HepG2, liver cancer, p21WAF1/CIP1, pcDNA3.1

  11. Expression of P53, P21 in Human Lung Adenocarcinoma A549 Cell Strains under Hypoxia Conditions and the Effect of TSA on Their Expression

    Institute of Scientific and Technical Information of China (English)

    黄宏; 张珍祥; 徐永健; 邵静芳

    2003-01-01

    This paper was designed to investigate the expression of p53, p21of A549 cell strains under hypoxic condition and the effect of trichostatin A (TSA), the inhibitor of histone deacetylasel (HDAC1) on their expression. The authors designed 1 normoxia group (control group) and 6 hypoxia groups (experiemntal group): hypoxia 6 h group (A), TSA+ hypoxia 6 h (B), hypoxia 12 h group (C) ,hypoxia 24 h group (D), TSA+hypoxia 24 h (E), hypoxia 48 h group (F). The expression of HDAC1 in A549 cells was examined by using Western blot and the expression of p53,p21 in A549 cells and the effect of TSA on them were determined by using immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The A value expressed by HDAC1 in A549 cell strains was 138±11 in the control group, 78±4, 86±5, 124±3, 120±9 in experimental groups A, C, D, F, respectively. The A value of the expression of the protein and mRNA of p53 in A549 cell strains were 0. 12±0.02, 0. 62±0.02 in the control group, 0. 10±0.03, 0.32±0.03; 0. 11±0.01, 0. 33±0.02; 0. 13±0.03, 0. 58±0.01; 0. 12±0. 02, 0. 56±0.02 in experimental group A, B, D, E, respectively. The A value of the expression of the protein and mRNA of p21 in A549 cell strains were 0. 17±0.03, 0. 62±0. 03 in the control group, 0. 16±0.02, 0. 50±0.02; 0. 14±0.02, 0. 36±0.02; 0. 15±0.03, 0. 49±0.03; 0. 13±0.02, 0. 33 ± 0. 02 in experimental groups A, B, D, E, respectively. These results indicate that the expression of HDAC1 is regulated by hypoxia and the effect of TSA is closely related to the expression of P21 under hypoxia condition.

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

    International Nuclear Information System (INIS)

    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)

  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

    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......-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal...

  14. CRIF1 interacting with CDK2 regulates bone marrow microenvironment-induced G0/G1 arrest of leukemia cells.

    Directory of Open Access Journals (Sweden)

    Qian Ran

    Full Text Available BACKGROUND: To assess the level of CR6-interacting factor 1 (CRIF1, a cell cycle negative regulator, in patients with leukemia and investigate the role of CRIF1 in regulating leukemia cell cycle. METHODS: We compared the CRIF1 level in bone marrow (BM samples from healthy and acute myeloid leukemia (AML, iron deficiency anemia (IDA and AML-complete remission (AML-CR subjects. We also manipulated CRIF1 level in the Jurkat cells using lentivirus-mediated overexpression or siRNA-mediated depletion. Co-culture with the BM stromal cells (BMSCs was used to induce leukemia cell cycle arrest and mimic the BM microenvironment. RESULTS: We found significant decreases of CRIF1 mRNA and protein in the AML group. CRIF1 overexpression increased the proportion of Jurkat cells arrested in G0/G1, while depletion of endogenous CRIF1 decreased cell cycle arrest. Depletion of CRIF1 reversed BMSCs induced cell cycle arrest in leukemia cells. Co-immunoprecipitation showed a specific binding of CDK2 to CRIF1 in Jurkat cells during cell cycle arrest. Co-localization of two proteins in both nucleus and cytoplasm was also observed with immunofluorescent staining. CONCLUSION: CRIF1 may play a regulatory role in the BM microenvironment-induced leukemia cell cycle arrest possibly through interacting with CDK2 and acting as a cyclin-dependent kinase inhibitor.

  15. High apoptotic index correlates to p21 and p27 expression indicating a favorable outcome of primary breast cancer patients, but lacking prognostic significance in multivariate analysis.

    Science.gov (United States)

    Schöndorf, Thomas; Göhring, Uwe-Jochen; Becker, Martina; Hoopmann, Markus; Schmidt, Torsten; Rützel, Sabine; Rein, Daniel T; Ulrich, Uwe; Fechteler, Roland; Bersch, Alexander; Mallmann, Peter; Valter, Markus M

    2004-01-01

    This study was performed in order to investigate the role of the apoptotic index (AI) as a prediction parameter for the prognosis of patients with primary breast cancer. AI was determined by DNA fragmentation on 298 primary breast cancer samples and compared to clinically established breast cancer parameters. Additionally, we determined the expression of functional parameters including proliferating cell nuclear antigen, p21waf and p27kip by immunohistochemistry. The mean AI was found to be 11.9% (range, 0-90%). 189 tumors (63.4%) were negative for apoptosis, while 109 tissue samples (36.6%) were apoptotic with >5% positive cells. Using univariate analysis (chi2 test), the AI did not show any significant correlation to one of the established prognostic parameters of primary breast cancer (p > 0.05). In contrast, we found a significant positive correlation to the expression of the cell cycle inhibitors p21waf (p = 0.04) and p27kip (p = 0.024). During the clinical follow-up (median observation time for disease-free survival 87 months), several clinically established prognostic parameters including menopausal status, nodal status, tumor size, tumor grade, and hormone receptor expression could be confirmed and were analyzed with respect to the AI in the tumor. Furthermore, AI displayed a significant positive correlation to disease-free survival using Kaplan-Meier survival analysis (log-rank test, p = 0.04). However, AI lost its prognostic significance in multivariate analysis based on the Cox proportional hazard model (relative risk 0.8, confidence interval 0.52-1.33, p = 0.44). Our data indicate that high apoptotic rates in cancer tissues are indicative of a favorable patient outcome. However, the AI was not an independent factor. The study provides indirect evidence that this process may involve cell cycle inhibitors physiologically. PMID:15263811

  16. 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.

  17. Effect of cycline-dependent kinase and matrix metalloproteinase inhibitors on hematopoietic and leukemic cells

    OpenAIRE

    Song, Hairong

    2008-01-01

    Rapid advances in molecular and cellular biology have improved the understanding of the mechanisms involved in leukemia development. Cyclin-dependent kinases (CDKs) and matrix metalloproteinases (MMPs) have been suggested as potential therapeutic targets and a number of pharmacologic inhibitors of CDKs and MMPs have been developed. This thesis aimed to increase knowledge about pharmacokinetics and cytotoxic effects of the CDK inhibitor roscovitine and MMP inhibitors from...

  18. A New Case of an Extremely Rare 3p21.31 Interstitial Deletion.

    Science.gov (United States)

    Lovrecic, Luca; Bertok, Sara; Žerjav Tanšek, Mojca

    2016-05-01

    Interstitial 3p21.31 deletions have been very rarely reported. We describe a 7-year-old boy with global developmental delay, specific facial characteristics, hydronephrosis, and hypothyreosis with a de novo deletion of 3p21.31, encompassing 29 OMIM genes. Despite the wide use of microarrays, no similar case has been reported in the literature so far. Five overlapping cases are deposited in the DECIPHER database, 2 of which have significant overlapping chromosomal aberrations. They both share some phenotypic characteristics with our case, e.g. developmental delay, intellectual disability and facial dysmorphism (arched eyebrows, hypertelorism, low-set ears, and a large nose tip). In addition, loss-of-function mutations in the SETD2 gene (OMIM 612778) of the deleted region have been described in 3 patients, presenting with some similar clinical features, namely overgrowth, intellectual disability, speech delay, hypotonia, autism, and epilepsy. Therefore, SETD2 may explain part of the phenotype in our case. We focused on 3 other genes in the deleted region, based on their known functions, namely CSPG5 (OMIM 606775), PTH1R (OMIM 168468) and SMARCC1 (OMIM 601732), and assessed their potentially important role in describing the patient's phenotype. Additional cases with haploinsufficiency of this region are needed to elucidate further genotype-phenotype correlations. PMID:27385966

  19. CDK5RAP2 function during Zebrafish neurogenesis

    OpenAIRE

    Martins, Tiago Filipe Mendes

    2014-01-01

    Microcefalia de origem primária é uma doença caracterizada por afectar o desenvolvimento cerebral. Cdk5rap2, Aspm e Wdr62 são algumas das proteínas centrossomáis que têm sido descritas como sendo associadas a microcefalias. As proteínas associadas aos centrossomas são evidenciadas como reguladoras da divisão celular e tem sido sugerido que a saída prematura do ciclo celular e a interferência com o tipo de divisão de células progenitoras pode causar microcefalias. O objetivo des...

  20. Upregulation of CDK7 in gastric cancer cell promotes tumor cell proliferation and predicts poor prognosis.

    Science.gov (United States)

    Wang, Qiuhong; Li, Manhua; Zhang, Xunlei; Huang, Hua; Huang, Jianfei; Ke, Jing; Ding, Haifang; Xiao, Jinzhang; Shan, Xiaohang; Liu, Qingqing; Bao, Bojun; Yang, Lei

    2016-06-01

    CDK7 has been known as a component of CDK activating kinase (CAK) complex, the complex was composed of CDK7, Cyclin H and RING finger protein Mat1 that contribute to cell cycle progression by phosphorylating other CDKs. In addition, the complex is also an essential component of general transcription factor TFIIH which controls transcription via activating RNA polymerase II by serines 5 and 7 phosphorylation of the carboxyl-terminal domain (CTD) of its largest subunit. However, the role of CDK7 in the pathogenesis of gastric cancer has not been identified. Our study showed that CDK7 was significantly upregulated and positively correlated with tumor grade, infiltration depth, lymph node, Ki-67, and predicted poor prognosis in 173 gastric cancer specimens by immunohistochemistrical analyses. Furthermore, in vitro results indicated that CDK7 promoted proliferation of gastric cancer cells by CCK8, clone formation analyses and flow cytometric analyses, while CDK7 knockdown led to decreased cell proliferation. Our study will provide a theoretical basis for the study of CDK7 in gastric cancer. PMID:27155449

  1. 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.

  2. 55K isoform of CDK9 associates with Ku70 and is involved in DNA repair

    International Nuclear Information System (INIS)

    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.

  3. Cucurbitacin E Induces G2/M Phase Arrest through STAT3/p53/p21 Signaling and Provokes Apoptosis via Fas/CD95 and Mitochondria-Dependent Pathways in Human Bladder Cancer T24 Cells

    Directory of Open Access Journals (Sweden)

    Wen-Wen Huang

    2012-01-01

    Full Text Available Cucurbitacin E, a tetracyclic triterpenes compound extracted from cucurbitaceous plants, has been shown to exhibit anticancer and anti-inflammatory activities. The purpose of this study was to elucidate whether cucurbitacin E promotes cell cycle arrest and induces apoptosis in T24 cells and further to explore the underlying molecular mechanisms. The effects of cucurbitacin E on T24 cell's growth and accompanied morphological changes were examined by MTT assay and a phase-contrast microscope. DNA content, mitochondrial membrane potential (ΔΨm and annexin V/PI staining were determined by flow cytometry. The protein levels were measured by Western blotting. Our results demonstrated that cucurbitacin E-induced G2/M arrest was associated with a marked increase in the levels of p53, p21 and a decrease in phospho-signal transducer and activator of transcription 3 (STAT3, cyclin-dependent kinase 1 (CDK1 and cyclin B. Cucurbitacin E-triggered apoptosis was accompanied with up-regulation of Fas/CD95, truncated BID (t-BID and a loss of ΔΨm, resulting in the releases of cytochrome c, apoptotic protease activating factor 1 (Apaf-1 and apoptosis-inducing factor (AIF, and sequential activation of caspase-8, caspase-9, and caspase-3. Our findings provided the first evidence that STAT3/p53/p21 signaling, Fas/CD95 and mitochondria-dependent pathways play critical roles in cucurbitacin E-induced G2/M phase arrest and apoptosis of T24 cells.

  4. 肝细胞肝癌p21WAF1与p53的表达及其意义%EXPRESSION AND SIGNIFICANCE OF p21WAF1 AND p53 IN HEPATIC CELL CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    戴大英; 翟为溶; 万大方; 朱腾方; 叶圣龙

    2001-01-01

    Objective To explore the expression and significance of p21WAF1 and p53 in HCC. Methods Immunohistochemical method (IHC) was used to localize and semi-quantitate the proteins of p21WAF1 and p53 and to observe the relationship between the expression of p21WAF1 and the different histopathologic characters in 38 patients of HCC and their peri-cancer tissue as well as 5 normal liver tissue. Results Of all 38 cases, both p21WAF1 and p53 expression were significantly higher in tumor than that in corresponding non-tumors liver tissue; 14 (36.8 %) of 38 cases showed p21WAF1 positive staining, 28 cases (73.7 %) were p53 positive, p21WAF1+/p53+ or p21WAF1-/p53- were observed in 18, while 20 cases showed p53+/p21WAF1- or p53-/p21WAF1+. p21WAF1+ was seen in 1 of 38 (2.6 %) corresponding non-cancerous tissue and 2 of 5 normal liver tissue. p53 protein was not detected neither in the non-tumorous tissue nor in normal liver. No significant association was found between the expressions of p21WAF1 and p53(P>0.05) in HCC. Their was no significant correlation between p21WAF1 or p53 expression and the different histopathologic characters of tumor (differentiating grades, intrahepatic metastasis and/or cancerous thrombi within portal veins). Conclusion Both p21WAF1 and p53 proteins are over expressed in HCC than that in corresponding non-tumorous liver tissue, but there is no relationship between them. Both p53-independent and p53-dependent mechanism may play a role in regulating p21WAF1 expression in HCC. p21WAF1 immunostaining cannot be used to assess the status of p53 in any given cell or tissue.%目的探讨HCC中p21WAF1与p53的表达及其意义。方法应用免疫组化法检测38例手术切取的HCC及其配对癌旁肝组织、5例正常肝组织中p21WAF1和p53的表达,并分析它们与病理形态的关系。结果 p21WAF1、p53在HCC中的表达明显高于癌旁肝组织,p21WAF1阳性14例(36.8 %),p53阳性28例(73.7 %),其中两者

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

    International Nuclear Information System (INIS)

    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 (Cdk5KD or PARP-2KD 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 Cdk5KD and PARP-2KD cells, show that an

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

    International Nuclear Information System (INIS)

    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. 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. 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. p21 induction may significantly contribute to the response of colon adenocarcinomas cells to DOX treatment; and small molecules that can exploit p53-independent pathways for p21 induction, such as

  7. CDK2在非小细胞肺癌组织中的表达%Expression of CDK2 in Non-small Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    林炜明; 罗茂春; 陈彤; 尹会方

    2014-01-01

    探讨CDK2在非小细胞肺癌组织中的表达与肺癌转移关系.将50例非小细胞肺癌组织分为转移组和非转移组,采用免疫组织化学和Western blot检测癌组织中CDK2蛋白的表达.结果表明:CDK2蛋白在肺癌细胞中主要位于细胞核.CDK2蛋白在肺癌组织中的表达水平显著高于癌旁组织(P<0.05).CDK2蛋白高水平表达与肺癌淋巴结转移呈正相关(P<0.05),但与肿瘤类型无关(P>0.05).CDK2的过表达可能与肺癌的形成有关,并与淋巴转移有关.

  8. Unique catalytic activities and scaffolding of p21 activated kinase-1 in cardiovascular signaling

    Directory of Open Access Journals (Sweden)

    YunboKe

    2013-09-01

    Full Text Available P21 activated kinase-1 has diverse functions in mammalian cells. Although a large number of phosphoproteins have been designated as Pak1 substrates from in vitro studies,emerging evidence has indicated that Pak1 may function as a signaling molecule through a unique molecular mechanism—scaffolding. By scaffolding, Pak1 delivers signals through an auto-phosphorylation-induced conformational change without transfer of a phosphate group to its immediate downstream effector(s. Here we review evidence for this regulatory mechanism based on structural and functional studies of Pak1 in different cell types and research models as well as in vitro biochemical assays. We also discuss the implications of Pak1 scaffolding in disease-related signaling processes and the potential in cardiovascular drug development.

  9. THE EXPRESSION AND SIGNIFICANCE OF P53 AND P21(WAF1/CIP1) IN THYROID CARCINOMA

    Institute of Scientific and Technical Information of China (English)

    Huo Xiongwei; Ma Qingyong; Gao Yanfeng; Sun Xuejun; Liu Hao; Sheng Wei

    2005-01-01

    Objective To determine the expression of P53 and P21 (WAF1/CIP1) in thyroid carcinomas and its relationship with development and prognosis of the carcinoma. Methods 90 cases of thyroid tissues (60 thyroid carcinomas, 10 thyroid adenomas, 10 goitres and 10 normal thyroid tissues) were studied by SP immunohistochemical method. Results Positive immunoreactivity of P53 and P21(WAF1/CIP1) was found only in thyroid carcinomas. The positive rate of the P53 and P21 is 53.3% and 41.7% respectively. The positive-staining rates of P53 were higher in cases of undifferentiated carcinomas, positive metastasis lymph nodes or in stage Ⅲ, Ⅳ than those in the cases of well-differentiated, no metastasis lymph nodes, or in stage Ⅰ,Ⅱ. In addition, the positive-staining of P21(WAF1/CIP1) were lower in cases of undifferentiated carcinomas, positive metastasis lymph nodes or stage Ⅲ, Ⅳ than that in the cases of well-differentiated, no metastasis lymph nodes or in stage Ⅰ,Ⅱ. The P21 (WAF1/CIP1) expression rate in the P53 positive group was lower than that in the P53 negative group (P<0.05). Conclusion The expression of P21(WAF1/CIP1) protein in thyroid cancer is related to P53-depend pathway and P53-independent pathway, mainly the P53-depend pathway. Examination of expression of P53 and P21 (WAF1/ CIP1) proteins may be helpful to judge the thyroid cancers behavior and prognosis.

  10. Role of p53 and p21 polymorphisms in the risk of cervical cancer among Chinese women

    Institute of Scientific and Technical Information of China (English)

    Pei Jiang; Jianxin Liu; Wen Li; Xiaoxi Zeng; Jianxin Tang

    2010-01-01

    The objective of this study was to identify whether poly morphic variants of p53 at codon 72 and p21 at codon 31 were associated with increased risk for cervical cancer,either independently or jointly,among Chinese women from southern Han.We genotyped p53 codon 72 and p21 codon 31 polymorphisms of peripheral blood DNA from 104 cervical cancer patients and 160 controls.Genotyping was confirmed by polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)and direct DNA sequencing.We observed an increased risk of cervi cal cancer associated with the p53 Arg/Arg(OR,2.25;95% CI,1.11-4.54)or p21 Ser/Ser(OR,2.09;95% CI,1.04-4.19)genotype,compared with the p53 Pro/Pro or p21 Arg/Arg genotype,respectively.In additional,inter action between these pS3 and p21 polymorphisms increased the risk of cervical cancer in a multiplicative manner,with the OR being 3.96(95% CI,1.51-10.41)for subjects carrying both p53 Arg/Arg and p21 Ser/Ser genotypes.These findings suggest that there is a significant association between the genetic polymorphism of pS3,p21,and the risk of cervical cancer among Chinese southern women,and there is a possible gene-gene inter action in the incidence of cervical cancer.

  11. Histone Lysine Methylation in TGF-β1 Mediated p21 Gene Expression in Rat Mesangial Cells

    Science.gov (United States)

    Guo, Qiaoyan; Li, Xiaoxia; Han, Hongbo; Li, Chaoyuan; Liu, Shujun; Gao, Wenhui

    2016-01-01

    Transforming growth factor beta1- (TGF-β1-) induced p21-dependent mesangial cell (MC) hypertrophy plays a key role in the pathogenesis of chronic renal diseases including diabetic nephropathy (DN). Increasing evidence demonstrated the role of posttranscriptional modifications (PTMs) in the event; however, the precise regulatory mechanism of histone lysine methylation remains largely unknown. Here, we examined the roles of both histone H3 lysine 4 and lysine 9 methylations (H3K4me/H3K9me) in TGF-β1 induced p21 gene expression in rat mesangial cells (RMCs). Our results indicated that TGF-β1 upregulated the expression of p21 gene in RMCs, which was positively correlated with the increased chromatin marks associated with active genes (H3K4me1/H3K4me2/H3K4me3) and negatively correlated with the decreased levels of repressive marks (H3K9me2/H3K9me3) at p21 gene promoter. TGF-β1 also elevated the recruitment of the H3K4 methyltransferase (HMT) SET7/9 to the p21 gene promoter. SET7/9 gene silencing with small interfering RNAs (siRNAs) significantly abolished the TGF-β1 induced p21 gene expression. Taken together, these results reveal the key role of histone H3Kme in TGF-β1 mediated p21 gene expression in RMC, partly through HMT SET7/9 occupancy, suggesting H3Kme and SET7/9 may be potential renoprotective agents in managing chronic renal diseases.

  12. Loss of p21Sdi1 expression in senescent cells after DNA damage accompanied with increase of miR-93 expression and reduced p53 interaction with p21Sdi1 gene promoter

    International Nuclear Information System (INIS)

    Highlights: → Reduced p21 expression in senescent cells treated with DNA damaging agents. → Increase of [3H]thymidine and BrdU incorporations in DNA damaged-senescent cells. → Upregulation of miR-93 expression in senescent cells in response to DSB. → Failure of p53 binding to p21 promoter in senescent cells in response to DSB. → Molecular mechanism of increased cancer development in aged than young individuals. -- Abstract: To answer what is a critical event for higher incidence of tumor development in old than young individuals, primary culture of human diploid fibroblasts were employed and DNA damage was induced by doxorubicin or X-ray irradiation. Response to the damage was different between young and old cells; loss of p21sdi1 expression in spite of p53S15 activation in old cells along with [3H]thymidine and BrdU incorporation, but not in young cells. The phenomenon was confirmed by other tissue fibroblasts obtained from different donor ages. Induction of miR-93 expression and reduced p53 binding to p21 gene promoter account for loss of p21sdi1 expression in senescent cells after DNA damage, suggesting a mechanism of in vivo carcinogenesis in aged tissue without repair arrest.

  13. Expression of p21WAF1 and Ki-67 in the lesions of Bowen disease%Bowen病皮损中p21WAF1蛋白、Ki-67蛋白的表达及其意义

    Institute of Scientific and Technical Information of China (English)

    郭书萍; 白莉

    2007-01-01

    目的 探讨Bowen病中细胞周期调节蛋白p21WAF1及Ki-67蛋白的表达和意义.方法 用免疫组织化学SP法检测35例Bowen病和12名正常人皮肤黏膜组织中p21WAF1蛋白、Ki-67蛋白的表达和分布.结果 正常人皮肤黏膜组织中p21WAF1蛋白阴性表达、Ki-67蛋白表达仅见于基底层;Bowen病皮损中,p21WAF1蛋白、Ki-67蛋白的表达均增高,与正常人皮肤黏膜相比较差异均有统计学意义(P值均<0.01).结论 Bowen病中p21WAF1的高表达可能与肿瘤细胞的分化有关.

  14. 胃腺癌组织中P21、HER2、EGFR表达临床病理关系探讨%Clinicopathological study of P21, HER2, EGFR expression in gastric adenocarcinoma tissue

    Institute of Scientific and Technical Information of China (English)

    胡志雄; 韦世强; 谭敏华; 邓超桦; 杨海云; 曹贤东; 郭锦辉; 陈威; 邹绮嫦

    2013-01-01

    目的 探讨P21、HER2、EGFR蛋白在胃腺癌组织中的表达特点及其临床病理关系.方法 应用免疫组织化学技术对65例胃腺癌病理切片进行P21、HER2和EGFR的联合检测,并结合检测结果进行统计学分析.结果 65例胃腺癌组织中P21、HER2、EGFR阳性表达率分别为56.9%(37/65)、43.1%(28/65)、30.8%(20/65).经统计学分析,P21、HER2、EGFR蛋白的表达与胃腺癌组织的分化程度、浸润深度、是否有癌栓、淋巴结转移、远处转移和TNM分期密切相关(P<0.05,P<0.01).与性别、年龄、肿瘤直径、肿瘤部位和组织学类型均无统计意义(P>0.05).P21表达与HER2、EGFR阳性表达,及HER2表达与EGFR阳性表达均有统计意义(P<0.05).结论 P21、HER2、EGFR的表达结果表明胃腺癌存在着多基因表达,这些基因可能共同参与了胃腺癌的发生和发展.三者的联合检测有助于判断胃腺癌的生物学行为和预后,也可为临床选择分子靶向药物治疗提供依据.%Objective To explore P21,HER2,EGFR protein expression in gastric adenocarcinoma tissue and their clinical pathological relationship.Methods Immunohistochemical technique was used in P21,HER2 and EGFR joint detection for 65 cases of gastric adenocarcinoma biopsy.The result was statistically analyzed.Results The positive expression rates of P21,HER2,EGFR were 56.9% (37/65),43.1% (28/65),30.8% (20/65) respectively.Statistical analysis showed,P21,HER2,EGFR protein expression were related to gastric adenocarcinoma tissue differentiation,depth of invasion,with or without tumor thrombus,lymph node metastasis,distant metastasis and TNM stage (P < 0.05 or P < 0.01),not related to gender,age,tumor size,tumor location and histological type (P > 0.05).P21 expression was related to HER2 and EGFR expression,and HER2 expression was related to EGFR expression (P < 0.05).Conclusions P21,HER2,EGFR expression results in gastric adenocarcinoma show the existence of

  15. Cloning and Functional Analysis of Porcine Cdk2 Gene%猪Cdk2基因的克隆及其功能研究

    Institute of Scientific and Technical Information of China (English)

    唐青海; 张辉; 危艳武; 刘长明

    2013-01-01

    本研究旨在克隆猪Cdk2基因,并研究其编码蛋白CDK2的生物学功能.采用RT-PCR扩增猪Cdk2基因,运用生物信息学软件分析其核苷酸和编码氨基酸特征,并预测编码蛋白的生物学功能;利用半定量RT-PCR方法分析该基因在猪各个脏器和组织中的表达情况;共聚焦显微镜观察CDK2的亚细胞定位,采用过表达和shRNA干扰技术研究CDK2在细胞周期和细胞增殖中的调控作用.结果表明,猪Cdk2基因开放阅读框(ORF)为897 bp(GenBank:JX967576),该基因与绵羊、牛、山羊、人、金仓鼠、小鼠、仓鼠和沟鼠Cdk2的核苷酸相似性依次为94.2%、94.0%、93.8%、93.4%、91.8%、91.0%、90.6%和89.9%,与牛、山羊和绵羊的亲缘关系最近;Cdk2编码298 aa,CDK2分子质量为34 ku.Cdk2 mRNA在猪10个不同脏器和组织中均有表达.CDK2定位于细胞质和细胞核中,并通过蛋白酶体途径降解.猪CDK2在PK-15细胞中过表达引起S期细胞比例显著减少及G2/M期细胞比例显著增加(P<0.05),而G0/G1期无显著变化;相反,CDK2表达量降低引起S期细胞比例显著减少及G0/G1期细胞比例显著增加,而G2/M期无显著变化.本研究成功克隆了猪Cdk2基因并对其编码蛋白生物学功能进行了初步研究.

  16. CDK2在鼻咽癌中的表达及意义%The Expression and Significance of CDK-2 in Nasopharyngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    官树雄; 蒋月荷; 王继群; 山艳春

    2011-01-01

    目的 探讨细胞周期蛋白依赖性激酶2(CDK2)在鼻咽癌和慢性鼻咽炎黏膜组织中的表达水平,及其与鼻咽癌的临床病理关系.方法 免疫组化SP法检测CDK2蛋白在鼻咽癌和慢性鼻咽炎黏膜组织中的表达情况.结果 CDK2蛋白阳性表达主要定位于细胞核,少数有胞浆着色,呈棕黄色或棕褐色不同强度的染色.62例鼻咽癌组织中阳性表达率为69.4%(43/62),慢性鼻咽炎黏膜组织中阳性率为32.0%(9/28),两组间差异有统计学意义(P<0.05).CDK2蛋白表达与鼻咽癌分化程度,淋巴结转移范围,TNM分期有关(P<0.05).结论 CDK2在鼻咽癌组织中的表达明显高于慢性鼻咽炎黏膜组织中的表达,提示CDK2与鼻咽癌的发生、发展有关.CDK2与鼻咽癌分化程度,淋巴结转移范围,TNM分期有关.%Objective The propose of the research was focused on the expression levels of CDK2 in the tissues of nasopharyngeal carcinoma and chronic inflammation nasopharyngeal membrane. And it may relate to the pathogenesis and clinical significance of nasopharyngeal carcinoma which had been observed in the field of cell cycle. Methods Immunohistochemistry.(sp) was used to examine the expression levels of CDK2 protein in the tissues of nasopharyngeal carcinoma and chronic inflammation nasopharyngeal membrane. Results Most of the CDK2 protein positive expression was found in cell nucleus,but some was found in the kytoplasm and its color was brown yellow or dark brown. The positive expression rate of nasopharyngeal carcinoma from 62 patients was 69.40% (43/62), and that of the chronic inflammation nasopharyngeal membrane was 32.0% (9/28). The difference of the two groups was found statistical significance( P < 0.05 ) ;the expression levels of CDK2 were related to the differential degree, the range of lymph node metastases and clinical staging of nasopharyngeal carcinoma, the difference was been found statistical significance( P < 0.05 ). Conclusion The expression

  17. Cdk5-mediated mitochondrial fission: A key player in dopaminergic toxicity in Huntington's disease.

    Science.gov (United States)

    Cherubini, Marta; Puigdellívol, Mar; Alberch, Jordi; Ginés, Silvia

    2015-10-01

    The molecular mechanisms underlying striatal vulnerability in Huntington's disease (HD) are still unknown. However, growing evidence suggest that mitochondrial dysfunction could play a major role. In searching for a potential link between striatal neurodegeneration and mitochondrial defects we focused on cyclin-dependent kinase 5 (Cdk5). Here, we demonstrate that increased mitochondrial fission in mutant huntingtin striatal cells can be a consequence of Cdk5-mediated alterations in Drp1 subcellular distribution and activity since pharmacological or genetic inhibition of Cdk5 normalizes Drp1 function ameliorating mitochondrial fragmentation. Interestingly, mitochondrial defects in mutant huntingtin striatal cells can be worsened by D1 receptor activation a process also mediated by Cdk5 as down-regulation of Cdk5 activity abrogates the increase in mitochondrial fission, the translocation of Drp1 to the mitochondria and the raise of Drp1 activity induced by dopaminergic stimulation. In sum, we have demonstrated a new role for Cdk5 in HD pathology by mediating dopaminergic neurotoxicity through modulation of Drp1-induced mitochondrial fragmentation, which underscores the relevance for pharmacologic interference of Cdk5 signaling to prevent or ameliorate striatal neurodegeneration in HD. PMID:26143143

  18. Amygdalin Blocks Bladder Cancer Cell Growth In Vitro by Diminishing Cyclin A and cdk2

    Science.gov (United States)

    Makarević, Jasmina; Rutz, Jochen; Juengel, Eva; Kaulfuss, Silke; Reiter, Michael; Tsaur, Igor; Bartsch, Georg; Haferkamp, Axel; Blaheta, Roman A.

    2014-01-01

    Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25–10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug. PMID:25136960

  19. Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

    Directory of Open Access Journals (Sweden)

    Jasmina Makarević

    Full Text Available Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25-10 mg/ml on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP. Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug.

  20. INHIBITION OF FARNESYL PROTEIN TRANSFERASE AND P21RAS MEMEBRANE ASSOCIATION BY D-LIMONENE IN HUMAN PANCREAS TUMOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    陈晓光; YoshihisaYano; TadayoshiHasuma; ToshikoYoshimata; WangYinna; ShuzoOtani

    1999-01-01

    The monoterpene d-limonene inhibit the plasma membrane associated P21ras expresion and the post-translational isoprenylatlon of P21ras, a mechanism that may contribute to its efficacy in the ehemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study, the relative abilities of d-limonene to inhibit membrane associated P21ras expression in Imncreas tumorcell (PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FT-Pase ) activity during the Rns p~otebi isoprenylation and cell proliferation were determined. Concomitantly,the effects of d-limonene on P21ras localization hy immunohimcchemistry and H-ras oncogene expression in PaCe tutor cell line by Northern blotting were observed. The results showed that ddimonene inhibited FPTase activity, thus to reduce P21H-ras isoprenylation, d limonene couM decrease P21ras meanhrane asso-ciation and increase cytosolie accumulation of P21ras This phenomenon was also noted when d-llmmaene-treated PaCe cells were stained immunohistcchemieally with anti-P21ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21ras membrane assoclaticon aad the alteration of P21ras localization. Inhibition ot farnesylation of P21ras altered their intraeellulsr localization and, hence, disrupted their biological activity, hut no relationship with H ras oneogene expression was found.

  1. INHIBITION OF FARNESYL PROTEIN TRANSFERASE AND P21RAS MEMEBRANE ASSOCIATION BY D-LIMONENE IN HUMAN PANCREAS TUMOR CELLS IN VITRO

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ The monoterpene d-limonene inhibit the plasma-membrane associated P21ras expression and the posttranslational isoprenylation of P21ras, a mechanism that may contribute to its efficacy in the chemoprevention and therapy of chemically induced rodent cancers and some human solid tumor cells. In the present study,the relative abilities of d-limonene to inhibit membrane associated P21ras expression in pancreas tumor cell(PaCa) was carried out with Western blotting, and the inhibition of farnesyl protein transferase (FTPase) activity during the Ras protein isoprenylation and cell proliferation were determined.Concomitantly,the effects of d-limonene on P21ras localization by immunohistochemistry and H-ras oncogene expression in PaCa tumor cell line by Northern blotting were observed. The results showed that d-limonene inhibited FPTase activity, thus to reduce P21H-ras isoprenylation. d-limonene could decrease P21ras membrane association and increase cytosolic accumulation of P21ras. This phenomenon was also noted when d-limonene-treated PaCa cells were stained immunohistochemically with anti-P21ras antibody. It is suggested that the inhibition of FPTase activity was closely related with the inhibiton of P21ras membrane association and the alteration of P21ras localization. Inhibition of farnesylation of P21ras altered their intracellular localization and, hence, disrupted their biological activity,but no relationship with H-ras oncogene expression was found.

  2. 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)

    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

  3. Nuclear translocation of p21WAF1/CIP1 protein prior to its cytosolic degradation by UV enhances DNA repair and survival

    International Nuclear Information System (INIS)

    We previously reported that UV induced rapid proteasomal degradation of p21 protein in an ubiquitination-independent manner. Here, UV-induced p21 proteolysis was found to occur in the cytosol. Before cytosolic degradation, however, p21 protein translocated to and transiently accumulated in the nucleus. Nuclear translocation of p21 was not required for its degradation, but rather promoted DNA repair and cell survival. Overexpression of the wild type p21, but not the one with defective nuclear localization signal (NLS), reduced UV-induced DNA damage and cell death. Some of p21 protein translocated to the nucleus were associated with chromatin-bound PCNA and saved from UV-induced proteolysis. These data together show that p21 translocates to the nucleus to participate in DNA repair, while the rest is rapidly degraded in the cytosol. We propose that our findings reflect a mechanism to facilitate removal of damaged cells, enhancing DNA repair at the same time.

  4. The Effect of cdk- 5 Overexpression and Overactivation on Tau Hyperphosphorylation in Cultured N2a Cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Juan; LI Hong-lian; FENG You-mei; WANG Jian-zhi

    2005-01-01

    Neurofibrillary tangles (NFTs) are one of the neuropathological hallmarks of Alzheimer' s disease (AD) and abnormally hyperphosphorylated tau is the major protein of NFTs. It was reported that cyclin-dependent kinase5 (Cdk-5) could phosphorylate tau at most AD-related epitopes in vivo. In this study, we investigated the effect of cdk-5 overexpression on tau hyperphosphorylation in neuroblastoma N2a cells. We demonstrated that overexpression of cdk-5 which resulted in a 3.5-fold Cdk5 activation in the transfected cells induced a dramatic increase in phosphorylation of tau at several phosphorylation sites. Overexpression of cdk-5 led to a reduced staining with antibody Tau-1 and an enhanced staining with antibody PHF-1, suggesting hy perphosphorylation of tau at Ser199/202 and Ser396/404 sites. It implies that in vitro overexpression of cdk-5 leads to Cdk5 overactivation and tau hyperphosphorylation may be the underline mechanism.

  5. Adenosine induces cell cycle arrest and apoptosis via cyclinD1/Cdk4 and Bcl-2/Bax pathways in human ovarian cancer cell line OVCAR-3.

    Science.gov (United States)

    Shirali, Saeid; Aghaei, Mahmoud; Shabani, Mahdi; Fathi, Mojtaba; Sohrabi, Majid; Moeinifard, Marzieh

    2013-04-01

    Adenosine is a regulatory molecule with widespread physiological effects in almost every cells and acts as a potent regulator of cell growth. Adenosine has been shown to inhibit cell growth and induce apoptosis in the several cancer cells via caspase activation and Bcl-2/Bax pathway. The present study was designed to understand the mechanism underlying adenosine-induced apoptosis in the OVCAR-3 human ovarian cancer cells. MTT viability, BrdU and cell counting assays were used to study the cell proliferation effect of adenosine in presence of adenosine deaminase inhibitor and the nucleoside transporter inhibitor. Cell cycle analysis, propidium iodide and annexin V staining, caspase-3 activity assay, cyclinD1, Cdk4, Bcl-2 and Bax protein expressions were assessed to detect apoptosis. Adenosine significantly inhibited cell proliferation in a concentration-dependent manner in OVCAR-3 cell line. Adenosine induced cell cycle arrest in G0/G1 phase via Cdk4/cyclinD1-mediated pathway. Adenosine induced apoptosis, which was determined by Annexin V-FITC staining and increased sub-G1 population. Moreover, down-regulation of Bcl-2 protein expression, up-regulation of Bax protein expression and activation of caspase-3 were observed in response to adenosine treatment. The results of this study suggest that extracellular adenosine induced G1 cell cycle arrest and apoptosis in ovarian cancer cells via cyclinD1/ Cdk4 and Bcl-2/Bax pathways and caspase-3 activation. These data might suggest that adenosine could be used as an agent for the treatment of ovarian cancer. PMID:23345014

  6. Fabrication of functionally graded materials between P21 tool steel and Cu by using laser aided layered manufacturing

    Energy Technology Data Exchange (ETDEWEB)

    Jeong, Jong Seol; Shin, Ki Hoon [Seoul Nat' l Univ., Seoul (Korea, Republic of)

    2013-01-15

    With the development of layered manufacturing, thermally conductive molds or molds embedding conformal cooling channels can be directly fabricated. Although P21 tool steel is widely used as a mold material because of its dimensional stability, it is not efficient for cooling molds owing to its low thermal conductivity. Hence, the use of functionally graded materials (FGMs) between P21 and Cu may circumvent a tradeoff between the strength and the heat transfer rate. As a preliminary study for the layered manufacturing of thermally conductive molds having FGM structures, one dimensional P21 Cu FGMs were fabricated by using laser aided direct metal tooling (DMT), and then, material properties such as the thermal conductivity and specific heat that are related to the heat transfer were measured and analyzed.

  7. Fabrication of functionally graded materials between P21 tool steel and Cu by using laser aided layered manufacturing

    International Nuclear Information System (INIS)

    With the development of layered manufacturing, thermally conductive molds or molds embedding conformal cooling channels can be directly fabricated. Although P21 tool steel is widely used as a mold material because of its dimensional stability, it is not efficient for cooling molds owing to its low thermal conductivity. Hence, the use of functionally graded materials (FGMs) between P21 and Cu may circumvent a tradeoff between the strength and the heat transfer rate. As a preliminary study for the layered manufacturing of thermally conductive molds having FGM structures, one dimensional P21 Cu FGMs were fabricated by using laser aided direct metal tooling (DMT), and then, material properties such as the thermal conductivity and specific heat that are related to the heat transfer were measured and analyzed

  8. Mutations in CDK5RAP2 cause Seckel syndrome

    OpenAIRE

    Karabey Kayserili, Hülya; Yiğit, G.; Brown, KE.; Pohl, E.; Caliebe, A.; Zahnleiter, D.; Rosser, E.; Bögershausen, N.; Uyguner, ZO.; Altunoğlu, U.; Nürnberg, G.; Nürnberg, P.; Rauch, A.; Li, Y.; Thiel, CT.; Wollnik, B.

    2015-01-01

    ORIGINAL ARTICLE Mutations in CDK5RAP2 cause Seckel syndrome Go¨ khan Yigit1,2,3,a, Karen E. Brown4,a, Hu¨ lya Kayserili5, Esther Pohl1,2,3, Almuth Caliebe6, Diana Zahnleiter7, Elisabeth Rosser8, Nina Bo¨ gershausen1,2,3, Zehra Oya Uyguner5, Umut Altunoglu5, Gudrun Nu¨ rnberg2,3,9, Peter Nu¨ rnberg2,3,9, Anita Rauch10, Yun Li1,2,3, Christian Thomas Thiel7 & Bernd Wollnik1,2,3 1Institute of Human Genetics, University of Cologne, Cologne, Germany 2Center for Molecular Medic...

  9. 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.

  10. CDKN2B expression and subcutaneous adipose tissue expandability: Possible influence of the 9p21 atherosclerosis locus

    International Nuclear Information System (INIS)

    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

  11. Effects of CDK2 on DNA ploidy in laryngeal squamous cell carcinoma%喉鳞癌中CDK2表达对DNA倍体的作用

    Institute of Scientific and Technical Information of China (English)

    刘荣; 皇甫辉

    2008-01-01

    目的 研究喉鳞癌组织中CDK2表达在引起DNA异倍体发生过程中的作用. 方法 取手术中获得的50例喉鳞癌组织和30例声带息肉组织,用γ-微管蛋白抗体标记中心体,用免疫组织化学的方法检测CDK2激酶和γ-微管蛋白的表达;用流式细胞术检测喉鳞癌组织DNA倍体. 结果 在喉鳞癌组织中CDK2激酶和γ-微管蛋白阳性率表达[分别为68.0%(34/50)和78.0%(39/50)]都显著高于在声带息肉组织中(P<0.05)[分别为20.0%(6/30)和33.3%(10/30)],而且CDK2激酶的表达与γ-微管蛋白的表达具有相关性.21例CDK2表达阳性的喉鳞癌组织其DI为1.76±0.36;9例CDK2表达阴性的喉鳞癌组织其DI为1.05±0.07,CDK2阳性的喉鳞癌组织较阴性表达的组织DI增高(P<0.05). 结论 喉鳞癌中CDK2过度表达导致肿瘤细胞DNA异倍体发生.在诊断和治疗喉鳞癌中,CDK2可能是一个有重要作用的指标.

  12. Loss of chromosome 9p21 and decreased p16 expression correlate with malignant gastrointestinal stromal tumor

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    AIM: To investigate loss of heterozygosity (LOH) of chromosome 9p21 and the prognostic relevance of p16 expression in gastrointestinal stromal tumor (GIST). METHODS: Fifty-one GIST patients (30 men and 21 women; median age 59 years; range 29-80 years) treated surgically within a 10-year period were grouped by aggressive behavior risk (17 with very low and low, 14 intermediate, and 20 high risk). GISTs were characterized immunohistochemically and evaluated for LOH of 9p21 by microsatellite analysis at D9S175...

  13. Functional analyses of coronary artery disease associated variation on chromosome 9p21 in vascular smooth muscle cells

    OpenAIRE

    Motterle, Anna; Pu, Xiangyuan; Wood, Harriet; Xiao, Qingzhong; Gor, Shivani; Liang Ng, Fu; Chan, Kenneth; Cross, Frank; Shohreh, Beski; Poston, Robin N.; Tucker, Arthur T.; Caulfield, Mark J; Ye, Shu

    2012-01-01

    Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16INK4a, p14ARF and p15INK4b and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs...

  14. Common variants at 7p21 are associated with frontotemporal lobar degeneration with TDP-43 inclusions

    Science.gov (United States)

    Van Deerlin, Vivianna M.; Sleiman, Patrick M. A.; Martinez-Lage, Maria; Chen-Plotkin, Alice; Wang, Li-San; Graff-Radford, Neill R; Dickson, Dennis W.; Rademakers, Rosa; Boeve, Bradley F.; Grossman, Murray; Arnold, Steven E.; Mann, David M.A.; Pickering-Brown, Stuart M.; Seelaar, Harro; Heutink, Peter; van Swieten, John C.; Murrell, Jill R.; Ghetti, Bernardino; Spina, Salvatore; Grafman, Jordan; Hodges, John; Spillantini, Maria Grazia; Gilman, Sid'; Lieberman, Andrew P.; Kaye, Jeffrey A.; Woltjer, Randall L.; Bigio, Eileen H; Mesulam, Marsel; al-Sarraj, Safa; Troakes, Claire; Rosenberg, Roger N.; White, Charles L.; Ferrer, Isidro; Lladó, Albert; Neumann, Manuela; Kretzschmar, Hans A.; Hulette, Christine Marie; Welsh-Bohmer, Kathleen A.; Miller, Bruce L; Alzualde, Ainhoa; de Munain, Adolfo Lopez; McKee, Ann C.; Gearing, Marla; Levey, Allan I.; Lah, James J.; Hardy, John; Rohrer, Jonathan D.; Lashley, Tammaryn; Mackenzie, Ian R.A.; Feldman, Howard H.; Hamilton, Ronald L.; Dekosky, Steven T.; van der Zee, Julie; Kumar-Singh, Samir; Van Broeckhoven, Christine; Mayeux, Richard; Vonsattel, Jean Paul G.; Troncoso, Juan C.; Kril, Jillian J; Kwok, John B.J.; Halliday, Glenda M.; Bird, Thomas D.; Ince, Paul G.; Shaw, Pamela J.; Cairns, Nigel J.; Morris, John C.; McLean, Catriona Ann; DeCarli, Charles; Ellis, William G.; Freeman, Stefanie H.; Frosch, Matthew P.; Growdon, John H.; Perl, Daniel P.; Sano, Mary; Bennett, David A.; Schneider, Julie A.; Beach, Thomas G.; Reiman, Eric M.; Woodruff, Bryan K.; Cummings, Jeffrey; Vinters, Harry V.; Miller, Carol A.; Chui, Helena C.; Alafuzoff, Irina; Hartikainen, Päivi; Seilhean, Danielle; Galasko, Douglas; Masliah, Eliezer; Cotman, Carl W.; Tuñón, M. Teresa; Martínez, M. Cristina Caballero; Munoz, David G.; Carroll, Steven L.; Marson, Daniel; Riederer, Peter F.; Bogdanovic, Nenad; Schellenberg, Gerard D.; Hakonarson, Hakon; Trojanowski, John Q.; Lee, Virginia M.-Y.

    2010-01-01

    Frontotemporal lobar degeneration (FTLD) is the second most common cause of presenile dementia. The predominant neuropathology is FTLD with TAR DNA binding protein (TDP-43) inclusions (FTLD-TDP)1. FTLD-TDP is frequently familial resulting from progranulin (GRN) mutations. We assembled an international collaboration to identify susceptibility loci for FTLD-TDP, using genome-wide association (GWA). We found that FTLD-TDP associates with multiple SNPs mapping to a single linkage disequilibrium (LD) block on 7p21 that contains TMEM106B in a GWA study (GWAS) on 515 FTLD-TDP cases. Three SNPs retained genome-wide significance following Bonferroni correction; top SNP rs1990622 (P=1.08×10−11; odds ratio (OR) minor allele (C) 0.61, 95% CI 0.53-0.71). The association replicated in 89 FTLD-TDP cases (rs1990622; P=2×10−4). TMEM106B variants may confer risk by increasing TMEM106B expression. TMEM106B variants also contribute to genetic risk for FTLD-TDP in patients with GRN mutations. Our data implicate TMEM106B as a strong risk factor for FTLD-TDP suggesting an underlying pathogenic mechanism. PMID:20154673

  15. 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.

  16. [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. PMID:18338576

  17. 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

  18. 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

  19. Methods Of Using Chemical Libraries To Search For New Kinase Inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Gray, Nathanael S. (Berkeley, CA), Schultz, Peter (Oakland, CA), Wodicka, Lisa (Santa Clara, CA), Meijer, Laurent (Roscoff, FR), Lockhart, David J. (Mountain View, CA)

    2003-06-03

    The generation of selective inhibitors for specific protein kinases would provide new tools for analyzing signal transduction pathways and possibly new therapeutic agents. We have invented an approach to the development of selective protein kinase inhibitors based on the unexpected binding mode of 2,6,9-trisubstituted purines to the ATP binding site of human CDK2. The most potent inhibitor, purvalanol B (IC.sub.50 =6 nM), binds with a 30-fold greater affinity than the known CDK2 inhibitor, flavopiridol. The cellular effects of this class of compounds were examined and compared to those of flavopiridol by monitoring changes in mRNA expression levels for all genes in treated cells of Saccharomyces cerevisiae using high-density oligonucleotide probe arrays.

  20. Expression of p21ras and p73 in endometrioid adenocarcinoma and its clinical significance%p21ras、p73在子宫内膜腺癌中的表达及意义

    Institute of Scientific and Technical Information of China (English)

    陈雪; 李美蓉

    2011-01-01

    目的 探讨p21ras、p73在子宫内膜腺癌中的表达及意义.方法 用免疫组化SP法在15例正常增生期子宫内膜、19例子宫内膜非典型增生及61例子宫内膜腺癌组织中检测p21ras、p73的表达情况.结果 随着子宫内膜病变的恶性进展,p21ras和p73的阳性表达率上调,且两者在子宫内膜腺癌中表达的相关性呈正相关(P<0.01).p21ras和p73表达与子宫内膜腺癌病理学分级有关(P<0.05),与子宫内膜腺癌的临床分期、肌层浸润、淋巴结转移无关.结论 p21ras、p73与子宫内膜腺癌的发生和发展有关,且两者存在协同作用.联合检测p21ras、p73的表达可能成为子宫内膜腺癌高危人群早期筛查、病理诊断的参考指标.%Objective To investigate the expression of p2lras and p73 in endometrioid adenocarcinoma and its clinical significance.Methds Immunohistochemistry was applied to detect the expression of p2lras and p73 protein in 15 cases of normal proliferating endometrial tissues, 19 cases of atypical hyperplasia and 61 cases of endometrioid adenocarcinoma. Results The positive rates of p21ras and p73 gradually increased from normal proliferating endometrial tissues to endometrioid adenocarcinoma. There was a positive correlation between p73 and p2lras in endometrioid adenocarcinoma ( P < 0. 01 ). Expression of p2lras and p73 was associated with the histological grade( P < 0. 05 ) .but not related with clinical stage, myometrial invasion and lymph node metastasis. Conclusion The results suggest that the activation of p2lras and p73 may be correlated with the genesis and development of endometrioid adenocarcinoma. The combined detection of p2lras and p73 may provide a reference for early screening of high-risk subjects,pathological diagnosis of endometrioid adenocarcinoma.

  1. CDK5 is essential for TGF-β1-induced epithelial-mesenchymal transition and breast cancer progression.

    Science.gov (United States)

    Liang, Qian; Li, Lili; Zhang, Jianchao; Lei, Yang; Wang, Liping; Liu, Dong-Xu; Feng, Jingxin; Hou, Pingfu; Yao, Ruosi; Zhang, Yu; Huang, Baiqu; Lu, Jun

    2013-01-01

    Epithelial-mesenchymal transition is a change of cellular plasticity critical for embryonic development and tumor metastasis. CDK5 is a proline-directed serine/threonine kinase playing important roles in cancer progression. Here we show that CDK5 is commonly overexpressed and significantly correlated with several poor prognostic parameters of breast cancer. We found that CDK5 participated in TGF-β1-induced EMT. In MCF10A, TGF-β1 upregulated the CDK5 and p35 expression, and CDK5 knockdown inhibited TGF-β1-induced EMT. CDK5 overexpression also exhibited a potential synergy in promoting TGF-β1-induced EMT. In mesenchymal breast cancer cells MDA-MB-231 and BT549, CDK5 knockdown suppressed cell motility and tumorigenesis. We further demonstrated that CDK5 modulated cancer cell migration and tumor formation by regulating the phosphorylation of FAK at Ser-732. Therefore, CDK5-FAK pathway, as a downstream step of TGF-β1 signaling, is essential for EMT and motility in breast cancer cells. This study implicates the potential value of CDK5 as a molecular marker for breast cancer. PMID:24121667

  2. 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 membra...

  3. Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies

    International Nuclear Information System (INIS)

    Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131I-RASK-3 and 125I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers

  4. Zinc Induced G2/M Blockage is p53 and p21 Dependent in Normal Human Bronchial Epithelial Cells

    Science.gov (United States)

    The involvement of the p53 and p21 signal pathway in the G2/M cell cycle progression of zinc supplemented normal human bronchial epithelial (NHBE) cells was examined using the siRNA approach. Cells were cultured for one passage in different concentrations of zinc: <0.4 microM (ZD) as zinc-deficient;...

  5. IL-6 modulates hepatocyte proliferation via induction of HGF/p21cip1: Regulation by SOCS3

    International Nuclear Information System (INIS)

    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 p21cip1 protein expression in primary mouse hepatocytes. Disruption of the p21cip1 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 p21cip1 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 p21cip1-dependent mechanism and indirectly enhances hepatocyte proliferation via stimulating nonparenchymal cells to produce HGF. SOCS3 negatively regulates liver regeneration

  6. GADD45 and p21 gene expression up regulated by X-ray in human peripheral blood lymphocytes

    International Nuclear Information System (INIS)

    SYBR real time RT-PCR method was applied to study the expression profiles of the Growth Arrest and DNA Damage gene 45 (GADD45) and p21 induced by X-rays in human peripheral blood lymphocytes in vivo. Results show that GADD45 and p21 gene expressions in human PBLs at 24h after 1, 2, 3, 5 Gy X-ray irradiation were increased significantly. A linear dose response relationship could be established for the GADD45 gene expression over the dose range from 1.0 Gy to 5.0 Gy and for the p21 gene expression over the dose range from 1.0 Gy to 3.0 Gy 24 h after X-ray irradiation respectively. Results suggest that the relative level of the GADD45 and p21 gene expression in human PBLs is dose dependent under the experimental conditions, indicating that the GADD45 gene may serve as a potential biological dosimeter in future radiation accident dose assessment

  7. The same sequence variant on 9p21 associates with myocardial infarction, abdominal aortic aneurysm and intracranial aneurysm

    DEFF Research Database (Denmark)

    Helgadottir, Anna; Thorleifsson, Gudmar; Magnusson, Kristinn P;

    2008-01-01

    Recently, two common sequence variants on 9p21, tagged by rs10757278-G and rs10811661-T, were reported to be associated with coronary artery disease (CAD) and type 2 diabetes (T2D), respectively. We proceeded to further investigate the contributions of these variants to arterial diseases and T2D....

  8. CDK4 Amplification Predicts Recurrence of Well-Differentiated Liposarcoma of the Abdomen

    OpenAIRE

    Lee, Sanghoon; Park, Hyojun; Ha, Sang Yun; Paik, Kwang Yeol; Lee, Seung Eun; Kim, Jong Man; Park, Jae Berm; Kwon, Choon Hyuck David; Joh, Jae-Won; Choi, Yoon-La; Kim, Sung Joo

    2014-01-01

    Background 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. Methods 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 quantit...

  9. On the Wegener granulomatosis associated region on chromosome 6p21.3

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    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

  10. 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 to ...

  11. A p21-activated kinase is required for conidial germination in Penicillium marneffei.

    Science.gov (United States)

    Boyce, Kylie J; Andrianopoulos, Alex

    2007-11-01

    Asexual spores (conidia) are the infectious propagules of many pathogenic fungi, and the capacity to sense the host environment and trigger conidial germination is a key pathogenicity determinant. Germination of conidia requires the de novo establishment of a polarised growth axis and consequent germ tube extension. The molecular mechanisms that control polarisation during germination are poorly understood. In the dimorphic human pathogenic fungus Penicillium marneffei, conidia germinate to produce one of two cell types that have very different fates in response to an environmental cue. At 25 degrees C, conidia germinate to produce the saprophytic cell type, septate, multinucleate hyphae that have the capacity to undergo asexual development. At 37 degrees C, conidia germinate to produce the pathogenic cell type, arthroconidiating hyphae that liberate uninucleate yeast cells. This study shows that the p21-activated kinase pakA is an essential component of the polarity establishment machinery during conidial germination and polarised growth of yeast cells at 37 degrees C but is not required for germination or polarised growth at 25 degrees C. Analysis shows that the heterotrimeric G protein alpha subunit GasC and the CDC42 orthologue CflA lie upstream of PakA for germination at both temperatures, while the Ras orthologue RasA only functions at 25 degrees C. These findings suggest that although some proteins that regulate the establishment of polarised growth in budding yeast are conserved in filamentous fungi, the circuitry and downstream effectors are differentially regulated to give rise to distinct cell types. PMID:17983267

  12. Transcriptional targeting in cancer gene therapy using the p21/WAF1 promoter

    International Nuclear Information System (INIS)

    Lack of target cell specificity is the main limitation with gene therapy. Tissue-specific receptors (Kashara et al. 1994) and enhancers (Manome et al. 1994) have been used to localise therapy to the desired area. However low levels of gene expression are often associated with these methods. There has been a great deal of interest in the use of exogenous inducible promoters that operate at the transcriptional level, such as the Egr-1 promoter (Manome et al. 1998). This investigation focuses on the promoter for the x-ray inducible gene, p21/WAF1 (wild type p53 activated fragment). The WAF1 promoter has a p53 binding site which has been shown to confer p53 dependent inducibility upon the gene (El-Deiry et al. 1993). However a p53 independent activation pathway also exists (Wu et al. 2000). The current study examines the radiation and hypoxia inducibility of the WAF1 promoter in several human tumour cell lines of which the p53 status is known. Using the pEGFP reporter gene, we have established that the promoter is induced by radiation at clinically relevant doses (Worthington et al, 2000) and also by hypoxia. Results indicate that the WAF1 promoter is not leaky in normal human cells but is leaky in human tumour cells regardless of p53 status, indicating a level of tumour specificity not yet fully explored. This observation, coupled with its hypoxia and radiation inducibility at clinically relevant radiation doses, suggest that the WAF1 promoter could have significant advantages in overcoming the target specificity associated with gene therapy

  13. 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.

  14. PLK1-dependent activation of LRRK1 regulates spindle orientation by phosphorylating CDK5RAP2.

    Science.gov (United States)

    Hanafusa, Hiroshi; Kedashiro, Shin; Tezuka, Motohiro; Funatsu, Motoki; Usami, Satoshi; Toyoshima, Fumiko; Matsumoto, Kunihiro

    2015-08-01

    Correct formation of the cell division axis requires the initial precise orientation of the mitotic spindle. Proper spindle orientation depends on centrosome maturation, and Polo-like kinase 1 (PLK1) is known to play a crucial role in this process. However, the molecular mechanisms that function downstream of PLK1 are not well understood. Here we show that LRRK1 is a PLK1 substrate that is phosphorylated on Ser 1790. PLK1 phosphorylation is required for CDK1-mediated activation of LRRK1 at the centrosomes, and this in turn regulates mitotic spindle orientation by nucleating the growth of astral microtubules from the centrosomes. Interestingly, LRRK1 in turn phosphorylates CDK5RAP2(Cep215), a human homologue of Drosophila Centrosomin (Cnn), in its γ-tubulin-binding motif, thus promoting the interaction of CDK5RAP2 with γ-tubulin. LRRK1 phosphorylation of CDK5RAP2 Ser 140 is necessary for CDK5RAP2-dependent microtubule nucleation. Thus, our findings provide evidence that LRRK1 regulates mitotic spindle orientation downstream of PLK1 through CDK5RAP2-dependent centrosome maturation. PMID:26192437

  15. Sumoylation of p35 modulates p35/cyclin-dependent kinase (Cdk) 5 complex activity.

    Science.gov (United States)

    Büchner, Anja; Krumova, Petranka; Ganesan, Sundar; Bähr, Mathias; Eckermann, Katrin; Weishaupt, Jochen H

    2015-03-01

    Cyclin-dependent kinase (Cdk) 5 is critical for central nervous system development and neuron-specific functions including neurite outgrowth as well as synaptic function and plasticity. Cdk5 activity requires association with one of the two regulatory subunits, called p35 and p39. p35 redistribution as well as misregulation of Cdk5 activity is followed by cell death in several models of neurodegeneration. Posttranslational protein modification by small ubiquitin-related modifier (SUMO) proteins (sumoylation) has emerged as key regulator of protein targeting and protein/protein interaction. Under cell-free in vitro conditions, we found p35 covalently modified by SUMO1. Using both biochemical and FRET-/FLIM-based approaches, we demonstrated that SUMO2 is robustly conjugated to p35 in cells and identified the two major SUMO acceptor lysines in p35, K246 and K290. Furthermore, different degrees of oxidative stress resulted in differential p35 sumoylation, linking oxidative stress that is encountered in neurodegenerative diseases to the altered activity of Cdk5. Functionally, sumoylation of p35 increased the activity of the p35/Cdk5 complex. We thus identified a novel neuronal SUMO target and show that sumoylation is a likely candidate mechanism for the rapid modulation of p35/Cdk5 activity in physiological situations as well as in disease. PMID:25391294

  16. Significant difference in p53 and p21 protein immunoreactivity in HPV 16 positive and HPV negative breast carcinomas

    International Nuclear Information System (INIS)

    Human papillomavirus (HPV) 16 has previously been found in 19/41 breast carcinomas (46%) in women with a history of HPV 16 positive CIN III lesions. There was no significant difference in distribution of histological subtypes, mean or median tumour diameter or number of regional lymph node metastases in the HPV positive and HPV negative breast carcinoma groups. P53, p21 and c-erbB-2 proteins were analyzed by immunohistochemistry in the HPV 16 positive and HPV negative breast carcinomas. There was a significant difference in p53 and p21 protein immunoreactivity between HPV 16 positive and HPV negative breast carcinomas (p=0.0091 and p=0.0040), with a significant less detectable p53 and p21 protein immunoreactivity in the HPV 16 positive cases. There was also a significant difference in the coexpression of p53/p21 between the HPV 16 positive and HPV 16 negative breast carcinomas (p=0.002). No significant difference in immunostaining for c-erbB-2 protein in the two groups was found (p=0.15), or for the coexpression of p53/c-erbB-2 (p=0.19). The significantly lower expression of p53 and p21 proteins in HPV 16 positive than in HPV 16 negative breast carcinomas supports the hypothesis of inactivation and degradation of wild-type p53 proteins by HPV 16 E6 and that p53 mutation is not necessary for transformation in the HPV 16 positive cases. (orig.)

  17. Prevalence of p21 immunohistochemical expression in esophageal adenocarcinoma Prevalência da expressão imunoistoquímica da proteína p21 em adenocarcinoma do esôfago

    Directory of Open Access Journals (Sweden)

    Maitê de Mello Villwock

    2006-09-01

    Full Text Available BACKGROUND: In western societies, the prevalence of adenocarcinoma of the gastroesophageal junction has increased in recent years. It is commonly accepted today that esophageal adenocarcinoma develops from a premalignant lesion: Barrett's esophagus. This type of carcinoma is hardly diagnosed at early stages, which results in significant mortality. Molecular biology studies have shown that most malignant tumors originate from the interaction between inherited characteristics and external factors, which may cause genetic changes that interfere with the control over the differentiation and growth of cells in susceptible individuals. p21 (WAF1/CIP1 has a key role in the regulation of the cell cycle, and its immunohistochemical expression has been investigated in several tumors, showing that it influences the prognosis of various neoplasms. AIM: To check the prevalence of p21 protein expression in patients with esophageal adenocarcinoma diagnosed in the last 5 years by the Group for Surgeries of the Esophagus and Stomach of "Hospital de Clínicas de Porto Alegre", RS, Brazil. METHODS: The study population consisted of 42 patients with esophageal adenocarcinoma diagnosed by the Group for Surgeries of the Esophagus and Stomach between January 1998 and December 2002. The expression of p21 protein was determined by immunohistochemistry using primary antibody, p21, clone SX118, code M7202 (Dako, and assessed according to the immunoreactive scoring system. RESULTS: Of 42 analyzed patients, 83.3% were male and older than 40 years. Among these, 56.2% were submitted to curative resection: total gastrectomy and transhiatal esophagogastrectomy. The remaining patients were submitted to palliative surgery or did not undergo any surgical treatment. Only five patients received adjuvant chemotherapy and radiation therapy, either alone or combined. Advanced disease (stages III and IV was detected in 78.6% of the patients. Only nine patients were positive for p21

  18. 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...

  19. Iron Chelators of the Di-2-pyridylketone Thiosemicarbazone and 2-Benzoylpyridine Thiosemicarbazone Series Inhibit HIV-1 Transcription: Identification of Novel Cellular Targets—Iron, Cyclin-Dependent Kinase (CDK) 2, and CDK9S⃞

    OpenAIRE

    Debebe, Zufan; Ammosova, Tatyana; Breuer, Denitra; Lovejoy, David B.; Kalinowski, Danuta S.; Karla, Pradeep K.; Kumar, Krishna; Jerebtsova, Marina; Ray, Patricio; KASHANCHI, FATAH; Gordeuk, Victor R; Richardson, Des R.; Nekhai, Sergei

    2011-01-01

    HIV-1 transcription is activated by HIV-1 Tat protein, which recruits cyclin-dependent kinase 9 (CDK9)/cyclin T1 and other host transcriptional coactivators to the HIV-1 promoter. Tat itself is phosphorylated by CDK2, and inhibition of CDK2 by small interfering RNA, the iron chelator 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), and the iron chelator deferasirox (ICL670) inhibits HIV-1 tran...

  20. miR-6734 Up-Regulates p21 Gene Expression and Induces Cell Cycle Arrest and Apoptosis in Colon Cancer Cells

    Science.gov (United States)

    Kang, Moo Rim; Park, Ki Hwan; Yang, Jeong-Ook; Lee, Chang Woo; Oh, Soo Jin; Yun, Jieun; Lee, Myeong Youl; Han, Sang-Bae; Kang, Jong Soon

    2016-01-01

    Recently, microRNAs have been implicated in the regulation of gene expression in terms of both gene silencing and gene activation. Here, we investigated the effects of miR-6734, which has a sequence homology with a specific region of p21WAF1/CIP1 (p21) promoter, on cancer cell growth and the mechanisms involved in this effect. miR-6734 up-regulated p21 expression at both mRNA and protein levels and chromatin immunoprecipitation analysis using biotin-labeled miR-6734 confirmed the association of miR-6734 with p21 promoter. Moreover, miR-6734 inhibited cancer cell growth and induced cell cycle arrest and apoptosis in HCT-116 cells, which was abolished by knockdown of p21. The phosphorylation of Rb and the cleavage of caspase 3 and PARP were suppressed by miR-6734 transfection in HCT-116 cells and these effects were also reversed by p21 knockdown. In addition, miR-6734 transfection caused prolonged induction of p21 gene and modification of histones in p21 promoter, which are typical aspects of a phenomenon referred to as RNA activation (RNAa). Collectively, our results demonstrated that miR-6734 inhibits the growth of colon cancer cells by up-regulating p21 gene expression and subsequent induction of cell cycle arrest and apoptosis, suggesting its role as an important endogenous regulator of cancer cell proliferation and survival. PMID:27509128

  1. 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.

  2. 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.

  3. Triptolide Induces Cell Killing in Multidrug-Resistant Tumor Cells via CDK7/RPB1 Rather than XPB or p44.

    Science.gov (United States)

    Yi, Jun-Mei; Huan, Xia-Juan; Song, Shan-Shan; Zhou, Hu; Wang, Ying-Qing; Miao, Ze-Hong

    2016-07-01

    Multidrug resistance (MDR) is a major cause of tumor treatment failure; therefore, drugs that can avoid this outcome are urgently needed. We studied triptolide, which directly kills MDR tumor cells with a high potency and a broad spectrum of cell death. Triptolide did not inhibit P-glycoprotein (P-gp) drug efflux and reduced P-gp and MDR1 mRNA resulting from transcription inhibition. Transcription factors including c-MYC, SOX-2, OCT-4, and NANOG were not correlated with triptolide-induced cell killing, but RPB1, the largest subunit of RNA polymerase II, was critical in mediating triptolide's inhibition of MDR cells. Triptolide elicited antitumor and anti-MDR activity through a universal mechanism: by activating CDK7 by phosphorylating Thr170 in both parental and MDR cell lines and in SK-OV-3 cells. The CDK7-selective inhibitor BS-181 partially rescued cell killing induced by 72-hour treatment of triptolide, which may be due to partial rescue of RPB1 degradation. We suggest that a precise phosphorylation site on RPB1 (Ser1878) was phosphorylated by CDK7 in response to triptolide. In addition, XPB and p44, two transcription factor TFIIH subunits, did not contribute to triptolide-driven RPB1 degradation and cell killing, although XPB was reported to covalently bind to triptolide. Several clinical trials are underway to test triptolide and its analogues for treating cancer and other diseases, so our data may help expand potential clinical uses of triptolide, as well as offer a compound that overcomes tumor MDR. Future investigations into the primary molecular target(s) of triptolide responsible for RPB1 degradation may suggest novel anti-MDR target(s) for therapeutic development. Mol Cancer Ther; 15(7); 1495-503. ©2016 AACR. PMID:27197304

  4. Theoretical studies on human cyclin-dependent kinase 2 complexed with inhibitor roscovitine%人细胞周期蛋白依赖性激酶2与抑制剂roscovitine作用的理论研究

    Institute of Scientific and Technical Information of China (English)

    赵宏涛; 黎云燕; 徐为人

    2012-01-01

    目的:研究人细胞周期蛋白依赖性激酶2(CDK2)与抑制剂roscovitine相互作用的机制.方法:利用分子动力学模拟的方法研究roscovitine与CDK2之间的相互作用.结果:和空载CDK2相比,roscovitine的存在会使RMSD和RMSF值略微降低,但roscovitine会显著影响CDK2活性位点残基侧链二面角.Roscovitine在分子动力学模拟过程中会与Ile10和Leu83形成稳定的氢键作用.结论:Roscovitine对CDK2骨架运动影响不大,但会使CDK2活性位点残基的侧链构象发生变化.Roscovitine与Ile10和Leu83之间的氢键作用是CDK2对roscovitine进行识别的重要途径.%Objective: To study the interaction mechanisms of inhibitor roscovitine with human cyclin-dependent kinase 2 (CDK2). Methods: Molecular dynamics simulation was used to investigate the interaction between roscovitine and CDK2. Results: The existence of roscovitine made the RMSD and RMSF values little larger than the apo-form of CDK2, but had significant impact on the side chain dihedrals of residues in the active site of CDK2. Roscovitine also could form stable hydrogen bonding with Ile10 and Leu83. Conclusion: Roscovitine do not have significant influence on the backbone movement of CDK2, but significant change is found in side chain conformation of residues in the active site of CDK2 due to the roscovitine bonding. The hydrogen bonds between roscovitine and Ile10, Leu83 are key pathways for CDK2 to recognize roscovitine.

  5. Expression of cyclin D1, p21WAF1, p53 and Ki-67 in hepatocellular carcinoma: a pathological study

    Directory of Open Access Journals (Sweden)

    Yu-lan WANG

    2014-03-01

    Full Text Available Objective To explore the expression of cyclin D1, p21WAF1, p53 and Ki-67 protein in hepatocellular carcinoma (HCC and its relationship with prognosis of HCC patients. Methods Liver specimens were collected from 80 HCC patients who received hepatectomy in the General Hospital of PLA from Jan. 2000 to Jan. 2005, and the expressions of cyclin D1, p21WAF1, p53 and Ki-67 protein were determined by immunohistochemical staining (EliVision method to investigate the relationship between the protein expressions and the clinicopathologic characteristics of HCC, and their relationship with the survival rate of the patients was analyzed. Results The positive expression rates of cyclin D1, p21WAF1, p53 and Ki-67 in HCC were 38.8%, 40.5%, 65.4% and 80.0% respectively, and they were significantly higher than those in matched normal tissues (19.0%, 11.5%, 0.0% and 6.3% respectively, P<0.005. Correlation analysis showed that the expression of cyclin D1 was positively related to the nuclear grade (P=0.041, the expression of p21WAF1 and p53 were positively related to the tumor differentiation (P=0.032, P=0.031 and vascular invasion (P=0.036, P=0.011, the expression of Ki-67 was positively related to the tumor differentiation (P=0.004, nuclear grade (P=0.045 and vascular invasion (P=0.001. Survival analysis showed the prognosis was poor in patients with high expression of cyclin D1 or/and Ki-67. The expression of Ki-67 was significantly related to the expression of p53 (P=0.000 and p21WAF1 (P=0.047, but no significant relation was found among the expression of other proteins. Cox regression analysis showed that the tumor size (P=0.042, tumor number (P=0.004 and vascular invasion (P=0.000 were independent prognostic factors of HCC. Conclusions The cyclin D1, p21WAF1, p53 and Ki-67 protein may be involved in the biological process of HCC. The positive expression of cyclin D1 and Ki-67 may be used to evaluate the prognosis of patients with HCC. DOI: 10.11855/j

  6. 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; Xie, Xu; Schultz, Christiane; Gussio, Rick; Zaharevitz, Daniel; Leost, Maryse; Meijer, Laurent; Weber, Alexander; Jørgensen, Flemming Steen; Lemcke, Thomas

    2004-01-01

    data of 52 paullone entities, which were aligned by a docking routine into the ATP-binding cleft of a CDK1/cyclin B homology model. Variation of grid spacing and column filtering were used during the optimization of the models. The predictive ability of the models was shown by a leave-one-out cross...

  7. CDK5 activator protein p25 preferentially binds and activates GSK3β.

    Science.gov (United States)

    Chow, Hei-Man; Guo, Dong; Zhou, Jie-Chao; Zhang, Guan-Yun; Li, Hui-Fang; Herrup, Karl; Zhang, Jie

    2014-11-11

    Glycogen synthase kinase 3β (GSK3β) and cyclin-dependent kinase 5 (CDK5) are tau kinases and have been proposed to contribute to the pathogenesis of Alzheimer's disease. The 3D structures of these kinases are remarkably similar, which led us to hypothesize that both might be capable of binding cyclin proteins--the activating cofactors of all CDKs. CDK5 is normally activated by the cyclin-like proteins p35 and p39. By contrast, we show that GSK3β does not bind to p35 but unexpectedly binds to p25, the calpain cleavage product of p35. Indeed, overexpressed GSK3β outcompetes CDK5 for p25, whereas CDK5 is the preferred p35 partner. FRET analysis reveals nanometer apposition of GSK3β:p25 in cell soma as well as in synaptic regions. Interaction with p25 also alters GSK3β substrate specificity. The GSK3β:p25 interaction leads to enhanced phosphorylation of tau, but decreased phosphorylation of β-catenin. A partial explanation for this situation comes from in silico modeling, which predicts that the docking site for p25 on GSK3β is the AXIN-binding domain; because of this, p25 inhibits the formation of the GSK3β/AXIN/APC destruction complex, thus preventing GSK3β from binding to and phosphorylating β-catenin. Coexpression of GSK3β and p25 in cultured neurons results in a neurodegeneration phenotype that exceeds that observed with CDK5 and p25. When p25 is transfected alone, the resulting neuronal damage is blocked more effectively with a specific siRNA against Gsk3β than with one against Cdk5. We propose that the effects of p25, although normally attributed to activate CDK5, may be mediated in part by elevated GSK3β activity. PMID:25331900

  8. 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

  9. 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.

  10. Induction of alternative lengthening of telomeres-associated PML bodies by p53/p21 requires HP1 proteins

    OpenAIRE

    Jiang, Wei-Qin; Zhong, Ze-Huai; Nguyen, Akira; Henson, Jeremy D.; Toouli, Christian D.; Braithwaite, Antony W.; Reddel, Roger R

    2009-01-01

    Alternative lengthening of telomeres (ALT) is a recombination-mediated process that maintains telomeres in telomerase-negative cancer cells. In asynchronously dividing ALT-positive cell populations, a small fraction of the cells have ALT-associated promyelocytic leukemia nuclear bodies (APBs), which contain (TTAGGG)n DNA and telomere-binding proteins. We found that restoring p53 function in ALT cells caused p21 up-regulation, growth arrest/senescence, and a large increase in cells containing ...

  11. Increased p53 and decreased p21 accompany apoptosis induced by ultraviolet radiation in the nervous system of a crustacean.

    Science.gov (United States)

    Hollmann, Gabriela; Linden, Rafael; Giangrande, Angela; Allodi, Silvana

    2016-04-01

    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 the number of mitotic cells in the same groups decreased. In conclusion, environmental doses of UV can cause apoptosis by increasing p53 and decreasing p21, revealing an UV-damage pathway for U. cordatus. PMID:26807499

  12. Cigarette Smoke Extract Inhibits the Proliferation of Alveolar Epithelial Cells and Augments the Expression of P21WAF1

    Institute of Scientific and Technical Information of China (English)

    Zongxian JIAO; Qilin AO; Xiaona GE; Mi XIONG

    2008-01-01

    Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke ex- tract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱand its relationship with P21WAF1, the alveolar epithelial type Ⅱ cell line (A549) cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke ex-tract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic mi-croscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1phases after the cells were incubated with cigarette smoke extract. The expression of p21WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracellular accumulation of P21WAF1 may be one of the mechanisms which contribute to cigarette smoke ex-tract-induced inhibition of cell proliferation.

  13. 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.

  14. Potentiality of phosphorylation of BRCA1 at ser 1524 to activate p21 in response to X-ray irradiation

    International Nuclear Information System (INIS)

    The breast and ovarian cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein, which functions as a tumor suppressor gene. Many studies suggested that multiple functions of BRCA1 may contribute to its tumor suppressor activity, including roles in cell cycle checkpoints, apoptosis and transcription. It is postulated that phosphorylation of BRCA1 is an important means by which its cellular functions are regulated. In this study, we employed phospho-Ser-specific antibody recognizing Ser-1524 to study BRCA1 phosphorylation under conditions of DNA damage and the effects of phosphorylation on BRCA1 functions. The results showed that 10 Gy X-ray treatment significantly induced phosphorylation of Ser-1524 but not total BRCA1 protein levels. The expression both of p53 and p21 increased after irradiation, but ionizing radiation (IR) -induced activation of p21 was prior to that of p53. The pementages of G0/G1 phase remarkably increased after IR. In addition, no detectable levels of 89 kDa fragment of PARP, a marker of apoptotic cells, were observed. Data implied that IR-induced phosphorylation of BRCA1 at Ser-1524 might activate p21 protein, by which BRCA1 regulated cell cycle, but play no role in apoptosis. (authors)

  15. Wnt/β-catenin signaling induces the aging of mesenchymal stem cells through the DNA damage response and the p53/p21 pathway.

    Directory of Open Access Journals (Sweden)

    Da-yong Zhang

    Full Text Available Recent studies have demonstrated the importance of cellular extrinsic factors in the aging of adult stem cells. However, the effects of an aged cell-extrinsic environment on mesenchymal stem cell (MSC aging and the factors involved remain unclear. In the current study, we examine the effects of old rat serum (ORS on the aging of MSCs, and explore the effects and mechanisms of Wnt/β-catenin signaling on MSC aging induced by ORS treatment. Senescence-associated changes in the cells are examined with SA-β-galactosidase staining and ROS staining. The proliferation ability is detected by MTT assay. The surviving and apoptotic cells are determined using AO/EB staining. The results suggest that ORS promotes MSC senescence and reduces the proliferation and survival of cells. The immunofluorescence staining shows that the expression of β-catenin increases in MSCs of old rats. To identify the effects of Wnt/β-catenin signaling on MSC aging induced with ORS, the expression of β-catenin, GSK-3β, and c-myc are detected. The results show that the Wnt/β-catenin signaling in the cells is activated after ORS treatment. Then we examine the aging, proliferation, and survival of MSCs after modulating Wnt/β-catenin signaling. The results indicate that the senescence and dysfunction of MSCs in the medium containing ORS is reversed by the Wnt/β-catenin signaling inhibitor DKK1 or by β-catenin siRNA. Moreover, the expression of γ-H2A.X, a molecular marker of DNA damage response, p16(INK4a, p53, and p21 is increased in senescent MSCs induced with ORS, and is also reversed by DKK1 or by β-catenin siRNA. In summary, our study indicates the Wnt/β-catenin signaling may play a critical role in MSC aging induced by the serum of aged animals and suggests that the DNA damage response and p53/p21 pathway may be the main mediators of MSC aging induced by excessive activation of Wnt/β-catenin signaling.

  16. A conditional mouse mutant in the tumor suppressor SdhD gene unveils a link between p21(WAF1/Cip1 induction and mitochondrial dysfunction.

    Directory of Open Access Journals (Sweden)

    Africa Millán-Uclés

    Full Text Available Mutations in mitochondrial complex II (MCII; succinate dehydrogenase, Sdh genes cause familiar pheochromocytoma/paraganglioma tumors. Several mechanisms have been proposed to account for Sdh-mutation-induced tumorigenesis, the most accepted of which is based on the constitutive expression of the hypoxia-inducible factor 1α (Hif1α at normal oxygen tension, a theory referred to as "pseudo-hypoxic drive". Other molecular processes, such as oxidative stress, apoptosis, or chromatin remodeling have been also proposed to play a causative role. Nevertheless, the actual contribution of each of these mechanisms has not been definitively established. Moreover, the biological factors that determine the tissue-specificity of these tumors have not been identified. In this work, we made use of the inducible SDHD-ESR mouse, a conditional mutant in the SdhD gene, which encodes the small subunit of MCII, and that acts as a tumor suppressor gene in humans. The analysis of the Hif1α pathway in SDHD-ESR tissues and in two newly derived cell lines after complete SdhD loss -a requirement for hereditary paraganglioma type-1 tumor formation in humans- partially recapitulated the "pseudo-hypoxic" response and rendered inconsistent results. Therefore, we performed microarray analysis of adrenal medulla and kidney in order to identify other early gene expression changes elicited by SdhD deletion. Our results revealed that each mutant tissue displayed different variations in their gene expression profiles affecting to different biological processes. However, we found that the Cdkn1a gene was up-regulated in both tissues. This gene encodes the cyclin-dependent kinase inhibitor p21(WAF1/Cip1, a factor implicated in cell cycle, senescence, and cancer. The two SDHD-ESR cell lines also showed accumulation of this protein. This new and unprecedented evidence for a link between SdhD dysfunction and p21(WAF1/Cip1 will open new avenues for the study of the mechanisms that cause

  17. Experimental Approaches to Study Mitochondrial Localization and Function of a Nuclear Cell Cycle Kinase, Cdk1.

    Science.gov (United States)

    Candas, Demet; Qin, Lili; Fan, Ming; Li, Jian-Jian

    2016-01-01

    Although mitochondria possess their own transcriptional machinery, merely 1% of mitochondrial proteins are synthesized inside the organelle. The nuclear-encoded proteins are transported into mitochondria guided by their mitochondria targeting sequences (MTS); however, a majority of mitochondrial localized proteins lack an identifiable MTS. Nevertheless, the fact that MTS can instruct proteins to go into the mitochondria provides a valuable tool for studying mitochondrial functions of normally nuclear and/or cytoplasmic proteins. We have recently identified the cell cycle kinase CyclinB1/Cdk1 complex in the mitochondria. To specifically study the mitochondrial functions of this complex, mitochondrial overexpression and knock-down of this complex without interfering with its nuclear or cytoplasmic functions were essential. By tagging CyclinB1/Cdk1 with MTS, we were able to achieve mitochondrial overexpression of this complex to study its mitochondrial targets as well as functions. Via tagging dominant-negative Cdk1 with MTS, inhibition of Cdk1 activity was accomplished particularly in the mitochondria. Potential mitochondrial targets of CyclinB1/Cdk1 complex were identified using a gel-based proteomics approach. Unlike traditional 2D gel analysis, we employed 2-dimensional difference gel electrophoresis (2D-DIGE) technology followed by phosphoprotein staining to fluorescently label differentially phosphorylated proteins in mitochondrial Cdk1 expressing cells. Identification of phosphoprotein spots that were altered in wild type versus dominant negative Cdk1 bearing mitochondria revealed the identity of mitochondrial targets of Cdk1. Finally, to determine the effect of CyclinB1/Cdk1 mitochondrial localization in cell cycle progression, a cell proliferation assay using a synthetic thymidine analogue EdU (5-ethynyl-2'-deoxyuridine) was used to monitor the cells as they go through the cell cycle and replicate their DNA. Altogether, we demonstrated a variety of approaches

  18. 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. PMID:26104286

  19. Znhit1 causes cell cycle arrest and down-regulates CDK6 expression

    International Nuclear Information System (INIS)

    Cyclin-dependent kinase 6 (CDK6) is the key element of the D-type cyclin holoenzymes which has been found to function in the regulation of G1-phase of the cell cycle and is presumed to play important roles in T cell function. In this study, Znhit1, a member of a new zinc finger protein family defined by a conserved Zf-HIT domain, induced arrest in the G1-phase of the cell cycle in NIH/3T3 cells. Of the G1 cell cycle factors examined, the expression of CDK6 was found to be strongly down-regulated by Znhit1 via transcriptional repression. This effect may have correlations with the decreased acetylation level of histone H4 in the CDK6 promoter region. In addition, considering that CDK6 expression predominates in T cells, the negative regulatory role of Znhit1 in TCR-induced T cell proliferation was validated using transgenic mice. These findings identified Znhit1 as a CDK6 regulator that plays an important role in cell proliferation.

  20. Effect of berberine on Cdk9 and cyclin T1 expressions in myocardium of diabetic rats

    Institute of Scientific and Technical Information of China (English)

    Zhou Jiyin; Zhou Shiwen; Tang Jianlin; Xu Ying; Ying Yi

    2008-01-01

    Objective: To investigate the effect of berberine, one of the main alkaloids of Rhizoma coptidis, on myocardial orphology and the expressions of cyclin-dependent kinase 9 (Cdk9) and cyclin T1 protein in the myocardium of type diabetic rats. Methods: Type 2 diabetes mellitus rats were induced by an injection of 35 mg/kg streptozotocin (STZ) nd a high-carbohydrate/high-fat diet for 16 weeks. Diabetic rats were given low-, middle-, high-dose berberine (75,150, 300 mg/kg), fenofibrate (100 mg/kg) and rosiglitazone (4 mg/kg) for another 16 weeks, respectively. The myocardium structure was observed with hematoxylin & eosin (H&E) staining and Cdk9 and cyclin T1 protein expressions were detected by immunohistochemistry. Results: Middle-dose, high-dose berberine improved myocardial hypertrophy and interstitial fibrosis of diabetic rats. Cdk9 and cyclin T1 protein were significantly lower in diabetic myocardium than in control one (P<0.01), and middle-dose, high-dose berberine and fenofibrate obviously increased oth Cdk9 and cyclin T1 expression to near control level (P<0.01). Conclusion: Berberine modulates Cdk9 and cyclin I protein expression in diabetic myocardium which may contribute to ameliorate myocardium damage.

  1. Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses vasculogenic mimicry and proliferation of highly aggressive pancreatic cancer PaTu8988 cells

    International Nuclear Information System (INIS)

    Pancreatic cancer is one of the most aggressive human malignancies with a extremely low 5-year survival rate. Hence, the search for more effective anti-pancreatic cancer agents is urgent. PaTu8988 pancreatic cancer cells were treated with different concentrations of suberoylanilide hydroxamic acid (SAHA), cell survival, proliferation, migration and vasculogenic mimicry (VM) were analyzed. Associated signaling changes were also analyzed by RT-PCR and Western blots. Here, we reported that SAHA, a histone deacetylase inhibitor (HDACi), exerted significant inhibitory efficiency against pancreatic cancer cell survival, proliferation, migration and VM. SAHA dose-dependently inhibited PaTu8988 pancreatic cancer cell growth with the IC-50 of 3.4 ± 0. 7 μM. Meanwhile, SAHA suppressed PaTu8988 cell cycle progression through inducing G2/M arrest, which was associated with cyclin-dependent kinase 1 (CDK-1)/cyclin-B1 degradation and p21/p27 upregulation. Further, SAHA induced both apoptotic and non-apoptotic death of PaTu8988 cells. Significantly, SAHA suppressed PaTu8988 cell in vitro migration and cell-dominant tube formation or VM, which was accompanied by semaphorin-4D (Sema-4D) and integrin-β5 down-regulation. Our evidences showed that Akt activation might be important for Sema-4D expression in PaTu8988 cells, and SAHA-induced Sema-4D down-regulation might be associated with Akt inhibition. This study is among the first to report the VM formation in cultured human pancreatic cancer cells. And we provided strong evidence to suggest that SAHA executes significant anti-VM efficiency in the progressive pancreatic cancer cells. Thus, SAHA could be further investigated as a promising anti-pancreatic cancer agent

  2. The p21-activated kinase, PAK2, is important in the activation of numerous pancreatic acinar cell signaling cascades and in the onset of early pancreatitis events.

    Science.gov (United States)

    Nuche-Berenguer, Bernardo; Ramos-Álvarez, Irene; Jensen, R T

    2016-06-01

    In a recent study we explored Group-1-p21-activated kinases (GP.1-PAKs) in rat pancreatic acini. Only PAK2 was present; it was activated by gastrointestinal-hormones/neurotransmitters and growth factors in a PKC-, Src- and small-GTPase-mediated manner. PAK2 was required for enzyme-secretion and ERK/1-2-activation. In the present study we examined PAK2's role in CCK and TPA-activation of important distal signaling cascades mediating their physiological/pathophysiological effects and analyzed its role in pathophysiological processes important in early pancreatitis. In rat pancreatic acini, PAK2-inhibition by the specific, GP.1.PAK-inhibitor, IPA-3-suppressed cholecystokinin (CCK)/TPA-stimulated activation of focal-adhesion kinases and mitogen-activated protein-kinases. PAK2-inhibition reversed the dual stimulatory/inhibitory effect of CCK/TPA on the PI3K/Akt/GSK-3β pathway. However, its inhibition did not affect PKC activation. PAK2-inhibition protected acini from CCK-induced ROS-generation; caspase/trypsin-activation, important in early pancreatitis; as well as from cell-necrosis. Furthermore, PAK2-inhibition reduced proteolytic-activation of PAK-2p34, which is involved in programmed-cell-death. To ensure that the study did not only rely in the specificity of IPA-3 as a PAK inhibitor, we used two other approaches for PAK inhibition, FRAX597 a ATP-competitive-GP.1-PAKs-inhibitor and infection with a PAK2-dominant negative(DN)-Advirus. Those two approaches confirmed the results obtained with IPA-3. This study demonstrates that PAK2 is important in mediating CCK's effect on the activation of signaling-pathways known to mediate its physiological/pathophysiological responses including several cellular processes linked to the onset of pancreatitis. Our results suggest that PAK2 could be a new, important therapeutic target to consider for the treatment of diseases involving deregulation of pancreatic acinar cells. PMID:26912410

  3. 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}.

  4. 哺乳动物细胞CDK2系列表达载体的构建与表达%Construction and expression of the CDK2 mammalian cell expression vectors

    Institute of Scientific and Technical Information of China (English)

    吴健谊; 蒋太峰; 谢剑君; 张锴; 杜则澎; 许丽艳

    2011-01-01

    目的:构建一系列细胞周期蛋白依赖激酶2(cyclin-dependent kinase 2,CDK2)在哺乳动物细胞的表达载体,为研究CDK2的功能和修饰提供实验材料.方法:从食管癌细胞中提取总RNA,逆转录PCR扩增CDK2编码区,然后将PCR产物克隆到T载体;扩增后的CDK2片段分别亚克隆入pcDNA3、pcDNA4、pNTAP和pEGFP等4种哺乳动物表达载体;最后,将获得的表达载体PEGFP/CDK2转染小鼠成纤维细胞NIH3T3进行初步的CDK2表达分析.结果:RT-PCR扩增获得约900 bp的目的片段,经T载体克隆和DNA序列分析,显示重组片段是人CDK2基因序列;CDK2片段分别亚克隆入上述4种载体后获得相应表达载体;运用构建的PEGFP/CDK2表达载体,在NIH3T3细胞中表达出CDK2蛋白.结论:成功构建了CDK2的哺乳动物细胞系列表达载体,并在NIH3T3细胞中成功表达目的蛋白.%OBJECTIVE: To construct a series of cyclin-dependent kinase 2 (CDK2) mammalian cell expression vectors and to assess CDK2 expression in NIH3T3 cells. METHODS: RNA was isolated from esophageal cancer cells and the full coding sequence of CDK2 gene was obtained by RT-PCR. The PCR product was then cloned into T vector and subsequently subcloned into four eukaryotic expression vectors (pcDNA3, pcDNA4, pNTAP and pEGFP). The expressing plasmids were transfected into NIH3T3 cells and the expression of CDK2 was detected by western blot. RESULTS: The PCR product was about 900 bp and the sequence analysis showed that it was the full coding sequence of CDK2 gene. The product was subcloned into the eukaryotic expression vectors and four CDK2 expression vectors were constructed. Western blot showed that CDK2 could be expressed in the expression vector-transfected cells.CONCLUSION: Four CDK2 eukaryotic expression vectors were successfully constructed and CDK2 was effectively expressed in NIH3T3 cells.

  5. 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.

  6. Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion

    OpenAIRE

    Ganuza, Miguel; Sáiz-Ladera, Cristina; Cañamero, Marta; Gómez, Gonzalo; Schneider, Ralph; Blasco, María A.; Pisano, David; Paramio, Jesús M.; Santamaría, David; Barbacid, Mariano

    2012-01-01

    Employing a conditionally inactive gene trap allele, Cdk7's function in regulating cellular proliferation by Cdk1/2-phosphorylation is convincingly dissected from alternative notions on CTD-phosphorylation of RNA Pol II. Premature aging phenotypes caused by stem cell depletion lend the necessary functional support.

  7. Aberrant cytological localization of p16 and CDK4 in colorectal epithelia in the normal adenoma carcinoma sequence

    Institute of Scientific and Technical Information of China (English)

    Po Zhao; Xin Mao; Ian C Talbot

    2006-01-01

    AIM: To study the correlation between the patterns of subcellular expression of p16 and CDK4 in colorectal epithelia in the normal-adenoma-carcinoma sequence.METHODS: Paraffin sections of 43 cases of normal colorectal epithelia and corresponding adenomas as well as carcinomas were analysed immunocytochemically for subcellular expression of p16 and CDK4 proteins.RESULTS: Most carcinomas showed more cytoplasmic overexpression for p16 and CDK4 than the adenomas from which they arised or the adjacent normal mucosa.Most normal or non-neoplastic epithelia showed more p16 and CDK4 expression in the nucleus than their adjacent adenomas and carcinomas. There was a significant difference between the subcellular expression pattern of p16 and CDK4 in normal-adenoma-carcinoma sequence epithelia (P < 0.001). Neither p16 nor CDK4 subcellular patterns correlated with histological grade or Dukes' stage.CONCLUSION: Interaction of expression of p16 and CDK4 plays an important role in the Rb/p16 pathway.Overexpression of p16 and CDK4 in the cytoplasm, as well as loss expression of p16 in the nucleus might be important in the evolution of colorectal carcinoma from adenoma and, of adenoma from normal epithelia.

  8. Essential role of the Cdk2 activator RingoA in meiotic telomere tethering to the nuclear envelope

    Science.gov (United States)

    Mikolcevic, Petra; Isoda, Michitaka; Shibuya, Hiroki; del Barco Barrantes, Ivan; Igea, Ana; Suja, José A.; Shackleton, Sue; Watanabe, Yoshinori; Nebreda, Angel R.

    2016-01-01

    Cyclin-dependent kinases (CDKs) play key roles in cell cycle regulation. Genetic analysis in mice has revealed an essential role for Cdk2 in meiosis, which renders Cdk2 knockout (KO) mice sterile. Here we show that mice deficient in RingoA, an atypical activator of Cdk1 and Cdk2 that has no amino acid sequence homology to cyclins, are sterile and display meiotic defects virtually identical to those observed in Cdk2 KO mice including non-homologous chromosome pairing, unrepaired double-strand breaks, undetectable sex-body and pachytene arrest. Interestingly, RingoA is required for Cdk2 targeting to telomeres and RingoA KO spermatocytes display severely affected telomere tethering as well as impaired distribution of Sun1, a protein essential for the attachment of telomeres to the nuclear envelope. Our results identify RingoA as an important activator of Cdk2 at meiotic telomeres, and provide genetic evidence for a physiological function of mammalian Cdk2 that is not dependent on cyclins. PMID:27025256

  9. 非小细胞肺癌组织中CDK2及β-catenin的表达%Expression of CDK2 and3-Catenin in Non Small-Cell Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    杨海燕; 李晓虹; 张映红

    2011-01-01

    Objective: To observe the expression of CDK2 and b-catenin in non small-cell lung cancer tissue and to investigate the relationship of CDK2 and b-catenin with metastatic lung cancer. Methods: 48 non small-cell lung cancer patient were divided into metachoresis and non metachoresis groups. Real-time FQ-PCR and western blot were applied respectively to detect the protein and mRNA expression of CDK2 and b-catenin in carcinoma tissue. Results: The protein and mRNA expression of CDK2 and b-catenin was obviously higher in metachoresis group than in non metachoresis group. Conclusion: The expression of CDK2 and b-catenin may be correlated with lung cancer metastasis.%目的:观察非小细胞肺癌组织中CDK2及β-catenin的表达,探讨CDK2及β-catenin与肺癌转移的关系.方法:48例非小细胞肺癌患者分为转移组和未转移组.手术取肺癌组织,分别采用实时荧光定量PCR法和western blot法检测脑组织中CDK2及β-catenin蛋白和mRNA的表达.结果:转移组肺癌组织中CDK2及β-catenin蛋白和mRNA的表达明显高于未转移组(P<0.01).结论:CDK2及β-catenin与肺癌转移有关.

  10. CDK2-AP1通过调控细胞周期抑制乳腺癌生长

    Institute of Scientific and Technical Information of China (English)

    何向明; 黄润; 俞洋; 向华; 杨红健; 宗祥云

    2015-01-01

    目的:探讨CDK2-AP1在乳腺癌的作用及其机制。方法分别在正常乳腺组织及不同分期乳腺癌组织中检测CDK2-AP1的表达情况;进行CDK2-AP1的LOF & GOF细胞功能实验;接种CDK2-AP1干扰或过表达的乳腺癌细胞及对照细胞在裸鼠观察成瘤及相应指标。结果在乳腺癌存在CDK2-AP1表达降低/缺失而CDK2/CyclinD1表达升高的情况,且CDK2-AP1的表达在正常乳腺组织细胞、乳腺导管原位癌、侵袭性乳腺癌、复发转移性乳腺癌渐次降低(P<0.001),与CDK2/CyclinD1相反。体内、外实验均发现抑制CDK2-AP1表达后乳腺癌细胞周期后移、增殖加快;过表达CDK2-AP1的乳腺癌细胞周期阻滞在G0/G1和G2/M期,生长受抑制、裸鼠成瘤速度及大小均受抑制。结论 CDK2-AP1的表达降低以至缺失促进乳腺细胞进入恶性增殖形成肿瘤,缺乏细胞周期负性调控的乳腺癌细胞增殖能力增强。%Objective To observe the role of CDK2-AP1 in breast cancer.Methods Expressions of CDK2-AP1,CDK2 and CyclinD1 were examined in 209 cases of pathological specimens using IHC staining. Lost-of-function and Gain-of-function assays were performed in vivo and in vitro to assess the specific role of CDK2-AP1 in breast cancer. ResultsThe positive ratio of CDK2-AP1 expression was reduced successively in normal breast tissue,DCIS,invasive breast cancer and relapsed breast cancer,suggesting that CDK2-AP1 was correlated closely with the tumor’s genesis and progress and might work as a tumor suppressor. After down-regulating CDK2-AP1 in breast cancer cells,the cell cycle was accelerated and the cell proliferation was promoted. The cell cycle was arrested in G0/G1 phase and G2/M phase after up-regulating CDK2-AP1 in breast cancer cells,resulting in inhibited cell proliferation. The same results were obtained by animal assays.Conclusions CDK2-AP1 affects tumor genesis and tumor growth by cell cycle regulation,which has the potential to be

  11. CDK11p58 represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    International Nuclear Information System (INIS)

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11p58 as a novel protein involved in the regulation of VDR. CDK11p58, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11p58 interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11p58 decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11p58 is involved in the negative regulation of VDR.

  12. CDK11{sup p58} represses vitamin D receptor-mediated transcriptional activation through promoting its ubiquitin-proteasome degradation

    Energy Technology Data Exchange (ETDEWEB)

    Chi, Yayun; Hong, Yi; Zong, Hongliang; Wang, Yanlin; Zou, Weiying; Yang, Junwu; Kong, Xiangfei; Yun, Xiaojing [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China); Gu, Jianxin, E-mail: jxgu@shmu.edu.cn [Gene Research Center, Shanghai Medical College and Institutes of Biomedical, Shanghai 200032 (China)

    2009-08-28

    Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and regulates transcription of target genes. In this study, we identified CDK11{sup p58} as a novel protein involved in the regulation of VDR. CDK11{sup p58}, a member of the large family of p34cdc2-related kinases, is associated with cell cycle progression, tumorigenesis, and apoptotic signaling. Our study demonstrated that CDK11{sup p58} interacted with VDR and repressed VDR-dependent transcriptional activation. Furthermore, overexpression of CDK11{sup p58} decreased the stability of VDR through promoting its ubiquitin-proteasome-mediated degradation. Taken together, these results suggest that CDK11{sup p58} is involved in the negative regulation of VDR.

  13. Expression of cdk4 and p16 in Oral Lichen Planus

    OpenAIRE

    Sinny Goel; Nita Khurana; Akanksha Marvah; Sunita Gupta

    2015-01-01

    ABSTRACT Objectives 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. Material and Methods 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 sq...

  14. The lethal response to Cdk1 inhibition depends on sister chromatid alignment errors generated by KIF4 and isoform 1 of PRC1

    NARCIS (Netherlands)

    E. Voets (Erik); J. Marsman (Judith); J.A.A. Demmers (Jeroen); R.L. Beijersbergen (Roderick); R. Wolthuis (Rob)

    2015-01-01

    textabstractCyclin-dependent kinase 1 (Cdk1) is absolutely essential for cell division. Complete ablation of Cdk1 precludes the entry of G2 phase cells into mitosis, and is early embryonic lethal in mice. Dampening Cdk1 activation, by reducing gene expression or upon treatment with cell-permeable Cd

  15. 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. PMID:27185864

  16. Discovery of [4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-6-methoxyphenyl)methanone (R547), A Potent and Selective Cyclin-Dependent Kinase Inhibitor with Significiant in Vivo Antitumor Activity

    Energy Technology Data Exchange (ETDEWEB)

    Chu,X.; DePinto, W.; Bartkovitz, D.; So, S.; Vu, B.; Packman, K.; Lukacs, C.; Ding, Q.; Jiang, N.; et al.

    2006-01-01

    The cyclin-dependent kinases (CDKs) and their cyclin partners are key regulators of the cell cycle. Since deregulation of CDKs is found with high frequency in many human cancer cells, pharmacological inhibition of CDKs with small molecules has the potential to provide an effective strategy for the treatment of cancer. The 2,4-diamino-5-ketopyrimidines 6 reported here represent a novel class of potent and ATP-competitive inhibitors that selectively target the cyclin-dependent kinase family. This diaminopyrimidine core with a substituted 4-piperidine moiety on the C2-amino position and 2-methoxybenzoyl at the C5 position has been identified as the critical structure responsible for the CDK inhibitory activity. Further optimization has led to a good number of analogues that show potent inhibitory activities against CDK1, CDK2, and CDK4 but are inactive against a large panel of serine/threonine and tyrosine kinases (K{sub i} > 10 {mu}M). As one of these representative analogues, compound 39 (R547) has the best CDK inhibitory activities (K{sub i} = 0.001, 0.003, and 0.001 M for CDK1, CDK2, and CDK4, respectively) and excellent in vitro cellular potency, inhibiting the growth of various human tumor cell lines including an HCT116 cell line (IC{sub 50} = 0.08 {mu}M). An X-ray crystal structure of 39 bound to CDK2 has been determined in this study, revealing a binding mode that is consistent with our SAR. Compound 39 demonstrates significant in vivo efficacy in the HCT116 human colorectal tumor xenograft model in nude mice with up to 95% tumor growth inhibition. On the basis of its superior overall profile, 39 was chosen for further evaluation and has progressed into Phase I clinical trial for the treatment of cancer.

  17. PET32a-CDK2重组质粒的构建与表达%The construction and expression of recombinant plasmid PET32a-CDK2 in E. Coli

    Institute of Scientific and Technical Information of China (English)

    黄宪章; 张战锋; 陈炜烨; 何敏; 庄俊华

    2011-01-01

    Objective To construct recomhinant plasmid PET32a - CDK2 in E. Coli, expressing human CDK2 protein. Methods Total RNA of human white blood cell was extracted for RT - PCR. CDK2 gene fragment was amplified by PCR, recombined into recombinant plasmid, and transformed into E. coli DH5α for cloning. Subsequently, recombinant plasmid was transformed into the competent cells BL21. The CDK2 proteins were induced with isopropy - β -D - thiogalactoside (IPTG) and detected with SDS - PAGE and Western - Blot. Results CDK2 gene recombinant plasmid PET32a was successfully constructed and expressed in E. coli according to DNA sequencing. IPTG - induced prokaryotic protein of 52 KD was detected in Western - blot. Conclusion Recombinant plasmid is constructed successfully.with expression of full - length protein of CDK2 in E. coli.%目的 构建含有细胞周期依赖性激酶2(CDK2)的PET32a-CDK2重组质粒,利用原核表达体系表达CDK2蛋白.方法 从人白细胞中提取总RNA, 采用聚合酶链反应从总RNA中扩增出CDK2基因,并将其插入PET32a质粒,构建重组质粒,化学法转化大肠杆菌DH5α进行克隆.将克隆得到的PET32a-CDK2重组质粒转化入表达菌株BL21,通过异丙基-β-D-硫代半乳糖苷(IPTG)诱导其蛋白表达,SDS-PAGE和Western-Blot鉴定蛋白表达情况.结果 菌落PCR及DNA测序证实CDK2基因已正确克隆到载体中;重组质粒成功转入表达菌株BL21(DE3),SDS-PAGE和Western-Blot结果显示表达菌经IPTG诱导后表达出52 kD左右的蛋白.结论 成功构建重组质粒,并且CDK2全长蛋白在原核表达菌BL21中成功表达.

  18. CCNG2和CDK2在结肠癌中的表达及意义%The Significance and the Expression of CCNG2 and CDK2 in colon cancer

    Institute of Scientific and Technical Information of China (English)

    王赛; 曾亚

    2015-01-01

    ObjectiveTo investigate the roles of Cyclin G2(CCNG2) and cycle protein dependent activating enzyme2(CDK2)in pathogenesis o f colon cancer and their relationships with tumor biological behavior.Methods Immunohis-tochemical methods were adopted to examine expressions of cyclin G2 and CDK2.Results In 89 cases CCNG2 in colonl cancer was significantly lower than that in normal colon tissues, while the expression of CDK2 was just in opposite. The expression of both CCNG2 and CDK2 were significantly related with tumor differention degree, lymph node metastasis and tumor TNM stage. The CCNG2 level was negatively related to the CDK2 level in human colon cancer tissues.Conclusion Abnormal expressions of CCNG2 and CDK2 play important roles in pathogenesis of colon cancer. Inverse correlation between CCNG2 and CDK2 ex-pression in human colon cancer tissues. The deficiency of CCNG2 could promote the invasion and metastasis of colon cancer by disinhibiting the expression of CDK2 protein.%目的:研究CCNG2及CDK2在结肠癌组织中的表达及其与结肠癌生物学行为的关系。方法:应用免疫组化法检测89例结肠癌组织中CCNG2及CDK2的表达。结果:89例结肠癌中,CCNG2蛋白阳性表达31例34.8%(31/89),CDK2蛋白阳性表达55例61.8%(55/89),两者与结肠癌的分化程度、淋巴结转移、临床分期分别呈负相关和正相关。且CCNG2与CDK2蛋白在结肠癌组织中的表达呈负相关性。结论:CCNG2的低表达可能导致CDK2表达的增强促进了结肠癌的发生发展。

  19. INHIBITION OF BREAST CANCER CELL PROLIFERATION AND TUMORIGENECITY BY cdk2 ANTISENSE RNA%cdk2反义RNA对乳腺癌细胞增殖及致瘤性的抑制作用

    Institute of Scientific and Technical Information of China (English)

    桑建利; 边昕; 王永潮

    2001-01-01

    为了研究cdk2对乳腺癌细胞生长及cyclinA, cyclinB1和cdk1(cdc2) mRNA表达水平的影响,利用真核表达载体pXJ41-neo构建了表达cdk2反义RNA的重组载体,并用此载体转染了人乳腺癌细胞系Bcap37,获得了cdk2表达受到抑制的细胞模型Bcap37-CDK2AS,然后将Bcap37-CDK2AS细胞的生长能力及cyclinA, cyclinB1和cdk1 mRNA的水平与转入空载体的对照细胞进行了对比分析.结果显示cdk2表达受到抑制时,细胞生长速率下降,根据测定出的细胞生长曲线,细胞培养至第7天时,细胞生长抑制率为64%.在流式细胞术的分析结果中,G1期细胞占的百分比从39%增加到47%,S期细胞由51%下降到39%.裸鼠接种的实验表明,Bcap37-CDK2AS的致瘤性明显减弱.在对cyclinA, cyclinB1和cdk1 mRNA的分析中发现,Bcap37-CDK2AS中这3种基因的mRNA水平均有不同程度的下降,依据这些结果可以推测,cdk2反义RNA可使乳腺癌细胞生长及致瘤性受到抑制,并且cdk2表达的抑制将影响cyclinA, cyclinB和cdk1的表达水平.

  20. Structural Relaxation of Pd39Ni10Cu30P21 Bulk Metallic Glass Under High Pressure

    Institute of Scientific and Technical Information of China (English)

    WANG Li-Min; ZHAN Zai-Ji; SUN Li-Ling; LI Gong; WANG Wen-Kui

    2001-01-01

    The Pd39Ni10Cu30P21 bulk metallic glass is isothermally relaxed under various pressures. The degree of thestructural relaxation is evaluated in terms of the enthalpy recovery behaviours involved in the irreversible glasstransition processes by using a temperature-modulated differential scanning calorimetry technique. A roughlylinear increase of the recovery enthalpy is observed within the experimental pressure range from 2.67 to 4.45 Gpa,whih reflects the release of the frozen-in enthalpy in the as-quenched glass withincreasing relaxation pressure.The pressure dependence of the timescale of the enthalpy recovery processes is also exhibited.

  1. 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 ostatní: 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

  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. Changes Of P21 And Bcl-2 During Induction Of Bladder Inflammation And/Or Bladder Carcinogenesis

    Directory of Open Access Journals (Sweden)

    A.A.Sayed1; M.A.El-Desoky 2; S.Shaarawy, 3&A.M.Ashmawy4

    2012-10-01

    Full Text Available The effect of E.coli infection in association with dibutylamine and sodium nitrate in induction of bladder inflammation and /or bladder carcinogenesis were investigated in 150 male albino rats, divided into five groups, as follows: The first group(G1 infected by E. coli , The second group (G2, given nitrosamine precursors in the diet , The third group(G3 infected by E. coli and given nitrosamine precursors in the diet ,The fourth group(G4 infected by E. coli , given nitrosamine precursors in the diet and received standard diet containing 1% Curcumin powder mixed in the diet and the fifth group (G5 , served as control group .Results:The worst histopathological changes are in G3 . A highly significant decrease in the mean cell lysate level of p21was found in different studied groups(G1,G2&G3 especially in(G3 where P values in these groups when compared to control group were (P < 0.0001 . In curcumin treated group(G4, there were downregulation in the mean cell lysate level of p21 gene when compared to control group but higher than other studied groups (G1,G2&G3 . A highly significant increase in the mean serum level of Bcl-2 was found in different studied groups(G1,G2&G3 especially in(G3 where P values in these groups (G1,G2,G3 when compared to control group were (P < 0.0001. (G4 showed higher Bcl-2 serum level when compared to control group, this difference was insignificant but this level is lower than other studied groups (G1,G2&G3 .Conclusion:The results in the present study indicate that both P21 and Bcl-2 can be used as biological markers in the diagnosis of bladder cancer . Curcumin have the ability to overcome the decrease in p21WAF1/CIP1 protein and the increase of Bcl-2 protein and reduce the induction of carcinogenic effect .

  4. Foxg1 Haploinsufficiency Reduces the Population of Cortical Intermediate Progenitor Cells: Effect of Increased p21 Expression

    OpenAIRE

    Julie A. Siegenthaler; Tremper-Wells, Barbara A.; Miller, Michael W

    2007-01-01

    Foxg1 is a transcription factor that is critical for forebrain development. Foxg1+/Cre mice were used to test the hypotheses 1) that the subventricular zone (SZ) generates supragranular neurons, 2) that Foxg1-regulated activities define the output from the SZ, and 3) that Foxg1 is involved in the suppression of p21-initiated cell-cycle exit. Foxg1+/Cre mice have thinner neocortices than wild-type controls, specifically in the supragranular layers, as detected by Brn2 immunostaining. Cell prol...

  5. Estrogen-related receptor γ is upregulated in liver cancer and its inhibition suppresses liver cancer cell proliferation via induction of p21 and p27

    Science.gov (United States)

    Kim, Ji-Hyun; Choi, Yeon-Kyung; Byun, Jun-Kyu; Kim, Mi-Kyung; Kang, Yu Na; Kim, Seong Heon; Lee, Sungwoo; Jang, Byoung Kuk; Park, Keun-Gyu

    2016-01-01

    Orphan nuclear receptor estrogen-related receptor γ (ERRγ) regulates cell growth and tumorigenesis in various cancers. However, the clinical relevance of ERRγ to hepatocellular carcinoma (HCC) remains unclear. Here we examined the clinical significance of ERRγ in HCC and its potential as a therapeutic target. ERRγ levels in tissues from completely resected specimens from 190 HCC patients were examined immunohistochemically and their association with clinical stage and pathological grade was analyzed. Small interfering RNA (siRNA)-mediated knockdown of ERRγ (siRNA-ERRγ) or an ERRγ inverse agonist, GSK5182, were also used to examine the effects of ERRγ inhibition on the proliferation and growth of a human hepatoma cell line, PLC/PRF/5. Immunohistochemical analysis revealed that tumor tissues showed higher levels of ERRγ-positivity than adjacent non-tumor lesions. Tumors showing high levels of ERRγ immunoreactivity also had advanced tumor node metastasis (TNM) and Barcelona Clinic Liver Cancer stages and a higher Edmondson–Steiner grade. In addition, high-level expression of ERRγ in tumors of advanced TNM stage correlated with poorer overall survival. Treatment of PLC/PRF/5 cells with siRNA-ERRγ or GSK5182 inhibited proliferation through G1 arrest, increased expression of p21 and p27 and decreased expression of phosphorylated retinoblastoma protein. GSK5182-induced reactive oxygen species also suppressed the proliferation of PLC/PRF/5 cells. The present study showed that ERRγ expression is clinically significant in HCC; therefore, it can be considered a biomarker for HCC diagnosis. Moreover, the results provide a rationale for the use of ERRγ inhibitors such as GSK5182 as potential therapeutic agents. PMID:26940882

  6. Preliminary Analysis of CDK2 Sequence and Its Nuclear Import%CDK2蛋白质分子结构与其入核转运过程关系的初步分析

    Institute of Scientific and Technical Information of China (English)

    刘琦; 罗阳; 姜莉; 周伟强; 满晓辉; 张学

    2004-01-01

    应用重组技术构建野生型及缺失型CDK2基因的真核表达载体,分别使野生型及缺失型CDK2蛋白与增强型绿色荧光蛋白(EnhRnced-green Fluorescent Protein,EGFP)形成融合蛋白.通过脂质体介导的方法将载体转染人宫颈癌细胞系HeLa和中华仓鼠卵巢细胞系CHO,经过细胞周期同步化处理后于荧光显微镜下观察EGFP的亚细胞定位以示踪野生型及缺失型CDK2基因的表达.结果表明,野生型CDK2基因的表达产物定位于细胞核,而两种缺失型CDK2基因分别编码的CDK2蛋白N-端1~201及98~298多肽均主要定位于细胞质.以上结果提示,CDK2蛋白序列中不含有与核定位直接相关的信号,其入核过程可能是由其N-端1~97及202~298多肽范围内的部分氨基酸共同形成高级结构,并依赖此高级结构与其他含有入核信号的蛋白形成复合物,从而被带动进入细胞核的.%We constructed the plasmids encoding enhanced green fluorescent protein (EGFP)-tagged wild type cyclin-de pendent kinase 2 (CDK2) (pEGFP-CDK2) and CDK2 deletion mutants ( pEGFP-CDK2N and pEGFP-CDK2C, lacking the last C-terminal and the first N-terminal 97 amino acids of CDK2 ,respectively) and transfected them into HeLa cell line and CHO cell line. After synchronization,green fluorescent signals were detected mainly in nucleus of the cells transfected with pEGFP-CDK2 and predominantly in cytoplasm of the cells transfected with the two mutant CDK2 constructs. Our results sug gested that there were no nuclear-import signals in CDK2 and that CDK2 nuclear import might be mediated by association with other proteins through the three-dimensional structure formed by amino acids including those from the N- and C-termi nal regions of CDK2.

  7. CDK2蛋白在胆管细胞癌中的表达及临床意义%Expression and Clinical Significance of CDK2 Protein in Cholangiocarcinoma

    Institute of Scientific and Technical Information of China (English)

    胡逸林; 张端莲

    2010-01-01

    目的 探讨细胞周期蛋白依赖性激酶2(cycle protein dependent activating enzyme 2, CDK2)在胆管细胞癌中的表达及临床意义.方法 收集武汉大学人民医院病理科2002/2008年胆管细胞癌存档蜡块40例,其中男性20例,女性20例.另取胆管细胞癌周围正常组织5例作对照.采用免疫组织化学方法检测各组中CDK2蛋白的表达,利用HPIAS-1000图像分析系统测定各组中CDK2蛋白表达的平均光密度和平均阳性面积率.结果 胆管细胞癌中CDK2蛋白呈高表达,对照组中CDK2蛋白呈低表达,两组CDK2表达的平均光密度及阳性面积率有显著性差异(P<0.05).结论 CDK2蛋白在胆管细胞癌的发生和发展过程中起了重要作用.

  8. Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Bin [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Hu, Zhiqiang, E-mail: zhiqhutg@126.com [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Huang, Hui; Zhu, Guangtong; Xiao, Zhiyong [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Wan, Weiqing; Zhang, Peng; Jia, Wang; Zhang, Liwei [Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050 (China)

    2014-11-07

    Highlights: • KDM5B is overexpressed in glioma samples. • KDM5B stimulated proliferation of glioma cells. • Inhibition of p21contributes to KDM5B-induced proliferation. - Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management.

  9. Salt and stress synergize H. pylori-induced gastric lesions, cell proliferation, and p21 expression in Mongolian gerbils.

    Science.gov (United States)

    Gamboa-Dominguez, Armando; Ubbelohde, Tom; Saqui-Salces, Milena; Romano-Mazzoti, Luis; Cervantes, Minerva; Domínguez-Fonseca, Claudia; de la Luz Estreber, Maria; Ruíz-Palacios, Guillermo M

    2007-06-01

    Our aim was to determine if salt and stress enhance Helicobacter pylori (Hp) lesions in Meriones unguiculatus. Two hundred seventy-eight pathogen-free gerbils were allocated to seven groups: Hp-Sydney strain (45), 8% higher-salt diet (38), stress (60% space reduction/water immersion; 36), Hp + salt (33), Hp + stress (34), N-methyl-N-nitro-N-nitrosoguanidine (34), and sham (58). Gerbils were sacrificed at 1 week (67), 12 weeks (73), 52 weeks (65), and 68 weeks (73). Sydney, Padova, and Lauren classifications were blindly used. Proliferation, p53, p21, and apoptosis were assessed. Follicular active gastritis (grade 2/3) was observed in 10% of Hp gerbils, 38% of Hp + salt gerbils, and 29% of Hp + stress gerbils at 52 weeks and 67%, 83%, and 43% at 68 weeks (P < 0.05). Heterotopic proliferative glands were identified in synergy groups from 52 weeks, with increases in their number and size by 68 weeks. Higher proliferative rates were observed in Hp+salt gerbils (P < 0.0001), and p21 overexpression in Hp+salt and Hp+stress gerbils (both P's < 0.0001), by 68 weeks, without p53 increases. We conclude that salt and stress synergize Hp damage and increase pseudo-invasive gland foci. PMID:17404882

  10. Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

    International Nuclear Information System (INIS)

    Highlights: • KDM5B is overexpressed in glioma samples. • KDM5B stimulated proliferation of glioma cells. • Inhibition of p21contributes to KDM5B-induced proliferation. - Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management

  11. Phosphorylation by Cdk1 induces Plk1-mediated vimentin phosphorylation during mitosis

    NARCIS (Netherlands)

    Yamaguchi, Tomoya; Goto, Hidemasa; Yokoyama, Tomoya; Silljé, Herman; Hanisch, Anja; Uldschmid, Andreas; Takai, Yasushi; Oguri, Takashi; Nigg, Erich A; Inagaki, Masaki

    2005-01-01

    Several kinases phosphorylate vimentin, the most common intermediate filament protein, in mitosis. Aurora-B and Rho-kinase regulate vimentin filament separation through the cleavage furrow-specific vimentin phosphorylation. Cdk1 also phosphorylates vimentin from prometaphase to metaphase, but its si

  12. Up-regulation of Tumor Suppressor Gene p21WAF1/CIP1 in Human Cells by Small Double Strand RNA%小分子双链RNA对人类细胞中抑癌基因p21表达的上调作用

    Institute of Scientific and Technical Information of China (English)

    胡嘏; 陈忠; 吴嘉; 张勇; 徐华; 杨为民; 叶章群

    2012-01-01

    Objective To screen more human cell lines susceptible to dsP21 322 mediated tumor suppressor gene p21WAF1/CIP1 activation and investigate whether this process is dependent on p53 expression. Methods A small double strand RNA,dsP21 322,targeting the p21 promoter at position 322 relative to the transcription start site was synthesized. A dsCon trol lacking significant homology to all known human sequences was also synthesized and used as a negative control. Four kinds of human cell lines which expressed different types of p53 protein were chosen,including human osteosarcoma cell line U2 OS (p53 wild type) ,human embryonic kidney cell line 293T(p53 wild type) ,human cervical cancer cell line HeLa(p53 mutant type) and human lung cancer cell line NCI H1299(p53 null). All above cell lines were cultured in vitro and transfected with dsControl or dsP21 322 by Entranster R. RT qPCR and Western blot were applied to detect the expression levels of p21 and p53 mRNA and protein, respectively. Results Seventy two h after transfections, dsP21 322 did not affect the expression of p53 in above four kinds of cell lines,but caused a significant induction in p21 mRNA expression in p53 wild type or p53 mutant cell lines. Compared with dsControl transfections,induction of p21mRNA was 3. 97 ,4. 94 ,and 4. 64 fold in U2 OS,HeLa and 293T cell lines, respectively. Western blot revealed that the elevated levels of p21 protein were strongly correlated to the increase in p21 mRNA expression in these three cell lines. The p21 protein level in dsP21 322 transfections was significantly higher than in ds Control transfections(P<0. 05). However,dsP21 322 was unable to elevate the p21 mRNA and protein levels in p53 null NCI H1299 cell line. Conclusion Activation of p21 expression by dsP21 322 in human cell lines is a pervasive phenomenon. Further more,dsP21 322 failed to elevate the p21 expression in p53 null cell,indicating this process might rely on the expression of p53.%目的 筛选更多的对小分子双链RNA(dsP

  13. Decreased apoptosis in advanced-stage/high-grade hepatocellular carcinoma complicating chronic hepatitis C is mediated through the downregulation of p21 ras

    Institute of Scientific and Technical Information of China (English)

    Nahed Baddour; Ebtehal Farrag; Ahmed Zeid; Essam Bedewy; Yousry Taher

    2013-01-01

    Objective and background:Although p21 ras has been reported to be upregulated in hepatocellular carcinoma complicating chronic hepatitis C type Ⅰ,p21 ras has a different role in advanced stages,as it has been found to be downregulated.The goal of this study was to investigate the status of p21 ras in early-stage/low-grade and late-stage/high-grade hepatocellular carcinoma and its possible link to apoptosis.Material and methods:Thirty-five cases each of chronic HCV hepatitis type 4 (group Ⅰ) and cirrhosis with hepatocellular carcinoma (HCC) complicating chronic HCV hepatitis (groups Ⅱ and Ⅲ) were immunohistochemically evaluated using a p21 ras polyclonal antibody.The apoptotic index was determined in histologic sections using the terminal deoxynucleotidyl transferase-mediated d-UTP biotin nick end labeling (TUNEL) assay.Results:Significant differences (P=0.001) were detected in p21 ras protein expression between the three groups.A near 2-fold increase in p21 ras staining was observed in the cirrhotic cases compared to the hepatitis cases,and p21 ras expression was decreased in the HCC group.p21 ras expression correlated with stage (r=0.64,P=0.001) and grade (r=-0.65,P=0.001) in the HCC group and grade in the HCV group (r=0.44,P=0.008).Both p21 ras expression and TUNEL-LI were significantly lower in large HCCs compared to small HCCs (P=0.01 each).The TUNEL values were negatively correlated with stage in the HCC group (r=-0.85,P=0.001).The TUNEL values were also negatively correlated with grade in both the HCV and HCC groups (r=0.89,P=0.001 and r=-0.53,P=0.001,respectively).The p21 ras scores were significantly correlated with the TUNEL-LI values in the HCC group (r=0.63,P=0.001) and HCV group (r=0.88,P=0.001).Conclusions:p21 ras acts as an initiator in HCC complicating type 4 chronic HCV and is downregulated with HCC progression,which most likely promotes tumor cell survival because it facilitates the downregulation of apoptosis with tumor progression.

  14. Effects on CDK2 Gene Expression in H22 Cells with Ganoderma Appanatum Polysacharides GF%树舌多糖GF对小鼠H22瘤CDK2基因表达的影响

    Institute of Scientific and Technical Information of China (English)

    于英君; 李雪松; 李和伟

    2010-01-01

    目的:探明树舌多糖GF对H22瘤细胞CDK2基因表达的影响.方法:运用Elisa法测定H22瘤细胞中CDK2蛋白的表达量.结果:树舌多糖组中CDK2蛋白的表达量均显著低于荷瘤对照组(P0.05).结论:树舌多糖GF可通过降低CDK2蛋白的表达,抑制H22瘤细胞的增殖.

  15. The antitumor efficacy of a novel adenovirus-mediated anti-p21Ras single chain fragment variable antibody on human cancers in vitro and in vivo.

    Science.gov (United States)

    Yang, Ju-Lun; Pan, Xin-Yan; Zhao, Wen-Xing; Hu, Qi-Chan; Ding, Feng; Feng, Qiang; Li, Gui-Yun; Luo, Ying

    2016-03-01

    Activated ras genes are found in a large number of human tumors, and therefore are one of important targets for cancer therapy. This study investigated the antitumor effects of a novel single chain fragment variable antibody (scFv) against ras protein, p21Ras. The anti-p21Ras scFv gene was constructed by phage display library from hybridoma KGHR1, and then subcloned into replication-defective adenovirus vector to obtain recombinant adenovirus KGHV100. Human tumor cell lines with high expression of p21Ras SW480, MDA-MB‑231, OVCAR-3, BEL-7402, as well as tumor cell line with low expression of p21Ras, SKOV3, were employed to investigate antitumor effects in vitro and in vivo. Fluorescence microscopy demonstrated that KGHV100 was able to express intracellularly anti-p21Ras scFv antibody in cultured tumor cells and in transplantation tumor cells. MTT, Transwell, colony formation, and flow cytometry analysis showed that KGHV100 led to significant growth arrest in tumor cells with high p21Ras expression, and induced G0/G1 cell cycle arrest in the studied tumor cell lines. In vivo, KGHV100 significantly inhibited tumor growth following intratumoral injection, and the survival rates of the mice were higher than the control group. These results indicate that the adenovirus-mediated intracellular expression of the novel anti-p21Ras scFv exerted strong antitumoral effects, and may be a potential method for therapy of cancers with p21Ras overexpression. PMID:26780944

  16. Spontaneous γH2AX Foci in Human Solid Tumor-Derived Cell Lines in Relation to p21WAF1 and WIP1 Expression

    Directory of Open Access Journals (Sweden)

    Razmik Mirzayans

    2015-05-01

    Full Text Available Phosphorylation of H2AX on Ser139 (γH2AX after exposure to ionizing radiation produces nuclear foci that are detectable by immunofluorescence microscopy. These so-called γH2AX foci have been adopted as quantitative markers for DNA double-strand breaks. High numbers of spontaneous γH2AX foci have also been reported for some human solid tumor-derived cell lines, but the molecular mechanism(s for this response remains elusive. Here we show that cancer cells (e.g., HCT116; MCF7 that constitutively express detectable levels of p21WAF1 (p21 exhibit low numbers of γH2AX foci (<3/nucleus, whereas p21 knockout cells (HCT116p21−/− and constitutively low p21-expressing cells (e.g., MDA-MB-231 exhibit high numbers of foci (e.g., >50/nucleus, and that these foci are not associated with apoptosis. The majority (>95% of cells within HCT116p21−/− and MDA-MB-231 cultures contain high levels of phosphorylated p53, which is localized in the nucleus. We further show an inverse relationship between γH2AX foci and nuclear accumulation of WIP1, an oncogenic phosphatase. Our studies suggest that: (i p21 deficiency might provide a selective pressure for the emergence of apoptosis-resistant progeny exhibiting genomic instability, manifested as spontaneous γH2AX foci coupled with phosphorylation and nuclear accumulation of p53; and (ii p21 might contribute to positive regulation of WIP1, resulting in dephosphorylation of γH2AX.

  17. 5-Aza-2'-deoxycytidine Activates the p53/p21waf1/Cip1 Pathway to Inhibit Cell Proliferation

    Institute of Scientific and Technical Information of China (English)

    Wei-GuoZhu; TheresaHileman; YangKe; PeichangWang; ShaoliLu; WenruiDuan; ZunyanDai; TanjunTong; MiguelA.Villalona-Calero; ChristophPlass; GregoryA.Otterson

    2005-01-01

    In addition to its demethylating function, 5-aza-2'-de- oxycytidine (5-aza-CdR) also plays an important role in inducing cell cycle arrest, differentiation, and cell death. However, the mechanism by which 5-aza-CdR in. duces antineoplastic activity is not clear. In this study, we found that 5-aza-CdR at limited concentrations(0.01-5μM) induces inhibition of cell proliferation as well as increased p53/p21waf1/Cip1 expression in A549 cells (wild-type p53) but not in H1299 (p53-null) and H719 cells (p53 mutant). The p53-dependent p21wafa/Cip1 expression induced by 5-aza-CdR was not seen in A549 cells transfected with the wild-type human papilloma virus type-16 E6 gene that induces p53 degradation. Furthermore, deletion analysis and site-directed mutagenesis of the p21 promoter reveals that 5-aza-CdR induces p21wafa/Cip1 expression through two p53 binding sites in the p21 promoter. Finally, 5-aza-CdR-induced p21waf1/cip1 expression was dependent on DNA damage but not on DNA demethylation as demonstrated by comet assay and bisulfite sequencing, respectively. Our data provide useful clues for judging the therapeutic efficacy of 5-aza-CdR in the treatment of human cancer cells.

  18. 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.

  19. Anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in human hepatocellular carcinoma cell lines

    Institute of Scientific and Technical Information of China (English)

    Zhi-ming WU; Gang CHEN; Chun DAI; Ying HUANG; Cui-fang ZHENG; Qiong-zhu DONG; Guan WANG; Xiao-wen LI; Xiao-fei ZHANG; Bin LI

    2011-01-01

    Aim: To investigate the anti-cancer effects of p21WAF1/CIP1 transcriptional activation induced by dsRNAs in hepatocellular carcinoma (HCC) cell lines.Methods: HCC cell lines BEL7402, SMMC-7721, MHCC97L, MHCC97H, and MHCCLM3 were used. HCC ceils were treated with dsP21322 (50 nmol/L), dsControl (50 nmol/L), siP21 (50 nmol/L), or mock transfection. The expression of p21 was detected using quantitative PCR and Western blot. The effects of RNA activation on HCC cells were determined using cell viability assays, apoptosis analyses and clonogenic survival assays. Western blot was also conducted to detect the expression of Bcl-xL, survivin, cleaved caspase-3,cleaved caspase-9 and cleaved PARP.Results: At 72 to 120 h following the transfection, dsP21-322 markedly inhibited the viability of HCC cells and clone formation. At the same times, dsP21-322 caused a significant increase in HCC cell apoptosis, as demonstrated with cytometric analysis. The phenomena were correlated with decreased expression levels of the anti-apoptotic proteins Bcl-xL, surviving, and increased expression of cleaved caspase-3, cleaved caspase-9 and cleaved PARP.Conclusion: RNA-induced activation of p21 gene expression may have significant therapeutic potential for the treatment of hepatocellular carcinoma and other cancers.

  20. Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

    OpenAIRE

    Nabti, Ibtissem; Marangos, Petros; Bormann, Jenny; Kudo, Nobuaki R; Carroll, John

    2014-01-01

    Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates...

  1. Update on Aromatase Inhibitors

    Directory of Open Access Journals (Sweden)

    Seifert-Klauss V

    2015-01-01

    therapies with AI include monotherapy over 5 years (in postmenopausal women, combination with GnRHanalogue (in women who are not postmenopausal and also the switch-concept (2 years of tamoxifene, followed by aromatase inhibitor for 3–5 years or vice versa. High risk situations may warrant „extended use“ with continuation of the therapy after 5 years (up to 10 years. In the metastasized situation, AI are applied in first- as well as in second-line therapy, if there is not a rapid disease progression in vital organs (lung, liver, or as maintenance therapy after chemotherapy. As in the adjuvant setting, in premenopausal women AI must be combined with GnRH analogues. For postmenopausal women with Her2 neu-positive carcinomas, a combination therapy of aromatase inhibitors with trastuzumab or lapatinib has recently been approved. For Her2 neu-negative, hormone receptor positive disease, a combination of exemestane with the m-TOR inhibitor everolimus can be applied after failureof aromatase monotherapy with non-steroidal AI.br Future perspectives:The combination of aromatase inhibitors with the anti-estrogen fulvestrant was not more effective than each substance on its own (SoFEA investigators. Further ongoing trials explore the combination of aromatase inhibitors with neutralising antibodies against IGF-1 or its receptor (e.g. ganitumab, metformin and inhibitors of PI3k and/or Akt. Some of these targeted therapy approaches try to overcome resistance to endocrine therapy, e. g. combinations with mTOR inhibitors are being investigated in clinical trials. Also, the inhibition of PI3k and the new class of CDK4/6 inhibitors represent new promising approaches of combination therapy with aromatase inhibitors.

  2. Study of Expression of CDK2 in Hamster Buckle Pouch Carcinogenesis%CDK2在金黄地鼠颊囊癌变过程中的表达的研究

    Institute of Scientific and Technical Information of China (English)

    孙淑芬; 高文信; 刘岩; 刘敏; 李晓丽; 刘树泰

    2004-01-01

    目的:探讨CDK2在金黄地鼠颊囊黏膜从正常黏膜到单纯增生、异常增生及鳞癌的表达变化.方法:采用DMBA诱导48只金黄地鼠颊囊癌变动物模型,SABC免疫组化法检测CDK2蛋白的表达.结果:CDK2在异常增生上皮及鳞癌的表达与正常和单纯增生组相比明显提高(P<0.05),阳性染色等级随病理等级改变提高(P<0.05).结论:CDK2参与了口腔黏膜癌前病变和鳞癌的发生与发展.

  3. Expression of p21WAF1 is related to acetylation of histone H3 in total chromatin in human colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Ying-Xuan Chen; Jing-Yuan Fang; Rong Lu; De-Kai Qiu

    2007-01-01

    AIM: To explore the relationship between acetylation of histone in total chromatin and p21WAF1 expression regulation in human colorectal carcinoma.METHODS: We analyzed the expression of tumor suppressor gene p21WAF1 mRNA by RT-PCR or realtime PCR in 33 samples of colorectal cancerous tissue,corresponding para-cancerous tissue and normal colorectal mucosa, and also examined the level of acetylated histone H3 in total chromatin using Western blotting.RESULTS: The expression level of p21WAF1 mRNA was significantly lower in colorectal cancerous tissue from 33 patients than in para-cancerous tissue and normal colorectal mucosa (2377.95 ± 865.80 vs 3216.58 ±1149.42 and 3541.61 ± 1433.17 respectively, P <0.01). In addition, when p21WAF1 mRNA expression was undectectable or at very low level (50% less than that in adjacent tissue and normal colorectal mucosa) in all tissues, the level of acetylated histone H3 in colorectal cancerous tissue was significantly lower than that in corresponding para-cancerous tissue and normal colorectal mucosa in five of seven (71.43%) cases. The transcriptional level of p21WAF1 in colorectal carcinoma might not be associated with its biological behaviors.CONCLUSION: The down-regulation of p21WAF1 transcription is involved in the tumorigenesis and development of colorectal carcinoma. The down-expression of p21WAF1 mRNA in colorectal carcinoma might be associated with histone hypoacetylation in chromatin but not with biological behaviors.

  4. EGFR and SYNE2 are associated with p21 expression and SYNE2 variants predict post-operative clinical outcomes in HBV-related hepatocellular carcinoma

    Science.gov (United States)

    Han, Chuangye; Liao, Xiwen; Qin, Wei; Yu, Long; Liu, Xiaoguang; Chen, Gang; Liu, Zhengtao; Lu, Sicong; Chen, Zhiwei; Su, Hao; Zhu, Guangzhi; Lu, Zili; Liu, Zhiming; Qin, Xue; Gui, Ying; Mo, Zengnan; Li, Lequn; Peng, Tao

    2016-01-01

    This study was to explore the association between gene variants and p21 expression and investigate the TP53-independent p21 regulation in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients from Guangxi by genome-wide association study. 426 HBV-related HCC patients were enrolled. Results showed that, after quality control, a total of 21,643 SNPs were identified in 107 p21 positive and 298 p21 negative patients. The variants of epidermal growth factor receptor (EGFR; rs2227983 and rs6950826) and spectrin repeat containing, nuclear envelope 2 (SYNE2; rs8010699, rs4027405 and rs1890908) were associated with p21 expression. Moreover the haplotype block (rs2227983 and rs6950826, r2 = 0.378) in EGFR and the haplotype block in SYNE2 (rs8010699 was in strong LD with rs4027405 and rs1890908 (r2 = 0.91 and 0.70, respectively)) were identified, and the haplotype A-G of EGFR and haplotype G-A-A of SYNE2 were significantly associated with p21 expression (P < 0.01). rs4027405 and rs1890908 were significantly associated with overall survival, and patients with AG/GG genotypes of SYNE2 gene had a worse overall survival (P = 0.001, P = 0.002). Our findings indicate that variants of EGFR and SYNE2 play an important role in p21 regulation and are associated with the clinical outcome of HBV-related HCC in a TP53-indenpdent manner. PMID:27502069

  5. EGFR and SYNE2 are associated with p21 expression and SYNE2 variants predict post-operative clinical outcomes in HBV-related hepatocellular carcinoma.

    Science.gov (United States)

    Han, Chuangye; Liao, Xiwen; Qin, Wei; Yu, Long; Liu, Xiaoguang; Chen, Gang; Liu, Zhengtao; Lu, Sicong; Chen, Zhiwei; Su, Hao; Zhu, Guangzhi; Lu, Zili; Liu, Zhiming; Qin, Xue; Gui, Ying; Mo, Zengnan; Li, Lequn; Peng, Tao

    2016-01-01

    This study was to explore the association between gene variants and p21 expression and investigate the TP53-independent p21 regulation in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) patients from Guangxi by genome-wide association study. 426 HBV-related HCC patients were enrolled. Results showed that, after quality control, a total of 21,643 SNPs were identified in 107 p21 positive and 298 p21 negative patients. The variants of epidermal growth factor receptor (EGFR; rs2227983 and rs6950826) and spectrin repeat containing, nuclear envelope 2 (SYNE2; rs8010699, rs4027405 and rs1890908) were associated with p21 expression. Moreover the haplotype block (rs2227983 and rs6950826, r(2) = 0.378) in EGFR and the haplotype block in SYNE2 (rs8010699 was in strong LD with rs4027405 and rs1890908 (r(2) = 0.91 and 0.70, respectively)) were identified, and the haplotype A-G of EGFR and haplotype G-A-A of SYNE2 were significantly associated with p21 expression (P < 0.01). rs4027405 and rs1890908 were significantly associated with overall survival, and patients with AG/GG genotypes of SYNE2 gene had a worse overall survival (P = 0.001, P = 0.002). Our findings indicate that variants of EGFR and SYNE2 play an important role in p21 regulation and are associated with the clinical outcome of HBV-related HCC in a TP53-indenpdent manner. PMID:27502069

  6. 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

  7. 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...

  8. Effects of CDK2 on cell cycle in laryngeal squamous cell carcinoma and its clinical significance%喉鳞癌中CDK2对细胞周期的影响及其意义

    Institute of Scientific and Technical Information of China (English)

    刘荣; 皇甫辉

    2008-01-01

    目的 探讨喉鳞癌组织CDK2激酶表达与肿瘤细胞增殖之间的关系. 方法 取手术中获得的50例喉鳞癌组织,12例非典型增生组织和30例声带息肉组织,用免疫组化的方法检测CDK2与PCNA的表达;用流式细胞术检测喉鳞癌组织细胞周期比率. 结果 在喉鳞癌组织中CDK2与PCNA的阳性率表达分别为68.0%和86.0%,显著高于声带息肉组织(P<0.05);并且CDK2的表达与临床分期、病理分级、淋巴转移密切相关,与患者的年龄、性别和原发部位无关. 结论 喉鳞癌中CDK2过度表达可能与肿瘤细胞增殖异常密切相关.在诊断和治疗喉鳞癌中,CDK2可能是一个有重要作用的指标.

  9. p21-activated kinase group II small compound inhibitor GNE-2861 perturbs estrogen receptor alpha signaling and restores tamoxifen-sensitivity in breast cancer cells

    OpenAIRE

    Zhuang, Ting; Zhu, Jian; Li, Zhilun; Lorent, Julie; Zhao, Chunyan; Dahlman-Wright, Karin; Strömblad, Staffan

    2015-01-01

    Estrogen receptor alpha (ERα) is highly expressed in most breast cancers. Consequently, ERα modulators, such as tamoxifen, are successful in breast cancer treatment, although tamoxifen resistance is commonly observed. While tamoxifen resistance may be caused by altered ERα signaling, the molecular mechanisms regulating ERα signaling and tamoxifen resistance are not entirely clear. Here, we found that PAK4 expression was consistently correlated to poor patient outcome in endocrine treated and ...

  10. pPICZαA-CDK2重组质粒的构建及表达

    Institute of Scientific and Technical Information of China (English)

    黄宪章; 张战锋; 谢诗园; 李朝霞; 李林; 陈炜烨; 何敏; 庄俊华

    2011-01-01

    目的:构建含有细胞周期依赖性激酶2(CDK2)的胞外分泌型pPICZαA-CDK2重组质粒,利用毕赤酵母表达体系表达CDK2蛋白.方法:从人白细胞中提取总RNA,逆转录后采用聚合酶链反应扩增出CDK2基因,并将其插入pPICZαA质粒,构建重组质粒,化学法转化大肠杆菌JM109进行克隆.重组质粒pPICZαA-CDK2转化毕赤酵母菌株GS115,甲醇诱导酵母细胞进行蛋白表达,SDS-PAGE和Western-Blot鉴定蛋白表达情况及抗原性.结果:PCR电泳及DNA测序证实CDK2基因已正确克隆到表达载体中;重组质粒转入酵母菌GS115,酵母经甲醇诱导表达后经SDS-PAGE检测发现在34 000左右有条带,Western-Blot检测发现有与CDK2单抗结合蛋白.结论:成功构建重组质粒,初步判断CDK2全长蛋白在毕赤酵母中表达成功且抗原性良好.

  11. Cooperative Action of Cdk1/cyclin B and SIRT1 Is Required for Mitotic Repression of rRNA Synthesis

    OpenAIRE

    Voit, Renate; Seiler, Jeanette; Grummt, Ingrid

    2015-01-01

    Author Summary In metazoans, transcription is arrested during mitosis. Previous studies have established that mitotic repression of cellular transcription is mediated by Cdk1/cyclin B-dependent phosphorylation of basal transcription factors that nucleate transcription complex formation. Repression of rDNA transcription at the onset of mitosis is brought about by inactivation of the TBP-containing transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of the TAFI110 subunit, which...

  12. CDK10/cyclin M is a protein kinase that controls ETS2 degradation and is deficient in STAR syndrome.

    Science.gov (United States)

    Guen, Vincent J; Gamble, Carly; Flajolet, Marc; Unger, Sheila; Thollet, Aurélie; Ferandin, Yoan; Superti-Furga, Andrea; Cohen, Pascale A; Meijer, Laurent; Colas, Pierre

    2013-11-26

    Cyclin-dependent kinases (CDKs) regulate a variety of fundamental cellular processes. CDK10 stands out as one of the last orphan CDKs for which no activating cyclin has been identified and no kinase activity revealed. Previous work has shown that CDK10 silencing increases ETS2 (v-ets erythroblastosis virus E26 oncogene homolog 2)-driven activation of the MAPK pathway, which confers tamoxifen resistance to breast cancer cells. The precise mechanisms by which CDK10 modulates ETS2 activity, and more generally the functions of CDK10, remain elusive. Here we demonstrate that CDK10 is a cyclin-dependent kinase by identifying cyclin M as an activating cyclin. Cyclin M, an orphan cyclin, is the product of FAM58A, whose mutations cause STAR syndrome, a human developmental anomaly whose features include toe syndactyly, telecanthus, and anogenital and renal malformations. We show that STAR syndrome-associated cyclin M mutants are unable to interact with CDK10. Cyclin M silencing phenocopies CDK10 silencing in increasing c-Raf and in conferring tamoxifen resistance to breast cancer cells. CDK10/cyclin M phosphorylates ETS2 in vitro, and in cells it positively controls ETS2 degradation by the proteasome. ETS2 protein levels are increased in cells derived from a STAR patient, and this increase is attributable to decreased cyclin M levels. Altogether, our results reveal an additional regulatory mechanism for ETS2, which plays key roles in cancer and development. They also shed light on the molecular mechanisms underlying STAR syndrome. PMID:24218572

  13. 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.; Chytil, A.; Forrester, E.; Nørgaard, Peter Henrik; Teoh, N.; Law, B.

    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...... development of therapeutic approaches to block the stromal desmoplastic reaction that likely plays an important role in the progression of multiple types of human tumors...

  14. In vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by EGFR targeted non-viral vector GE7 system

    Institute of Scientific and Technical Information of China (English)

    陈永新; 许秀兰; 张光霁; 王韦; 金海英; 卢亦成; 朱诚; 顾健人

    2003-01-01

    Objective: To construct the EGFR targeted non-viral vector GE7 system and explore the in vitro effect of p21WAF-1/CIP1 gene on growth of human glioma cells mediated by the GE7 system. Methods: The EGFR targeted non-viral vector GE7 gene delivery system was constructed. The malignant human glioma cell line U251MG was transfected in vitro with β-galactosidase gene(reporter gene) and p21WAF-1/CIP1 gene (therapeutic gene) using the GE7 system. By means of X-gal staining, MTS and FACS, the transfection efficiency of exogenous gene and apoptosis rate of tumor cells were examined. The expression of p21WAF-1/CIP1 gene in transfected U251MG cell was examined by immunohistochemistry staining. Results: The highest transfer rate of exogenous gene was 70%. After transfection with p21WAF-1/CIP1 gene, the expression of WAF-1 increased remarkably and steadily; the growth of U251MG cells were inhibited evidently. FACS examination showed G1 arrest. The average apoptosis rate was 25.2%. Conclusion: GE7 system has the ability to transfer exogenous gene to targeted cells efficiently, and expression of p21WAF-1/CIP1 gene can induce apoptosis of glioma cell and inhibit its growth.

  15. 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.

  16. 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.

  17. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    Science.gov (United States)

    Putz, Eva Maria; Gotthardt, Dagmar; Hoermann, Gregor; Csiszar, Agnes; Wirth, Silvia; Berger, Angelika; Straka, Elisabeth; Rigler, Doris; Wallner, Barbara; Jamieson, Amanda M.; Pickl, Winfried F.; Zebedin-Brandl, Eva Maria; Müller, Mathias; Decker, Thomas; Sexl, Veronika

    2013-01-01

    Summary The transcription factor STAT1 is important in natural killer (NK) cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A) enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8). Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance. PMID:23933255

  18. CDK8-Mediated STAT1-S727 Phosphorylation Restrains NK Cell Cytotoxicity and Tumor Surveillance

    Directory of Open Access Journals (Sweden)

    Eva Maria Putz

    2013-08-01

    Full Text Available The transcription factor STAT1 is important in natural killer (NK cells, which provide immediate defense against tumor and virally infected cells. We show that mutation of a single phosphorylation site (Stat1-S727A enhances NK cell cytotoxicity against a range of tumor cells, accompanied by increased expression of perforin and granzyme B. Stat1-S727A mice display significantly delayed disease onset in NK cell-surveilled tumor models including melanoma, leukemia, and metastasizing breast cancer. Constitutive phosphorylation of S727 depends on cyclin-dependent kinase 8 (CDK8. Inhibition of CDK8-mediated STAT1-S727 phosphorylation may thus represent a therapeutic strategy for stimulating NK cell-mediated tumor surveillance.

  19. CDK-mediated RNF4 phosphorylation regulates homologous recombination in S-phase.

    Science.gov (United States)

    Luo, Kuntian; Deng, Min; Li, Yunhui; Wu, Chenming; Xu, Ziwen; Yuan, Jian; Lou, Zhenkun

    2015-06-23

    There are the two major pathways responsible for the repair of DNA double-strand breaks (DSBs): non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ operates throughout the cell-cycle, while HR is primarily active in the S/G2 phases suggesting that there are cell cycle-specific mechanisms that regulate the balance between NHEJ and HR. Here we reported that CDK2 could phosphorylate RNF4 on T26 and T112 and enhance RNF4 E3 ligase activity, which is important for MDC1 degradation and proper HR repair during S phase. Mutation of the RNF4 phosphorylation sites results in MDC1 stabilization, which in turn compromised HR during S-phase. These results suggest that in addition to drive cell cycle progression, CDK also targets RNF4, which is involved in the regulatory network of DSBs repair. PMID:25948581

  20. CDK5RAP2 Regulates Centriole Engagement and Cohesion in Mice

    OpenAIRE

    Barrera, Jose A.; Kao, Ling-Rong; Robert E Hammer; Seemann, Joachim; Fuchs, Jannon L.; Megraw, Timothy L.

    2010-01-01

    Centriole duplication occurs once per cell cycle, ensuring that each cell contains two centrosomes, each containing a mother-daughter pair of tightly engaged centrioles at mitotic entry. Loss of the tight engagement between mother and daughter centrioles appears to license the next round of centriole duplication. However, the molecular mechanisms regulating this process remain largely unknown. Mutations in CDK5RAP2, which encodes a centrosomal protein, cause autosomal recessive primary microc...

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

    OpenAIRE

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

    2014-01-01

    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, w...

  2. 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

  3. MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

    International Nuclear Information System (INIS)

    Highlights: ► miR-191 expression is upregulated in senescencent human epidermal keratinocytes. ► miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. ► SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. ► 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-β-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′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.

  4. Stereospecific phosphorylation by the central mitotic kinase Cdk1-cyclin B.

    Science.gov (United States)

    Etzkorn, Felicia A; Zhao, Song

    2015-04-17

    The cis vs trans conformation, or shape, of phosphoserine-proline (pSer-Pro), a prevalent motif in cell cycle proteins, may play a significant role in regulating mitosis. We demonstrate that Cdk1-cyclin B, the central mitotic kinase, is specific for the trans conformation, not cis, of synthetic, locked Ser-Pro 11-residue peptide substrates, using LC-MSMS detection and sequencing of phosphorylated products. This substrate stereospecificity may contribute an additional level of mitotic regulation. PMID:25603287

  5. New developments on the cheminformatics open workflow environment CDK-Taverna

    OpenAIRE

    Truszkowski Andreas; Jayaseelan Kalai; Neumann Stefan; Willighagen Egon L; Zielesny Achim; Steinbeck Christoph

    2011-01-01

    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 fre...

  6. 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)

    We previously reported that 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), 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-PGJ2-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-PGJ2-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-PGJ2, but p16 and p27 were not changed, suggesting that the involvement of p21 in inhibition of cell proliferation. Activation of caspase-3 by 15d-PGJ2 was partly, but not completely, blocked by PPARγ antagonist (GW9662) suggesting the 15d-PGJ2 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-PGJ2 in regulation of cell cycle and inhibition of Bcl-xL expression might be beneficial in the development of novel pharmacological agents for chondrosarcoma

  7. SUMOylation of Rb enhances its binding with CDK2 and phosphorylation at early G1 phase.

    Science.gov (United States)

    Meng, Fengxi; Qian, Jiang; Yue, Han; Li, Xiaofeng; Xue, Kang

    2016-07-01

    Retinoblastoma protein (Rb) is a prototypical tumor suppressor that is vital to the negative regulation of the cell cycle and tumor progression. Hypo-phosphorylated Rb is associated with G0/G1 arrest by suppressing E2F transcription factor activity, whereas Rb hyper-phosphorylation allows E2F release and cell cycle progression from G0/G1 to S phase. However, the factors that regulate cyclin-dependent protein kinase (CDK)-dependent hyper-phosphorylation of Rb during the cell cycle remain obscure. In this study, we show that throughout the cell cycle, Rb is specifically small ubiquitin-like modifier (SUMO)ylated at early G1 phase. SUMOylation of Rb stimulates its phosphorylation level by recruiting a SUMO-interaction motif (SIM)-containing kinase CDK2, leading to Rb hyper-phosphorylation and E2F-1 release. In contrast, a SUMO-deficient Rb mutant results in reduced SUMOylation and phosphorylation, weakened CDK2 binding, and attenuated E2F-1 sequestration. Furthermore, we reveal that Rb SUMOylation is required for cell proliferation. Therefore, our study describes a novel mechanism that regulates Rb phosphorylation during cell cycle progression. PMID:27163259

  8. A human ribonuclease induces apoptosis associated with p21WAF1/CIP1 induction and JNK inactivation

    Directory of Open Access Journals (Sweden)

    Vilanova Maria

    2011-01-01

    Full Text Available Abstract Background Ribonucleases are promising agents for use in anticancer therapy. Among the different ribonucleases described to be cytotoxic, a paradigmatic example is onconase which manifests cytotoxic and cytostatic effects, presents synergism with several kinds of anticancer drugs and is currently in phase II/III of its clinical trial as an anticancer drug against different types of cancer. The mechanism of cytotoxicity of PE5, a variant of human pancreatic ribonuclease carrying a nuclear localization signal, has been investigated and compared to that of onconase. Methods Cytotoxicity was measured by the MTT method and by the tripan blue exclusion assay. Apoptosis was assessed by flow cytometry, caspase enzymatic detection and confocal microscopy. Cell cycle phase analysis was performed by flow cytometry. The expression of different proteins was analyzed by western blot. Results We show that the cytotoxicity of PE5 is produced through apoptosis, that it does not require the proapoptotic activity of p53 and is not prevented by the multiple drug resistance phenotype. We also show that PE5 and onconase induce cell death at the same extent although the latter is also able to arrest the cell growth. We have compared the cytotoxic effects of both