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Sample records for cd44 variant isoforms

  1. Serum levels of soluble CD44 variant isoforms are elevated in rheumatoid arthritis.

    Science.gov (United States)

    Kittl, E M; Haberhauer, G; Ruckser, R; Selleny, S; Rech-Weichselbraun, I; Hinterberger, W; Bauer, K

    1997-01-01

    Serum levels of soluble CD44 variant proteins including sequences encoded by exon v5 and exon v6 (sCD44v5, sCD44v6) were determined in patients with inflammatory rheumatic diseases: 56 with rheumatoid arthritis (RA+) and 31 with miscellaneous inflammatory rheumatic diseases (MIRD). There were very significantly higher serum levels of sCD44v5 and sCD44v6 in patients with RA+ than in those with MIRD (RA+ to MIRD: sCD44v5: 81 +/- 54 ng/ml to 33 +/- 13 ng/ml; sCD44v6: 237 +/- 124 ng/ml to 166 +/- 53 ng/ml; both P < 0.001). In RA+ elevated serum levels of sCD44v5 were correlated with the inflammatory activity of disease. In 17 patients with RA+ three or four follow-up measurements of sCD44v5 were performed within 6 months. The development of sCD44v5 serum levels reflected the clinical course of disease in the patients investigated. PMID:9032816

  2. Li-Fraumeni syndrome with simultaneous osteosarcoma and liver cancer: Increased expression of a CD44 variant isoform after chemotherapy

    International Nuclear Information System (INIS)

    Li-Fraumeni syndrome (LFS) is a hereditary cancer predisposition syndrome that is commonly associated with a germline mutation in the tumor suppressor gene p53. Loss of p53 results in increased expression of CD44, a cancer stem cell (CSC) marker, which is involved in the scavenging of reactive oxygen species (ROS). Here, we report a change in the expression of a CD44 variant isoform (CD44v8-10) in an 8-year-old female LFS patient with osteosarcoma and atypical liver cancer after chemotherapy. The patient visited a clinic with a chief complaint of chronic pain in a bruise on her right knee. Magnetic resonance imaging (MRI) raised the possibility of a bone malignancy. Biochemical testing also revealed significantly elevated levels of AFP, which strongly suggested the existence of a primary malignancy in the liver. MRI imaging showed the simultaneous development of osteosarcoma and liver cancer, both of which were confirmed upon biopsy. Combined therapy with surgical resection after chemotherapy was successful in this patient. Regardless of the absence of a familial history of hereditary cancer, a germline mutation in p53 was identified (a missense mutation defined as c.722 C>T, p.Ser241Phe). To better understand the cancer progression and response to treatment, immunohistochemical (IHC) analysis of biopsy specimens obtained before and after chemotherapy was performed using a specific antibody against CD44v8-10. This case demonstrates the ectopic up-regulation of CD44v8-10 in a biopsy sample obtained after cytotoxic chemotherapy, which confers high levels of oxidative stress on cancer cells. Because the alternative splicing of CD44 is tightly regulated epigenetically, it is possible that micro-environmental stress resulting from chemotherapy caused the ectopic induction of CD44v8-10 in vivo

  3. [Serum soluble CD44 isoform variant 5 level in patients with seropositive rheumatoid arthritis treated with cyclosporin A].

    Science.gov (United States)

    Feyertag, J; Haberhauer, G; Skoumal, M; Kittl, E M; Bauer, K; Dunky, A

    2000-01-01

    CD44 is a widely expressed cell surface glycoprotein which is involved in many cell-cell and cell-matrix interactions. Expression of soluble CD44 splice variants is strictly regulated and is linked to a high rate of cell division. Serum levels of soluble CD44 variant 5 (sCD44v5) were determined in 14 patients with erosive RA. Patients were divided into two groups. In group 1 cyclosporin A treatment (CYA) was initiated after the first visit. In group 2 preliminary CYA was continued. Controls were performed after 6 months. We found a significant decrease of swollen joint count (SJC) and sCD44v5 in group 1. No effect of CYA was found on c-reactive protein, erythrocyte sedimentation rate and IgM-rheumatoid factor (IgM-RF). In group 2 a significant decrease of CRP was found. Therefore we conclude that measurement of sCD44v5 might be useful in monitoring RA+ patients with CYA. PMID:11261266

  4. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    International Nuclear Information System (INIS)

    CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding. Finally, I

  5. Conditional expression of CD44 isoforms in lymphoma cells: influence on hyaluronate binding and tumor growth

    Energy Technology Data Exchange (ETDEWEB)

    Fu, J.

    2002-03-01

    CD44 describes a family of surface proteins consisting of many isoforms due to alternative splice of ten 'variant' exons. Members of this family are involved in various processes including hematopoiesis, lymphocyte activation and homing, limb development, wound healing and tumor progression. Clinically, CD44 has been shown to be a prognostic factor for several human cancers. To answer the question which isoform might be relevant for tumor progression and to gain an insight into the mechanism of its function, I established transfectants of the LB lymphoma cell line in which the expression of four CD44 isoforms, namely CD44v3-10, CD44v4-10, CD44v8-10 and CD44s, was controlled by the Tet-off promoter. In the presence of Doxycycline, the expression was repressed. Removal of Doxycycline switched on expression and the maximal CD44 amount was obtained within two days. The transfectants were characterized regarding their ability to bind to the extracellular matrix component hyaluronate (HA). Overexpression of all four CD44 isoforms conferred the ability to bind HA on LB cells. Other glycosaminoglycans (GAGs) were bound in an isotype-specific fashion. CD44v3-10, CD44v4-10 and CD44v8-10 showed high binding affinity to chondroitin A, B and C, and low affinity to heparin, heparan sulfate and keratan sulfate. CD44s could not bind to these GAGs. Among these three variants, the binding ability of CD44v3-10 was the strongest. CD44 clustering seemed to play a crucial role for HA binding. Both CD44s and CD44v8-10 formed reduction-sensitive complexes in LB cells. The complexes are homooligomers or heterooligomers composed of different isoforms. Cys286 in CD44 transmember domain was not responsible for the formation of reduction-sensitive oligomer or for the enhanced HA binding in LB cell line. Using a conditional dimerization system the requirement of CD44 oligomerization for HA binding was directly demonstrated. The induction of oligomerization increased HA binding

  6. CD44 variant expression in cutaneous T-cell lymphoma.

    Science.gov (United States)

    Orteu, C H; Li, W; Allen, M H; Smith, N P; Barker, J N; Whittaker, S J

    1997-07-01

    Expression of the lymphocyte homing receptor CD44 and its splice variants have been linked to tumour dissemination and poor prognosis in non-Hodgkin's lymphoma. Specifically, the in vitro expression of variant exon V6 confers metastatic potential in rat pancreatic carcinoma cell lines. In this study, we investigated the expression of CD44 splice variants in cutaneous T-cell lymphomas, including patients with mycosis fungoides (MF), Sezary syndrome (SS), large-cell anaplastic lymphoma (LCAL) and HTLV1-associated cutaneous lymphoma. In addition, 4 involved lymph nodes from 2 patients with MF and 1 patient with SS were examined. Inflammatory dermatoses, lichen planus and psoriasis, and normal skin were also studied. Immunohistochemistry was performed using a panel of monoclonal antibodies, including those with specificity for CD44H (standard isoform) and variant exons V3, V6 and V8-9. Normal epidermal keratinocytes were consistently CD44H and CD44 V3, V6 and V8-9 positive. In all the different clinicopathological subtypes and stages of cutaneous T-cell lymphomas, including involved lymph nodes, tumour cells consistently expressed CD44H, but were CD44 V3 and V6 negative. CD44 V8-9 was expressed on a majority of tumour cells in 2/5 LCAL and on occasional tumour cells in 2/5 LCAL. Occasional V8-9 positive tumour cells were also identified in 6/13 MF, 1/4 SS and 3/4 HTLV1. In 2/3 lymph node samples from 2 patients with tumour-stage MF, CD44 V8-9 expression was found on a small percentage of atypical mononuclear cells. Scattered V8-9 positive dermal mononuclear cells were present in sections of lichen planus and psoriasis. We have found no evidence to suggest that the metastasis-associated CD44 variant exon (V6) is expressed in cutaneous T-cell lymphoma, or that CD44H expression is associated with an adverse prognostic group. It is not clear whether the strong expression of CD44 V8-9 in 2 patients with CD30 positive LCAL reflects activation status or metastatic potential.

  7. Production and Characterization of a Peptide-based Monoclonal Antibody Against CD44 Variant 6

    OpenAIRE

    ZAREI, Saeed; Bayat, Ali Ahmad; Hadavi, Reza; Mahmoudi, Ahmad R.; Tavangar, Banafsheh; VOJGANI, Yasaman; Jeddi-Tehrani, Mahmood; Amirghofran, Zahra

    2015-01-01

    The gene that codes for the CD44 family members consists of 20 exons, nine of which encode the standard form of the molecule. The other exons can be inserted in various combinations into the membrane proximal region of the extracellular domain of the protein, giving rise to variant isoforms (CD44v). CD44 variants, especially the CD44v6, have been reported to regulate tumor invasion, progression, and metastasis of carcinomas. Producing a high affinity monoclonal antibody against human CD44v6 p...

  8. CD44 isoforms are heterogeneously expressed in breast cancer and correlate with tumor subtypes and cancer stem cell markers

    International Nuclear Information System (INIS)

    The CD44 cell adhesion molecule is aberrantly expressed in many breast tumors and has been implicated in the metastatic process as well as in the putative cancer stem cell (CSC) compartment. We aimed to investigate potential associations between alternatively spliced isoforms of CD44 and CSCs as well as to various breast cancer biomarkers and molecular subtypes. We used q-RT-PCR and exon-exon spanning assays to analyze the expression of four alternatively spliced CD44 isoforms as well as the total expression of CD44 in 187 breast tumors and 13 cell lines. ALDH1 protein expression was determined by IHC on TMA. Breast cancer cell lines showed a heterogeneous expression pattern of the CD44 isoforms, which shifted considerably when cells were grown as mammospheres. Tumors characterized as positive for the CD44+/CD24- phenotype by immunohistochemistry were associated to all isoforms except the CD44 standard (CD44S) isoform, which lacks all variant exons. Conversely, tumors with strong expression of the CSC marker ALDH1 had elevated expression of CD44S. A high expression of the CD44v2-v10 isoform, which retain all variant exons, was correlated to positive steroid receptor status, low proliferation and luminal A subtype. The CD44v3-v10 isoform showed similar correlations, while high expression of CD44v8-v10 was correlated to positive EGFR, negative/low HER2 status and basal-like subtype. High expression of CD44S was associated with strong HER2 staining and also a subgroup of basal-like tumors. Unsupervised hierarchical cluster analysis of CD44 isoform expression data divided tumors into four main clusters, which showed significant correlations to molecular subtypes and differences in 10-year overall survival. We demonstrate that individual CD44 isoforms can be associated to different breast cancer subtypes and clinical markers such as HER2, ER and PgR, which suggests involvement of CD44 splice variants in specific oncogenic signaling pathways. Efforts to link CD44 to CSCs

  9. Significant elevation of tumour-associated isoforms of soluble CD44 in serum of normal individuals caused by cigarette smoking.

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    Kittl, E M; Ruckser, R; Rech-Weichselbraun, I; Hinterberger, W; Bauer, K

    1997-02-01

    While performing a prospective study on sCD44 variant isoforms as tumour markers in certain malignancies, we detected relevant differences in the control group between non-smokers and smokers. For a detailed evaluation of these findings, serum levels of sCD44 variant proteins, including sequences encoded by exon v5 and exon v6, respectively, were adjusted to sex, age and smoking habit. We were able to demonstrate a significant elevation of serum levels of sCD44v5 and sCD44v6 in normal individuals due to cigarette smoking (non-smokers to smokers: sCD44v5: 33 +/- 11 microg/l to 62 +/- 30 microg/l; sCD44v6: 142 +/- 34 microg/l to 232 +/- 86 microg/l). Stepwise multiple linear regression analysis of the concentrations of sCD44v5 and sCD44v6 on the possible influence factors sex, age and smoking habit revealed cigarette smoking as the only factor influencing these isoforms (both p < 0.001). Further investigations have to elucidate a possible clinical importance of these findings in smokers. However, in patients with suspected or proven malignancy the diagnostic specifity of sCD44v5 and sCD44v6 is diminished due to this observation. PMID:9056747

  10. Increased expression of CD44 in hypertrophied ligamentum flavum and relevance of splice variants CD44v5 and CD44v6

    OpenAIRE

    Lakemeier, Stefan; Schmid, Raphael; Foltz, Lisa; Rohlfs, Jochen; Fuchs-Winkelmann, Susanne; Efe, Turgay; Foelsch, Christian; Jürgen R.J. Paletta

    2011-01-01

    Background The most common spinal disorder in the elderly is lumbar spinal stenosis (LSS), which results in part from ligamentum flavum (LF) hypertrophy. Although prior histologic and immunochemical studies have been performed in this area, the pathophysiology of loss of elasticity and hypertrophy is not completely understood. The purpose of this immunohistological study is to elucidate the role of CD44 and its splice variants CD44v5 and CD44v6 in the hypertrophied LF ...

  11. Production and characterization of a peptide-based monoclonal antibody against CD44 variant 6.

    Science.gov (United States)

    Zarei, Saeed; Bayat, Ali Ahmad; Hadavi, Reza; Mahmoudi, Ahmad R; Tavangar, Banafsheh; Vojgani, Yasaman; Jeddi-Tehrani, Mahmood; Amirghofran, Zahra

    2015-02-01

    The gene that codes for the CD44 family members consists of 20 exons, nine of which encode the standard form of the molecule. The other exons can be inserted in various combinations into the membrane proximal region of the extracellular domain of the protein, giving rise to variant isoforms (CD44v). CD44 variants, especially the CD44v6, have been reported to regulate tumor invasion, progression, and metastasis of carcinomas. Producing a high affinity monoclonal antibody against human CD44v6 provides a powerful tool to monitor and trace CD44v6 function in different biological fluids. In this study, a synthetic peptide from CD44v6 was conjugated to keyhole limpet hemocyanin (KLH) and injected into BALB/c mice. Splenocytes from the immunized mice were fused with murine SP2/0 myeloma cells followed by selection of antibody producing hybridoma cells. After screening of hybridoma colonies by ELISA, high affinity antibodies were selected and purified by affinity chromatography. Western blot, immunocytochemistry, and flow cytometry experiments were used to characterize the antibodies. Six stable hybridoma cell lines, designated as 1H1, 1H2, 2A12, 2G11, 3H3, and 3H7, were obtained. Flow cytometry and immunocytochemistry results showed that the new monoclonal antibodies recognized CD44v6 on the cell surface. This novel panel of anti-CD44v6 antibodies has the potential for investigating the role of CD44v6 in cancer pathogenesis. PMID:25723282

  12. A STUDY OF ADHESIVE MOLECULE CD44S CD44V VARIANT EXPRESSION IN GASTRIC CARCINOMA WITH RT-PCR

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Objective:To investigate the correlation between transcript CD44s,CD44v and gastric carcinoma invasion metastasis,and to know what role the CD44 gene's abnormal activity plays in the genesis and development of gastric cancer.Methose RT-PCR was used to detect mRNA of CD44v and CD44s in 10 gastric normal mucosa,8 gastricprecancerous mucosa,10 tissue adjacent to cancer,40 gastric carcinoma(including metastatic focus) with different pathological features,Result:All gastric carcinomas and non-cancerous tissues expressed CD44s,but CD44v expression rate were0%,12.5%,20%and 72.5%.respectively,in normal gastric mucosa,precancerous tissue,tissue adjacent to cancer,carcinoma,Cancer invasion limited to submucosal layer showed lower CD44v expression rate than invasion extended beyond the muscle layer(P0.05),COnclusion:There is a close relation between CD44v and genesis and development of gastric cancer,CD44 gene variant splicing is and early genetic affair in gastric cancer.It Could be a diagnostic tool to screen high0risk people for gastric carcinoma.CD44v can be regard as a parameter in evaluating the potential of cancer cell invasion and metastasis ,as well as prognosis.

  13. TNFalpha and IL-8 regulate the expression and function of CD44 variant proteins in human colon carcinoma cells.

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    Barshishat, Michal; Ariel, Amiram; Cahalon, Liora; Chowers, Yehuda; Lider, Ofer; Schwartz, Betty

    2002-01-01

    The mechanisms underlying the inflammatory and metastatic processes share a number of similar pathways, such as those involving adhesion, migration and extravasation. In this article, the effects of pro-inflammatory cytokines on metastatic-related activities of colon cancer cells were tested. The expression and biological activity of the proteoglycan CD44 in low (LS174T) and high metastatic (HM7) cell lines following exposure to TNFalpha and IL-8 were assessed. Treated cells expressed more CD44 splice variants (CD44v), while CD44 standard protein (CD44s) expression remained unchanged. Treatment with TNFalpha induced IL-8 secretion and IL-8 gene transcription in a time-dependent manner. Both cytokines enhanced the ability of the cells to adhere to the CD44-specific ligand hyaluronic acid, an effect that was specifically blocked by an anti-IL-8 antibody. These results suggest that the effect of TNFalpha on IL-8 is responsible for the regulation of the expression of CD44 isoforms. Additional experiments showed that neither of the cytokines tested regulate the expression of CD44 gene regulation via activation of a well-characterized specific 22-bp epidermal growth factor regulatory element present in the CD44 promoter sequence, suggesting that this is not the mechanism of activation. We conclude that immuno-modulatory mediators can modify the expression of cell-to-cell or cell-to-matrix adhesion proteins, implicated in the determination of phenotypes associated with aggressiveness and metastasis of colon cancer cells. PMID:12090473

  14. Evaluation of soluble CD44 splice variant v5 in the diagnosis and follow-up in breast cancer patients.

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    Kittl, E M; Ruckser, R; Selleny, S; Samek, V; Hofmann, J; Huber, K; Reiner, A; Ogris, E; Hinterberger, W; Bauer, K

    1997-01-01

    Aberrant expression of CD44 splice variants has been detected on a variety of human tumor cells. Overexpression of specific isoforms has been shown to be associated with metastasis and poor prognosis in breast cancer. We evaluated the possible utility of soluble CD44 splice variant v5 (sCD44v5) as a circulating, tumor-associated marker in breast cancer patients. Serum levels of sCD44v5 were determined in 147 healthy volunteers, in 53 patients with nonmalignant breast disease, in 85 patients with breast cancer at presentation, in 13 patients with recurrence and in 73 patients with active metastatic disease. Statistically, the levels at presentation in stages I-IV, in benign disease, and in a female control group were not significantly different. First longitudinal studies over 1-2 years in the follow-up of 28 patients who have remained tumor-free showed considerable between-patient variation while the intrapatient levels remained within relatively narrow limits. In patients with active metastatic disease, elevated levels of sCD44v5 (> 58 ng.ml-1) were detected in 50% of the cases with marked elevation in only 26%. In these cases, sCD44v5 correlated with the extent of metastatic disease and fell during clinical response to cytoreductive therapy. In comparison with CA15-3 in the patients' follow-up serum levels of sCD44v5 proved to be much less sensitive concerning lead time, percentage of raised serum levels at the time of recurrence and in metastatic disease. The value of sCD44v5 determinations in breast cancer patients was further limited by the poor diagnostic specificity of this marker due to elevated levels in smokers and chronic inflammatory disease. PMID:9523162

  15. Lack of CD44 variant 6 expression in rectal cancer invasive front associates with early recurrence

    Institute of Scientific and Technical Information of China (English)

    Suvi Tuulia Avoranta; Eija Annika Korkeila; Kari Juhani Syrj(a)nen; Seppo Olavi Pyrh(o)nen; Jari Toivo Tapio Sundstr(o)m

    2012-01-01

    AIM:To investigate the prognostic value of CD44 variant 6 (CD44v6),a membranous adhesion molecule,in rectal cancer.METHODS:Altogether,210 rectal cancer samples from 214 patients treated with short-course radiotherapy (RT,n =90),long-course (chemo) RT (n =53) or surgery alone (n =71) were studied with immunohistochemistry for CD44v6.The extent and intensity of membranous and cytoplasmic CD44v6 staining,and the intratumoral membranous staining pattern,were analyzed.RESULTS:Membranous CD44v6 expression was seen in 84% and cytoplasmic expression in 81% of the cases.In 59% of the tumors with membranous CD44v6 expression,the staining pattern in the invasive front was determined as "front-positive" and in 41% as "front-negative".The latter pattern was associated with narrower circumferential margin (P =0.01),infiltrative growth pattern (P < 0.001),and shorter disease-free survival in univariate survival analysis (P =0.022) when compared to the "front-positive" tumors.CONCLUSION:The lack of membranous CD44v6 in the rectal cancer invasive front could be used as a method to identify patients at increased risk for recurrent disease.

  16. Potential of sulfasalazine as a therapeutic sensitizer for CD44 splice variant 9-positive urogenital cancer.

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    Takayama, Tatsuya; Kubo, Taro; Morikawa, Ai; Morita, Tatsuo; Nagano, Osamu; Saya, Hideyuki

    2016-05-01

    Cancer stem-like cells (CSCs) with high expression of CD44 splice variant (CD44v) have an enhanced capacity for intracellular reduced glutathione synthesis and defense against reactive oxygen species, resulting in resistance to various therapeutic stresses. Sulfasalazine (SSZ), a drug used in the treatment of rheumatoid arthritis (RA), inhibits glutamate-cystine transport, and suppressed CD44v-dependent tumor growth and increased sensitivity to cytotoxic drugs in an in vivo study. Here, we present two cases of CD44v9-positive urogenital cancer with concomitant treatment with SSZ for RA. Patient 1 was a 62-year-old man who had received SSZ for RA beginning 2 months before the diagnosis of urinary bladder cancer. Although he had multiple metastases to the bladder, abdominal, left cervical and left axillary lymph nodes, and brain, complete response with multidisciplinary therapy was maintained for more than 2 years. Patient 2 was a 74-year-old man with castration-resistant prostate cancer who was diagnosed with RA during chemotherapy and a gradual increase in prostate-specific antigen (PSA) level. When SSZ was added, his PSA value (ng/mL) decreased from 12.93 to 5.58 in only 2 weeks and then quickly rebounded, whereas levels of neuron-specific enolase, a neuroendocrine differentiator and CSC marker, remained almost unchanged. We therefore speculate that SSZ treatment may represent a new adjuvant treatment option for patients with CD44v9-positive urogenital cancer. PMID:27044355

  17. CD44 function as a growth factor co-receptor

    Energy Technology Data Exchange (ETDEWEB)

    Chen, L.

    2003-06-01

    CD44 splice variant proteins containing exon v6 encoded sequence have been implicated in tumour metastasis. The work presented in this thesis shows that CD44 isoforms containing exon v6 encoded sequences act as coreceptor for the c-Met receptor, a tyrosine kinase receptor that is involved in growth control and invasive growth. The c-Met receptor and its ligand hepatocyte growth factor (HGF) have also been implicated in tumour metastasis. My results show the cooperation between CD44, HGF and c-Met. A CD44 isoform containing variant exon v6 encoded sequences is strictly required for c-Met activation by HGF/SF in rat and human carcinoma cells. In a non-metastatic cell line BSp73AS cells which only expressed CD44 standard form, HGF can not activate c-Met. Upon transfection with the CD44 bearing v6 encoded sequences, the cells become HGF inducible. Antibodies against two CD44 exon v6-encoded epitopes inhibit autophosphorylation of c-Met. The CD44 isoform is required for the assembly of signalling complex containing at least HGF, c-Met and CD44 v6 bearing isoform. Furthermore, this growth factor co-receptor function could be a more general mechanism. I have investigated the involvement of CD44 isoforms in the signalling by the EGF receptor family. My results show that HB-EGF, EGF and Amphiregulin activate their receptors in a CD44 dependent manner. CD44 v6 specific antibodies can interfere with HB-EGF, EGF and Amphiregulin signalling both at Erk level and at receptor level. (orig.) [German] In der nicht metastasierenden Zelllinie Bsp73 AS, die ausschliesslich die CD44 Standardform exprimiert, fuehrt HGF nicht zur Aktivierung von c-Met. Durch Transfektion mit unterschiedlichen CD44 v6 enthaltenden Isoformen, werden die Zellen HGF-induzierbar. Antikoerper gegen zwei von CD44 Exon v6 kodierte Epitope verhindern die Autophosphorylierung von c-Met. Die CD44 Isoform wird zur Bildung eines Signalkomplexes benoetigt, der zumindest HGF, c-Met und CD44v6 tragende Isoformen

  18. Endothelial adhesion of synchronized gastric tumor cells changes during cell cycle transit and correlates with the expression level of CD44 splice variants

    Institute of Scientific and Technical Information of China (English)

    Anton Oertl; Jens Castein; Tobias Engl; Wolf-Dietrich Beecken; Dietger Jonas; Richard Melamed; Roman A. Blaheta

    2005-01-01

    AIM: To study adhesion capacity and CD44 expression of human gastric adenocarcinoma MKN45 cells at different stages of a first cell cycle.METHODS: MKN45 cells were synchronized by aphidicolin and assayed for adhesion to an endothelial cell (HUVEC)monolayer. Surface expression of CD44 and CD44 splice variants on MKN45 cells was evaluated by flow cytometry.Functional relevance of CD44 adhesion receptors was investigated by blocking studies using anti CD44 monoclonal antibodies or by hyaluronan digestion.RESULTS: Adhesion of MKN45 to HUVEC was increased during G2/M transit, after which adhesion returned to baseline levels with cell cycle completion. In parallel, CD44splice variants CD44v4, CD44v5, and CD44v7 were all upregulated on MKN45 during cell cycle progression with a maximum effect in G2/M. The function of CD44 surface receptors was assessed with specific receptor blocking monodonal antibodies or removal of hyaluronan by digestion with hyaluronidase. Both strategies inhibited tumor cell adhesion to HUVEC by nearly 50%, which indicates that MKN45-HUVEC-interaction is CD44 dependent.CONCLUSION: CD44 expression level is linked to the cell cycle in gastrointestinal tumor cells, which in turn leads to cell cyde dependent alterations of their adhesion behaviour to endothelium.

  19. Anti-CD44-mediated blockade of leukocyte migration in skin-associated immune diseases.

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    Zöller, Margot; Gupta, Pooja; Marhaba, Rachid; Vitacolonna, Mario; Freyschmidt-Paul, Pia

    2007-07-01

    CD44 plays an important role in leukocyte extravasation, which is fortified in autoimmune diseases and delayed-type hypersensitivity (DTH) reactions. There is additional evidence that distinct CD44 isoforms interfere with the extravasation of selective leukocyte subsets. We wanted to explore this question in alopecia areata (AA), a hair-follicle centric autoimmune disease, and in a chronic eczema. The question became of interest because AA is treated efficiently by topical application of a contact sensitizer, such that a mild DTH reaction is maintained persistently. Aiming to support the therapeutic efficacy of a chronic eczema in AA by anti-CD44 treatment, it became essential to control whether a blockade of migration, preferentially of AA effector cells, could be achieved by CD44 isoform-specific antibodies. Anti-panCD44 and anti-CD44 variant 10 isoform (CD44v10) inhibited in vitro migration of leukocytes from untreated and allergen-treated, control and AA mice. In vivo, both antibodies interfered with T cell and monocyte extravasation into the skin; only anti-panCD44 prevented T cell homing into lymph nodes. Contributing factors are disease-dependent alterations in chemokine/chemokine receptor expression and a blockade of CD44 on endothelial cells and leukocytes. It is important that CD44 can associate with several integrins and ICAM-1. Associations depend on CD44 activation and vary with CD44 isoforms and leukocyte subpopulations. CD44 standard isoform preferentially associates with CD49d in T cells and CD44v10 with CD11b in monocytes. Accordingly, anti-panCD44 and anti-CD49d inhibit T cell, anti-CD11b, and anti-CD44v10 macrophage migration most efficiently. Thus, allergen treatment of AA likely can be supported by targeting AA T cells selectively via a panCD44-CD49d-bispecific antibody. PMID:17442857

  20. Genetic variant in CD44 confer susceptibility to acute skin reaction in breast cancer patients undergoing radiotherapy

    International Nuclear Information System (INIS)

    Heterogeneity in toxicity to normal tissue is observed in 10% of cancer patients after radiotherapy (RT) which limits the therapeutic outcome. Response to RT is manifested from alterations in gene of vivid pathways involving DNA damage-repair, inflammatory cytokine, cell cycle regulation, antioxidant response etc. Therefore, the common sequence variants in these radioresponsive genes may modify the severity of normal tissue toxicity and identification of the same may have clinical relevance as a predictive biomarker. The present study was aimed to evaluate the potential modifying role of genetic variants in NFE2L2, OGG1, NEIL3, RAD17, PTTG1, REV3L, ALAD, CD44, RAD9A, LIG3, SH3GL1, BAXS, XRCC1, MAD2L2 and TGFBR3 on the individual susceptibility to RT induced acute skin reactions. All the 132 breast cancer patients were treated with a total dose of 50 Gy in case of mastectomy and 60 Gy in breast conservation surgery. The severity of skin damage was scored according to the Radiation Therapy Oncology Group (RTOG) criteria and the toxicity scores were dichotomized as non-over-responders (NOR; RTOG<2) and over-responders (NOR;RTOG>2) for analysis. Out of the 132 subjects, 44 were ORs. Among the 20 studied SNPs of indicated genes, the rs8193 (CD44) polymorphism lying in the miRNA binding site was significantly (p<0.05) associated with the RT induced adverse skin reactions. The non-coding CD44 3'-UTR serves as a competitor for miRNA binding and subsequently inactivates miRNA functions, by freeing the target mRNAs from being repressed. Therefore, though the role of CD44 in radiosensitivity is unknown, the change in the miRNA binding to CD44mRNA transcripts may regulate expression of several genes involved in pathophysiology of normal tissue radiosensitivity leading to the observed outcome. (author)

  1. Evaluation of cancer stem cell markers CD133, CD44, CD24: association with AKT isoforms and radiation resistance in colon cancer cells.

    Directory of Open Access Journals (Sweden)

    Sara Häggblad Sahlberg

    Full Text Available The cell surface proteins CD133, CD24 and CD44 are putative markers for cancer stem cell populations in colon cancer, associated with aggressive cancer types and poor prognosis. It is important to understand how these markers may predict treatment outcomes, determined by factors such as radioresistance. The scope of this study was to assess the connection between EGFR, CD133, CD24, and CD44 (including isoforms expression levels and radiation sensitivity, and furthermore analyze the influence of AKT isoforms on the expression patterns of these markers, to better understand the underlying molecular mechanisms in the cell. Three colon cancer cell-lines were used, HT-29, DLD-1, and HCT116, together with DLD-1 isogenic AKT knock-out cell-lines. All three cell-lines (HT-29, HCT116 and DLD-1 expressed varying amounts of CD133, CD24 and CD44 and the top ten percent of CD133 and CD44 expressing cells (CD133high/CD44high were more resistant to gamma radiation than the ten percent with lowest expression (CD133low/CD44low. The AKT expression was lower in the fraction of cells with low CD133/CD44. Depletion of AKT1 or AKT2 using knock out cells showed for the first time that CD133 expression was associated with AKT1 but not AKT2, whereas the CD44 expression was influenced by the presence of either AKT1 or AKT2. There were several genes in the cell adhesion pathway which had significantly higher expression in the AKT2 KO cell-line compared to the AKT1 KO cell-line; however important genes in the epithelial to mesenchymal transition pathway (CDH1, VIM, TWIST1, SNAI1, SNAI2, ZEB1, ZEB2, FN1, FOXC2 and CDH2 did not differ. Our results demonstrate that CD133high/CD44high expressing colon cancer cells are associated with AKT and increased radiation resistance, and that different AKT isoforms have varying effects on the expression of cancer stem cell markers, which is an important consideration when targeting AKT in a clinical setting.

  2. CD44v6 regulates growth of brain tumor stem cells partially through the AKT-mediated pathway.

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    Mayumi Jijiwa

    Full Text Available Identification of stem cell-like brain tumor cells (brain tumor stem-like cells; BTSC has gained substantial attention by scientists and physicians. However, the mechanism of tumor initiation and proliferation is still poorly understood. CD44 is a cell surface protein linked to tumorigenesis in various cancers. In particular, one of its variant isoforms, CD44v6, is associated with several cancer types. To date its expression and function in BTSC is yet to be identified. Here, we demonstrate the presence and function of the variant form 6 of CD44 (CD44v6 in BTSC of a subset of glioblastoma multiforme (GBM. Patients with CD44(high GBM exhibited significantly poorer prognoses. Among various variant forms, CD44v6 was the only isoform that was detected in BTSC and its knockdown inhibited in vitro growth of BTSC from CD44(high GBM but not from CD44(low GBM. In contrast, this siRNA-mediated growth inhibition was not apparent in the matched GBM sample that does not possess stem-like properties. Stimulation with a CD44v6 ligand, osteopontin (OPN, increased expression of phosphorylated AKT in CD44(high GBM, but not in CD44(low GBM. Lastly, in a mouse spontaneous intracranial tumor model, CD44v6 was abundantly expressed by tumor precursors, in contrast to no detectable CD44v6 expression in normal neural precursors. Furthermore, overexpression of mouse CD44v6 or OPN, but not its dominant negative form, resulted in enhanced growth of the mouse tumor stem-like cells in vitro. Collectively, these data indicate that a subset of GBM expresses high CD44 in BTSC, and its growth may depend on CD44v6/AKT pathway.

  3. Increased serum levels of soluble CD44-isoform v5 in rheumatic diseases are restricted to seropositive rheumatoid arthritis.

    Science.gov (United States)

    Haberhauer, G; Kittl, E M; Skoumal, M; Hübl, W; Wagner, E; Bayer, P M; Bauer, K; Dunky, A

    1997-01-01

    Serum levels of sCD44v5 were measured in 134 patients with definite inflammatory rheumatic diseases (IRD) using a sandwich type ELISA. 94 patients suffered from erosive IgM-rheumatoid factor positive rheumatoid arthritis (RA+), 20 with undifferentiated seronegative polyarthritis, 12 with osteoarthropathia psoriatica and psoriasis vulgaris, 3 with systemic lupus erythematosus, 3 with scleroderma and 2 with reactive arthritis. Elevated serum levels (> 58 ng/ml to 221 ng/ml; median: 93 ng/ml) were only detected in 54/94 (57%) patients with RA+, but not in other IRD. They correlated with advanced stages of disease (Steinbrocker stages III + IV; p < 0.05), elevated CRP-levels (p < 0.01) and higher measurements of IgM rheumatoid factor. PMID:9150806

  4. CD44 expression in oro-pharyngeal carcinoma tissues and cell lines.

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    Abirami Rajarajan

    Full Text Available Expression of CD44, a transmembrane hyaluronan-binding glycoprotein, is variably considered to have prognostic significance for different cancers, including oral squamous cell carcinoma. Although unclear at present, tissue-specific expression of particular isoforms of CD44 might underlie the different outcomes in currently available studies. We mined public transcriptomics databases for gene expression data on CD44, and analyzed normal, immortalized and tumour-derived human cell lines for splice variants of CD44 at both the transcript and protein levels. Bioinformatics readouts, from a total of more than 15,000 analyses, implied an increased CD44 expression in head and neck cancer, including increased expression levels relative to many normal and tumor tissue types. Also, meta-analysis of over 260 cell lines and over 4,000 tissue specimens of diverse origins indicated lower CD44 expression levels in cell lines compared to tissue. With minor exceptions, reverse transcribed polymerase chain reaction identified expression of the four main isoforms of CD44 in normal oral keratinocytes, transformed lines termed DT and HaCaT, and a series of paired primary and metastasis-derived cell lines from oral or pharyngeal carcinomas termed HN4/HN12, HN22/HN8 and HN30/HN31. Immunocytochemistry, Western blotting and flow cytometric assessments all confirmed the isoform expression pattern at the protein level. Overall, bioinformatic processing of large numbers of global gene expression analyses demonstrated elevated CD44 expression in head and neck cancer relative to other cancer types, and that the application of standard cell culture protocols might decrease CD44 expression. Additionally, the results show that the many variant CD44 exons are not fundamentally deregulated in a diverse range of cultured normal and transformed keratinocyte lines.

  5. CD44 alternative splicing in gastric cancer cells is regulated by culture dimensionality and matrix stiffness.

    Science.gov (United States)

    Branco da Cunha, Cristiana; Klumpers, Darinka D; Koshy, Sandeep T; Weaver, James C; Chaudhuri, Ovijit; Seruca, Raquel; Carneiro, Fátima; Granja, Pedro L; Mooney, David J

    2016-08-01

    Two-dimensional (2D) cultures often fail to mimic key architectural and physical features of the tumor microenvironment. Advances in biomaterial engineering allow the design of three-dimensional (3D) cultures within hydrogels that mimic important tumor-like features, unraveling cancer cell behaviors that would not have been observed in traditional 2D plastic surfaces. This study determined how 3D cultures impact CD44 alternative splicing in gastric cancer (GC) cells. In 3D cultures, GC cells lost expression of the standard CD44 isoform (CD44s), while gaining CD44 variant 6 (CD44v6) expression. This splicing switch was reversible, accelerated by nutrient shortage and delayed at lower initial cell densities, suggesting an environmental stress-induced response. It was further shown to be dependent on the hydrogel matrix mechanical properties and accompanied by the upregulation of genes involved in epithelial-mesenchymal transition (EMT), metabolism and angiogenesis. The 3D cultures reported here revealed the same CD44 alternative splicing pattern previously observed in human premalignant and malignant gastric lesions. These findings indicate that fundamental features of 3D cultures - such as soluble factors diffusion and mechanical cues - influence CD44 expression in GC cells. Moreover, this study provides a new model system to study CD44 dysfunction, whose role in cancer has been in the spotlight for decades.

  6. CD44 alternative splicing in gastric cancer cells is regulated by culture dimensionality and matrix stiffness.

    Science.gov (United States)

    Branco da Cunha, Cristiana; Klumpers, Darinka D; Koshy, Sandeep T; Weaver, James C; Chaudhuri, Ovijit; Seruca, Raquel; Carneiro, Fátima; Granja, Pedro L; Mooney, David J

    2016-08-01

    Two-dimensional (2D) cultures often fail to mimic key architectural and physical features of the tumor microenvironment. Advances in biomaterial engineering allow the design of three-dimensional (3D) cultures within hydrogels that mimic important tumor-like features, unraveling cancer cell behaviors that would not have been observed in traditional 2D plastic surfaces. This study determined how 3D cultures impact CD44 alternative splicing in gastric cancer (GC) cells. In 3D cultures, GC cells lost expression of the standard CD44 isoform (CD44s), while gaining CD44 variant 6 (CD44v6) expression. This splicing switch was reversible, accelerated by nutrient shortage and delayed at lower initial cell densities, suggesting an environmental stress-induced response. It was further shown to be dependent on the hydrogel matrix mechanical properties and accompanied by the upregulation of genes involved in epithelial-mesenchymal transition (EMT), metabolism and angiogenesis. The 3D cultures reported here revealed the same CD44 alternative splicing pattern previously observed in human premalignant and malignant gastric lesions. These findings indicate that fundamental features of 3D cultures - such as soluble factors diffusion and mechanical cues - influence CD44 expression in GC cells. Moreover, this study provides a new model system to study CD44 dysfunction, whose role in cancer has been in the spotlight for decades. PMID:27187279

  7. Correlation of matrix metalloproteinase-2, -9, tissue inhibitor-1 of matrix metalloproteinase and CD44 variant 6 in head and neck cancer metastasis

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    This study aimed to explore the molecular mechanism in tumor invasion and metastasis. The expression of matrix metalloproteinase-2,-9 (MMP-2, MMP-9), tissue inhibitor-1 of matrix metalloproteinase (TIMP-1), cell adhesion molecule 44 variant 6 (CD44v6), HER2/neu and p53 was investigated in 154 patients with head and neck squamous cell carcinoma (SCC) by ABC and ImmunoMax immunohistochemical method. Their clinical relevance and correlation were analysed. The expression of MMP-2, MMP-9, TIMP-1, CD44v6, HER2/neu and p53 was found in cancer cells in 87.01%, 85.71%, 68.18%, 98.05%, 55.19% and 50.65% cases respectively. Linear regression and correlation analysis revealed that there was close positive relationship (P<0.05) between the expression of MMP-2 and MMP-9, TIMP-1 and CD44v6, HER2/neu and MMP-9, MMP-2 and p53. Up-regulation of MMP-2 was accompanied by advanced T stage(P<0.01). There was also a trend of MMP-2 expression being related with tumor metastasis. Increased expression of HER2/neu was found in patients with tumor recurrence(P<0.05). The expression of TIMP-1 was higher in laryngeal cancer than that in pharyngeal cancer, and higher in keratinizing and non-keratinizing SCC than that in basaloid SCC(P<0.05). These findings suggested that MMP-2 and MMP-9, HER2/neu and MMP-9, MMP-2 and p53 had a coordinate function in aggression of tumor; that MMP-2 had a more important function than MMP-9 in tumor invasion and metastasis; and that HER2/neu might serve as a biomarker for poor prognosis in HNSCC.

  8. Correlation of matrix metalloproteinase-2, -9, tissue inhibitor-1 of matrix metalloproteinase and CD44 variant 6 in head and neck cancer metastasis

    Institute of Scientific and Technical Information of China (English)

    徐娅苹; 赵学群; SOMMER,K.; MOUBAYED,P.

    2003-01-01

    This study aimed to explore the molecular mechanism in tumor invasion and metastasis. The ex-pression of matrix metalloproteinase-2,-9 (MMP-2,MMP-9), tissue inhibitor-1 of matrix metalloprote inase(TIMP-1) , cell adhesion molecule 44 variant 6 (CD44v6) , HER2/neu and p53 was investigated in 154 pa-tients with head and neck squamous cell carcinoma (SCC) by ABC and ImmunoMax immunohistochemical method. Their clinical relevance and correlation were analysed. The expression of MMP-2, MMP-9, TIMP-1,CD44v6, HER2/neu and p53 was found in cancer cells in 87.01%, 85.71%, 68. 18%, 98.05%,55.19% and 50.65% cases respectively. Linear regression and correlation analysis revealed that there wasclose positive relationship ( P < 0.05) between the expression of MMP-2 and MMP-9, TIMP-1 and CD44v6,HER2/neu and MMP-9, MMP-2 and p53. Up-regulation of MMP-2 was accompanied by advanced T stage( P < 0.01 ) . There was also a trend of MMP-2 expression being related with tumor metastasis. Increased ex-pression of HER2/neu was found in patients with tumor recurrence( P < 0.05 ) . The expression of TIMP-1 washigher in laryngeal cancer than that in pharyngeal cancer, and higher in keratinizing and non-keratlnizing SCC than that in basaloid SCC ( P < 0.05 ) . These findings suggested that MMP-2 and MMP-9, HER2/neu andMMP-9, MMP-2 and p53 had a coordinate function in aggression of tumor; that MMP-2 had a more important function than MMP-9 in tumor invasion and metastasis; and that HER2/neu might serve as a biomarker forpoor prognosis in HNSCC.

  9. EXPRESSION AND CLINICAL SIGNIFICANCE OF CD44 IN THE PERIPHERAL BLOOD OF PATIENTS WITH CASTRIC CANCER

    Institute of Scientific and Technical Information of China (English)

    XU; Shen-hua

    2001-01-01

    [1]Li XY, Hu JL. Relationship between the expression of CD44 and tumor of digestive tract & metastasis [J]. Chin J Dig 1999; 19:196.[2]Washington K, Gottfried MR, Telen MJ, et al. Expression of the cell adhesion molecule CD44 in gastric adenocarcinomas [J]. Human Pathol 1994; 25:1043.[3]Tran TA, Kallakury BV, Sheehan CE, et al. Expression of CD44 standard from and variant isoforms in non-small cell lung carcinomas [J]. Human Pathol 1997; 28:809.[4]Wimmel A, Schilli M, Kaiser U, et al. Preferential histiotypic expression of CD44 isoforms in human lung cancer [J]. Lung Cancer 1997; 16:151.[5]Lu GO, Xu SH, Feng JG, et al. Expression and clinical significance of CD44 in peripheral blood in esophageal cancer [J]. Chin J Clin Oncol 1992; 26:500.[6]Xu SH, Feng JG, Li DC, et al. Relationship between CD44 in the peripheral blood of patients with colorectal cancer and clinico-pathological features [J]. Shijie Huaren Xiaohua Zazhi 2000; 8:432.[7]Matsumura Y, Hanbury D, Smith J, et al. Non-invasive detection of malignancy by identification of unusual CD44 gene activity in exfoliated cancer cells [J]. BMJ 1994; 308:619.[8]Reter Herrlich, Margot Zoller, Steven T Pals, et al. CD44 splice variants: metastases meet lymphocytes [J]. Immunology Today 1993; 14:395.[9]Pituch-Noworolska-A, et al. Evaluation of circulating tumor cells expressing CD44 variants in the blood of gastric cancer patients by flow cytometry [J]. Anticancer Res 1998; 18:3747.[10]Guo YJ, Liu GL, Wang XN, et al. Potential use of soluble CD44 in serum as indicator of tumor burden and metastasis in patients with gastric or colon cancer [J]. Cancer Res 1994; 54: 422.[11]Harn HJ, Ho LI, Chang JY, et al. Soluble CD44 isoforms in serum as potential markers of metastatic gastric carcinoma [J]. J Clin Gastroenterol 1996; 22:107.[12]Harn HJ, Ho LI, Chang JY, et al. Differential expression of the human metastasis adhesion molecule CD44 in normal and carcinomatous stomach mucosa

  10. Levels of v5 and v6 CD44 splice variants in serum of patients with colorectal cancer are not correlated with pT stage,histopathological grade of malignancy and clinical features

    Institute of Scientific and Technical Information of China (English)

    Bogdan Zalewski

    2004-01-01

    AIM: This study was designed to compare the levels of v5and v6 splice variants of CD44 evaluated using ELISA test in the serum of patients with colorectal cancer in different stages of progression of the disease estimated in pT stage according to WHO score, histopathological grade of malignancy and some clinicopathological features.METHODS: The serum obtained from 114 persons with colorectal adenocarcinomas was examined using ELISA method. pT stage and grade of malignancy of the tumour were examined in formalin fixed and paraffin embedded materials obtained during operation.RESULTS: Only the level of CD44 v5 in the serum of patients before operation with G2 pT4 tumour was lower than that in other probes and the difference was statistically significant.We did not find any other correlations between the level of v5 and v6 CD44 variants and other evaluated parameters.CONCLUSION: The level of CD44 v5 and v6 estimated by ELISA test in the serum can not be used as a prognostic factor in colorectal cancer.

  11. Expression of proliferating cell nuclear antigen and CD44 variant exon 6 in primary tumors and corresponding lymph node metastases of colorectal carcinoma with Dukes'stage C or D

    Institute of Scientific and Technical Information of China (English)

    Ji-Cheng Zhang; Zuo-Ren Wang; Yan-Juan Cheng; Ding-Zhong Yang; Jing-Sen Shi; Ai-Lin Liang; Ning-Na Liu; Xiao-Min Wang

    2003-01-01

    AIM: To study changes in characteristics of colorectal carcinoma during the metastatic process and to investigate the correlation between cell proliferation activity and metastatic ability of patients with Dukes′ stage C or D.METHODS: Formalin fixed and paraffin embedded materials of primary tumors and corresponding lymph node metastases resected from 56 patients with Dukes′ stage C or D of colorectal carcinoma were stained immunohistochemically with proliferating cell nuclear antigen (PCNA) and CD44variant exon 6 (CD44v6).RESULTS: Thirty-one of 56 patients (55.4 %) expressed PCNA in the primary sites and 36 of 56 patients (64.3 %)expressed PCNA in the metastatic lymph nodes. A significant relation in PCNA expression was observed between the primary site and the metastatic lymph node (0.010<P<0.025).Forty-one of 56 patients (73.2 %) expressed CD44v6 in the primary site and 39 of 56 patients (69.6 %) expressed CD44v6 in the metastatic lymph node. There was also an significant relationship of CD44v6 between the primary site and the metastatic lymph node (0.005<P<0.010). No difference was observed between expression of CD44v6 and PCNA in the primary site (0.250<P<0.500).CONCLUSION: This study partially demonstrates that tumor cells in metastatic lymph node of colorectal carcinoma still possess cell proliferation activity and metastatic ability of tumor cells in primary site. There may be no association between cell proliferation activity and metastatic ability in colorectal carcinoma.

  12. Selective hyaluronan-CD44 signaling promotes miRNA-21 expression and interacts with vitamin D function during cutaneous squamous cell carcinomas progression following UV irradiation

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    Lilly YW Bourguignon

    2015-05-01

    Full Text Available Hyaluronan (HA, the major extracellular matrix component, is often anchored to CD44 isoforms, a family of structurally/functionally important cell surface receptors. Our recent results indicate that UV irradiation (UVR-induced cutaneous squamous cell carcinomas (SCC overexpress a variety of CD44 variant isoforms (CD44v, with different CD44v isoforms appear to confer malignant SCC properties. UVR also stimulates HA degradation in epidermal keratinocytes. Both large HA polymers and their UVR-induced catabolic products (small HA selectively activate CD44 isoform-mediated cellular signaling in normal keratinocytes and SCC cells, with all of the downstream processes being mediated by RhoGTPases (e.g., RhoA and Rac1. Importantly, we found that the hormonally active form of vitamin D (1,25(OH2D3 not only prevents the UVR-induced small HA activation of abnormal keratinocyte behavior and SCC progression, but also enhances large HA stimulation of normal keratinocyte activities and epidermal function(s. Furthermore, we found that HA and its UVR-induced catabolic products (e.g., large and small HA selectively activate CD44-mediated Rac and RhoA signaling. Specifically, large HA-CD44 interaction promotes Rac/PKNγ-dependent normal keratinocyte differentiation, DNA repair and keratinocyte survival. Conversely, small HA-CD44v isoform interaction stimulates RhoA/ROK-dependent NFκB signaling and microRNA-21 (miR-21 production, leading to inflammation, proliferation (following acute UVR response and SCC progression (following chronic UVR exposure. Active vitamin D inhibits small HA-CD44v-mediated RhoA/ROK signaling and SCC progression; and it also enhances large HA-CD44-mediated differentiation, DNA repair and normal epidermal function. Selective applications of large HA and vitamin D will be used to improve the UVR-induced HA (small vs. large HA-CD44 isoform interaction with RhoGTPase signaling and skin inflammation as a potential therapeutic treatment for skin

  13. Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes.

    Science.gov (United States)

    Assayag-Asherie, Nathalie; Sever, Dror; Bogdani, Marika; Johnson, Pamela; Weiss, Talya; Ginzberg, Ariel; Perles, Sharon; Weiss, Lola; Sebban, Lora Eshkar; Turley, Eva A; Okon, Elimelech; Raz, Itamar; Naor, David

    2015-01-01

    CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting β cells renders them susceptible to the autoimmune attack, and is associated with a diminution in β-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on β cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in β cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing β cells is implicated in β-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated. PMID:26624007

  14. Simvastatin inhibits CD44 fragmentation in chondrocytes.

    Science.gov (United States)

    Terabe, Kenya; Takahashi, Nobunori; Takemoto, Toki; Knudson, Warren; Ishiguro, Naoki; Kojima, Toshihisa

    2016-08-15

    In human osteoarthritic chondrocytes, the hyaluronan receptor CD44 undergoes proteolytic cleavage at the cell surface. CD44 cleavage is thought to require transit of CD44 into cholesterol-rich lipid rafts. The purpose of this study was to investigate whether statins exert a protective effect on articular chondrocytes due to diminution of cholesterol. Three model systems of chondrocytes were examined including human HCS-2/8 chondrosarcoma cells, human osteoarthritic chondrocytes and normal bovine articular chondrocytes. Treatment with IL-1β + Oncostatin M resulted in a substantial increase in CD44 fragmentation in each of the three chondrocyte models. Pre-incubation with simvastatin prior to treatment with IL-1β + Oncostatin M decreased the level of CD44 fragmentation, decreased the proportion of CD44 that transits into the lipid raft fractions, decreased ADAM10 activity and diminished the interaction between CD44 and ADAM10. In HCS-2/8 cells and bovine articular chondrocytes, fragmentation of CD44 was blocked by the knockdown of ADAM10. Inhibition of CD44 fragmentation by simvastatin also resulted in improved retention of pericellular matrix. Addition of cholesterol and farnesyl-pyrophosphate reversed the protective effects of simvastatin. Thus, the addition of simvastatin exerts positive effects on chondrocytes including reduced CD44 fragmentation and enhanced the retention of pericellular matrix. PMID:27242325

  15. The Role of CD44 in Disease Pathophysiology And Targeted Treatment

    Directory of Open Access Journals (Sweden)

    Andre R. Jordan

    2015-04-01

    Full Text Available The cell-surface glycoprotein CD44 is involved in a multitude of important physiological functions including cell proliferation, adhesion, migration, hematopoiesis, and lymphocyte activation. The diverse physiological activity of CD44 is manifested in the pathology of a number of diseases including cancer, arthritis, bacterial and viral infections, interstitial lung disease, vascular disease and wound healing. This diversity in biological activity is conferred by both a variety of distinct CD44 isoforms generated through complex alternative splicing, posttranslational modifications (e.g., N- and O-glycosylation, interactions with a number of different ligands and the abundance and spatial distribution of CD44 on the cell surface. The extracellular matrix glycosaminoglycan hyaluronan (HA is the principle ligand of CD44. This review focuses both CD44-hyaluronan dependent and independent CD44 signaling and the role of CD44-HA interaction in various pathophysiologies. The review also discusses recent advances in novel treatment strategies that exploit the CD44-HA interaction either for direct targeting or for drug delivery.

  16. Brain Metastasis is Predetermined in Early-Stages of Cutaneous Melanoma by CD44v6 Expression through Epigenetic Regulation of the Spliceosome

    OpenAIRE

    Marzese, Diego M.; Liu, Michelle; Huynh, Jamie L.; Hirose, Hajime; Donovan, Nicholas C.; Huynh, Kelly T.; Kiyohara, Eiji; Chong, Kelly; Cheng, David; Tanaka, Ryo; Morton, Donald L.; Barkhoudarian, Garni; Daniel F. Kelly; Hoon, Dave S.B.

    2014-01-01

    Melanoma brain metastasis (MBM) is frequent and has a very poor prognosis with no current predictive factors or therapeutic molecular targets. Our study unravels the molecular alterations of cell-surface glycoprotein CD44 variants during melanoma progression to MBM. High expression of CD44 splicing variant 6 (CD44v6) in primary melanoma (PRM) and regional lymph node metastases from AJCC Stage IIIC patients significantly predicts MBM-development. The expression of CD44v6 also enhances the migr...

  17. Downregulation of CD44 reduces doxorubicin resistance of CD44+CD24- breast cancer cells

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    Phuc PV

    2011-06-01

    Full Text Available Pham Van Phuc, Phan Lu Chinh Nhan, Truong Hai Nhung, Nguyen Thanh Tam, Nguyen Minh Hoang, Vuong Gia Tue, Duong Thanh Thuy, Phan Kim NgocLaboratory of Stem Cell Research and Application, University of Science, Vietnam National University, Ho Chi Minh, VietnamBackground: Cells within breast cancer stem cell populations have been confirmed to have a CD44+CD24- phenotype. Strong expression of CD44 plays a critical role in numerous types of human cancers. CD44 is involved in cell differentiation, adhesion, and metastasis of cancer cells.Methods: In this study, we reduced CD44 expression in CD44+CD24- breast cancer stem cells and investigated their sensitivity to an antitumor drug. The CD44+CD24- breast cancer stem cells were isolated from breast tumors; CD44 expression was downregulated with siRNAs followed by treatment with different concentrations of the antitumor drug.Results: The proliferation of CD44 downregulated CD44+CD24- breast cancer stem cells was decreased after drug treatment. We noticed treated cells were more sensitive to doxorubicin, even at low doses, compared with the control groups.Conclusions: It would appear that expression of CD44 is integral among the CD44+CD24- cell population. Reducing the expression level of CD44, combined with doxorubicin treatment, yields promising results for eradicating breast cancer stem cells in vitro. This study opens a new direction in treating breast cancer through gene therapy in conjunction with chemotherapy.Keywords: antitumor drugs, breast cancer stem cells, CD44, CD44+CD24- cells, doxorubicin

  18. CD44: molecular interactions, signalling and functions in the nervous system.

    Directory of Open Access Journals (Sweden)

    Grzegorz Marek Wilczynski

    2015-05-01

    Full Text Available CD44 is the major surface hyaluronan receptor implicated in intercellular and cell-matrix adhesion, cell migration and signalling. It is a transmembrane, highly glycosylated protein with several isoforms resulting from alternative gene splicing. The CD44 molecule consists of several domains serving different functions: the N-terminal extracellular domain, the stem region, the transmembrane domain and the C-terminal tail. In the nervous system, CD44 expression occurs in both glial and neuronal cells. The role of CD44 in the physiology and pathology of the nervous system is not entirely understood, however, there exists evidence suggesting it might be involved in the axon guidance, cytoplasmic Ca2+ clearance, dendritic arborization, synaptic transmission, epileptogenesis, oligodendrocyte and astrocyte differentiation, post-traumatic brain repair and brain tumour development.

  19. Fibronectin Extra Domain A (EDA) Sustains CD133+/CD44+ Subpopulation of Colorectal Cancer Cells

    Science.gov (United States)

    Ou, Juanjuan; Deng, Jia; Wei, Xing; Xie, Ganfeng; Zhou, Rongbin; Yu, Liqing; Liang, Houjie

    2013-01-01

    Fibronectin is a major extracellular matrix glycoprotein with several alternatively spliced variants, including extra domain A (EDA), which was demonstrated to promote tumorigenesis via stimulating angiogenesis and lymphangiogenesis. Given that CD133+/CD44+ cancer cells are critical in tumorigenesis of colorectal cancer (CRC), we hypothesize that fibronectin EDA may promote tumorigenesis by sustaining the properties of CD133+/CD44+ colon cancer cells. We found that tumor tissue and serum EDA levels are substantially higher in advanced versus early stage human CRC. Additionally we showed that tumor tissue EDA levels are positively correlated with differentiation status and chemoresistance, and correlated with a poor prognosis of CRC patients. We also showed that in colon cancer cells SW480, CD133+/CD44+ versus CD133−/CD44− cells express significantly elevated EDA receptor integrin α9β1. Silencing EDA in SW480 cells reduces spheroid formation and cells positive for CD133 or CD44, which is associated with reduced expressions of embryonic stem cell markers and increased expressions of differentiation markers. Blocking integrin α9β1 function strongly reversed the effect of EDA overexpression. We also provided evidence suggesting that EDA sustains Wnt/β-catenin signaling activity via activating integrin/FAK/ERK pathway. In xenograft models, EDA-silenced SW480 cells exhibit reduced tumorigenic and metastatic capacity. In conclusions, EDA is essential for the maintenance of the properties of CD133+/CD44+ colon cancer cells. PMID:23811539

  20. Fibronectin extra domain A (EDA) sustains CD133(+)/CD44(+) subpopulation of colorectal cancer cells.

    Science.gov (United States)

    Ou, Juanjuan; Deng, Jia; Wei, Xing; Xie, Ganfeng; Zhou, Rongbin; Yu, Liqing; Liang, Houjie

    2013-09-01

    Fibronectin is a major extracellular matrix glycoprotein with several alternatively spliced variants, including extra domain A (EDA), which was demonstrated to promote tumorigenesis via stimulating angiogenesis and lymphangiogenesis. Given that CD133(+)/CD44(+) cancer cells are critical in tumorigenesis of colorectal cancer (CRC), we hypothesize that fibronectin EDA may promote tumorigenesis by sustaining the properties of CD133(+)/CD44(+) colon cancer cells. We found that tumor tissue and serum EDA levels are substantially higher in advanced versus early stage human CRC. Additionally we showed that tumor tissue EDA levels are positively correlated with differentiation status and chemoresistance, and correlated with a poor prognosis of CRC patients. We also showed that in colon cancer cells SW480, CD133(+)/CD44(+) versus CD133(-)/CD44(-) cells express significantly elevated EDA receptor integrin α9β1. Silencing EDA in SW480 cells reduces spheroid formation and cells positive for CD133 or CD44, which is associated with reduced expressions of embryonic stem cell markers and increased expressions of differentiation markers. Blocking integrin α9β1 function strongly reversed the effect of EDA overexpression. We also provided evidence suggesting that EDA sustains Wnt/β-catenin signaling activity via activating integrin/FAK/ERK pathway. In xenograft models, EDA-silenced SW480 cells exhibit reduced tumorigenic and metastatic capacity. In conclusion, EDA is essential for the maintenance of the properties of CD133(+)/CD44(+) colon cancer cells. PMID:23811539

  1. Expression and Function of CD44 in Epithelial Ovarian Carcinoma

    Directory of Open Access Journals (Sweden)

    Joelle D. Sacks

    2015-11-01

    Full Text Available CD44, a cell surface glycoprotein, has been increasingly implicated in the pathogenesis and progression of epithelial ovarian cancer, the deadliest gynecologic malignancy in women. Here, we review recent reports on the expression and function of CD44 in epithelial ovarian carcinoma. Further functional data for CD44 in peritoneal adhesion and metastatic progression and its association with stem cells is highlighted. Recent studies utilizing CD44 for therapeutic targeting are also discussed.

  2. Effect of CD44 gene polymorphisms on risk of transitional cell carcinoma of the urinary bladder in Taiwan.

    Science.gov (United States)

    Weng, Wei-Chun; Huang, Yu-Hui; Yang, Shun-Fa; Wang, Shian-Shiang; Kuo, Wu-Hsien; Hsueh, Chao-Wen; Huang, Ching-Hsuan; Chou, Ying-Erh

    2016-05-01

    The carcinogenesis of transitional cell carcinoma (TCC) of the urinary bladder involves etiological factors, such as ethnicity, the environment, genetics, and diet. Cluster of differentiation (CD44), a well-known tumor marker, plays a crucial role in regulating tumor cell differentiation and metastasis. This study investigated the effect of CD44 single nucleotide polymorphisms (SNPs) on TCC risk and clinicopathological characteristics. Five SNPs of CD44 were analyzed through real-time polymerase chain reaction in 275 patients with TCC and 275 participants without cancer. In this study, we observed that CD44 rs187115 polymorphism carriers with the genotype of at least one G were associated with TCC risk. Furthermore, TCC patients who carried at least one G allele at CD44 rs187115 had a higher stage risk than did patients carrying the wild-type allele (p TCC. In conclusion, our results suggest that CD44 SNPs influence the risk of TCC. Patients with CD44 rs187115 variant genotypes (AG + GG) exhibited a higher risk of TCC; these patients may possess chemoresistance to developing late-stage TCC compared with those with the wild-type genotype. The CD44 rs187115 SNP may predict poor prognosis in patients with TCC.

  3. Fibroblast invasive migration into fibronectin/fibrin gels requires a previously uncharacterized dermatan sulfate-CD44 proteoglycan

    DEFF Research Database (Denmark)

    Clark, Richard A F; Lin, Fubao; Greiling, Doris;

    2004-01-01

    After tissue injury, fibroblast migration from the peri-wound collagenous stroma into the fibrin-laden wound is critical for granulation tissue formation and subsequent healing. Recently we found that fibroblast transmigration from a collagen matrix into a fibrin matrix required the presence of...... migration into a fibronectin/fibrin gel. This conclusion was based on beta-xyloside inhibition of glycanation and specific glycosaminoglycan degradation. CD44, a cell surface receptor known to bind hyaluronan, not infrequently exists as a proteoglycan, decorated with various glycosaminoglycan chains...... including heparan sulfate and chondroitin sulfate, and as such can bind fibronectin. We found that CD44H, the non-spliced isoform of CD44, was necessary for fibroblast invasion into fibronectin/fibrin gels. Resting fibroblasts expressed mostly nonglycanated CD44H core protein, which became glycanated with...

  4. miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Henry, Jon C. [Department of Surgery, Ohio State University Medical Center, Columbus, OH (United States); Park, Jong-Kook; Jiang, Jinmai; Kim, Ji Hye [College of Pharmacy, Ohio State University, Columbus, OH (United States); Nagorney, David M.; Roberts, Lewis R. [Divisions of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, MN (United States); Banerjee, Soma [Center for Liver Research, School of Digestive and Liver Diseases, Institute of Post Graduate Medical Education and Research, Kolkata (India); Schmittgen, Thomas D., E-mail: Schmittgen.2@osu.edu [College of Pharmacy, Ohio State University, Columbus, OH (United States)

    2010-12-03

    Research highlights: {yields} miR-199a-3p targets CD44 in HCC. {yields} Proliferation and invasion are reduced by miR-199a-3p in CD44+ HCC. {yields} miR-199a-3p is reduced and CD44 protein is increased in HCC tissues. {yields} The duplex form of miR-199a-3p mimetic is required for activity. -- Abstract: Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced

  5. Research of CD44 and Breast Cancer%CD44与乳腺癌的研究进展

    Institute of Scientific and Technical Information of China (English)

    赵书

    2011-01-01

    CD44基因蛋白是一种细胞表面跨膜糖蛋白,属于黏附分子家族.CD44基因外显子根据表达方式可分为标准型CD44(CD44s)和变异型CD44(CD44v)两种类型.大量研究发现,CD44蛋白参与细胞-细胞、细胞-胞外基质之间的特异性粘附,CD44及其亚型在乳腺癌的异常表达可能与癌症的发生、发展和转移密切相关.此外,乳腺癌干细胞表面高度表达CD44分子,针对CD44分子与乳腺癌干细胞关系的研究,将会为临床上对乳腺癌的诊断、治疗选择以及预后预测提供更充分的理论依据.

  6. Expression of CD44 in pancreatic cancer and its significance

    OpenAIRE

    Li, Xiao-Ping; Zhang, Xiao-Wei; Zheng, Lei-Zhen; Guo, Wei-Jian

    2015-01-01

    Background: CD44 is a potentially interesting prognostic marker and therapeutic target in pancreatic cancer. The expression of CD44 has been reported to correlate with poor prognosis of pancreatic cancer in most literatures. The purpose of this study is to investigate the roles of CD44 in pancreatic caner, and their correlation with the prognosis of pancreatic cancer patients. Methods: 67 pancreatic cancer samples were collected in Xinhua hospital affiliated to Shanghai Jiaotong University da...

  7. Osteopontin increases hepatocellular carcinoma cell growth in a CD44 dependant manner

    Institute of Scientific and Technical Information of China (English)

    Renee J Phillips; Karla J Helbig; Kylie H Van der Hoek; Devanshi Seth; Michael R Beard

    2012-01-01

    AIM:To investigate the role of osteopontin (OPN) and its splice variants in the proliferation of hepatocellular carcinoma (HCC).METHODS:The expression of OPN variants in HCC cell lines as well as HCC tissue samples and nontumour tissue was studied using polymerase chain reaction.OPN variant cDNAs were cloned into a mammalian expression vector allowing both transient expression and the production of stable OPN expressing cell lines.OPN expression was studied in these cells using Western blotting,immunofluoresnce and enzyme linked immunosorbent assay.A CD44 blocking antibody and siRNA targeting of CD44 were used to examine the role of this receptor in the OPN stimulated cell growth observed in culture.Huh-7 cells stably expressing either OPN-A,-B or-C were injected subcutaneously into the flanks of nude mice to observe in vivo tumour growth.Expression of OPN mRNA and protein in these tumours was examined using reverse transcriptionpolymerase chain reaction and immunohistochemistry.RESULTS:OPN is expressed in HCC in 3 forms,the full length OPN-A and 2 splice variants OPN-B and-C.OPN variant expression was noted in HCC tissue as well as cognate surrounding cirrhotic liver tissue.Expression of these OPN variants in the HCC derived cell line Huh-7resulted in secretion of OPN into the culture medium.Transfer of OPN conditioned media to naive Huh-7 and HepG2 cells resulted in significant cell growth suggesting that all OPN variants can modulate cell proliferation in a paracrine manner.Furthermore the OPN mediated increase in cellular proliferation was dependent on CD44 as only CD44 positive cell lines responded to OPN conditioned media while siRNA knockdown of CD44 blocked the proliferative effect.OPN expression also increased the proliferation of Huh-7 cells in a subcutaneous nude mouse tumour model,with Huh-7 cells expressing OPN-A showing the greatest proliferative effect.CONCLUSION:This study demonstrates that OPN plays a significant role in the proliferation of HCC

  8. Expression of CD44 in Cultured Human Trabecular Meshwork Cells

    Institute of Scientific and Technical Information of China (English)

    Zhongguo Li; Hong Zhang

    2004-01-01

    Purpose:To determine whether cultured human trabecular meshwork cells express CD44 and to discuss their possible relationship with primary open angle glaucoma.Methods:Human trabecular meshwork cells were cultured in DMEM/F12 media. Total RNAs from the cells were extracted with Trizol reagent. Messenger RNA expression of CD44 in human trabecular meshwork cells was examined by using reverse transcriptasepolymerase chain reaction ( RT-PCR ) analysis. Expression of CD44 was confirmed by Western-blotting and immunofiuorescent microscopy. Effect of CD44-specific antisense oligonucleotide on adhesion of trabecular meshwork cells to hyaluronate was determined by MTT assay.Results:A single RT-PCR product whose size was 471bp was obtained.A band about 80kD was stained by Western-blot. Immunofiuorescent examination of expression of CD44 on the cell surface was positive and reactions were mainly localized in cell membranes.Adhesion of trabecular meshwork cells to hyaluronate was inhibited by CD44-specific antisense oligonucleotide.Conclusions: Cultured human trabecular meshwork cells express CD44. CD44 may play a role in pathogenesis of primary open angle glaucoma. Eye Science 2004;20:52-56.

  9. Preparation and bioevaluation of {sup 177}Lu-labelled anti-CD44 for radioimmunotherapy of colon cancer

    Energy Technology Data Exchange (ETDEWEB)

    Lee, So Young; Hong, Young Don; Jung, Sung Hee; Choi, Sun Ju [Radioisotope Research Division, Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2015-12-15

    CD44 is a particular adhesion molecule and facilitates both cell-cell and cell-matrix interactions. In particular, splice variants of CD44 are particularly overexpressed in a large number of malignancies and carcinomas. In this study, the {sup 177}Lu-labelled CD44 targeting antibody was prepared and bioevaluated in vitro and in vivo. Anti-CD44 was immunoconjugated with the equivalent molar ratio of cysteine-based dtPA-ncS and radioimmunoconjugated with {sup 177}Lu at room temperature within 15 minutes. the stability was tested in human serum. An in vitro study was carried out in Ht-29 human colon cancer cell lines. For the biodistribution study {sup 177}Lu-labelled anti-CD44 was injected in xenograft mice. Anti-CD44 was immunoconjugated with cysteinebased dtPA-ncS and purified by a centricon filter system having a molecular cut-off of 50 kda. radioimmunoconjugation with {sup 177}Lu was reacted for 15 min at room temperature. the radiolabeling yield was >99%, and it was stable in human serum without any fragmentation or degradation. The radioimmunoconjugate showed a high binding affinity on HT-29 colon cancer cell surfaces. In a biodistribution study, the tumor-to-blood ratio of the radioimmunoconjugate was 43 : 1 at 1 day post injection (p.i) in human colon cancer bearing mice. the anti-CD44 monoclonal antibody for the targeting of colon cancer was effectively radioimmunoconjugated with {sup 177}Lu. the in vitro high immunoactivity of this radioimmunoconjugate was determined by a cell binding assay. In addition, the antibody's tumor targeting ability was demonstrated with very high uptake in tumors. this radioimmunoconjugate is applicable to therapy in human colon cancer with highly expressed CD44.

  10. The hyaluronan receptors CD44 and RHAMM (CD168) form complexeswith ERK1,2, which sustain high basal motility in breast cancercells

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, Sara R.; Fard, Shireen F.; Paiwand, Frouz F.; Tolg,Cornelia; Veiseh, Mandana; Wang, Chao; McCarthy, James B.; Bissell, MinaJ.; Koropatnick, James; Turley, Eva A.

    2007-03-28

    CD44 is an integral hyaluronan receptor that can promote or inhibit motogenic signaling in tumor cells. Rhamm is a non-integral cell surface hyaluronan receptor (CD168) and intracellular protein that promotes cell motility in culture and its expression is strongly upregulated in diseases like arthritis and aggressive cancers. Here we describe an autocrine mechanism utilizing cell surface Rhamm/CD44 interactions to sustain rapid basal motility in invasive breast cancer cell lines. This mechanism requires endogenous hyaluronan synthesis and the formation of Rhamm/CD44/ERK1, 2 complexes. Motile/ invasive MDA-MB-231 and Ras-MCF10A cells produce more endogenous hyaluronan, cell surface CD44 and Rhamm, an oncogenic Rhamm isoform, and exhibit elevated basal activation of ERK1, 2 than less invasive MCF7 and MCF10A breast cancer cells. Furthermore, CD44, Rhamm and ERK1, 2 uniquely co-immunoprecipitate and co-localize in MDA-MB-231 and Ras-MCF10A cells. Rapid motility of the invasive cell lines requires interaction of hyaluronan with cells, activation of ERK1, 2 and the participation of both cell surface CD44 and Rhamm. Combinations of anti-CD44, anti-Rhamm antibodies and a MEK1 inhibitor (PD098059) have less-than-additive blocking effects, suggesting action of all three proteins on a common motogenic signaling pathway. Collectively, these results show that cell surface Rhamm and CD44 act together in a hyaluronan-dependent, autocrine mechanism to coordinate sustained signaling through ERK1, 2 leading to high basal motility of invasive breast cancer cells. Since CD44/Rhamm complexes are not evident in less motile cells, an effect of CD44 on tumor cell motility may depend in part on its ability to partner with additional proteins, in this case cell surface Rhamm.

  11. Expression and significance of CD44s, CD44v6, and nm23 mRNA in human cancer

    Institute of Scientific and Technical Information of China (English)

    Yong-Jun Liu; Pei-Song Yan; Jun Li; Jing-Fen Jia

    2005-01-01

    AIM: To investigate the relationship between the expression levels of nm23 mRNA, CD44s, and CD44v6,and oncogenesis, development and metastasis of human gastric adenocarcinoma, colorectal adenocarcinoma,intraductal carcinoma of breast, and lung cancer.METHODS: Using tissue microarray by immuhistochemical (IHC) staining and in situ hybri-dization (ISH), we examined the expression levels of nm23mRNA, CD44s, and CD44v6 in 62 specimens of human gastric adenocarcinoma and 62 specimens of colorectal adenocarcinoma; the expression of CD44s and CD44v6in 120 specimens of intraductal carcinoma of breast and 20 specimens of normal breast tissue; the expression of nm23 mRNA in 72 specimens of human lung cancer and 23 specimens of normal tissue adjacent to cancer.RESULTS: The expression of nm23 mRNA in the tissues of gastric and colorectal adenocarcinoma was not significantly different from that in the normal tissues adjacent to cancer (P>0.05), and was not associated with the invasion of tumor and the pathology grade of adenocarcinoma (P>0.05). However, the expression of nm23 mRNA was correlated negatively to the lymph node metastasis of gastric and colorectal adenocarcinoma (r = -0.49, P<0.01; r = -4.93, P<0.01). The expression of CD44s in the tissues of gastric and colorectal adenocarcinoma was significantly different from that in the normal tissues adjacent to cancer (P<0.05;P<0.01). CD44v6 was expressed in the tissues of gastric and colorectal adenocarcinoma only, the expression of CD44v6 was significantly associated with the lymph node metastasis, invasion and pathological grade of the tumor (r = 0.47, P<0.01; r = 5.04, P<0.01). CD44sand CD44v6 were expressed in intraductal carcinoma of breast, the expression of CD44s and CD44v6 was significantly associated with lymph node metastases and invasion (P<0.01). However, neither of them was expressed in the normal breast tissue. In addition, the expression of CD44v6 was closely related to the degree of cell

  12. Modulation of CD44 Activity by A6-Peptide

    Directory of Open Access Journals (Sweden)

    Malcolm eFinlayson

    2015-03-01

    Full Text Available AbstractHyaluronan (HA is a nonsulfated glycosaminoglycan distributed throughout the extracellular matrix that plays a major role in cell adhesion, migration, and proliferation. CD44, a multifunctional cell surface glycoprotein, is a receptor for HA. In addition, CD44 is known to interact with other receptors and ligands, and to mediate a number of cellular functions as well as disease progression. Studies have shown that binding of HA to CD44 in cancer cells activates survival pathways resulting in cancer cell survival. This effect can be blocked by anti-CD44 monoclonal antibodies. A6 is a capped, 8 L-amino acid peptide (Ac-KPSSPPEE-NH2 derived from the biologically active connecting peptide domain of the serine protease, human urokinase plasminogen activator (uPA. A6 does not bind to the uPA receptor (uPAR nor interfere with uPA/uPAR binding. A6 binds to CD44 resulting in the inhibition of migration, invasion, and metastasis of tumor cells, and the modulation of CD44-mediated cell signaling. A6 has been shown to have no dose-limiting toxicity in animal studies. A6 has demonstrated efficacy and an excellent safety profile in Phase 1a, 1b, and 2 clinical trials. In animal models, A6 has also exhibited promising results for the treatment of diabetic retinopathy and wet age-related macular degeneration through the reduction of retinal vascular permeability and inhibition of choroidal neovascularization, respectively. Recently, A6 has been shown to be directly cytotoxic for B-lymphocytes obtained from patients with chronic lymphocytic leukemia (CLL expressing the kinase, ZAP-70. This review will discuss the activity of A6, A6 modulation of HA and CD44, and a novel strategy for therapeutic intervention in disease.

  13. Induction of interactions between CD44 and hyaluronic acid by a short exposure of human T cells to diverse pro-inflammatory mediators.

    Science.gov (United States)

    Ariel, A; Lider, O; Brill, A; Cahalon, L; Savion, N; Varon, D; Hershkoviz, R

    2000-07-01

    Migration of T cells into extravascular sites of inflammation is mediated by cell-cell and cell-matrix adhesion receptors, including the hyaluronan-binding glycoprotein, CD44. The biochemical nature of CD44 variants and the ligand specificity, function and the regulation of activation of CD44 expressed on various cell types have been extensively studied. However, little is still known about the short-term influence of cytokines and chemokines on the activation of CD44 on human T cells. Therefore, we studied the role of inflammatory mediators in regulating the adhesion of T cells from human peripheral blood to immobilized hyaluronan under static or shear stress conditions. We found that the CD44-dependent adhesion, under static and shear stress (i.e. relative gradual resistance to flow of 150 and 1500 s-1) conditions, of T cells to hyaluronan requires a T-cell activation of 2-3 hr and is regulated by the cross-linking of CD3, cytokines (e.g. interleukin-2 and tumour necrosis factor-alpha), and chemokines (e.g. MIP-1beta, interleukin-8, and RANTES). This T-cell adhesion was manifested by polarization, spreading and co-localization of cell surface CD44 with a rearranged actin cytoskeleton in hyaluronan-bound T cells. Thus, cytokines and chemokines present in the vicinities of blood vessel walls or present intravascularly in tissues where immune reactions take place, can rapidly activate the CD44 molecules expressed on T cells. PMID:10929056

  14. The role of a new CD44st in increasing the invasion capability of the human breast cancer cell line MCF-7

    Directory of Open Access Journals (Sweden)

    Jiang Wei

    2011-07-01

    Full Text Available Abstract Background CD44, a hyaluronan (HA receptor, is a multistructural and multifunctional cell surface molecule involved in cell proliferation, cell differentiation, cell migration, angiogenesis, presentation of cytokines, chemokines and growth factors to the corresponding receptors, and docking of proteases at the cell membrane, as well as in signaling for cell survival. The CD44 gene contains 20 exons that are alternatively spliced, giving rise to many CD44 isoforms, perhaps including tumor-specific sequences. Methods Reverse transcriptase polymerase chain reaction (RT-PCR and Western blotting were used to detect CD44st mRNA and CD44 protein in sensitive MCF-7, Lovo, K562 and HL-60 cell lines as well as their parental counterparts, respectively. The full length cDNA encoding CD44st was obtained from the total RNA isolated from MCF-7/Adr cells by RT-PCR, and subcloned into the pMD19-T vector. The CD44st gene sequence and open reading frame were confirmed by restriction enzyme analysis and nucleotide sequencing, and then inserted into the eukaryotic expression vector pcDNA3.1. The pcDNA3.1-CD44st was transfected into MCF-7 cells using Lipofectamine. After transfection, the positive clones were obtained by G418 screening. The changes of the MMP-2 and MMP-9 genes and protein levels were detected by RT-PCR and gelatin zymography, respectively. The number of the cells penetrating through the artificial matrix membrane in each group (MCF-7, MCF-7+HA, MCF-7/neo, MCF-7/neo+HA, MCF-7/CD44st, MCF-7/CD44st+HA and MCF-7/CD44st+Anti-CD44+HA was counted to compare the change of the invasion capability regulated by the CD44st. Erk and P-Erk were investigated by Western blotting to approach the molecular mechanisms of MMP-2 and MMP-9 expression regulated by the CD44st. Results Sensitive MCF-7, Lovo, K562 and HL-60 cells did not contain CD44st mRNA and CD44 protein. In contrast, the multidrug resistance MCF-7/Adr, Lovo/Adr, K562/Adr and HL-60/Adr cells

  15. Tunable CD44-specific cellular retargeting with hyaluronic acid nanoshells

    DEFF Research Database (Denmark)

    Ebbesen, Morten F.; Olesen, Morten T. J.; Gjelstrup, Mikkel C.;

    2014-01-01

    Purpose In this work we specifically investigate the molecular weight (Mw) dependent combinatorial properties of hyaluronic acid (HA) for exhibiting stealth and targeting properties using different Mw HA nanoshells to tune nanoparticle retargeting to CD44-expressing cancer cells. Methods HA of di...

  16. Lipid raft association restricts CD44-ezrin interaction and promotion of breast cancer cell migration.

    LENUS (Irish Health Repository)

    Donatello, Simona

    2012-12-01

    Cancer cell migration is an early event in metastasis, the main cause of breast cancer-related deaths. Cholesterol-enriched membrane domains called lipid rafts influence the function of many molecules, including the raft-associated protein CD44. We describe a novel mechanism whereby rafts regulate interactions between CD44 and its binding partner ezrin in migrating breast cancer cells. Specifically, in nonmigrating cells, CD44 and ezrin localized to different membranous compartments: CD44 predominantly in rafts, and ezrin in nonraft compartments. After the induction of migration (either nonspecific or CD44-driven), CD44 affiliation with lipid rafts was decreased. This was accompanied by increased coprecipitation of CD44 and active (threonine-phosphorylated) ezrin-radixin-moesin (ERM) proteins in nonraft compartments and increased colocalization of CD44 with the nonraft protein, transferrin receptor. Pharmacological raft disruption using methyl-β-cyclodextrin also increased CD44-ezrin coprecipitation and colocalization, further suggesting that CD44 interacts with ezrin outside rafts during migration. Conversely, promoting CD44 retention inside lipid rafts by pharmacological inhibition of depalmitoylation virtually abolished CD44-ezrin interactions. However, transient single or double knockdown of flotillin-1 or caveolin-1 was not sufficient to increase cell migration over a short time course, suggesting complex crosstalk mechanisms. We propose a new model for CD44-dependent breast cancer cell migration, where CD44 must relocalize outside lipid rafts to drive cell migration. This could have implications for rafts as pharmacological targets to down-regulate cancer cell migration.

  17. Hyaluronan-CD44 Interactions in Cancer: Paradoxes and Possibilities

    OpenAIRE

    Toole, Bryan P.

    2009-01-01

    Hyaluronan is a prominent component of the micro-environment in most malignant tumors and can be prognostic for tumor progression. Extensive experimental evidence in animal models implicates hyaluronan interactions in tumor growth and metastasis, but it is also evident that a balance of synthesis and turnover by hyaluronidases is critical. CD44, a major hyaluronan receptor, is commonly but not uniformly associated with malignancy, and is frequently used as a marker for cancer stem cells in hu...

  18. CD44 and hyaluronan expression in human cutaneous scar fibroblasts.

    OpenAIRE

    Messadi, D. V.; Bertolami, C. N.

    1993-01-01

    Fibrotic disorders of skin and other organs are typically associated with an abnormal accumulation of extracellular matrix. This study focuses on a matrix constituent, hyaluronan-which is known to be altered in fibrotic disorders of skin- and on CD44, a cell adhesion molecule and putative receptor for hyaluronan. Tissue samples were obtained from biopsies of human normal skin, normal cutaneous scar; and hypertrophic cutaneous scar. After culturing, cells were studied by single- and double-lab...

  19. Expression of CD44v6 and Its Association with Prognosis in Epithelial Ovarian Carcinomas

    Directory of Open Access Journals (Sweden)

    Dang-xia Zhou

    2012-01-01

    Full Text Available The aim of this study was to evaluate CD44v6 protein expression and its prognostic value of CD44v6 in ovarian carcinoma. The expression of CD44v6 was analyzed in 62 patients with ovarian carcinoma by immunohistochemical method. The data obtained were analyzed by univariate and multivariate analyses. The present study clearly demonstrates that tumor tissues from 41 (66.1% patients showed positive expression with CD44v6. The expression of CD44v6 was significantly correlated with histological type, FIGO stage and histological grade of ovarian carcinomas. Concerning the prognosis, the survival period of patients with CD44v6 positive was shorter than that of patients with CD44v6 negative (36.6% versus 66.7%, 5-year survival, P<0.05. Univariate analysis showed that CD44v6 expression, histological type, FIGO stage and histological grade were associated with 5-year survival, and CD44v6 expression was associated with histological type, FIGO stage and histological grade and 5-year survival. In multivariate analysis, using the COX-regression model, CD44v6 expression was important prognostic factor. In conclusion, these results suggest that CD44v6 may be related to histological type, FIGO stage and histological grade of ovarian carcinomas, and CD44v6 may be an important molecular marker for poor prognosis in ovarian carcinomas.

  20. CD44 Plays a Critical Role in Regulating Diet-Induced Adipose Inflammation, Hepatic Steatosis, and Insulin Resistance

    OpenAIRE

    Hong Soon Kang; Grace Liao; DeGraff, Laura M.; Kevin Gerrish; Bortner, Carl D.; Stavros Garantziotis; Jetten, Anton M.

    2013-01-01

    CD44 is a multifunctional membrane receptor implicated in the regulation of several biological processes, including inflammation. CD44 expression is elevated in liver and white adipose tissue (WAT) during obesity suggesting a possible regulatory role for CD44 in metabolic syndrome. To study this hypothesis, we examined the effect of the loss of CD44 expression on the development of various features of metabolic syndrome using CD44 null mice. Our study demonstrates that CD44-deficient mice (CD...

  1. Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells

    Science.gov (United States)

    Aires, Antonio; Ocampo, Sandra M.; Simões, Bruno M.; Josefa Rodríguez, María; Cadenas, Jael F.; Couleaud, Pierre; Spence, Katherine; Latorre, Alfonso; Miranda, Rodolfo; Somoza, Álvaro; Clarke, Robert B.; Carrascosa, José L.; Cortajarena, Aitziber L.

    2016-02-01

    Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

  2. Tumor-initiating cells are enriched in CD44(hi population in murine salivary gland tumor.

    Directory of Open Access Journals (Sweden)

    Shukun Shen

    Full Text Available Tumor-initiating cells (T-ICs discovered in various tumors have been widely reported. However, T-IC populations in salivary gland tumors have yet to be elucidated. Using the established Pleomorphic Adenoma Gene-1 (Plag1 transgenic mouse model of a salivary gland tumor, we identified CD44(high (CD44(hi tumor cells, characterized by high levels of CD44 cell surface expression, as the T-ICs for pleomorphic adenomas. These CD44(hi tumor cells incorporated 5-bromo-2-deoxyuridine (BrdU, at a lower rate than their CD44(negative (CD44(neg counterparts, and also retained BrdU for a long period of time. Cell surface maker analysis revealed that 25% of the CD44(hi tumor cells co-express other cancer stem cell markers such as CD133 and CD117. As few as 500 CD44(hi tumor cells were sufficient to initiate pleomorphic adenomas in one third of the wildtype mice, whereas more than 1×10(4 CD44(neg cells were needed for the same purpose. In NIH 3T3 cells, Plag1 was capable of activating the gene transcription of Egr1, a known upregulator for CD44. Furthermore, deletion of sequence 81-96 in the Egr1 promoter region abolished the effect of Plag1 on Egr1 upregulation. Our results establish the existence of T-ICs in murine salivary gland tumors, and suggest a potential molecular mechanism for CD44 upregulation.

  3. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation

    Science.gov (United States)

    Cuff, Carolyn A.; Kothapalli, Devashish; Azonobi, Ijeoma; Chun, Sam; Zhang, Yuanming; Belkin, Richard; Yeh, Christine; Secreto, Anthony; Assoian, Richard K.; Rader, Daniel J.; Puré, Ellen

    2001-01-01

    Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD44 to atherosclerosis, we bred CD44-null mice to atherosclerosis-prone apoE-deficient mice. We found a 50–70% reduction in aortic lesions in CD44-null mice compared with CD44 heterozygote and wild-type littermates. We demonstrate that CD44 promotes the recruitment of macrophages to atherosclerotic lesions. Furthermore, we show that CD44 is required for phenotypic dedifferentiation of medial smooth muscle cells to the “synthetic” state as measured by expression of VCAM-1. Finally, we demonstrate that hyaluronan, the principal ligand for CD44, is upregulated in atherosclerotic lesions of apoE-deficient mice and that the low-molecular-weight proinflammatory forms of hyaluronan stimulate VCAM-1 expression and proliferation of cultured primary aortic smooth muscle cells, whereas high-molecular-weight forms of hyaluronan inhibit smooth muscle cell proliferation. We conclude that CD44 plays a critical role in the progression of atherosclerosis through multiple mechanisms. PMID:11581304

  4. CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity

    Science.gov (United States)

    Li, Yuheng; Zhong, Guohui; Sun, Weijia; Zhao, Chengyang; Zhang, Pengfei; Song, Jinping; Zhao, Dingsheng; Jin, Xiaoyan; Li, Qi; Ling, Shukuan; Li, Yingxian

    2015-01-01

    The CD44 is cellular surface adhesion molecule that is involved in physiological processes such as hematopoiesis, lymphocyte homing and limb development. It plays an important role in a variety of cellular functions including adhesion, migration, invasion and survival. In bone tissue, CD44 is widely expressed in osteoblasts, osteoclasts and osteocytes. However, the mechanisms underlying its role in bone metabolism remain unclear. We found that CD44 expression was upregulated during osteoclastogenesis. CD44 deficiency in vitro significantly inhibited osteoclast activity and function by regulating the NF-κB/NFATc1-mediated pathway. In vivo, CD44 mRNA levels were significantly upregulated in osteoclasts isolated from the hindlimb of tail-suspended mice. CD44 deficiency can reduce osteoclast activity and counteract cortical bone loss in the hindlimb of unloaded mice. These results suggest that therapeutic inhibition of CD44 may protect from unloading induced bone loss by inhibiting osteoclast activity. PMID:26530337

  5. CD44 deficiency inhibits unloading-induced cortical bone loss through downregulation of osteoclast activity.

    Science.gov (United States)

    Li, Yuheng; Zhong, Guohui; Sun, Weijia; Zhao, Chengyang; Zhang, Pengfei; Song, Jinping; Zhao, Dingsheng; Jin, Xiaoyan; Li, Qi; Ling, Shukuan; Li, Yingxian

    2015-01-01

    The CD44 is cellular surface adhesion molecule that is involved in physiological processes such as hematopoiesis, lymphocyte homing and limb development. It plays an important role in a variety of cellular functions including adhesion, migration, invasion and survival. In bone tissue, CD44 is widely expressed in osteoblasts, osteoclasts and osteocytes. However, the mechanisms underlying its role in bone metabolism remain unclear. We found that CD44 expression was upregulated during osteoclastogenesis. CD44 deficiency in vitro significantly inhibited osteoclast activity and function by regulating the NF-κB/NFATc1-mediated pathway. In vivo, CD44 mRNA levels were significantly upregulated in osteoclasts isolated from the hindlimb of tail-suspended mice. CD44 deficiency can reduce osteoclast activity and counteract cortical bone loss in the hindlimb of unloaded mice. These results suggest that therapeutic inhibition of CD44 may protect from unloading induced bone loss by inhibiting osteoclast activity.

  6. Epigenetic regulation of CD44 in Hodgkin and non-Hodgkin lymphoma

    International Nuclear Information System (INIS)

    Epigenetic inactivation of tumor suppressor genes (TSG) by promoter CpG island hypermethylation is a hallmark of cancer. To assay its extent in human lymphoma, methylation of 24 TSG was analyzed in lymphoma-derived cell lines as well as in patient samples. We screened for TSG methylation using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) in 40 lymphoma-derived cell lines representing anaplastic large cell lymphoma, Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), Hodgkin lymphoma and mantle cell lymphoma (MCL) as well as in 50 primary lymphoma samples. The methylation status of differentially methylated CD44 was verified by methylation-specific PCR and bisulfite sequencing. Gene expression of CD44 and its reactivation by DNA demethylation was determined by quantitative real-time PCR and on the protein level by flow cytometry. Induction of apoptosis by anti-CD44 antibody was analyzed by annexin-V/PI staining and flow cytometry. On average 8 ± 2.8 of 24 TSG were methylated per lymphoma cell line and 2.4 ± 2 of 24 TSG in primary lymphomas, whereas 0/24 TSG were methylated in tonsils and blood mononuclear cells from healthy donors. Notably, we identified that CD44 was hypermethylated and transcriptionally silenced in all BL and most FL and DLBCL cell lines, but was usually unmethylated and expressed in MCL cell lines. Concordant results were obtained from primary lymphoma material: CD44 was not methylated in MCL patients (0/11) whereas CD44 was frequently hypermethylated in BL patients (18/29). In cell lines with CD44 hypermethylation, expression was re-inducible at mRNA and protein levels by treatment with the DNA demethylating agent 5-Aza-2'-deoxycytidine, confirming epigenetic regulation of CD44. CD44 ligation assays with a monoclonal anti-CD44 antibody showed that CD44 can mediate apoptosis in CD44+ lymphoma cells. CD44 hypermethylated, CD44- lymphoma cell lines were consistently

  7. Targeting CD44 expressing cancer cells with anti-CD44 monoclonal antibody improves cellular uptake and antitumor efficacy of liposomal doxorubicin.

    Science.gov (United States)

    Arabi, Leila; Badiee, Ali; Mosaffa, Fatemeh; Jaafari, Mahmoud Reza

    2015-12-28

    Although liposomes improve the safety and pharmacokinetic properties of free drugs, they have not sufficiently enhanced the therapeutic efficacy compared to them. To address this problem, targeted therapy of tumor cells holds great promise to further enhance therapeutic index and decreases off-target effects compared with non-targeted liposomes. In the context of antibody-mediated targeted cancer therapy, we evaluated the anti-tumor activity and therapeutic efficacy of Doxil, and that of Doxil modified with a monoclonal antibody (mAb) against CD44, which is one of the most well-known surface markers associated with Cancer Stem Cells (CSCs). Flow cytometry analyses and confocal laser scanning microscopy results showed significant enhanced cellular uptake of CD44-targeted Doxil (CD44-Doxil) in CD44-positive C-26 cells compared to Doxil. However, CD44-negative NIH-3T3 cells showed a similar uptake and in vitro cytotoxicity with both CD44-Doxil and non-targeted Doxil. In BALB/c mice bearing C-26 murine carcinoma, CD44-Doxil groups exhibited significantly higher doxorubicin concentration (than Doxil) inside the tumor cells, while their circulation time and distribution profile remained comparable. CD44-Doxil at doses of either 10 or 15 mg/kg resulted in superior tumor growth inhibition and higher inclination to tumor, indicating the potential of anti-CD44 mAb targeting in therapeutic efficacy improvement. This study provides proof-of-principle for actively tumor-targeting concept and merits further investigations. PMID:26518722

  8. Expression of p53 and CD44 in Canine Breast Tumor

    Institute of Scientific and Technical Information of China (English)

    LIU Yun; CUI Wen; CHENG Xi; FENG Xinchang

    2008-01-01

    The p53 and CD44 expression of 10 cases in canine breast tumor were examined utilizing immunohistochemical assay with rabbit anti-mouse polyclonal antibodies against p53 or CD44,respectively.The p53 expression was significantly higher in malignant than in benign breast tumor.The expression of CD44 was not significantly different in malignant breast cancer and benign breast tumor.This suggests that p53 can be used as an indicator for animal prognosis.

  9. The adhesion receptor CD44 promotes atherosclerosis by mediating inflammatory cell recruitment and vascular cell activation

    OpenAIRE

    Cuff, Carolyn A.; Kothapalli, Devashish; Azonobi, Ijeoma; Chun, Sam; Zhang, Yuanming; Belkin, Richard; Yeh, Christine; Secreto, Anthony; Richard K Assoian; Rader, Daniel J; Puré, Ellen

    2001-01-01

    Atherosclerosis causes most acute coronary syndromes and strokes. The pathogenesis of atherosclerosis includes recruitment of inflammatory cells to the vessel wall and activation of vascular cells. CD44 is an adhesion protein expressed on inflammatory and vascular cells. CD44 supports the adhesion of activated lymphocytes to endothelium and smooth muscle cells. Furthermore, ligation of CD44 induces activation of both inflammatory and vascular cells. To assess the potential contribution of CD4...

  10. CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

    Science.gov (United States)

    Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong; Zhou, Xianguang; Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing; Wang, Jiandong; Zhang, Longjiang; Teng, Zhaogang; Lu, Guangming

    2015-03-01

    Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer.

  11. Tumor-Initiating Cells Are Enriched in CD44hi Population in Murine Salivary Gland Tumor

    OpenAIRE

    Shukun Shen; Wenjun Yang; Zhugang Wang; Xia Lei; Liqun Xu; Yang Wang; Lizhen Wang; Lei Huang; Zhiwei Yu; Xinhong Zhang; Jiang Li; Yan Chen; Xiaoping Zhao; Xuelai Yin; Chenping Zhang

    2011-01-01

    Tumor-initiating cells (T-ICs) discovered in various tumors have been widely reported. However, T-IC populations in salivary gland tumors have yet to be elucidated. Using the established Pleomorphic Adenoma Gene-1 (Plag1) transgenic mouse model of a salivary gland tumor, we identified CD44(high) (CD44(hi)) tumor cells, characterized by high levels of CD44 cell surface expression, as the T-ICs for pleomorphic adenomas. These CD44(hi) tumor cells incorporated 5-bromo-2-deoxyuridine (BrdU), at a...

  12. Phototheranostics of CD44-positive cell populations in triple negative breast cancer

    Science.gov (United States)

    Jin, Jiefu; Krishnamachary, Balaji; Mironchik, Yelena; Kobayashi, Hisataka; Bhujwalla, Zaver M.

    2016-01-01

    Triple-negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer that has limited treatment options. Its high rates of recurrence and metastasis have been associated, in part, with a subpopulation of breast cancer stem-like cells that are resistant to conventional therapies. A compendium of markers such as CD44high/CD24low, and increased expression of the ABCG2 transporter and increased aldehyde dehydrogenase (ALDH1), have been associated with these cells. We developed a CD44-targeted monoclonal antibody photosensitizer conjugate for combined fluorescent detection and photoimmunotherapy (PIT) of CD44 expressing cells in TNBC. The CD44-targeted conjugate demonstrated acute cell killing of breast cancer cells with high CD44 expression. This cell death process was dependent upon CD44-specific cell membrane binding combined with near-infrared irradiation. The conjugate selectively accumulated in CD44-positive tumors and caused dramatic tumor shrinkage and efficient elimination of CD44-positive cell populations following irradiation. This novel phototheranostic strategy provides a promising opportunity for the destruction of CD44-positive populations that include cancer stem-like cells, in locally advanced primary and metastatic TNBC. PMID:27302409

  13. CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

    International Nuclear Information System (INIS)

    Graphical abstract: - Highlights: • CD44-engineered mesoporous silica nanoparticles are synthesized. • The mechanism of CD44-engineered mesoporous silica nanoparticles is revealed. • This new delivery system increased the drug accumulation in vitro and in vivo. • This new delivery system offers an effective approach to treat multidrug resistance. - Abstract: Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer

  14. CD44 antibodies and immune thrombocytopenia in the amelioration of murine inflammatory arthritis.

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    Patrick J Mott

    Full Text Available Antibodies to CD44 have been used to successfully ameliorate murine models of autoimmune disease. The most often studied disease model has been murine inflammatory arthritis, where a clear mechanism for the efficacy of CD44 antibodies has not been established. We have recently shown in a murine passive-model of the autoimmune disease immune thrombocytopenia (ITP that some CD44 antibodies themselves can induce thrombocytopenia in mice, and the CD44 antibody causing the most severe thrombocytopenia (IM7, also is known to be highly effective in ameliorating murine models of arthritis. Recent work in the K/BxN serum-induced model of arthritis demonstrated that antibody-induced thrombocytopenia reduced arthritis, causing us to question whether CD44 antibodies might primarily ameliorate arthritis through their thrombocytopenic effect. We evaluated IM7, IRAWB14.4, 5035-41.1D, KM201, KM114, and KM81, and found that while all could induce thrombocytopenia, the degree of protection against serum-induced arthritis was not closely related to the length or severity of the thrombocytopenia. CD44 antibody treatment was also able to reverse established inflammation, while thrombocytopenia induced by an anti-platelet antibody targeting the GPIIbIIIa platelet antigen, could not mediate this effect. While CD44 antibody-induced thrombocytopenia may contribute to some of its therapeutic effect against the initiation of arthritis, for established disease there are likely other mechanisms contributing to its efficacy. Humans are not known to express CD44 on platelets, and are therefore unlikely to develop thrombocytopenia after CD44 antibody treatment. An understanding of the relationship between arthritis, thrombocytopenia, and CD44 antibody treatment remains critical for continued development of CD44 antibody therapeutics.

  15. CD44-engineered mesoporous silica nanoparticles for overcoming multidrug resistance in breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xin; Liu, Ying; Wang, Shouju; Shi, Donghong [Department of Radiology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002 (China); Zhou, Xianguang [National Clinical Research Center of Kidney Diseases, Jinling Hospital, Nanjing 210016 (China); Wang, Chunyan; Wu, Jiang; Zeng, Zhiyong; Li, Yanjun; Sun, Jing [Department of Radiology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002 (China); Wang, Jiandong [Department of Pathology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002 (China); Zhang, Longjiang [Department of Radiology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002 (China); Teng, Zhaogang, E-mail: tzg@fudan.edu.cn [Department of Radiology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002 (China); State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China); Lu, Guangming, E-mail: cjr.luguangming@vip.163.com [Department of Radiology, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing 210002 (China); State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 (China)

    2015-03-30

    Graphical abstract: - Highlights: • CD44-engineered mesoporous silica nanoparticles are synthesized. • The mechanism of CD44-engineered mesoporous silica nanoparticles is revealed. • This new delivery system increased the drug accumulation in vitro and in vivo. • This new delivery system offers an effective approach to treat multidrug resistance. - Abstract: Multidrug resistance is a major impediment for the successful chemotherapy in breast cancer. CD44 is over-expressed in multidrug resistant human breast cancer cells. CD44 monoclonal antibody exhibits anticancer potential by inhibiting proliferation and regulating P-glycoprotein-mediated drug efflux activity in multidrug resistant cells. Thereby, CD44 monoclonal antibody in combination with chemotherapeutic drug might be result in enhancing chemosensitivity and overcoming multidrug resistance. The purpose of this study is to investigate the effects of the CD44 monoclonal antibody functionalized mesoporous silica nanoparticles containing doxorubicin on human breast resistant cancer MCF-7 cells. The data showed that CD44-modified mesoporous silica nanoparticles increased cytotoxicity and enhanced the downregulation of P-glycoprotein in comparison to CD44 antibody. Moreover, CD44-engineered mesoporous silica nanoparticles provided active target, which promoted more cellular uptake of DOX in the resistant cells and more retention of DOX in tumor tissues than unengineered counterpart. Animal studies of the resistant breast cancer xenografts demonstrated that CD44-engineered drug delivery system remarkably induced apoptosis and inhibited the tumor growth. Our results indicated that the CD44-engineered mesoporous silica nanoparticle-based drug delivery system offers an effective approach to overcome multidrug resistance in human breast cancer.

  16. MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

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    Chia-Hui Wang

    Full Text Available Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

  17. Evaluation of Stem Cell Markers, CD44/CD24 in Breast Cancer Cell Lines

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    Masoud Hashemi Arabi

    2014-05-01

    Four breast cancer cell lines, MCF-7 ، T47D ، MDA-MB231 and MDA-MB468 were purchased from National cell Bank of Iran based in Iran Pasture Institute and were cultured in high glucose DMEM supplemented with 10% FCS. Cells were stained with antiCD44-PE and antiCD24-FITC antibodies and Status of CD44 and CD24 as markers of breast cancer stem cells were evaluated using flow cytometer and fluorescent microscopy.Evaluation of CD44 and CD24 as markers of breast cancer stem cells showed that MDA-MB231 with 97±1.2% CD44+/CD24-/low cells is significantly different from the others that they were mainly CD44 and CD24 positive cells(p

  18. Expression of CD44v6 gene in normal human peripheral blood

    Institute of Scientific and Technical Information of China (English)

    Jian Song; Dong-Sheng Zhang; Jie Zheng

    2005-01-01

    AIM: To investigate if CD44v6 could be used as a molecular marker of cancer progression and metastasis through the detection of CD44v6 gene expression in normal human peripheral blood.METHODS: RNA was extracted from the peripheral blood mononuclear cells of 50 healthy donors, the expression of CD44v6 was investigated using reverse transcriptasepolymerase chain reaction (RT-PCR).RESULTS: CD44v6 mRNA was detected in 58% of healthy volunteers under the proper controls.CONCLUSION: Our results suggest that the measurement of CD44v6 expression in peripheral blood by RT-PCR is not suitable for detection of circulating tumor cells.

  19. PCBP-1 regulates alternative splicing of the CD44 gene and inhibits invasion in human hepatoma cell line HepG2 cells

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    Ge Changhui

    2010-04-01

    Full Text Available Abstract Background PCBP1 (or alpha CP1 or hnRNP E1, a member of the PCBP family, is widely expressed in many human tissues and involved in regulation of transcription, transportation process, and function of RNA molecules. However, the role of PCBP1 in CD44 variants splicing still remains elusive. Results We found that enforced PCBP1 expression inhibited CD44 variants expression including v3, v5, v6, v8, and v10 in HepG2 cells, and knockdown of endogenous PCBP1 induced these variants splicing. Invasion assay suggested that PCBP1 played a negative role in tumor invasion and re-expression of v6 partly reversed the inhibition effect by PCBP1. A correlation of PCBP1 down-regulation and v6 up-regulation was detected in primary HCC tissues. Conclusions We first characterized PCBP1 as a negative regulator of CD44 variants splicing in HepG2 cells, and loss of PCBP1 in human hepatic tumor contributes to the formation of a metastatic phenotype.

  20. CD44v6和MRP在卵巢癌中的表达%Expression and Significance of CD44v6 and MRP in Ovarian Carcinoma

    Institute of Scientific and Technical Information of China (English)

    谭文福; 吴绪峰; 陈惠祯

    2005-01-01

    目的探讨CD44v6和 MRP在上皮性卵巢癌组织中表达与化疗疗效和预后的关系.方法采用免疫组织化学S-P法,检测10例正常卵巢组织、20例卵巢良性肿瘤和50例卵巢恶性肿瘤上皮性组织中CD44v6和 MRP的表达.结果①CD44v6和MRP在卵巢恶性肿瘤中的表达明显高于在良性及正常组织中的表达(P<0.05);②CD44v6和MRP强阳性率与卵巢癌的临床分期、组织学分级和淋巴结转移有关(P<0.05);③卵巢癌中CD44v6和MRP的表达呈正相关(r=0.557, P<0.05);④CD44v6和MRP阳性表达的病例,化疗疗效和预后差. 结论检测卵巢癌中的CD44v6和MRP表达可反映肿瘤细胞的化疗疗效及预测预后.

  1. Expression of cell adhesion molecule CD44 in gastric adenocarcinoma and its prognostic importance

    Institute of Scientific and Technical Information of China (English)

    Kamran Ghaffarzadehgan; Mostafa Jafarzadeh; Hamid Reza Raziee; Harold Reza Sima; Ehsan Esmaili-Shandiz; Hanieh Hosseinnezhad; Ail Taghizadeh Kermani; Omeed Moaven; Maryam Bahrani

    2008-01-01

    AIM: To evaluate the relation of cluster of differentiation 44 (CD44) expression with clinicopathological features of gastric adenocarcinoma, and also its effect on prognosis with an emphasis on the differences between intestinal and diffuse types. METHODS: From 2000 to 2006, 100 patients with gastric adenocarcinoma, who had undergone total or subtotal gastrectomy without any prior treatment, were studied. Haematoxylin & eosin (HE) staining was used for histological evaluation, including the type (Lauren's classification) and grading of the tumor. The expression of CD44 in the gastric adenocarcinoma mucosa and the adjacent mucosa were determined by immunohistochemistry. The survival analysis was obtained using the Kaplan-Meier test. RESULTS: Of 100 patients, 74 (74%) patients were male. The tumors were categorized as intestinal type (78%) or diffuse type (22%). Sixty-five percent of patients were CD44-positive. CD44 expression was not detected in normal gastric mucosa. Rather, CD44 was more commonly expressed in the intestinal subtype (P = 0.002). A significant relation was seen between the grade of tumor and the expression of CD44 (P=0.014). The survival analysis showed a poor prognosis of patients with CD44-positive tumors (P = 0.008); and this was more prominent in the intestinal (P = 0.001) rather than diffuse type. CONCLUSION: Cell adhesion molecule CD44 is highly expressed in gastric adenocarcinoma. CD44 expression is correlated with a poor prognosis in patients with the intestinal type of gastric adenocarcinoma. CD44 can, therefore, be utilized as a prognostic marker for this group of patients.

  2. Expression of the CD44 Protein in the Heart Valves Affected with Rheumatic Heart Disease

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    R. Vijayalakshmi

    2010-01-01

    Full Text Available Problem statement: The primary aim was to study the expression of CD44 protein in the heart valves removed surgically for either stenosis or regurgitation and to study the morphology of valves using histochemical staining. Approach: We studied 107 valves which were collected from the International Centre for Cardio Thoracic and Vascular Diseases. Results: Aortic and mitral valves were obtained from the centre and processed in research pathology lab. CD44 protein is a receptor for the ligand hyaluronic acid which causes inflammation in the heart valves. A total of 107 valves were studied. Of the 61 mitral valves studied 38 (62.2% showed CD44 positivity as brownish granules within the cytoplasm. Of the 46 aortic valves studied 19 (41.3% showed CD44 positivity as brownish granules within the cytoplasm. The valves showed evidence of past inflammation showing thick and thin walled blood vessels and lymphocytes. CD44 protein increased in 62.2% of mitral valves, of which 60.5% were from patients presenting when they were less than 40 years old. CD44 protein increased in 41.3% of aortic valves, of which 63.15% were from patients presenting when they were less than 40 years old. CD44 positivity was seen in 57 valves as brownish granules within the cytoplasm of the cell. CD44 protein increased in 53.27% of mitral and aortic valves, of which 63.15% were from the males patients. CD44 protein increased in 53.27% of mitral and aortic valves, of which 36.84% were from the females patients. Conclusion/Recommendations: The results showed CD44 is over expressed in the heart valves removed surgically for stenosis or regurgitation where the hyaluronic acid content is high.

  3. Ligand binding to anti-cancer target CD44 investigated by molecular simulations.

    Science.gov (United States)

    Nguyen, Tin Trung; Tran, Duy Phuoc; Pham Dinh Quoc Huy; Hoang, Zung; Carloni, Paolo; Van Pham, Phuc; Nguyen, Chuong; Li, Mai Suan

    2016-07-01

    CD44 is a cell-surface glycoprotein and receptor for hyaluronan, one of the major components of the tumor extracellular matrix. There is evidence that the interaction between CD44 and hyaluronan promotes breast cancer metastasis. Recently, the molecule F-19848A was shown to inhibit hyaluronan binding to receptor CD44 in a cell-based assay. In this study, we investigated the mechanism and energetics of F-19848A binding to CD44 using molecular simulation. Using the molecular mechanics/Poisson Boltzmann surface area (MM-PBSA) method, we obtained the binding free energy and inhibition constant of the complex. The van der Waals (vdW) interaction and the extended portion of F-19848A play key roles in the binding affinity. We screened natural products from a traditional Chinese medicine database to search for CD44 inhibitors. From combining pharmaceutical requirements with docking and molecular dynamics simulations, we found ten compounds that are potentially better or equal to the F-19848A ligand at binding to CD44 receptor. Therefore, we have identified new candidates of CD44 inhibitors, based on molecular simulation, which may be effective small molecules for the therapy of breast cancer. PMID:27342250

  4. Differentiation of breast cancer stem cells by knockdown of CD44: promising differentiation therapy

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    Pham Phuc V

    2011-12-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs are the source of breast tumors. Compared with other cancer cells, cancer stem cells show high resistance to both chemotherapy and radiotherapy. Targeting of BCSCs is thus a potentially promising and effective strategy for breast cancer treatment. Differentiation therapy represents one type of cancer stem-cell-targeting therapy, aimed at attacking the stemness of cancer stem cells, thus reducing their chemo- and radioresistance. In a previous study, we showed that down-regulation of CD44 sensitized BCSCs to the anti-tumor agent doxorubicin. This study aimed to determine if CD44 knockdown caused BCSCs to differentiate into breast cancer non-stem cells (non-BCSCs. Methods We isolated a breast cancer cell population (CD44+CD24- cells from primary cultures of malignant breast tumors. These cells were sorted into four sub-populations based on their expression of CD44 and CD24 surface markers. CD44 knockdown in the BCSC population was achieved using small hairpin RNA lentivirus particles. The differentiated status of CD44 knock-down BCSCs was evaluated on the basis of changes in CD44+CD24- phenotype, tumorigenesis in NOD/SCID mice, and gene expression in relation to renewal status, metastasis, and cell cycle in comparison with BCSCs and non-BCSCs. Results Knockdown of CD44 caused BCSCs to differentiate into non-BCSCs with lower tumorigenic potential, and altered the cell cycle and expression profiles of some stem cell-related genes, making them more similar to those seen in non-BCSCs. Conclusions Knockdown of CD44 is an effective strategy for attacking the stemness of BCSCs, resulting in a loss of stemness and an increase in susceptibility to chemotherapy or radiation. The results of this study highlight a potential new strategy for breast cancer treatment through the targeting of BCSCs.

  5. Prognostic significance of CD44s expression in resected non-small cell lung cancer

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    Ko Yoon

    2011-08-01

    Full Text Available Abstract Background CD44s is a cell adhesion molecule known to mediate cellular adhesion to the extracellular matrix, a prerequisite for tumor cell migration. CD44s plays an important role in invasion and metastasis of various cancers. In the present study, we sought to determine whether CD44s is involved in clinical outcomes of patients with resected non-small cell lung cancer (NSCLC. Methods Using immunohistochemical staining, we investigated CD44s protein expression using tissue array specimens from 159 patients with resected NSCLC (adenocarcinoma (AC; n = 82 and squamous cell carcinoma (SCC; n = 77. Additionally, the immunoreactivity of cyclooxygenase (COX-2 was also studied. The clinicopathological implications of these molecules were analyzed statistically. Results High CD44s expression was detected more frequently in NSCLC patients with SCC (66/72; 91.7% than in those with AC histology (P 0.001. Additionally, high CD44s expression was significant correlated with more advanced regional lymph node metastasis (P = 0.021. In multivariate analysis of survival in NSCLC patients with AC histology, significant predictors were lymph node metastasis status (P P = 0.046, and high CD44s expression (P = 0.014. For NSCLC patients with SCC histology, the significant predictor was a more advanced tumor stage (P = 0.015. No significant association was found between CD44s and clinical outcome (P = 0.311. Conclusions High CD44s expression was a negative prognostic marker with significance in patients with resected NSCLC, particularly those with AC histology, and was independent of tumor stage.

  6. ADAM17-mediated CD44 cleavage promotes orasphere formation or stemness and tumorigenesis in HNSCC

    International Nuclear Information System (INIS)

    CD44, an extracellular matrix (ECM) receptor, has been described as a cancer stem cell marker in multiple cancers, including head and neck squamous cell carcinoma (HNSCC). HNSCC orasphere formation or stemness was characterized by cleavage of CD44, and thus we hypothesized that this proteolytic processing may be critical to stemness and tumorigenesis. We tested this hypothesis by examining the mechanisms that regulate this process in vitro and in vivo, and by exploring its clinical relevance in human specimens. Sphere assays have been used to evaluate stemness in vitro. Spheres comprised of HNSCC cells or oraspheres and an oral cancer mouse model were used to examine the significance of CD44 cleavage using stable suppression and inhibition approaches. These mechanisms were also examined in HNSCC specimens. Oraspheres exhibited increased levels of CD44 cleavage compared to their adherent counterparts. Given that disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) is a major matrix metalloproteinase known to cleave CD44, we chemically inhibited and stably suppressed ADAM17 expression in HNSCC cells and found that these treatments blocked CD44 cleavage and abrogated orasphere formation. Furthermore, stable suppression of ADAM17 in HNSCC cells also diminished tumorigenesis in an oral cancer mouse model. Consistently, stable suppression of CD44 in HNSCC cells abrogated orasphere formation and inhibited tumorigenesis in vivo. The clinical relevance of these findings was confirmed in matched primary and metastatic human HNSCC specimens, which exhibited increased levels of ADAM17 expression and concomitant CD44 cleavage compared to controls. CD44 cleavage by ADAM17 is critical to orasphere formation or stemness and HNSCC tumorigenesis

  7. Expression and significance of CD44 and p-AKT in pancreatic head cancer

    OpenAIRE

    Xiaoping, Li; Xiaowei, Zhang; Leizhen, Zheng; Weijian, Guo

    2015-01-01

    Background CD44 and phosphorylated AKT (p-AKT) is a potentially interesting prognostic marker and therapeutic target in pancreatic cancer. The expression of CD44 and p-AKT has been reported to correlate with poor prognosis of pancreatic cancer in most literatures. The purpose of this study is to investigate the roles of CD44 and p-AKT in pancreatic head cancer and their correlation with the prognosis of pancreatic head cancer patients. Methods Forty-eight pancreatic head cancer samples were c...

  8. Allogeneic reconstitution after nonmyeloablative conditioning: mitigation of graft-versus-host and host-versus-graft reactivity by anti-CD44v6

    OpenAIRE

    Christ, Oliver; Günthert, Ursula; Schmidt, Dirk-Steffen; Zöller, Margot

    2002-01-01

    T-cell maturation is accelerated in transgenic mice expressing rat CD44v4-v7 on T cells, the effect being blocked by anti-CD44v6. This finding suggested functional activity of CD44v6 in thymocyte development. We tested the hypothesis by antibody blocking and using mice with targeted deletion of CD44v6/v7 exon products (CD44v6/v7(-/-)). When lethally irradiated CD44v6/v7-competent (CD44v6/v7(+/+)) mice were reconstituted syngeneically, higher numbers of CD44v6/v7(-/-) than CD44v6/v7(+/+) BMC w...

  9. The Role of CD44 in Disease Pathophysiology and Targeted Treatment

    OpenAIRE

    Jordan, Andre R.; Racine, Ronny R.; Hennig, Martin J. P.; Lokeshwar, Vinata B.

    2015-01-01

    The cell-surface glycoprotein CD44 is involved in a multitude of important physiological functions including cell proliferation, adhesion, migration, hematopoiesis, and lymphocyte activation. The diverse physiological activity of CD44 is manifested in the pathology of a number of diseases including cancer, arthritis, bacterial and viral infections, interstitial lung disease, vascular disease, and wound healing. This diversity in biological activity is conferred by both a variety of distinct C...

  10. Functionalizing Liposomes with anti-CD44 Aptamer for Selective Targeting of Cancer Cells.

    Science.gov (United States)

    Alshaer, Walhan; Hillaireau, Hervé; Vergnaud, Juliette; Ismail, Said; Fattal, Elias

    2015-07-15

    CD44 receptor protein is found to be overexpressed by many tumors and is identified as one of the most common cancer stem cell surface markers including tumors affecting colon, breast, pancreas, and head and neck, making this an attractive receptor for therapeutic targeting. In this study, 2'-F-pyrimidine-containing RNA aptamer (Apt1), previously selected against CD44, was successfully conjugated to the surface of PEGylated liposomes using the thiol-maleimide click reaction. The conjugation of Apt1 to the surface of liposomes was confirmed by the change in size and zeta potential and by migration on agarose gel electrophoresis. The binding affinity of Apt1 was improved after conjugation compared to free-Apt1. The cellular uptake for Apt1-Lip was tested by flow cytometry and confocal imaging using the two CD44(+) cell lines, human lung cancer cells (A549) and human breast cancer cells (MDA-MB-231), and the CD44(-) cell line, mouse embryonic fibroblast cells (NIH/3T3). The results showed higher sensitivity and selectivity for Apt1-Lip compared to the blank liposomes (Mal-Lip). In conclusion, we demonstrate a successful conjugation of anti-CD44 aptamer to the surface of liposome and binding preference of Apt1-Lip to CD44-expressing cancer cells and conclude to a promising potency of Apt1-Lip as a specific drug delivery system.

  11. Functionalizing Liposomes with anti-CD44 Aptamer for Selective Targeting of Cancer Cells.

    Science.gov (United States)

    Alshaer, Walhan; Hillaireau, Hervé; Vergnaud, Juliette; Ismail, Said; Fattal, Elias

    2015-07-15

    CD44 receptor protein is found to be overexpressed by many tumors and is identified as one of the most common cancer stem cell surface markers including tumors affecting colon, breast, pancreas, and head and neck, making this an attractive receptor for therapeutic targeting. In this study, 2'-F-pyrimidine-containing RNA aptamer (Apt1), previously selected against CD44, was successfully conjugated to the surface of PEGylated liposomes using the thiol-maleimide click reaction. The conjugation of Apt1 to the surface of liposomes was confirmed by the change in size and zeta potential and by migration on agarose gel electrophoresis. The binding affinity of Apt1 was improved after conjugation compared to free-Apt1. The cellular uptake for Apt1-Lip was tested by flow cytometry and confocal imaging using the two CD44(+) cell lines, human lung cancer cells (A549) and human breast cancer cells (MDA-MB-231), and the CD44(-) cell line, mouse embryonic fibroblast cells (NIH/3T3). The results showed higher sensitivity and selectivity for Apt1-Lip compared to the blank liposomes (Mal-Lip). In conclusion, we demonstrate a successful conjugation of anti-CD44 aptamer to the surface of liposome and binding preference of Apt1-Lip to CD44-expressing cancer cells and conclude to a promising potency of Apt1-Lip as a specific drug delivery system. PMID:25343502

  12. Oncolytic adenoviruses kill breast cancer initiating CD44+CD24-/low cells.

    Science.gov (United States)

    Eriksson, Minna; Guse, Kilian; Bauerschmitz, Gerd; Virkkunen, Pekka; Tarkkanen, Maija; Tanner, Minna; Hakkarainen, Tanja; Kanerva, Anna; Desmond, Renee A; Pesonen, Sari; Hemminki, Akseli

    2007-12-01

    Cancer stem cells have been indicated in the initiation of tumors and are even found to be responsible for relapses after apparently curative therapies have been undertaken. In breast cancer, they may reside in the CD44(+)CD24(-/low) population. The use of oncolytic adenoviruses presents an attractive anti-tumor approach for eradication of these cells because their entry occurs through infection and they are, therefore, not susceptible to those mechanisms that commonly render stem cells resistant to many drugs. We isolated CD44(+)CD24(-/low) cells from patient pleural effusions and confirmed stem cell-like features including oct4 and sox2 expression and Hoechst 33342 exclusion. CD44(+)CD24(-/low) cells, including the Hoechst excluding subpopulation, could be effectively killed by oncolytic adenoviruses Ad5/3-Delta24 and Ad5.pk7-Delta24. In mice, CD44(+)CD24(-/low) cells formed orthotopic breast tumors but virus infection prevented tumor formation. Ad5/3-Delta24 and Ad5.pk7-Delta24 were effective against advanced orthotopic CD44(+)CD24(-/low)-derived tumors. In summary, Ad5/3-Delta24 and Ad5.pk7-Delta24 can kill CD44(+)CD24(-/low), and also committed breast cancer cells, making them promising agents for treatment of breast cancer. PMID:17848962

  13. SLUG/SNAI2 and Tumor Necrosis Factor Generate Breast Cells With CD44+/CD24- Phenotype

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    Bhat-Nakshatri Poornima

    2010-08-01

    Full Text Available Abstract Background Breast cancer cells with CD44+/CD24- cell surface marker expression profile are proposed as cancer stem cells (CSCs. Normal breast epithelial cells that are CD44+/CD24- express higher levels of stem/progenitor cell associated genes. We, amongst others, have shown that cancer cells that have undergone epithelial to mesenchymal transition (EMT display the CD44+/CD24- phenotype. However, whether all genes that induce EMT confer the CD44+/CD24- phenotype is unknown. We hypothesized that only a subset of genes associated with EMT generates CD44+/CD24- cells. Methods MCF-10A breast epithelial cells, a subpopulation of which spontaneously acquire the CD44+/CD24- phenotype, were used to identify genes that are differentially expressed in CD44+/CD24- and CD44-/CD24+ cells. Ingenuity pathway analysis was performed to identify signaling networks that linked differentially expressed genes. Two EMT-associated genes elevated in CD44+/CD24- cells, SLUG and Gli-2, were overexpressed in the CD44-/CD24+ subpopulation of MCF-10A cells and MCF-7 cells, which are CD44-/CD24+. Flow cytometry and mammosphere assays were used to assess cell surface markers and stem cell-like properties, respectively. Results Two thousand thirty five genes were differentially expressed (p Conclusions EMT-mediated generation of CD44+/CD24- or CD44+/CD24+ cells depends on the genes that induce or are associated with EMT. Our studies reveal a role for TNF in altering the phenotype of breast CSC. Additionally, the CD44+/CD24+ phenotype, in the context of SLUG overexpression, can be associated with breast CSC "stemness" behavior based on mammosphere forming ability.

  14. ALDH/CD44 identifies uniquely tumorigenic cancer stem cells in salivary gland mucoepidermoid carcinomas.

    Science.gov (United States)

    Adams, April; Warner, Kristy; Pearson, Alexander T; Zhang, Zhaocheng; Kim, Hong Sun; Mochizuki, Daiki; Basura, Gregory; Helman, Joseph; Mantesso, Andrea; Castilho, Rogério M; Wicha, Max S; Nör, Jacques E

    2015-09-29

    A small sub-population of cells characterized by increased tumorigenic potential, ability to self-renew and to differentiate into cells that make up the tumor bulk, has been characterized in some (but not all) tumor types. These unique cells, namedcancer stem cells, are considered drivers of tumor progression in these tumors. The purpose of this work is to understand if cancer stem cells play a functional role in the tumorigenesis of salivary gland mucoepidermoid carcinomas. Here, we investigated the expression of putative cancer stem cell markers (ALDH, CD10, CD24, CD44) in primary human mucoepidermoid carcinomas by immunofluorescence, in vitro salisphere assays, and in vivo tumorigenicity assays in immunodeficient mice. Human mucoepidermoid carcinoma cells (UM-HMC-1, UM-HMC-3A, UM-HMC-3B) sorted for high levels of ALDH activity and CD44 expression (ALDHhighCD44high) consistently formed primary and secondary salispheres in vitro, and showed enhanced tumorigenic potential in vivo (defined as time to tumor palpability, tumor growth after palpability), when compared to ALDHlowCD44low cells. Cells sorted for CD10/CD24, and CD10/CD44 showed varying trends of salisphere formation, but consistently low in vivo tumorigenic potential. And finally, cells sorted for CD44/CD24 showed inconsistent results in salisphere formation and tumorigenic potential assays when different cell lines were evaluated. Collectively, these data demonstrate that salivary gland mucoepidermoid carcinomas contain a small population of cancer stem cells with enhanced tumorigenic potential and that are characterized by high ALDH activity and CD44 expression. These results suggest that patients with mucoepidermoid carcinoma might benefit from therapies that ablate these highly tumorigenic cells.

  15. Regulatory T Cells Resist Cyclosporine-Induced Cell Death via CD44-Mediated Signaling Pathways

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    Shannon M. Ruppert

    2015-01-01

    Full Text Available Cyclosporine A (CSA is an immunosuppressive agent that specifically targets T cells and also increases the percentage of pro-tolerogenic CD4+Foxp3+ regulatory T cells (Treg through unknown mechanisms. We previously reported that CD44, a receptor for the extracellular matrix glycosaminoglycan hyaluronan (HA, promotes Treg stability in IL-2-low environments. Here, we asked whether CD44 signaling also promotes Treg resistance to CSA. We found that CD44 cross-linking promoted Foxp3 expression and Treg viability in the setting of CSA treatment. This effect was IL-2 independent but could be suppressed using sc-355979, an inhibitor of Stat5-phosphorylation. Moreover, we found that inhibition of HA synthesis impairs Treg homeostasis but that this effect could be overcome with exogenous IL-2 or CD44-cross-linking. Together, these data support a model whereby CD44 cross-linking by HA promotes IL-2-independent Foxp3 expression and Treg survival in the face of CSA.

  16. Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

    Institute of Scientific and Technical Information of China (English)

    李中国; 张虹

    2004-01-01

    The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD4, specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

  17. Lung cancer tumorigenicity and drug resistance are maintained through ALDH(hi)CD44(hi) tumor initiating cells.

    Science.gov (United States)

    Liu, Jing; Xiao, Zhijie; Wong, Sunny Kit-Man; Tin, Vicky Pui-Chi; Ho, Ka-Yan; Wang, Junwen; Sham, Mai-Har; Wong, Maria Pik

    2013-10-01

    Limited improvement in long term survival of lung cancer patients has been achieved by conventional chemotherapy or targeted therapy. To explore the potentials of tumor initiating cells (TIC)-directed therapy, it is essential to identify the cell targets and understand their maintenance mechanisms. We have analyzed the performance of ALDH/CD44 co-expression as TIC markers and treatment targets of lung cancer using well-validated in vitro and in vivo analyses in multiple established and patient-derived lung cancer cells. The ALDH(hi)CD44(hi) subset showed the highest enhancement of stem cell phenotypic properties compared to ALDH(hi)CD44(lo), ALDH(lo)CD44(hi), ALDH(lo)CD44(lo) cells and unsorted controls. They showed higher invasion capacities, pluripotency genes and epithelial-mesenchymal transition transcription factors expression, lower intercellular adhesion protein expression and higher G2/M phase cell cycle fraction. In immunosuppressed mice, the ALDH(hi)CD44(hi)xenografts showed the highest tumor induction frequency, serial transplantability, shortest latency, largest volume and highest growth rates. Inhibition of sonic Hedgehog and Notch developmental pathways reduced ALDH+CD44+ compartment. Chemotherapy and targeted therapy resulted in higher AALDH(hi)CD44(hi) subset viability and ALDH(lo)CD44(lo) subset apoptosis fraction. ALDH inhibition and CD44 knockdown led to reduced stemness gene expression and sensitization to drug treatment. In accordance, clinical lung cancers containing a higher abundance of ALDH and CD44-coexpressing cells was associated with lower recurrence-free survival. Together, results suggested theALDH(hi)CD44(hi)compartment was the cellular mediator of tumorigenicity and drug resistance. Further investigation of the regulatory mechanisms underlying ALDH(hi)CD44(hi)TIC maintenance would be beneficial for the development of long term lung cancer control.

  18. CD44 plays a critical role in regulating diet-induced adipose inflammation, hepatic steatosis, and insulin resistance.

    Directory of Open Access Journals (Sweden)

    Hong Soon Kang

    Full Text Available CD44 is a multifunctional membrane receptor implicated in the regulation of several biological processes, including inflammation. CD44 expression is elevated in liver and white adipose tissue (WAT during obesity suggesting a possible regulatory role for CD44 in metabolic syndrome. To study this hypothesis, we examined the effect of the loss of CD44 expression on the development of various features of metabolic syndrome using CD44 null mice. Our study demonstrates that CD44-deficient mice (CD44KO exhibit a significantly reduced susceptibility to the development of high fat-diet (HFD-induced hepatic steatosis, WAT-associated inflammation, and insulin resistance. The decreased expression of genes involved in fatty acid synthesis and transport (Fasn and Cd36, de novo triglyceride synthesis (Mogat1, and triglyceride accumulation (Cidea, Cidec appears in part responsible for the reduced hepatic lipid accumulation in CD44KO(HFD mice. In addition, the expression of various inflammatory and cell matrix genes, including several chemokines and its receptors, osteopontin, and several matrix metalloproteinases and collagen genes was greatly diminished in CD44KO(HFD liver consistent with reduced inflammation and fibrogenesis. In contrast, lipid accumulation was significantly increased in CD44KO(HFD WAT, whereas inflammation as indicated by the reduced infiltration of macrophages and expression of macrophage marker genes, was significantly diminished in WAT of CD44KO(HFD mice compared to WT(HFD mice. CD44KO(HFD mice remained considerably more insulin sensitive and glucose tolerant than WT(HFD mice and exhibited lower blood insulin levels. Our study indicates that CD44 plays a critical role in regulating several aspects of metabolic syndrome and may provide a new therapeutic target in the management of insulin resistance.

  19. Expression of CD44 and P53 in renal cell carcinoma: Association with tumor subtypes

    Directory of Open Access Journals (Sweden)

    Farahnaz Noroozinia

    2014-01-01

    Full Text Available Renal cell carcinoma (RCC is a common malignancy of the kidney and accurate prediction of prognosis is valuable for the design of adjuvant therapy and counseling and effective scheduling of follow-up visits. Molecular genetic investigations of CD44 and P53 in RCC may be helpful in this regard. We studied the CD44 and P53 expressions semi-quantitatively on paraffin-embedded specimens of 64 RCC patients (37 male/27 female who underwent surgery from 2003 to 2008 by immunohistochemistry and analyzed the correlation of P53 and CD44 expression in RCC and outcome. Thirteen of 64 (20.3% specimens were P53 positive, 30/64 (46.9% were CD44 positive and five tumors with positive P53 expressed CD44 protein (P = 0.5. A statistically significant correlation was not found between CD44 and P53 expression (P = 0.5 and age (P = 0.07, sex (P= 0.3, tumor size (P = 0.7, grade (P = 0.23, vascular invasion (P = 1.00 and ureteral invasion (P = 1.00. Furthermore, a significant correlation was not found between P53 expression with age (P = 0.3, sex (P = 0.7, tumor size (P = 0.7, grade (P = 0.1, vascular inva-sion (P = 1.00 and ureteral invasion (P = 1.00. According to our findings, only P53 expression is generally accompanied by non-conventional subtype tumor.

  20. Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.

    Science.gov (United States)

    Ferrari, L F; Araldi, D; Bogen, O; Levine, J D

    2016-06-01

    We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation. PMID:26996509

  1. EXPRESSION AND CLINICAL SIGNIFICANCE OF CD44 IN THE PERIPHERAL BLOOD OF PATIENTS WITH CASTRIC CANCER

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective: To investigate the clinical significance of cell adhesive molecule (CD44) expression on periphery blood (PB) of patients with gastric cancer. Methods: Both the level of CD44 and the immunocyte phenotype of the lymphocytes of 110 patients with gastric cancer and 100 healthy subjects were examined by flow cytometry, and the results were analyzed pathologically and statistically. Results: The mean of the CD44% in PB of the healthy subjects was 46.14±13.4 and there were no statistic differences for their age and sex. Site of tumor growth: The significant difference (P 10 cm mass (P<0.0l) and 7-10 cm mass (P<0.05). Degree of tumor differentiation: The significant difference (P<0.01) was present between the patients with low differentiation gastric cancer and normal individuals. The significant difference (P<0.01) was present between patients with metastatic stage: The significant difference (P<0.0l) was present lymph node gastric cancer and normal individuals. Clinical between the patients with advanced or relapsed gastric cancer and normal individuals. Age: The significant difference (P<0.01) was present between the gastric cancer patients under 59 years and normal individuals. Conclusion: The increased level of CD44 in the PB of patients with gastric cancer indicated the possible existence of relapse, advance or metastasis of tumors. When the tumor was poorly differentiated and bigger in tumor mass, the level of CD44% would be higher. Examining the level of CD44 by flow cytometry in the periphery blood of patients with gastric cancer was useful for the prognosis.

  2. Tissue-specific promoters active in CD44+CD24-/low breast cancer cells.

    Science.gov (United States)

    Bauerschmitz, Gerd J; Ranki, Tuuli; Kangasniemi, Lotta; Ribacka, Camilla; Eriksson, Minna; Porten, Marius; Herrmann, Isabell; Ristimäki, Ari; Virkkunen, Pekka; Tarkkanen, Maija; Hakkarainen, Tanja; Kanerva, Anna; Rein, Daniel; Pesonen, Sari; Hemminki, Akseli

    2008-07-15

    It has been proposed that human tumors contain stem cells that have a central role in tumor initiation and posttreatment relapse. Putative breast cancer stem cells may reside in the CD44(+)CD24(-/low) population. Oncolytic adenoviruses are attractive for killing of these cells because they enter through infection and are therefore not susceptible to active and passive mechanisms that render stem cells resistant to many drugs. Although adenoviruses have been quite safe in cancer trials, preclinical work suggests that toxicity may eventually be possible with more active agents. Therefore, restriction of virus replication to target tissues with tissues-specific promoters is appealing for improving safety and can be achieved without loss of efficacy. We extracted CD44(+)CD24(-/low) cells from pleural effusions of breast cancer patients and found that modification of adenovirus type 5 tropism with the serotype 3 knob increased gene delivery to CD44(+)CD24(-/low) cells. alpha-Lactalbumin, cyclo-oxygenase 2, telomerase, and multidrug resistance protein promoters were studied for activity in CD44(+)CD24(-/low) cells, and a panel of oncolytic viruses was subsequently constructed. Each virus featured 5/3 chimerism of the fiber and a promoter controlling expression of E1A, which was also deleted in the Rb binding domain for additional tumor selectivity. Cell killing assays identified Ad5/3-cox2L-d24 and Ad5/3-mdr-d24 as the most active agents, and these viruses were able to completely eradicate CD44(+)CD24(-/low) cells in vitro. In vivo, these viruses had significant antitumor activity in CD44(+)CD24(-/low)-derived tumors. These findings may have relevance for elimination of cancer stem cells in humans. PMID:18632604

  3. Study on the Relationship between P-glycoprotein and CD44 Expression in Gastric Carcinoma

    Institute of Scientific and Technical Information of China (English)

    YU Chuanding; XU Shenhua; LING Yutian; ZHU Chihong; ZHOU Xinming; LIU Xianglin

    2006-01-01

    Objective: Investigation of the relationship between P-glycoprotein (P-gp) and adhesion molecule CD44 as well as their clinical significance in gastric carcinoma. Methods: To examine the expressed level of P-gp and CD44 in 98 cases with gastric carcinoma by flow cytometry and evaluate their relationships with clinicopathological factors. Results: Among the 98 gastric carcinomas, 40 cases (40.8%)were P-gp negative (positive cells <25%); 14 cases (14.2%) were 25%-40% expression of P-gp positive cells;17 cases (17.3%) were 41%-60% expression of P-gp positive cells; 27 cases (27.5%) were the high expression(positive cells >60%) of P-gp in all patients with gastric carcinoma. When the tumor sizes were more than6 cm, the P-gp positive of CD44 showed a significant difference (P<0.05) in 35 cases with P-gp positive,compared with it in 24 cases with P-gp negative. When the tumors were in low-moderate differentiated gastric carcinoma, the expression of CD44 showed a significant difference (P<0.05) in 44 cases with P-gp positive, as compared with it in 30 cases with P-gp negative. When the patients were in clinical Ⅲ-Ⅳ stage, the expression of CD44 showed a significant difference (P<0.05) in 42 cases with P-gp positive, as compared with it in 30 cases with P-gp negative. When the patients with lymph node metastasis, their CD44 expression showed a significant difference (P<0.05) in 46 cases with P-gp positive, compared with it in 32 cases with P-gp negative. When the tumors P-gp expressed positive, their CD44 expression will be increase. Conclusion: When the CD44 and P-gp both have the positive high expression, it will be significantly associated with the gastric carcinoma progression and metastasis, so both were a positive expression in gastric carcinoma, it might suggest a poor and unfavorable prognosis result.

  4. Expression of CD44v6 and Livin in gastric cancer tissue

    Institute of Scientific and Technical Information of China (English)

    LIANG Yi-zhi; FANG Tai-yong; XU Hai-gang; ZHUO Zhi-qiang

    2012-01-01

    Background CD44v6 plays an important role in invasion and metastasis of tumor,Livin has anti-apoptotic effects.The present study aimed to explore the expression and clinical significance of CD44v6 and Livin in gastric cancer tissue.Methods Streptavidin-peroxidase linked immunohistochemical method was used to determine the expression of CD44v6 and Livin in gastric cancer tissue and adjacent normal gastric tissues from 59 patients with histopathologically confirmed gastric cancer,and in gastric tissue specimens of 15 patients with gastric polyps,and 15 patients with chronic non-atrophic gastritis.The chi-square test was used for comparison of the relevant factors,Spearman's rank correlation test was applied for relationship among positive expression of the proteins.Results The expresion of CD44v6 was positive in 64.4% of the gastric cancer patients; 5.1%,0 and 13.3% in specimens of normal tissues adjacent to the cancer tissues,in gastric tissue specimens of patients with gastric polyps,and patients with chronic non-atrophic gastritis,respectively.The expression of Livin was positive in 52.5% of the gastric cancer tissues,6.8%,0 and 6.7% in the adjacent normal gastric tissue,specimens of patients with gastric polyps and chronic non-atrophic gastritis,respectively.The expression of CD44v6 was significantly correlated with the depth of invasion,the degree of differentiation,and lymphnode metastasis of gastric cancer (P <0.05).The positive expression rate of Livin protein was also significantly correlated with degree of differentiation of gastric cancer cells and metastasis to lymphnodes (P <0.05),but not correlated with the depth of invasion and pathological types (P >0.05).The expression of CD44v6 and Livin in the gastric cancer tissue was positively correlated (rs=0.286,P=-0.028).Conclusions The increased expression of CD44v6 and Livin in gastric cancer tissue may be closely related with development and progression of gastric cancer.CD44v6 and Livin

  5. CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation.

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    Nina Bertaux-Skeirik

    2015-02-01

    Full Text Available The cytotoxin-associated gene (Cag pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (∆CagA::cat. Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ∆CagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylori that was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the well-established Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique

  6. Identification of a novel splice variant of human PD-L1 Mrna encoding an isoform-lacking Igv-like domain

    Institute of Scientific and Technical Information of China (English)

    Xian-hui HE; Li-hui XU; Yi LIU

    2005-01-01

    Aim: To investigate the expression and regulation of PD-1 ligand 1 (PD-L1) in peripheral blood mononuclear cells (PBMC). Methods: The cDNA encoding human PD-L1 precursor was cloned from the total RNA extracted from the resting and phorbol dibutyrate plus ionomycin- or phytohemagglutinin-activated PBMC, by reverse transcription polymerase chain reaction (RT-PCR), and independent clones were sequenced and analyzed. The expression and subcellular localization were examined in transiently transfected cells. The PD-L1 gene expression in different PBMC was also analyzed by RT-PCR. Results: A novel human PD-L1 splice variant was identified from the activated PBMC. It was generated by splicing out exon 2 encoding an immunoglobulin variable domain (Igv)-like domain but retaining all other exons without a frame-shift. Consequently, the putative translated protein contained all other domains including the transmembrane region except for the Igv-like domain. Furthermore, the conventional isoform was expressed on the plasma surface whereas the novel isoform showed a pattern of intmcellular membrane distribution in transiently transfected K562 cells. In addition, the expression pattern of the PD-L1 splice variant was variable in different individuals and in different cellular status. Conclusion: PD-L1 expression may be regulated at the posttranscriptional level through alternative splicing, and modulation of the PD-L1 isoform expression may influence the outcome of specific immune responses in the peripheral tissues.

  7. Application of Collagen-Model Triple-Helical Peptide-Amphiphiles for CD44-Targeted Drug Delivery Systems

    OpenAIRE

    Ndinguri, Margaret W.; Alexander Zheleznyak; Lauer, Janelle L.; Anderson, Carolyn J.; Fields, Gregg B.

    2012-01-01

    Cancer treatment by chemotherapy is typically accompanied by deleterious side effects, attributed to the toxic action of chemotherapeutics on proliferating cells from nontumor tissues. The cell surface proteoglycan CD44 has been recognized as a cancer stem cell marker. The present study has examined CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44/chondroitin sulfate proteoglycan binds to a triple-helical sequence derived f...

  8. Impacts of CD44 knockdown in cancer cells on tumor and host metabolic systems revealed by quantitative imaging mass spectrometry.

    Science.gov (United States)

    Ohmura, Mitsuyo; Hishiki, Takako; Yamamoto, Takehiro; Nakanishi, Tsuyoshi; Kubo, Akiko; Tsuchihashi, Kenji; Tamada, Mayumi; Toue, Sakino; Kabe, Yasuaki; Saya, Hideyuki; Suematsu, Makoto

    2015-04-30

    CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue. PMID:25461272

  9. A bioengineered murine model using CD24+CD44+ pancreatic cancer stem cells for chemotherapy study

    International Nuclear Information System (INIS)

    In this work we first developed a murine pancreatic tumor model using CD24+CD44+ pancreatic cancer stem cells (CSC) supported by an electrospun scaffold. Unlike conventional models, the use of CSC and the scaffold, which were biologically and chemically defined, afforded scientists a reliable platform to evaluate novel chemotherapy regimens. CD24+CD44+ CSC successfully initiated tumorigenesis in vitro on the scaffold without suffering apoptosis, evidencing the lack of cytotoxicity of scaffolding materials. Also, the scaffold contributed to the acceleration of in vivo tumorigenesis and increased the likelihood of tumor formation. Using this model, we set out to explore the effectiveness of irinotecan/gemcitabine (IRIN-GEM), a chemotherapy regimen, for pancreatic cancer. Our study showed that IRIN-GEM induced a tumor regression whereas gemcitabine alone could only arrest the tumor growth. Further study suggested that the superior performance of IRIN-GEM could be attributed to its capacity to demolish the CD24+CD44+ CSC sub-population by inducing a large-scale apoptosis. The use of highly proliferative yet homogenous CD24+CD44+ CSC along with a chemically defined scaffold accelerated the tumor formation and significantly reduced the variability associated with conventional murine models. Armed with this new model, we discovered that IRIN-GEM would be a promising chemotherapy candidate for patients with advanced pancreatic cancer. (paper)

  10. Osteoactivin inhibition of osteoclastogenesis is mediated through CD44-ERK signaling.

    Science.gov (United States)

    Sondag, Gregory R; Mbimba, Thomas S; Moussa, Fouad M; Novak, Kimberly; Yu, Bing; Jaber, Fatima A; Abdelmagid, Samir M; Geldenhuys, Werner J; Safadi, Fayez F

    2016-01-01

    Osteoactivin is a heavily glycosylated protein shown to have a role in bone remodeling. Previous studies from our lab have shown that mutation in Osteoactivin enhances osteoclast differentiation but inhibits their function. To date, a classical receptor and a signaling pathway for Osteoactivin-mediated osteoclast inhibition has not yet been characterized. In this study, we examined the role of Osteoactivin treatment on osteoclastogenesis using bone marrow-derived osteoclast progenitor cells and identify a signaling pathway relating to Osteoactivin function. We reveal that recombinant Osteoactivin treatment inhibited osteoclast differentiation in a dose-dependent manner shown by qPCR, TRAP staining, activity and count. Using several approaches, we show that Osteoactivin binds CD44 in osteoclasts. Furthermore, recombinant Osteoactivin treatment inhibited ERK phosphorylation in a CD44-dependent manner. Finally, we examined the role of Osteoactivin on receptor activator of nuclear factor-κ B ligand (RANKL)-induced osteolysis in vivo. Our data indicate that recombinant Osteoactivin inhibits RANKL-induced osteolysis in vivo and this effect is CD44-dependent. Overall, our data indicate that Osteoactivin is a negative regulator of osteoclastogenesis in vitro and in vivo and that this process is regulated through CD44 and ERK activation. PMID:27585719

  11. Mitogen-activated protein kinases mediate Mycobacterium tuberculosis–induced CD44 surface expression in monocytes

    Indian Academy of Sciences (India)

    Natarajan Palaniappan; S Anbalagan; Sujatha Narayanan

    2012-03-01

    CD44, an adhesion molecule, has been reported to be a binding site for Mycobacterium tuberculosis (M. tuberculosis) in macrophages and it also mediates mycobacterial phagocytosis, macrophage recruitment and protective immunity against pulmonary tuberculosis in vivo. However, the signalling pathways that are involved in M. tuberculosis–induced CD44 surface expression in monocytic cells are currently unknown. Exposure of THP-1 human monocytes to M. tuberculosis H37Rv and H37Ra induced distinct, time-dependent, phosphorylation of mitogen-activated protein kinase kinase-1, extracellular signal regulated kinase 1/2, mitogen-activated protein kinase kinase 3/6, p38 mitogen-activated protein kinase and c-jun N-terminal kinases. The strains also differed in their usage of CD14 and human leukocyte antigen-DR (HLA-DR) receptors in mediating mitogen-activated protein kinase activation. M. tuberculosis H37Rv strain induced lower CD44 surface expression and tumour necrosis factor-alpha levels, whereas H37Ra the reverse. Using highly specific inhibitors of mitogen-activated protein kinase kinase-1, p38 mitogen-activated protein kinase and c-jun N-terminal kinase, we report that inhibition of extracellular signal regulated kinase 1/2 and c-jun N-terminal kinases increases, but that inhibition of p38 mitogen-activated protein kinase decreases M. tuberculosis–induced CD44 surface expression in THP-1 human monocytes.

  12. BRCA1 Protein Expression Level and CD44+ Phenotype in Breast Cancer Patients

    Directory of Open Access Journals (Sweden)

    Saadat Molanae

    2011-01-01

    Full Text Available Objective: CD44+/CD24-/low breast cancer cells have tumour-initiating properties with stemcell-like features. Breast cancer gene 1 (BRCA1 is a tumour suppressor gene that playsa crucial role in DNA repair and maintenance of chromosome stability. The clinicopathologicalfeatures of breast cancer in BRCA1 mutation carriers suggest that BRCA1 mayfunction as a stem-cell regulator.Materials and Methods: In the present experimental study we examined the expressionand localization of the BRCA1 protein and investigated the prognostic value aswell as its relationship with the putative cancer stem cell (CSC marker (CD44 in 156tumour samples from a well-characterized series of unselected breast carcinomas usingimmunohistochemistry. Statistical analysis of the data was performed using SPSSsoftware version 16 (Chicago, IL, USA.Results: In breast tumours, the loss of nuclear expression was detected in 23 cases(15%, whereas cytoplasmic expression of BRCA1 was observed in 133 breast carcinomas(85%. Altered BRCA1 expression was significantly associated with high grade and poorprognosis breast tumours (p=0.006. We further established an inverse significant correlationbetween BRCA1 expression levels and CD44+ cancer cell phenotype (p=0.02Conclusion: Loss of BRCA1 expression is a marker of tumour aggressiveness andcorrelates with CD44+ tumour cell phenotype. Taken together, the present study supportsthe idea that the loss of BRCA1 results in persistent errors in DNA replication inbreast stem cells and provides targets for additional carcinogenic events.

  13. Rb suppresses collective invasion, circulation and metastasis of breast cancer cells in CD44-dependent manner.

    Directory of Open Access Journals (Sweden)

    Kui-Jin Kim

    Full Text Available Basal-like breast carcinomas (BLCs present with extratumoral lymphovascular invasion, are highly metastatic, presumably through a hematogenous route, have augmented expression of CD44 oncoprotein and relatively low levels of retinoblastoma (Rb tumor suppressor. However, the causal relation among these features is not clear. Here, we show that Rb acts as a key suppressor of multiple stages of metastatic progression. Firstly, Rb suppresses collective cell migration (CCM and CD44-dependent formation of F-actin positive protrusions in vitro and cell-cluster based lymphovascular invasion in vivo. Secondly, Rb inhibits the release of single cancer cells and cell clusters into the hematogenous circulation and subsequent metastatic growth in lungs. Finally, CD44 expression is required for collective motility and all subsequent stages of metastatic progression initiated by loss of Rb function. Altogether, our results suggest that Rb/CD44 pathway is a crucial regulator of CCM and metastatic progression of BLCs and a promising target for anti-BLCs therapy.

  14. Hyaluronic acid-conjugated liposome nanoparticles for targeted delivery to CD44 overexpressing glioblastoma cells

    Science.gov (United States)

    Hayward, Stephen L.; Wilson, Christina L.; Kidambi, Srivatsan

    2016-01-01

    Glioblastoma Multiforme (GBM) is a highly prevalent and deadly brain malignancy characterized by poor prognosis and restricted disease management potential. Despite the success of nanocarrier systems to improve drug/gene therapy for cancer, active targeting specificity remains a major hurdle for GBM. Additionally, since the brain is a multi-cell type organ, there is a critical need to develop an approach to distinguish between GBM cells and healthy brain cells for safe and successful treatment. In this report, we have incorporated hyaluronic acid (HA) as an active targeting ligand for GBM. To do so, we employed HA conjugated liposomes (HALNPs) to study the uptake pathway in key cells in the brain including primary astrocytes, microglia, and human GBM cells. We observed that the HALNPs specifically target GBM cells over other brain cells due to higher expression of CD44 in tumor cells. Furthermore, CD44 driven HALNP uptake into GBM cells resulted in lysosomal evasion and increased efficacy of Doxorubicin, a model anti-neoplastic agent, while the astrocytes and microglia cells exhibited extensive HALNP-lysosome co-localization and decreased antineoplastic potency. In summary, novel CD44 targeted lipid based nanocarriers appear to be proficient in mediating site-specific delivery of drugs via CD44 receptors in GBM cells, with an improved therapeutic margin and safety. PMID:27120809

  15. Aggravation of Allergic Airway Inflammation by Cigarette Smoke in Mice Is CD44-Dependent.

    Directory of Open Access Journals (Sweden)

    Smitha Kumar

    Full Text Available Although epidemiological studies reveal that cigarette smoke (CS facilitates the development and exacerbation of allergic asthma, these studies offer limited information on the mechanisms involved. The transmembrane glycoprotein CD44 is involved in cell adhesion and acts as a receptor for hyaluronic acid and osteopontin. We aimed to investigate the role of CD44 in a murine model of CS-facilitated allergic airway inflammation.Wild type (WT and CD44 knock-out (KO mice were exposed simultaneously to house dust mite (HDM extract and CS. Inflammatory cells, hyaluronic acid (HA and osteopontin (OPN levels were measured in bronchoalveolar lavage fluid (BALF. Proinflammatory mediators, goblet cell metaplasia and peribronchial eosinophilia were assessed in lung tissue. T-helper (Th 1, Th2 and Th17 cytokine production was evaluated in mediastinal lymph node cultures.In WT mice, combined HDM/CS exposure increased the number of inflammatory cells and the levels of HA and OPN in BALF and Th2 cytokine production in mediastinal lymph nodes compared to control groups exposed to phosphate buffered saline (PBS/CS, HDM/Air or PBS/Air. Furthermore, HDM/CS exposure significantly increased goblet cell metaplasia, peribronchial eosinophilia and inflammatory mediators in the lung. CD44 KO mice exposed to HDM/CS had significantly fewer inflammatory cells in BALF, an attenuated Th2 cytokine production, as well as decreased goblet cells and peribronchial eosinophils compared to WT mice. In contrast, the levels of inflammatory mediators were similar or higher than in WT mice.We demonstrate for the first time that the aggravation of pulmonary inflammation upon combined exposure to allergen and an environmental pollutant is CD44-dependent. Data from this murine model of concomitant exposure to CS and HDM might be of importance for smoking allergic asthmatics.

  16. The Role of Hyaluronan and CD44 in the Pathogenesis of Lupus Nephritis

    Directory of Open Access Journals (Sweden)

    Susan Yung

    2012-01-01

    Full Text Available Systemic lupus erythematosus (SLE is a prototype autoimmune disease that affects multiorgan systems. Lupus nephritis is one of the most severe manifestations of SLE whereby immune-mediated inflammation can lead to permanent damage within the glomerular, tubulo-interstitial, and vascular compartments of the kidney, resulting in acute or chronic renal failure. The mechanisms that regulate host inflammatory responses and tissue injury are incompletely understood. Accumulating evidence suggests that hyaluronan and its interaction with its cell surface receptor CD44 plays an important role in mediating pathogenic mechanisms in SLE. This paper discusses the putative mechanisms through which hyaluronan and CD44 contribute to the pathogenesis of SLE, with particular emphasis on lupus nephritis.

  17. Preparation and reactivities of anti-porcine CD44 monoclonal antibodies.

    Science.gov (United States)

    Yang, H; Hutchings, A; Binns, R M

    1993-04-01

    Five monoclonal antibodies (MoAbs) were raised against porcine soluble CD44. The MoAbs recognized the same antigen on the surface of porcine lymphocytes as was recognized by anti-human CD44 MoAb Hermes-1, but identified five different epitopes. They bound to most porcine leucocytes but not to red cells. The epitopes were susceptible to treatment with papain or bromelain, whereas trypsinization of porcine leucocytes only reduced the antigen density. The epitopes seem to be co-expressed among various lymphoid tissues. The MoAbs also cross-reacted to various degrees with leucocytes of humans, dogs, sheep, cattle, goats and horses, suggesting that the corresponding epitopes are differentially conserved among species.

  18. Hyaluronic acid modified mesoporous carbon nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells

    Science.gov (United States)

    Wan, Long; Jiao, Jian; Cui, Yu; Guo, Jingwen; Han, Ning; Di, Donghua; Chang, Di; Wang, Pu; Jiang, Tongying; Wang, Siling

    2016-04-01

    In this paper, hyaluronic acid (HA) functionalized uniform mesoporous carbon spheres (UMCS) were synthesized for targeted enzyme responsive drug delivery using a facile electrostatic attraction strategy. This HA modification ensured stable drug encapsulation in mesoporous carbon nanoparticles in an extracellular environment while increasing colloidal stability, biocompatibility, cell-targeting ability, and controlled cargo release. The cellular uptake experiments of fluorescently labeled mesoporous carbon nanoparticles, with or without HA functionalization, demonstrated that HA-UMCS are able to specifically target cancer cells overexpressing CD44 receptors. Moreover, the cargo loaded doxorubicin (DOX) and verapamil (VER) exhibited a dual pH and hyaluronidase-1 responsive release in the tumor microenvironment. In addition, VER/DOX/HA-UMCS exhibited a superior therapeutic effect on an in vivo HCT-116 tumor in BALB/c nude mice. In summary, it is expected that HA-UMCS will offer a new method for targeted co-delivery of drugs to tumors overexpressing CD44 receptors.

  19. Whole-genome approach implicates CD44 in cellular resistance to carboplatin

    Directory of Open Access Journals (Sweden)

    Shukla Sunita J

    2009-01-01

    Full Text Available Abstract Carboplatin is a chemotherapeutic agent used in the management of many cancers, yet treatment is limited by resistance and toxicities. To achieve a better understanding of the genetic contribution to carboplatin resistance or toxicities, lymphoblastoid cell lines from 34 large Centre d'Etude du Polymorphisme Humain pedigrees were utilised to evaluate interindividual variation in carboplatin cytotoxicity. Significant heritability, ranging from 0.17-0.36 (p = 1 × 10-7 to 9 × 10-4, was found for cell growth inhibition following 72-hour treatment at each carboplatin concentration (10, 20, 40 and 80 μM and IC50 (concentration for 50 per cent cell growth inhibition. Linkage analysis revealed 11 regions with logarithm of odds (LOD scores greater than 1.5. The highest LOD score on chromosome 11 (LOD = 3.36, p = 4.2 × 10-5 encompasses 65 genes within the 1 LOD confidence interval for the carboplatin IC50. We further analysed the IC50 phenotype with a linkage-directed association analysis using 71 unrelated HapMap and Perlegen cell lines and identified 18 single nucleotide polymorphisms within eight genes that were significantly associated with the carboplatin IC50 (p -5; false discovery rate 50 values of the eight associated genes, which identified the most significant correlation between CD44 expression and IC50 (r2 = 0.20; p = 6 × 10-4. The quantitative real-time polymerase chain reaction further confirmed a statistically significant difference in CD44 expression levels between carboplatin-resistant and -sensitive cell lines (p = 5.9 × 10-3. Knockdown of CD44 expression through small interfering RNA resulted in increased cellular sensitivity to carboplatin (p CD44 as being important in conferring cellular resistance to carboplatin.

  20. Chronic UVR Causes Increased Immunostaining of CD44 and Accumulation of Hyaluronan in Mouse Epidermis

    OpenAIRE

    Siiskonen, Hanna; Törrönen, Kari; Kumlin, Timo; Rilla, Kirsi; Tammi, Markku I; Tammi, Raija H.

    2011-01-01

    Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure proto...

  1. Lipid Raft-Mediated Regulation of Hyaluronan–CD44 Interactions in Inflammation and Cancer

    OpenAIRE

    Murai, Toshiyuki

    2015-01-01

    Hyaluronan is a major component of the extracellular matrix and plays pivotal roles in inflammation and cancer. Hyaluronan oligomers are frequently found in these pathological conditions, in which they exert their effects via association with the transmembrane receptor CD44. Lipid rafts are cholesterol- and glycosphingolipid-enriched membrane microdomains that may regulate membrane receptors while serving as platforms for transmembrane signaling at the cell surface. This article focuses on th...

  2. The hyaluronan receptor (CD44) participates in the uptake and degradation of hyaluronan

    OpenAIRE

    1992-01-01

    The hyaluronan receptor belongs to the polymorphic family of CD44 glycoproteins, which have been implicated in a variety of cellular functions including adhesion to hyaluronan and collagen, the binding of lymphocytes to high endothelial cells during extravasation, and conferring metastatic potential to carcinoma cells. Here, we demonstrate that the receptor also participates in the uptake and degradation of hyaluronan by both transformed fibroblasts (SV-3T3 cells) and alveolar macrophages. Th...

  3. Chondroprotective Effect of Kartogenin on CD44-Mediated Functions in Articular Cartilage and Chondrocytes

    OpenAIRE

    Ono, Yohei; Ishizuka, Shinya; Knudson, Cheryl B.; Knudson, Warren

    2014-01-01

    Objective: A recent report identified the small molecule kartogenin as a chondrogenic and chondroprotective agent. Since changes in hyaluronan metabolism occur during cartilage degeneration in osteoarthritis, we began studies to determine whether there was a connection between extracellular hyaluronan, CD44–hyaluronan interactions and the effects of kartogenin on articular chondrocytes. Methods: Chondrocytes cultured in monolayers, bioengineered neocartilages, or cartilage explants were treat...

  4. WNT5A inhibits metastasis and alters splicing of Cd44 in breast cancer cells.

    Directory of Open Access Journals (Sweden)

    Wen Jiang

    Full Text Available Wnt5a is a non-canonical signaling Wnt. Low expression of WNT5A is correlated with poor prognosis in breast cancer patients. The highly invasive breast cancer cell lines, MDA-MB-231 and 4T1, express very low levels of WNT5A. To determine if enhanced expression of WNT5A would affect metastatic behavior, we generated WNT5A expressing cells from the 4T1 and MDA-MB-231 parental cell lines. WNT5A expressing cells demonstrated cobblestone morphology and reduced in vitro migration relative to controls. Cell growth was not altered. Metastasis to the lung via tail vein injection was reduced in the 4T1-WNT5A expressing cells relative to 4T1-vector controls. To determine the mechanism of WNT5A action on metastasis, we performed microarray and whole-transcriptome sequence analysis (RNA-seq to compare gene expression in 4T1-WNT5A and 4T1-vector cells. Analysis indicated highly significant alterations in expression of genes associated with cellular movement. Down-regulation of a subset of these genes, Mmp13, Nos2, Il1a, Cxcl2, and Lamb3, in WNT5A expressing cells was verified by semi-quantitative RT-PCR. Significant differences in transcript splicing were also detected in cell movement associated genes including Cd44. Cd44 is an adhesion molecule with a complex genome structure. Variable exon usage is associated with metastatic phenotype. Alternative spicing of Cd44 in WNT5A expressing cells was confirmed using RT-PCR. We conclude that WNT5A inhibits metastasis through down-regulation of multiple cell movement pathways by regulating transcript levels and splicing of key genes like Cd44.

  5. Prognostic evaluation of CD44 expression in correlation with bcl-2 and p53 in colorectal cancer

    Directory of Open Access Journals (Sweden)

    G. Peros

    2011-08-01

    Full Text Available To investigate the expression of CD44 in colorectal cancer and examine its association with clinicopathological features, bcl-2, p53 and long-term outcome, paraffin-embedded tumour specimens from 61 patients with Dukes stage B (AJCC/UICC stage I and 39 patients with Dukes stage C (AJCC/UICC stage III colorectal adenocarcinoma were assessed by immunohistochemistry. The expression of CD44, bcl-2 and p53 were correlated with 5-year follow-up. Low CD44 expression was present in 30%, moderate in 30% and extensive in 40% of cases. It was not related to patient sex and age but was related to tumour differentiation, stage and tumour site. No association was demonstrated between CD44 and bcl-2. However, there was significant evidence of an association between CD44 and p53 in 66 cases in which p53 was previously assessed. There was a trend towards increased survival in patients whose tumours expressed lower levels of CD44 protein. When entered into multivariate analysis model, which also included bcl-2 and p53, CD44 staining emerged as an indicator of poor prognosis in colorectal cancer patients.

  6. Aqueous humor level of sCD44 in patients with degenerative myopia and primary open-angle glaucoma

    Directory of Open Access Journals (Sweden)

    Akdogan Muberra

    2009-11-01

    Full Text Available Abstract Background The transmembrane glycoprotein CD44 is a major hyaluronan cell surface receptor widely distributed in eye tissues and fluids. The shed ectodomain of CD44 is termed soluble CD44 and is toxic to human retinal ganglion cells in cell culture. The purpose of this study was to investigate the concentration of sCD44 in the aqueous humor (AH of normal subjects, patients with primary open-angle glaucoma, and patients with degenerative myopia but without glaucoma, to determine if the molecule might serve as a protein marker of glaucoma. Findings In this case-control study, AH samples were collected from controls (n = 16, patients with primary open-angle glaucoma (n = 11, and patients with degenerative myopia (n = 11 who underwent phacoemulsification surgery to treat mature or immature cataracts. The sCD44 concentration in AH was measured using a commercial ELISA kit. In normal AH samples the sCD44 concentration was 5.40 ± 1.21 ng/mL, whereas in degenerative myopia patients the sCD44 concentration was 5.76 ± 1.15 ng/mL. There was thus no statistically significant difference between these two groups (p > 0.05. The aqueous sCD44 concentration in patients with primary open-angle glaucoma (12.2 ± 10.1 ng/mL was higher than that of the control group (p Conclusion sCD44 may be a protein marker of primary open-angle glaucoma.

  7. Expression and Significance of CD44, CD47 and c-met in Ovarian Clear Cell Carcinoma

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    Huimin Wang

    2015-02-01

    Full Text Available Aims: The aim of the present study is to investigate the differential expression of CD44, CD47 and c-met in ovarian clear cell carcinoma (OCCC, the correlation in their expression and their relationship with the biological behavior of OCCC. Methods: We used immunohistochemistry to examine the expression of CD44, CD47 and c-met in OCCC (86 cases and investigated the effects of the expression and interaction of these molecules on the development of OCCC. Results: CD44, CD47 and c-met expression was significantly high in OCCC. Expression of CD44 and CD47 correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05, and expression of c-met correlated with chemotherapy resistance and prognosis (all p < 0.05, but did not correlate with lymph node metastasis (all p > 0.05. The surgical stage, CD44, CD47 and c-met expression were independent risk factors for OCCC prognosis (all p < 0.05. Patients with low levels of CD44, CD47 and c-met showed better survival than those with high levels (all p < 0.05. There was a positive correlation between CD44 (or CD47 and c-met, as well as between CD44 and CD47 (the Spearman correlation coefficient rs was 0.783, 0.776 and 0.835, respectively, all p < 0.01. Additionally, pairwise correlation analysis of these three markers shows that the high expression of CD44/CD47, CD44/c-met and CD47/c-met were correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05, but did not correlate with lymph node metastasis (all p > 0.05. Conclusions: Expression of CD44, CD47 and c-met was upregulated in OCCC and pairwise correlation. CD44, CD47 and c-met may have synergistic effects on the development of OCCC and are prognostic factors for ovarian cancer.

  8. Prognostic evaluation of CD44 expression in correlation with bcl-2 and p53 in colorectal cancer

    OpenAIRE

    Peros, G; Elemenoglou, I.; G. Athanasas; D. Panousopoulos; A. Zizi-Sermpetzoglou; H.N. Zavrides

    2011-01-01

    To investigate the expression of CD44 in colorectal cancer and examine its association with clinicopathological features, bcl-2, p53 and long-term outcome, paraffin-embedded tumour specimens from 61 patients with Dukes stage B (AJCC/UICC stage I) and 39 patients with Dukes stage C (AJCC/UICC stage III) colorectal adenocarcinoma were assessed by immunohistochemistry. The expression of CD44, bcl-2 and p53 were correlated with 5-year follow-up. Low CD44 expression was present in 30%, moderate in...

  9. Lipid-based nanosystems for CD44 targeting in cancer treatment: recent significant advances, ongoing challenges and unmet needs.

    Science.gov (United States)

    Nascimento, Thais Leite; Hillaireau, Hervé; Vergnaud, Juliette; Fattal, Elias

    2016-07-01

    Extensive experimental evidence demonstrates the important role of hyaluronic acid (HA)-CD44 interaction in cell proliferation and migration, inflammation and tumor growth. Taking advantage of this interaction, the design of HA-modified nanocarriers has been investigated for targeting CD44-overexpressing cells with the purpose of delivering drugs to cancer or inflammatory cells. The effect of such modification on targeting efficacy is influenced by several factors. In this review, we focus on the impact of HA-modification on the characteristics of lipid-based nanoparticles. We try to understand how these modifications influence particle physicochemical properties, interaction with CD44 receptors, intracellular trafficking pathways, toxicity, complement/macrophage activation and pharmacokinetics. Our aim is to provide insight in tailoring particle modification by HA in order to design more efficient CD44-targeting lipid nanocarriers.

  10. CD44v6 in peripheral blood and bone marrow of patients with gastric cancer as micro-metastasis

    Institute of Scientific and Technical Information of China (English)

    Dao-Rong Wang; Guo-Yu Chen; Xun-Liang Liu; Yi Miao; Jian-Guo Xia; Lin-Hai Zhu; Dong Tang

    2006-01-01

    AIM: To detect the expression of CD44 correlated with the ability of micro-metastasis in peripheral blood and bone marrow of patients with gastric cancer and to deduce its clinical significance.METHODS: Preoperative peripheral blood and bone marrow specimens from 46 patients with gastric cancer and 6 controls were studied by semi-quantitative RTPCR amplification of CD44v6mRNA. Preoperative and postoperative peripheral blood specimens from 40patients with gastric cancer and 14 controls were studied by quantitative RT-PCR amplification of CD44v6mRNA in the corresponding period.RESULTS: Semi-quantitative RT-PCR amplification showed that CD44v6mRNA expression of peripheral blood and bone marrow was positive in 39 (84.8%)and 40 (86.9%) of 46 patients with gastric cancer,respectively. In peripheral blood, CD44v6mRNA expression was positive for diffuse type in 30 (93.8%)of 32 patients and for intestinal type in 9 (64.3%)of 14 patients. On the other hand, in bone marrow,CD44v6mRNA expression was positive for diffuse type in 31 (96.9%) of 32 patients and for intestinal type in 10 (71.4%) of 14 patients. There was a significant difference between the diffuse type and intestinal type.Quantitative RT-PCR amplification demonstrated that CD44v6mRNA was not expressed in the peripheral blood of controls and CD44v6mRNA expression was positive for preoperative peripheral blood in 40 patients with gastric cancer, the expression levels being from 4.9×102 to 3.2×105 copies/g RNA. The average expression level of CD44v6mRNA in peripheral blood was 3.9×1010copies/g RNA. The expression levels of CD44v6mRNA in peripheral blood in gastric cancer patients after curative operation increased from 5.5×100 to 7.6×10copies/g RNA (P=0.00496). After curative operation, the expression level decreased markedly.CONCLUSION: Semi-quantitative and quantitative RTPCR amplification for CD44v6mRNA is a sensitive and specific method for the detection of micro-metastasis in peripheral blood and bone

  11. CD44 Antibody Inhibition of Macrophage Phagocytosis Targets Fcγ Receptor- and Complement Receptor 3-Dependent Mechanisms.

    Science.gov (United States)

    Amash, Alaa; Wang, Lin; Wang, Yawen; Bhakta, Varsha; Fairn, Gregory D; Hou, Ming; Peng, Jun; Sheffield, William P; Lazarus, Alan H

    2016-04-15

    Targeting CD44, a major leukocyte adhesion molecule, using specific Abs has been shown beneficial in several models of autoimmune and inflammatory diseases. The mechanisms contributing to the anti-inflammatory effects of CD44 Abs, however, remain poorly understood. Phagocytosis is a key component of immune system function and can play a pivotal role in autoimmune states where CD44 Abs have shown to be effective. In this study, we show that the well-known anti-inflammatory CD44 Ab IM7 can inhibit murine macrophage phagocytosis of RBCs. We assessed three selected macrophage phagocytic receptor systems: Fcγ receptors (FcγRs), complement receptor 3 (CR3), and dectin-1. Treatment of macrophages with IM7 resulted in significant inhibition of FcγR-mediated phagocytosis of IgG-opsonized RBCs. The inhibition of FcγR-mediated phagocytosis was at an early stage in the phagocytic process involving both inhibition of the binding of the target RBC to the macrophages and postbinding events. This CD44 Ab also inhibited CR3-mediated phagocytosis of C3bi-opsonized RBCs, but it did not affect the phagocytosis of zymosan particles, known to be mediated by the C-type lectin dectin-1. Other CD44 Abs known to have less broad anti-inflammatory activity, including KM114, KM81, and KM201, did not inhibit FcγR-mediated phagocytosis of RBCs. Taken together, these findings demonstrate selective inhibition of FcγR and CR3-mediated phagocytosis by IM7 and suggest that this broadly anti-inflammatory CD44 Ab inhibits these selected macrophage phagocytic pathways. The understanding of the immune-regulatory effects of CD44 Abs is important in the development and optimization of therapeutic strategies for the potential treatment of autoimmune conditions. PMID:26944929

  12. Phototheranostics of CD44-positive cell populations in triple negative breast cancer

    OpenAIRE

    Jiefu Jin; Balaji Krishnamachary; Yelena Mironchik; Hisataka Kobayashi; Bhujwalla, Zaver M.

    2016-01-01

    Triple-negative breast cancer (TNBC) is one of the most lethal subtypes of breast cancer that has limited treatment options. Its high rates of recurrence and metastasis have been associated, in part, with a subpopulation of breast cancer stem-like cells that are resistant to conventional therapies. A compendium of markers such as CD44high/CD24low, and increased expression of the ABCG2 transporter and increased aldehyde dehydrogenase (ALDH1), have been associated with these cells. We developed...

  13. Silencing of CD44 gene expression in human 143-B osteosarcoma cells promotes metastasis of intratibial tumors in SCID mice.

    Directory of Open Access Journals (Sweden)

    Ana Gvozdenovic

    Full Text Available Osteosarcoma (OS is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA, is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.

  14. The Interplay of Antigen Affinity, Internalization, and Pharmacokinetics on CD44-Positive Tumor Targeting of Monoclonal Antibodies.

    Science.gov (United States)

    Glatt, Dylan M; Beckford Vera, Denis R; Parrott, Matthew C; Luft, J Christopher; Benhabbour, S Rahima; Mumper, Russell J

    2016-06-01

    Monoclonal antibodies (mAbs) offer promise as effective tumor targeting and drug delivery agents for cancer therapy. However, comparative biological and clinical characteristics of mAbs targeting the same tumor-associated antigen (TAA) often differ widely. This study examined the characteristics of mAbs that impact tumor targeting using a panel of mAb clones specific to the cancer-associated cell-surface receptor and cancer stem cell marker CD44. CD44 mAbs were screened for cell-surface binding, antigen affinity, internalization, and CD44-mediated tumor uptake by CD44-positive A549 cells. It was hypothesized that high-affinity, rapidly internalizing CD44 mAbs would result in high tumor uptake and prolonged tumor retention. Although high-affinity clones rapidly bound and were internalized by A549 cells in vitro, an intermediate-affinity clone demonstrated significantly greater tumor uptake and retention than high-affinity clones in vivo. Systemic exposure, rather than high antigen affinity or rapid internalization, best associated with tumor targeting of CD44 mAbs in A549 tumor-bearing mice. PMID:27079967

  15. Hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells

    Science.gov (United States)

    Yu, Meihua; Jambhrunkar, Siddharth; Thorn, Peter; Chen, Jiezhong; Gu, Wenyi; Yu, Chengzhong

    2012-12-01

    In this paper, a targeted drug delivery system has been developed based on hyaluronic acid (HA) modified mesoporous silica nanoparticles (MSNs). HA-MSNs possess a specific affinity to CD44 over-expressed on the surface of a specific cancer cell line, HCT-116 (human colon cancer cells). The cellular uptake performance of fluorescently labelled MSNs with and without HA modification has been evaluated by confocal microscopy and fluorescence-activated cell sorter (FACS) analysis. Compared to bare MSNs, HA-MSNs exhibit a higher cellular uptake via HA receptor mediated endocytosis. An anticancer drug, doxorubicin hydrochloride (Dox), has been loaded into MSNs and HA-MSNs as drug delivery vehicles. Dox loaded HA-MSNs show greater cytotoxicity to HCT-116 cells than free Dox and Dox-MSNs due to the enhanced cell internalization behavior of HA-MSNs. It is expected that HA-MSNs have a great potential in targeted delivery of anticancer drugs to CD44 over-expressing tumors.

  16. CD44 Influences Fibroblast Behaviors Via Modulation of Cell-Cell and Cell-Matrix Interactions, Affecting Survivin and Hippo Pathways.

    Science.gov (United States)

    Tsuneki, Masayuki; Madri, Joseph A

    2016-03-01

    CD44 has been studied in a wide variety of cell types, in a diverse array of cell behaviors and in a diverse range of signaling pathways. We now document a role for CD44 in mediating fibroblast behaviors via regulation of N-cadherin, extracellular matrix expression, Survivin and the Hippo pathway. Here, we report our findings on the roles of CD44 in modulating proliferation, apoptosis, migration and invasion of murine wild-type (WT-FB) and CD44 knockout dermal fibroblasts (CD44KO-FB). As we have documented in microvascular endothelial cells lacking CD44, we found persistent increased proliferation, reduced activation of cleaved caspase 3, increased initial attachment, but decreased strength of cell attachment in high cell density, post confluent CD44KO-FB cultures. Additionally, we found that siRNA knock-down of CD44 mimicked the behaviors of CD44KO-FB, restoring the decreases in N-cadherin, collagen type I, fibronectin, Survivin, nuclear fractions of YAP and phospho-YAP and decreased levels of cleaved caspase 3 to the levels observed in CD44KO-FB. Interestingly, plating CD44KO-FB on collagen type I or fibronectin resulted in significant decreases in secondary proliferation rates compared to plating cells on non-coated dishes, consistent with increased cell adhesion compared to their effects on WT-FB. Lastly, siRNA knockdown of CD44 in WT-FB resulted in increased fibroblast migration compared to WT-FB, albeit at reduced rates compared to CD44KO-FB. These results are consistent with CD44's pivotal role in modulating several diverse behaviors important for adhesion, proliferation, apoptosis, migration and invasion during development, growth, repair, maintenance and regression of a wide variety of mesenchymal tissues.

  17. Relationship between P-glycoprotein and CD44 expression in esophageal carcinoma%食管癌细胞P-糖蛋白与CD44表达相关性研究

    Institute of Scientific and Technical Information of China (English)

    许沈华; 凌雨田; 朱赤红

    2006-01-01

    Objective: To investigate the relationship between P-glycoprotein (P-gp) and adhesion molecule CD44 expression as well as their clinical significance in esophageal carcinoma.Methods: To examine the expressed level of P-gp and CD44 by flow cytometry (FCM) in the operated samples of 70 cases with esophageal carcinoma and their normal mucosa of esophageal incision,and to evaluate their relationship with clinicopathological factors.Results: Among the 70 cases with esophageal carcinoma,the expression of P-gp in the 27 cases (38.6%) was negative (positive cells <25%); 11 cases (15.7%) were 25%-40%expression of P-gp positive cells; 14 cases (20%) were 41%-60% expression of P-gp positive cells; 18 cases (25.7%) were the high expression (positive cells >60%) of P-gp.Of the cases with the tumor sizes being more than 4 cm,the expression of CD44showed a significant difference (P<0.05) in 25 cases with P-gp positive,compared with 19 cases with P-gp negative.Of the cases with high-mild differentiated esophageal carcinoma,the expression of CD44 showed a significant difference (P<0.05) in 22 cases with P-gp positive,compared with 17 cases with P-gp negative.Of the cases with clinical Ⅲ--Ⅳ stage,the expression of CD44 showed a significant difference (P<0.05) in 26 cases with P-gp positive,compared with 10 cases with P-gp negative.Of the cases with lymph node metastasis,the CD44 expression showed a significant difference (P=0.050)in 27 cases with P-gp positive,compared with 11 cases with P-gp negative.Of the cases of the patients' age being more than 56 years,the expression of CD44 showed a significant difference (P<0.01) in 27 cases with P-gp positive,compared with 12 cases with P-gp negative.When the P-gp and CD44 expression were positive,the clinical Ⅱ stage and Ⅲ-Ⅳ stage in esophageal carcinoma was showed a significant difference (P<0.05).Conclusion: When the CD44 and P-gp both have the positive high expression,it will be significantly associated with the

  18. Nanovesicle-mediated systemic delivery of microRNA-34a for CD44 overexpressing gastric cancer stem cell therapy.

    Science.gov (United States)

    Jang, Eunji; Kim, Eunjung; Son, Hye-Young; Lim, Eun-Kyung; Lee, Hwunjae; Choi, Yuna; Park, Kwangyeol; Han, Seungmin; Suh, Jin-Suck; Huh, Yong-Min; Haam, Seungjoo

    2016-10-01

    The cancer stem cell (CSC) hypothesis postulates that cancer cells overexpressing CD44 are marked as CSCs that cause tumorigenesis and recurrence. This hypothesis suggests that CD44 is a potential therapeutic target that can interfere with CSCs qualities. MicroRNA-34a (miR-34a) is a promising candidate for CD44 repression-based cancer therapy as it has been reported to inhibit proliferation, metastasis, and survival of CD44-positive CSCs. Here, we used nanovesicles containing PLI/miR complexes (NVs/miR) to systemically deliver miR-34a and induce miR-34a-triggered CD44 suppression in orthotopically and subcutaneously implanted tumors in nude mice. Poly(l-lysine-graft-imidazole) (PLI) condenses miRs and is functionally modified to deliver miRs to the site of action by buffering effect of imidazole residues under endosomal pH. Indeed, NVs/miR consisting of PEGylated lipids enveloping PLI/miR complexes greatly reduced inevitable toxicity of polycations by compensating their surface charge and markedly improved their in vivo stability and accumulation to tumor tissue compared to PLI/miR polyplexes. Our NVs-mediated miR-34a delivery system specifically increased endogenous target miR levels, thereby attenuating proliferation and migration of gastric cancer cells by repressing the expression of CD44 with decreased levels of Bcl-2, Oct 3/4 and Nanog genes. Our strategy led to a greater therapeutic outcome than PLI-based delivery with highly selective tumor cell death and significantly delayed tumor growth in CD44-positive tumor-bearing mouse models, thus providing a fundamental therapeutic window for CSCs. PMID:27497057

  19. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    Energy Technology Data Exchange (ETDEWEB)

    Wang Mu [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Ruan Yuxia [Department of Ophthalmology, The First Affiliated Hospital, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Xing Xiaobo; Chen Qian; Peng, Yuan [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China); Cai Jiye, E-mail: tjycai@jnu.edu.cn [Department of Chemistry, Jinan University, 601 Huangpu Road West, Tianhe District, Guangzhou 510632 (China)

    2011-07-04

    Graphical abstract: Highlights: > In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. > We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. > Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. > The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 {+-} 4.62 nm to 129.70 {+-} 43.72 nm) and the expression of CD44 decreased (99.79 {+-} 0.16% to 75.14 {+-} 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 {mu}M curcumin-treated) and 50-120 pN (20 {mu}M curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  20. Expression of CD44, CD24 and ESA in pancreatic adenocarcinoma cell lines varies with local microenvironment

    Institute of Scientific and Technical Information of China (English)

    Hong-JiWei; TaoYin; ZhuZhu; Peng-FeiShi; YuanTian; Chun-YouWang

    2011-01-01

    BACKGROUND: Emerging evidence suggests that pancreatic adenocarcinoma is hierarchically organized and sustained by pancreatic cancer stem cells. Furthermore, elimination of these cells is possible and therapeutically relevant. This study aimed to investigate the expression patterns of pancreatic cancer stem cell surface markers CD44, CD24 and ESA in pancreatic adenocarcinoma cell lines and explore the influence of their local microenvironment. METHODS: Flow cytometry was used to analyze the expression patterns of CD44, CD24 and ESA in five pancreatic adenocarcinoma cell lines (PANC-1, PC-2, MIA-Paca-2, AsPC-1 and BxPC-3). In addition, the capacity for sphere-formation in serum-free medium of four cell lines (PANC-1, PC-2, MIA-Paca-2 and BxPC-3) was assessed. Then, the same assays were performed when tumor cell spheres were developed. The role of sonic hedgehog (SHH) in cell spheres from PANC-1 and MIA-Paca-2 were also assessed by RT-PCR. RESULTS:  CD44 and CD24 were detected in PANC-1. Only CD44 expression was detected in PC-2, MIA-Paca-2 and AsPC-1. CD44, CD24 and ESA were all detected in BxPC-3. Tumor cell spheres developed in PANC-1 and MIA-Paca-2 in serum-free medium. This was accompanied by an increase in CD24 expression and a decrease in CD44 expression in PANC-1. Interestingly, the expression of CD44 and CD24 returned to initial levels once the medium was changed back from serum-free to serum-containing medium. No significant change in the expression of CD44 was detected in MIA-Paca-2. Furthermore, the relative quantification of SHH mRNA in PANC-1 cell spheres was significantly higher than that in cells cultured in the serum-containing medium. CONCLUSION: The expression patterns of the pancreatic cancer stem cell surface markers CD44, CD24 and ESA were diverse in different pancreatic adenocarcinoma cell lines and changed with their local microenvironment.

  1. Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44

    Energy Technology Data Exchange (ETDEWEB)

    Mori, Tomoyuki; Kitano, Ken; Terawaki, Shin-ichi; Maesaki, Ryoko; Hakoshima, Toshio, E-mail: hakosima@bs.naist.jp [Structural Biology Laboratory, Nara Institute of Science and Technology, Keihanna Science City, Nara 630-0192 (Japan)

    2007-10-01

    The radixin FERM domain complexed with the CD44 cytoplasmic tail peptide has been crystallized. A diffraction data set from the complex was collected to 2.1 Å. CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell–cell and cell–matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane–cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 Å.

  2. Knock-down of CD44 regulates endothelial cell differentiation via NFκB-mediated chemokine production.

    Directory of Open Access Journals (Sweden)

    Berit Olofsson

    Full Text Available A striking feature of microvascular endothelial cells is their capacity to fuse and differentiate into tubular structures when grown in three-dimensional (3D extracellular matrices, in collagen or Matrigel, mimicking the in vivo blood vessel formation. In this study we demonstrate that human telomerase-immortalised foreskin microvascular endothelial (TIME cells express high levels of the hyaluronan receptor CD44 and the hyaluronidase HYAL2. Knock-down of CD44 or HYAL2 resulted in an inability of TIME cells to form a tubular network, suggesting a key regulatory role of hyaluronan in controlling TIME cell tubulogenesis in 3D matrices. Knock-down of CD44 resulted in an upregulation of mRNA expression of the chemokines CXCL9 and CXCL12, as well as their receptors CXCR3 and CXCR4. This was accompanied by a defect maturation of the tubular structure network and increased phosphorylation of the inhibitor of NFκB kinase (IKK complex and thus translocation of NFκB into the nucleus and activation of chemokine targed genes. Furthermore, the interaction between CD44 and hyaluronan determines the adhesion of breast cancer cells. In summary, our observations support the notion that the interaction between CD44 and hyaluronan regulates microvascular endothelial cell tubulogenesis by affecting the expression of cytokines and their receptors, as well as breast cancer dissemination.

  3. Crystallographic characterization of the radixin FERM domain bound to the cytoplasmic tail of adhesion molecule CD44

    International Nuclear Information System (INIS)

    The radixin FERM domain complexed with the CD44 cytoplasmic tail peptide has been crystallized. A diffraction data set from the complex was collected to 2.1 Å. CD44 is an important adhesion molecule that specifically binds hyaluronic acid and regulates cell–cell and cell–matrix interactions. Increasing evidence has indicated that CD44 is assembled in a regulated manner into the membrane–cytoskeletal junction, a process that is mediated by ERM (ezrin/radixin/moesin) proteins. Crystals of a complex between the radixin FERM domain and the C-terminal cytoplasmic region of CD44 have been obtained. The crystal of the radixin FERM domain bound to the CD44 cytoplasmic tail peptide belongs to space group P212121, with unit-cell parameters a = 62.70, b = 66.18, c = 86.22 Å, and contain one complex in the crystallographic asymmetric unit. An intensity data set was collected to a resolution of 2.1 Å

  4. CD44、CD24和PCNA与乳腺癌腋窝淋巴结转移关系研究﹡%Relationship among CD44, CD24, PCNA and axillary lymph node metastasis in breast cancer

    Institute of Scientific and Technical Information of China (English)

    焦伟; 王人颢

    2016-01-01

    目的:探讨CD44、CD24和增殖细胞核抗原( PCNA)与乳腺癌淋巴结转移之间的关系。方法乳腺癌切除组织标本120例,分为①前哨淋巴结阴性病例组、②前哨淋巴结阳性腋窝淋巴结未转移病例组、③前哨淋巴结阳性腋窝淋巴结转移病例组。分别对乳腺癌病理石蜡存档组织标本进行CD44、CD24和PCNA免疫组化标记,分析CD44、CD24及PCNA与淋巴结转移之间的关系。结果腋窝淋巴结转移组中:PCNA及CD44+/CD24-/loW同时表达阳性细胞病例数与其他两组比较明显增高,差异具有统计学意义(P<0.05);CD44、PCNA、CD24阳性表达例数均显著高于无腋窝淋巴结转移的其他两组,差异具有统计学意义(P均<0.05)。结论乳腺癌组织中CD44、CD24、PCNA阳性表型细胞比例决定腋窝淋巴结转移程度。%Objective To explore the relationship among CD44, CD24, proliferating cell nuclear antigen (PCNA) and lymph node metastasis of breast cancer .Methods A total of 120 breast cancer samples were divided into the follow-ing groups:a sentinel lymph node negative group , a sentinel lymph node group where no metastasis was found in the posi-tive axillary lymph nodes , and a sentinel lymph node where metastasis was found in the positive axillary lymph nodes . Then, CD44, CD24 and PCNA within the samples were immunohistochemically stained , in order to analyze the relation-ship among CD44, CD24, PCNA and lymph node metastasis .Results Patients with metastasis in the axillary lymph node produced a remarkably higher number of cells with positive staining of PCNA and CD 44 +/CD24 -/low than those in other two groups (P<0.05) and their number of cells with positive CD 44, CD24 and PCNA staining was significantly higher than those in other two groups (P<0.05).Conclusion The proportion of cells with CD44, CD24 and PCNA positive phenotypes in breast cancer determines the degree of axillary lymph node metastasis .

  5. Activation of Hedgehog signal pathway in breast cancer CD+44 CD-24 cells%Hedgehog信号通路在乳腺癌CD+44CD-24细胞中激活

    Institute of Scientific and Technical Information of China (English)

    陶雅军; 毛俊; 张晴晴; 李连宏

    2011-01-01

    Objective To explore the effect of Hedgehog signal pathway in the progress of hreast cancer.Methods CD+44 CD-24 cells and non-CD+44 CD-24 cells were selected from cell suspension in the culture system of a serum-free medium by magnetic activated cell sorting system ( MACS) and injected in NOD/SCID mice, respectively.Real-time RT-PCR was employed to detect the expression of Hedgehog signal molecule SMO and GLI1 mRNA in CD+44CD-24 cells and non-CD+44CD-24cells, respectively.Results CD+44CD-24 cells accounted for 2.25% of hreast cancer cells.The expression levels of SMO and GLI1 mRNA in CD+44CD-24 cells were higher than those in non-CD+44CD-24 cells (P < 0.05 ) .The rate of tumor formation was 100% ( 3/3 ) in CD+44CD-24 cells contrast to 50% ( 2/4) in non-CD+44CD-24 cells.One mouse had lung metastasis, one mouse had lymph node and lung metastasis, and one mouse had liver and lymph node metastasis in CD+44CD-24 mice, while no organ metastasis was displayed in non-CD+44 CD-24 mice.Conclusion Hedgehog signal pathway is activated in breast cancer CD+44 CD-24 cells and plays an important role in breast carcinogenesis.%目的 了解Hedgehog信号通路在乳腺癌发生发展中的作用.方法 采用免疫磁珠法从无血清培养的乳腺癌悬浮细胞中分选CDCD细胞和非CDCD细胞,分别接种于NOD/SCID鼠乳腺脂肪垫内,观察成瘤情况.采用real-time RT-PCR技术检测Hedgehog信号通路分子SMO和GLI1在CDCD细胞和非CDCD细胞中的表达情况.结果 分选出的CDCD细胞约占细胞总数的2.25%,SMO mRNA和GLI1 mRNA在CDCD细胞中的表达均高于其在非CDCD细胞中的表达(P均CD组乳腺癌发生率为100%(3/3),发生肺转移、淋巴结和肺转移、肝脏和淋巴结转移各1只;非CDCD细胞组乳腺癌发生率为50%(2/4),未见有转移发生.结论 在乳腺癌CDCD细胞中Hedgehog信号通路分子表达增强,其与乳腺癌的浸润转移关系密切.

  6. A CD44high/EGFRlow subpopulation within head and neck cancer cell lines shows an epithelial-mesenchymal transition phenotype and resistance to treatment.

    Directory of Open Access Journals (Sweden)

    Linnea La Fleur

    Full Text Available Mortality in head and neck squamous cell carcinoma (HNSCC is high due to emergence of therapy resistance which results in local and regional recurrences that may have their origin in resistant cancer stem cells (CSCs or cells with an epithelial-mesenchymal transition (EMT phenotype. In the present study, we investigate the possibility of using the cell surface expression of CD44 and epidermal growth factor receptor (EGFR, both of which have been used as stem cell markers, to identify subpopulations within HNSCC cell lines that differ with respect to phenotype and treatment sensitivity. Three subpopulations, consisting of CD44(high/EGFR(low, CD44(high/EGFR(high and CD44(low cells, respectively, were collected by fluorescence-activated cell sorting. The CD44(high/EGFR(low population showed a spindle-shaped EMT-like morphology, while the CD44(low population was dominated by cobblestone-shaped cells. The CD44(high/EGFR(low population was enriched with cells in G0/G1 and showed a relatively low proliferation rate and a high plating efficiency. Using a real time PCR array, 27 genes, of which 14 were related to an EMT phenotype and two with stemness, were found to be differentially expressed in CD44(high/EGFR(low cells in comparison to CD44(low cells. Moreover, CD44(high/EGFR(low cells showed a low sensitivity to radiation, cisplatin, cetuximab and gefitinib, and a high sensitivity to dasatinib relative to its CD44(high/EGFR(high and CD44(low counterparts. In conclusion, our results show that the combination of CD44 (high and EGFR (low cell surface expression can be used to identify a treatment resistant subpopulation with an EMT phenotype in HNSCC cell lines.

  7. Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

    Science.gov (United States)

    Boere, Janneke; van de Lest, Chris H. A.; Libregts, Sten F. W. M.; Arkesteijn, Ger J. A.; Geerts, Willie J. C.; Nolte-'t Hoen, Esther N. M.; Malda, Jos; van Weeren, P. René; Wauben, Marca H. M.

    2016-01-01

    Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery. PMID:27511891

  8. Isoproterenol regulates CD44 expression in gastric cancer cells through STAT3/MicroRNA373 cascade.

    Science.gov (United States)

    Wei, Bo; Sun, Xiaoyan; Geng, Zhijun; Shi, Ming; Chen, Zhida; Chen, Lin; Wang, Yongan; Fu, Xiaobing

    2016-10-01

    Gastric cancer is a heterogeneous disease, and stem cells are thought to be the cell of origin contributed to this malignancy. However, studies with breast and intestinal cancer models show non-stem cancer cells can change their surface phenotype and convert into tumor-initiating cells induced by the signals emanating from surrounding tumor microenvironment. Here, we show that CD44 was expressed at different levels in gastric metastases compared with primary tumors, and also negatively correlated with the expression of miR-373. By using a panel of human gastric cancer cell lines and analysis of archived data from The Cancer Genomics Altas (TCGA) database, we verified the inverse correlation between CD44 and miR-373. Furthermore, the stress-associated hormone, isoproterenol, could increase the expression levels of "stem"-related proteins, such as CD44, Nanog, and Rex-1, and induce chemoresistance in gastric cancer cells. Transfection with miR-373, however, reversed not only the effect of isoproterenol on phenotypic conversion but also its effect on drug sensitivity. Isoproterenol triggered downstream target STAT3 mainly through β2-adrenergic receptors (β2-ARs). Activated STAT3 functioned as a miR-373 suppressor by binding to its promoter, which forms a positive feedback circuit to maintain CD44 activity and direct the phenotypic conversion from CD44(low) to CD44(hi) expression. Our data suggest an important role of β2-AR/STAT3/miR-373 signaling on the transformation of gastric cancer cells. This study also suggests a potential therapeutic or preventive treatment for gastric cancer patients who are especially prone to psychosocial stress. PMID:27512943

  9. Breast cancer stromal fibroblasts promote the generation of CD44+CD24- cells through SDF-1/CXCR4 interaction

    Directory of Open Access Journals (Sweden)

    Zhang Huanle

    2010-06-01

    Full Text Available Abstract Background Breast cancer stem cells (BCSCs have been recently identified in breast carcinoma as CD44+CD24- cells, which exclusively retain tumorigenic activity and display stem cell-like properties. Using a mammosphere culture technique, MCF7 mammosphere cells are found to enrich breast cancer stem-like cells expressing CD44+CD24-. The stromal cells are mainly constituted by fibroblasts within a breast carcinoma, yet little is known of the contributions of the stromal cells to BCSCs. Methods Carcinoma-associated fibroblasts (CAFs and normal fibroblasts (NFs were isolated and identified by immunohistochemistry. MCF7 mammosphere cells were co-cultured with different stromal fibroblasts by a transwell cocultured system. Flow cytometry was used to measure CD44 and CD24 expression status on MCF7. ELISA (enzyme-linked immunosorbent assay was performed to investigate the production of stromal cell-derived factor 1 (SDF-1 in mammosphere cultures subject to various treatments. Mammosphere cells were injected with CAFs and NFs to examine the efficiency of tumorigenity in NOD/SCID mice. Results CAFs derived from breast cancer patients were found to be positive for α-smooth muscle actin (α-SMA, exhibiting the traits of myofibroblasts. In addition, CAFs played a central role in promoting the proliferation of CD44+CD24- cells through their ability to secrete SDF-1, which may be mediated to SDF-1/CXCR4 signaling. Moreover, the tumorigenicity of mammosphere cells with CAFs significantly increased as compared to that of mammosphere cells alone or with NFs. Conclusion We for the first time investigated the effects of stromal fibroblasts on CD44+CD24- cells and our findings indicated that breast CAFs contribute to CD44+CD24- cell proliferation through the secretion of SDF-1, and which may be important target for therapeutic approaches.

  10. Overexpression of CD44 is associated with the occurrence and migration of non-small cell lung cancer

    Science.gov (United States)

    Li, Guanghu; Gao, Yufei; Cui, Yongsheng; Zhang, Tao; Cui, Rui; Jiang, Yang; Shi, Jingwei

    2016-01-01

    Non-small cell lung cancer (NSCLC) is a potentially fatal disease and the incidence is increasing annually. In order to diagnose and treat NSCLC effectively, greater understanding of its molecular mechanism is required. In the present study, 36 NSCLC tissues and 10 normal tissues were selected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to analyze the CD44 mRNA expression level in NSCLC tissue and DNA sequencing was performed to further verify the CD44 expression level. Differentially expressed genes between tumor tissues and controls were determined by DNA sequencing and the Gene_act_net between CD44 and its associated genes was constructed. Gene Ontology (GO) term enrichment analysis of the differentially expressed genes was performed by the Biological Networks Gene Ontology tool. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed based on the Expression Analysis Systematic Explorer test applied in the Database for Annotation, Visualization and Integrated Discovery. RT-qPCR results showed that CD34 was overexpressed in 21 of the 36 NSCLC tissues (58.3%). The Gene_act_net indicated that there were 20 differentially expressed genes with 17 upregulated and 3 downregulated. Among them, CD44, MET, ERBB2, EGFR, AKT1, IQGAP1 and STAT3 were associated with the occurrence and migration of NSCLC. In KEGG pathway analysis, extracellular matrix-receptor interaction and hematopoietic cell lineage pathways were the most affected by overexpressed CD44; and thus may be important in the development and migration of NSCLC. In conclusion, CD44 was overexpressed in NSCLC and the overexpression was associated with the occurrence of NSCLC and migration of NSCLC cells. PMID:27573351

  11. EXPRESSION OF INTRON 9 IN CD44 GENE IN CERVICAL CANCER AND CIN AND ITS CLINICAL SIGNIFICANCE

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To detect the retention of intron 9 in CD44 mRNA in cervical cancer tissue, CIN, cervicitis and their exfoliated cells, and to study their clinical significance in diagnosis and treatment of early-stage, non-invasive cervical cancer. Methods: RT-PCR methods were used to detect the retention of intron 9 in CD44 mRNA in 30 cases of cervical cancer tissue, 11 cases of CIN tissue, 30 cases of cervicitis tissue and their exfoliated cells. Results: The retention rate of intron 9 in CD44 gene transcripts were 76.7% in cervical cancer tissue, 89.8% in corresponding exfoliated cells, 70.8% in CIN tissue, and 60.0% in CIN exfoliated cells, but undetected in neither cervicitis tissue nor exfoliated cells. The relative quantity of intron 9 in CD44 gene transcripts was 1.10 ( 0.12 in cervical cancer tissue, 1.21 ( 0.11 in CIN tissue, 1.11 ( 0.19 in cervical cancer exfoliated cells, 1.17 ( 0.12 in CIN exfoliated cells respectively, but undetected in neither cervicitis tissue nor exfoliated cells. The retention rate and relative content of intron 9 in CD44 gene transcripts in cervical cancer and CIN tissue and their exfoliated cells were statistically higher than that in cervicitis and their exfoliated cells (P0.05). Conclusion: Detecting the retention of intron 9 in CD44 mRNA in cervical exfoliated cells was more sensitivity than traditional cytology exam for diagnosing cervical cancer, and the techniques was worth clinical application.

  12. Expression of S518 phosphorylated Merlin and its interaction with CD44 in vestibular schwannoma%听神经瘤S518磷酸化Merlin蛋白的表达及与CD44结合特性

    Institute of Scientific and Technical Information of China (English)

    蔡丽慧; 吴皓; 吕静荣; 汪照炎

    2008-01-01

    Objective To investigate the impact of S518 phosphorylation in Merlin on the interaction with CD44 in vestibular schwannoma and the tumor growth.Methods Thirty-five samples of vestibular schwannoma were identified by pathology.Immunohistopathology and western blot were employed to analyze the expression and localization of S518 phosphorylated Merlin in the tumor tissues.Nerve tissues that were collected during other surgical operation were used 118 control.The expression level of S518 phosphorylated Merlin was compared with clinical stages,tumor size,clinical course and cystic degeneration.Immunoprecipitation was used to evaluate the impact of S518 phosphorylation in Merlin on the interaction with CD44.Results In vestibular schwannoma,Merlin was phosphorylated at S518 and demonstrated perinuclear localization.The S518 phosphorylation level was much lower in the normal control nerve tissues than that in vestibular schwannoma tissues.There was no correlation between the phosphorylation level on Merlin and clinical stages,tumor size,clinical course and cystic degeneration.The S518 phosphorylated Merlin bound CD44 Was higher than wild-type Merlin bound CD44 in vestibular schwannoma tissues.Conclusions The affinity of Merlin to CD44 Was increased after phosphorylation at S518.Different cellular biological results might be triggered through binding to wild type Merlin and S518phosphorylated Merlin.%目的 探讨听神经瘤组织中S518磷酸化对Merlin蛋白与细胞表面糖蛋白CD44结合能力的影响及其与肿瘤生长的关系.方法 35例听神经瘤组织标本均经过病理学证实,用免疫组织化学技术和免疫印迹技术分析S518磷酸化Merlin蛋白的表达、组织学分布情况,以手术中切除的正常颅神经作对照.将各标本中S518磷酸化Merlin条带的相对灰度值与听神经瘤临床分期、瘤体大小、病程及囊性变进行相关分析.用免疫沉淀技术比较野生型Merlin蛋白及S518磷酸化Merlin蛋白与CD

  13. survivin和CD44v6在非小细胞肺癌中的表达及意义%Expression and significance of survivin and CD44v6 protein in non-small cell lung cancer

    Institute of Scientific and Technical Information of China (English)

    魏霞; 石志红; 嵇喜祥

    2009-01-01

    Objective To investigate the expression and significance of survivin and CD44v6 in non-small cell lung cancer (NSCLC) and their correlation. Methods SP immunohistochemical technique was used to detect the expression of survivin and CD44V6 protein in 53 cases of NSCLC and 13 cases of para-eaneer nor-mal tissues. Results The positive rate of survivin and CD44v6 in NSCLC was 60. 38% and 69. 81% respec-tively,which was higher than that of normal pulmonary tissues adjacent to carcinoma(P 0.05). The expression of survivin was related to TNM stages and cell differentiation (P 0. 05). The expression of CD44v6 in squamous carcinoma was significantly higher than that of adenocareinoma (P 0. 05). There was no correlation between the expression of survivin and C1)44v6 (r = -0. 058, P >0. 05). Conclusion Survivin might be used to evaluate NSCLC development;CD44v6 might be used for the differential diagnosis of squamous carcinoma in NSCLC;both of them might be helpful to predict the metastasis of NSCLC. They might be two independent events in the process of NSCLC genesis and develop-ment.%目的 探讨非小细胞肺癌(NSCLC)中survivin及CD44v6的表达及意义,以及二者的相关性.方法 采用SP免疫组化方法检测survivin及CD44v6在53例NSCLC组织、13例癌旁正常肺组织中的表达.结果 53例NSCLC癌组织中survivin、CD44v6阳性表达率分别是60.38%和69.81%,高于癌旁正常肺组织的表达(P0.05).survivin的表达与临床TNM分期及肿瘤分化程度相关(P0.05).CD44v6在鳞癌的表达率远高于腺癌(P0.05).survivin与CD44v6之间无相关性(r=-0.058,P>0.05).结论 survivin有望作为评估NSCLC病变进展的指标,CD44v6可用来鉴别诊断NSCLC中的鳞癌,二者有可能成为预测NSCLC转移的指标.survivin、CD44v6可能是NSCLC发生发展过程中的两个独立事件.

  14. Non-secreted clusterin isoforms are translated in rare amounts from distinct human mRNA variants and do not affect Bax-mediated apoptosis or the NF-κB signaling pathway.

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    Hans Prochnow

    Full Text Available Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU, which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU mRNA variants in non-cancer and cancer cell lines. In all cell lines variant 1 represents the most abundant mRNA, whereas all other variants collectively account for no more than 0.34% of total CLU mRNA, even under stressed conditions. Overexpression of CLU cDNAs combined with in vitro mutagenesis revealed distinct translational start sites including a so far uncharacterized non-canonical CUG start codon. We show that all exon 2-containing mRNAs encode sCLU and at least three non-glycosylated intracellular isoforms, CLU1‑449, CLU21‑449 and CLU34‑449, which all reside in the cytosol of unstressed and stressed HEK‑293 cells. The latter is the only form expressed from an alternatively spliced mRNA variant lacking exon 2. Functional analysis revealed that none of these cytosolic CLU forms modulate caspase-mediated intrinsic apoptosis or significantly affects TNF-α-induced NF-κB-activity. Therefore our data challenge some of the current ideas regarding the physiological functions of CLU isoforms in pathologies.

  15. CD44-deficiency attenuates the immunologic responses to LPS and delays the onset of endotoxic shock-induced renal inflammation and dysfunction.

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    Elena Rampanelli

    Full Text Available Acute kidney injury (AKI is a common complication during systemic inflammatory response syndrome (SIRS, a potentially deadly clinical condition characterized by whole-body inflammatory state and organ dysfunction. CD44 is a ubiquitously expressed cell-surface transmembrane receptor with multiple functions in inflammatory processes, including sterile renal inflammation. The present study aimed to assess the role of CD44 in endotoxic shock-induced kidney inflammation and dysfunction by using CD44 KO and WT mice exposed intraperitoneally to LPS for 2, 4, and 24 hours . Upon LPS administration, CD44 expression in WT kidneys was augmented at all time-points. At 2 and 4 hours, CD44 KO animals showed a preserved renal function in comparison to WT mice. In absence of CD44, the pro-inflammatory cytokine levels in plasma and kidneys were lower, while renal expression of the anti-inflammatory cytokine IL-10 was higher. The cytokine levels were associated with decreased leukocyte influx and endothelial activation in CD44 KO kidneys. Furthermore, in vitro assays demonstrated a role of CD44 in enhancing macrophage cytokine responses to LPS and leukocyte migration. In conclusion, our study demonstrates that lack of CD44 impairs the early pro-inflammatory cytokine response to LPS, diminishes leukocyte migration/chemotaxis and endothelial activation, hence, delays endotoxic shock-induced AKI.

  16. CD44v6修饰DC融合瘤苗体外抗结肠癌作用的研究%The Anticolon-cancer Research of Fusion Hybrid of Dendritic Cells Expressing CD44v6 in Vitro

    Institute of Scientific and Technical Information of China (English)

    赵光艳; 胡显芳; 李彦

    2013-01-01

    Objective To survey the antitumor mechanism and effect of fusion hybrid cells of DCs and colon cancer cell CT26.WT co-cultivating and expressing CD44v6 gene, DCs and colon cancer cell CT26.WT derived from mice were blended, and modified with CD44v6 gene, it will provide the experimental foundation on the prevention and immunotherapy for tumor patients. Methods①CT26.WT cells and DCs were blended by polyethylene glycal, and the fused cells were screened by HAT/HT system. The recombinant pBud-CD44v6 vector was transfected into the fused cells by lipofectamine, and to express;②The anticancer effect and cytotoxicty of fused–cell vaccine were studied in vitro. Result DC/CT26.WT fused cell vaccine modified with CD44v6 geng was constructed;the lymphocyte cytotoxicity T(CTL) of immune group was larger activity than that of the anainst group .Conclusion The fused cells of DC expressing CD44v6 geng can induce strong anticolon-cancer activity of T lymphocyte.%  目的小鼠提取的树突状细胞(DC)和小鼠结肠癌细胞CT26.WT共培养、融合,CD44v6基因来修饰融合细胞,研究CD44v6修饰DC融合瘤苗体外抗结肠癌的作用机制,为肿瘤预防及免疫治疗提供实验依据。方法①在聚乙二醇的作用下,DC细胞和CT26.WT细胞共培养、融合,利用HAT/HT筛选系统筛选出纯净DC融合细胞。在脂质体的作用下,将pBud-CD44v6转染到DC融合细胞中表达;②体外实验观察DC融合瘤苗对小鼠结肠癌细胞的杀灭作用。结果构建了基因CD44v6修饰的DC/CT26.WT肿瘤融合疫苗;实验组细胞的淋巴细胞毒(CTL)活性明显高于对照组。结论 CD44v6修饰的DC融合瘤苗可有效诱导T淋巴细胞产生强大抗结肠癌作用。

  17. Reactivation from latency displays HIV particle budding at plasma membrane, accompanying CD44 upregulation and recruitment

    Directory of Open Access Journals (Sweden)

    Sano Kouichi

    2009-07-01

    Full Text Available Abstract Background It has been accepted that HIV buds from the cell surface in T lymphocytes, whereas in macrophages it buds into intracellular endosomes. Recent studies, on the other hand, suggest that HIV preferentially buds from the cell surface even in monocytic cells. However, most studies are based on observations in acutely infected cells and little is known about HIV budding concomitant with reactivation from latency. Such studies would provide a better understanding of a reservoir for HIV. Results We observed HIV budding in latently infected T lymphocytic and monocytic cell lines following TNF-α stimulation and examined the upregulation of host factors that may be involved in particle production. Electron microscopy analysis revealed that reactivation of latently infected J1.1 cells (latently infected Jurkat cells with HIV-1 and U1 cells (latently infected U937 cells with HIV-1 displayed HIV particle budding predominantly at the plasma membrane, a morphology that is similar to particle budding in acutely infected Jurkat and U937 cells. When mRNA expression levels were quantified by qRT-PCR, we found that particle production from reactivated J1.1 and U1 cells was accompanied by CD44 upregulation. This upregulation was similarly observed when Jurkat and U937 cells were acutely infected with HIV-1 but not when just stimulated with TNF-α, suggesting that CD44 upregulation was linked with HIV production but not with cell stimulation. The molecules in endocytic pathways such as CD63 and HRS were also upregulated when U1 cells were reactivated and U937 cells were acutely infected with HIV-1. Confocal microscopy revealed that these upregulated host molecules were recruited to and accumulated at the sites where mature particles were formed at the plasma membrane. Conclusion Our study indicates that HIV particles are budded at the plasma membrane upon reactivation from latency, a morphology that is similar to particle budding in acute

  18. Hyaluronan-CD44 interaction promotes growth of decidual stromal cells in human first-trimester pregnancy.

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    Rui Zhu

    Full Text Available Hyaluronan (HA and its receptor CD44 are expressed at the maternal-fetal interface, but its role in early pregnancy remains unclear. Here, we found that primary decidual stromal cells (DSCs continuously secreted HA and expressed its receptor CD44. Pregnancy-associated hormones up-regulated HA synthetase (HAS 2 transcription and HA release from DSCs. High molecular weight-HA (HMW-HA, but not medium molecular weight (MMW-HA or low molecular weight (LMW-HA, promoted proliferation and inhibited apoptosis of DSCs in a CD44-dependent manner. The in-cell Western analysis revealed HMW-HA activated PI3K/AKT and mitogen-activated protein kinase (MAPK/ERK1/2 signaling pathways time-dependently. Blocking these pathways by specific inhibitor LY294002 or U0126 abrogated HMW-HA-regulated DSc proliferation and apoptosis. Finally, we have found that HA content, HA molecular weight, HAS2 mRNA level, and CD44 expression were significantly decreased in DSCs from unexplained miscarriage compared with the normal pregnancy. Collectively, our results indicate that higher level and greater molecular mass of HA at maternal-fetal interface contributes to DSc growth and maintenance of DSCs in human early pregnancy.

  19. αVβ5 and CD44 Are Oxygen-Regulated Human Embryonic Stem Cell Attachment Factors

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    Deepak Kumar

    2013-01-01

    Full Text Available Human embryonic stem cells (hESCs have great potential for clinical therapeutic use. However, relatively little is known of the mechanisms which dictate their specificity of adhesion to substrates through adhesion proteins including integrins. Previous observations demonstrated enhanced clonogenicity in reduced oxygen culture systems. Here, we demonstrated via antibody blocking experiments that αVβ5 and α6 significantly promoted hESC attachment in 2% O2 only, whereas blockage of CD44 inhibited cell attachment in 21% O2 alone. Immunofluorescence confirmed expression of αVβ5 and CD44 in both 2% O2 and 21% O2 cultured hESCs while flow cytometry revealed significantly higher αVβ5 expression in 2% O2 versus 21% O2 cultured hESCs and higher CD44 expression in 21% O2 versus 2% O2 cultured hESCs. Adhered hESCs following blockage of αVβ5 in 2% O2 displayed a reduction in nuclear colocalisation of Oct-4 and Nanog with little effect observed in 21% O2. Blockage of CD44 had the converse effect with dramatic reductions in nuclear colocalisation of Oct-4 and Nanog in 21% O2 cultured hESC which retained adherence, but not in 2% O2 cultured cells. Identification of oxygen-dependent substrate attachment mechanisms in hESCs has the potential to play a role in the development of novel substrates to improve hESC attachment and culture.

  20. Relationship between the Expression of CD44v6 and Development, Progress, Invasion and Metastasis of Laryngeal Carcinoma

    Institute of Scientific and Technical Information of China (English)

    LIU Banghua; KONG Weijia; GONG Shusheng; YANG Chengzhang; WANG Guangping; ZHU Lixin

    2005-01-01

    Summary: The expression of CD44v6 and its relationship with the development, progress, invasion and metastasis of laryngeal carcinoma was investigated. The expression and content of CD44v6 mRNA in tissuess were detected by both RT-PCR and FCM which were respectively extracted from normal laryngeal mucosa, leukoplakia of larynx, laryngeal papilloma, polyp of vocal cord, tissues of laryngeal carcinoma, metastatic and nonmetastatic lymph nodes of neck, and tissues close to carcinoma. The outcome of RT-PCR indicated that the expression rate of CD44v6 mRNA involved in tissues of laryngeal carcinoma and metastatic lymph nodes of neck was the highest (90 %-100 %) compared with that of leukoplakia of larynx, laryngeal papilloma, tissues close to carcinoma by 0.5 cm (55.56 %-60.00 %) and that of normal laryngeal mucosa, polyp of vocal cord, nonmetastatic lymph nodes and tissues close to carcinoma by 1.0 cm was the lowest ( 13.33 %-20 %). The result from FCM was highly consistent with that from RT-PCR. It was suggested that CD44v6 was closely related with the development, progress, invasion and metastasis of laryngeal carcinoma. The outcome from the tissues close to carcinoma by different distance could do help to the determination of incisal edge in surgery abstractly.

  1. Curcumin induced nanoscale CD44 molecular redistribution and antigen-antibody interaction on HepG2 cell surface

    International Nuclear Information System (INIS)

    Graphical abstract: Highlights: → In this study, we investigate the changes of CD44 expression and distribution on HepG2 cells after curcumin treatment. → We find curcumin is able to change the morphology and ultrastructure of HepG2 cells. → Curcumin can reduce the expression of CD44 molecules and induce the nanoscale molecular redistribution on cell surface. → The binding force between CD44-modified AFM tip and the HepG2 cell surface decreases after curcumin-treatment. - Abstract: The cell surface glycoprotein CD44 was implicated in the progression, metastasis and apoptosis of certain human tumors. In this study, we used atomic force microscope (AFM) to monitor the effect of curcumin on human hepatocellular carcinoma (HepG2) cell surface nanoscale structure. High-resolution imaging revealed that cell morphology and ultrastructure changed a lot after being treated with curcumin. The membrane average roughness increased (10.88 ± 4.62 nm to 129.70 ± 43.72 nm) and the expression of CD44 decreased (99.79 ± 0.16% to 75.14 ± 8.37%). Laser scanning confocal microscope (LSCM) imaging showed that CD44 molecules were located on the cell membrane. The florescence intensity in control group was weaker than that in curcumin treated cells. Most of the binding forces between CD44 antibodies and untreated HepG2 cell membrane were around 120-220 pN. After being incubated with curcumin, the major forces focused on 70-150 pN (10 μM curcumin-treated) and 50-120 pN (20 μM curcumin-treated). These results suggested that, as result of nanoscale molecular redistribution, changes of the cell surface were in response to external treatment of curcumin. The combination of AFM and LSCM could be a powerful method to detect the distribution of cell surface molecules and interactions between molecules and their ligands.

  2. Relationship between LYVE-1, VEGFR-3 and CD44 gene expressions and lymphatic metastasis in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    FusunOzmen; MahirOzmen; EvrenOzdemir; MunevverMoran; SeldaSeckin; DicleGUC; ErgunKaraagaoglu; EminKansu

    2011-01-01

    AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their levels and clinicopathological parameters in gastric cancer.METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic fea- tures such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the dis- tribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.

  3. Application of Collagen-Model Triple-Helical Peptide-Amphiphiles for CD44-Targeted Drug Delivery Systems

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    Margaret W. Ndinguri

    2012-01-01

    Full Text Available Cancer treatment by chemotherapy is typically accompanied by deleterious side effects, attributed to the toxic action of chemotherapeutics on proliferating cells from nontumor tissues. The cell surface proteoglycan CD44 has been recognized as a cancer stem cell marker. The present study has examined CD44 targeting as a way to selectively deliver therapeutic agents encapsulated inside colloidal delivery systems. CD44/chondroitin sulfate proteoglycan binds to a triple-helical sequence derived from type IV collagen, α1(IV1263–1277. We have assembled a peptide-amphiphile (PA in which α1(IV1263–1277 was sandwiched between 4 repeats of Gly-Pro-4-hydroxyproline and conjugated to palmitic acid. The PA was incorporated into liposomes composed of DSPG, DSPC, cholesterol, and DSPE-PEG-2000 (1 : 4 : 5 : 0.5. Doxorubicin-(DOX-loaded liposomes with and without 10% α1(IV1263–1277 PA were found to exhibit similar stability profiles. Incubation of DOX-loaded targeted liposomes with metastatic melanoma M14#5 and M15#11 cells and BJ fibroblasts resulted in IC50 values of 9.8, 9.3, and >100 μM, respectively. Nontargeted liposomes were considerably less efficacious for M14#5 cells. In the CD44+ B16F10 mouse melanoma model, CD44-targeted liposomes reduced the tumor size to 60% of that of the untreated control, whereas nontargeted liposomes were ineffective. These results suggest that PA targeted liposomes may represent a new class of nanotechnology-based drug delivery systems.

  4. The NF2 tumor suppressor gene product, merlin, mediates contact inhibition of growth through interactions with CD44

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    Morrison, H.L.

    2002-03-01

    The neurofibromatosis-2 (NF2) gene encodes merlin, an ezrin-radixin-moesin-(ERM)-related protein, that functions as a tumor suppressor. I found that merlin plays a critical role in the establishment and maintenance of contact inhibition of growth. At high cell density, merlin is activated and blocks profileration with corresponding changes in cell cycle parameters. Merlin interfered with growth factor receptor or Ras-dependent signal transduction of MAP kinase and the step of interference was located downstream of Ras and Raf and upstream of MEK. Merlins growth inhibiting function depended on interaction with a specific domain of the cytoplasmic tail of CD44. In addition merlin activity and phosphorylation status depended on the extracellular ligand associated with the N-terminus of CD44. At high cell densities, in the presence of the extracellular ligand HA, merlin was dephosphorylated and bound directly to a basic amino acid motif in the cytoplasmic tail of CD44. Ezrin and moesin, which are also known to bind to the same basic amino acid motif in CD44 were absent within this growth inhibitory complex. Alternatively in logarithmically growing cells, merlin was inactive, phosphorylated and in a complex with ezrin and moesin. This growth permissive complex was also associated with the cytoplasmic tail of CD44. My data provide not only significant clues about how merlin functions as a tumor suppressor but revealed the existence of a novel molecular switch that, under the influence of ligands in the microenvironment, controls a cell decision to proliferate or growth arrest. (orig.)

  5. Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

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    Hiscox Stephen

    2012-10-01

    Full Text Available Abstract Background Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. Methods CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression or MCF7 cells (± transfection with the CD44 gene were treated with the CD44 ligand, hyaluronon (HA, or heregulin and their in vitro growth (MTT, migration (Boyden chamber and wound healing and invasion (Matrigel transwell migration determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. Results TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2

  6. Suppression of human breast tumors in NOD/SCID mice by CD44 shRNA gene therapy combined with doxorubicin treatment

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    Pham PV

    2012-05-01

    Full Text Available Phuc Van Pham1, Ngoc Bich Vu1, Thuy Thanh Duong1, Tam Thanh Nguyen1, Nhung Hai Truong1, Nhan Lu Chinh Phan1, Tue Gia Vuong1, Viet Quoc Pham1, Hoang Minh Nguyen1, Kha The Nguyen1, Nhung Thi Nguyen1, Khue Gia Nguyen1, Lam Tan Khat1, Dong Van Le2, Kiet Dinh Truong1, Ngoc Kim Phan11Laboratory of Stem Cell Research and Application, University of Science, Vietnam National University, HCM City, 2Military Medical University, Ha Noi, VietnamBackground: Breast cancer stem cells with a CD44+CD24- phenotype are the origin of breast tumors. Strong CD44 expression in this population indicates its important role in maintaining the stem cell phenotype. Previous studies show that CD44 down-regulation causes CD44+CD24- breast cancer stem cells to differentiate into non-stem cells that are sensitive to antitumor drugs and lose many characteristics of the original cells. In this study, we determined tumor suppression in non-obese severe combined immunodeficiency mice using CD44 shRNA therapy combined with doxorubicin treatment.Methods: Tumor-bearing non-obese severe combined immunodeficiency mice were established by injection of CD44+CD24- cells. To track CD44+CD24- cells, green fluorescence protein was stably transduced using a lentiviral vector prior to injection into mice. The amount of CD44 shRNA lentiviral vector used for transduction was based on CD44 down-regulation by in vitro CD44 shRNA transduction. Mice were treated with direct injection of CD44 shRNA lentiviral vector into tumors followed by doxorubicin administration after 48 hours. The effect was evaluated by changes in the size and weight of tumors compared with that of the control.Results: The combination of CD44 down-regulation and doxorubicin strongly suppressed tumor growth with significant differences in tumor sizes and weights compared with that of CD44 down-regulation or doxorubicin treatment alone. In the combination of CD44 down-regulation and doxorubicin group, the tumor weight was

  7. Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

    International Nuclear Information System (INIS)

    Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor known to interact with growth factor receptors, is upregulated in tamoxifen-resistant (TamR) MCF7 breast cancer cells. The purpose of this study was to explore the consequences of CD44 upregulation in an MCF7 cell model of acquired tamoxifen resistance, specifically with respect to the hypothesis that CD44 may influence erbB activity to promote an adverse phenotype. CD44 expression in MCF7 and TamR cells was assessed by RT-PCR, Western blotting and immunocytochemistry. Immunofluorescence and immunoprecipitation studies revealed CD44-erbB associations. TamR cells (± siRNA-mediated CD44 suppression) or MCF7 cells (± transfection with the CD44 gene) were treated with the CD44 ligand, hyaluronon (HA), or heregulin and their in vitro growth (MTT), migration (Boyden chamber and wound healing) and invasion (Matrigel transwell migration) determined. erbB signalling was assessed using Western blotting. The effect of HA on erbB family dimerisation in TamR cells was determined by immunoprecipitation in the presence or absence of CD44 siRNA. TamR cells overexpressed CD44 where it was seen to associate with erbB2 at the cell surface. siRNA-mediated suppression of CD44 in TamR cells significantly attenuated their response to heregulin, inhibiting heregulin-induced cell migration and invasion. Furthermore, TamR cells exhibited enhanced sensitivity to HA, with HA treatment resulting in modulation of erbB dimerisation, ligand-independent activation of erbB2 and EGFR and induction of cell migration

  8. Interaction between Entamoeba histolytica and intestinal epithelial cells involves a CD44 cross-reactive protein expressed on the parasite surface.

    OpenAIRE

    Renesto, P; Sansonetti, P. J.; Guillén, N

    1997-01-01

    This study shows that Entamoeba histolytica binds hyaluronic acid. The binding molecule was identified as an 80-kDa membrane protein and was recognized by anti-CD44 monoclonal antibodies. These data indicate that a CD44 cross-reacting adherence molecule is expressed on E. histolytica.

  9. CD44 disruption prevents degeneration of the capillary network in obstructive nephropathy via reduction of TGF-beta 1-induced apoptosis

    NARCIS (Netherlands)

    K.M.A. Rouschop; N. Claessen; S.T. Pals; J.J. Weening; S. Florquin

    2006-01-01

    CD44 is a glycoprotein that is involved in inflammation and cell-cell/cell-matrix interactions, is upregulated in the kidney upon injury, and leads to fibrosis through enhancement of TGF-beta 1 signaling. Absence of CD44 prevents development of renal fibrosis in unilateral ureteral obstruction (UUO)

  10. CD44v6 expression in human skin keratinocytes as a possible mechanism for carcinogenesis associated with chronic arsenic exposure

    Directory of Open Access Journals (Sweden)

    S. Huang

    2013-01-01

    Full Text Available Inorganic arsenic is a well-known human skin carcinogen. Chronic arsenic exposure results in various types of human skin lesions, including squamous cell carcinoma (SCC. To investigate whether mutant stem cells participate in arsenic-associated carcinogenesis, we repeatedly exposed the HaCaT cells line to an environmentally relevant level of arsenic (0.05 ppm in vitro for 18 weeks. Following sodium arsenic arsenite administration, cell cycle, colony-forming efficiency (CFE, cell tumorigenicity, and expression of CD44v6, NF-κB and p53, were analyzed at different time points (0, 5, 10, 15, 20, 25 and 30 passages. We found that a chronic exposure of HaCaT cells to a low level of arsenic induced a cancer stem- like phenotype. Furthermore, arsenic-treated HaCaT cells also became tumorigenic in nude mice, their growth cycle was predominantly in G2/M and S phases. Relative to nontreated cells, they exhibited a higher growth rate and a significant increase in CFE. Western blot analysis found that arsenic was capable of increasing cell proliferation and sprouting of cancer stem-like phenotype. Additionally, immunohistochemical analysis demonstrated that CD44v6 expression was up-regulated in HaCaT cells exposed to a low level of arsenic during early stages of induction. The expression of CD44v6 in arsenic-treated cells was positively correlated with their cloning efficiency in soft agar (r=0.949, P=0.01. Likewise, the expressions of activating transcription factor NF-κB and p53 genes in the arsenic-treated HaCaT cells were significantly higher than that in non-treated cells. Higher expressions of CD44v6, NF-κB and p53 were also observed in tumor tissues isolated from Balb/c nude mice. The present results suggest that CD44v6 may be a biomarker of arsenic-induced neoplastic transformation in human skin cells, and that arsenic promotes malignant transformation in human skin lesions through a NF-κB signaling pathway-stimulated expression of CD44v6.

  11. 大肠腺瘤及癌组织中β-catenin、APC及CD44V6异常改变的意义%β-catenin, APC and CD44v6 genes in the colorectal adenoma and adenocarcinoma

    Institute of Scientific and Technical Information of China (English)

    王东旭; 苏铃; 房殿春; 李伟; 王建文; 闫晓初

    2003-01-01

    目的探讨β-catenin、APC基因及黏附分子CD44V6在大肠癌发生发展的作用.方法运用PCR-SSCP及免疫组化方法检测大肠腺瘤25例,大肠癌90例及癌旁正常粘膜45例中APC、β-catenin基因突变、蛋白表达及粘附分子CD44V6的表达.结果大肠粘膜中未见β-catenin、APC基因突变及CD44V6阳性表达(0%,0%,0%),而APC蛋白表达阳性率为100%(45/45).大肠腺瘤β-catenin突变率及蛋白异常表达率分别为24%(6/25),32%(8/25).APC突变率及蛋白表达率分别为12%(3/25)、80%(20/25).CD44V6异常表达率为4%(1/25).大肠癌中β-catenin突变率、蛋白表达率分别为27.8%(25/90)及32.2%(29/90),APC突变率、蛋白表达率分别为22.2%(20/90)及44.4%(40/90).CD44V6表达率72.2%(65/90).大肠癌及腺瘤中β-catenin突变率及蛋白异常表达率显著高于正常大肠黏膜(P<0.05,P<0.01),而大肠癌与腺瘤组织间无显著差别(P>0.05).大肠癌中APC突变率及蛋白表达缺失率、CD44V6异常表达率显著高于腺瘤及正常大肠黏膜(P<0.05,P<0.01),而大肠腺瘤与正常组织间无显著差别(P>0.05).CD44V6表达异常与大肠癌的淋巴结转移、临床分期密切相关(P<0.05).三种基因改变之间无显著相关性.结论β-catenin、APC基因改变在大肠腺瘤向癌的转化中起一定作用.黏附分子CD44V6表达异常与大肠癌的预后有关,可作为判断预后的指标.

  12. The interaction between aromatase, metalloproteinase 2,9 and cd44 in breast cancer A interação entre aromatase, metalloproteinase 2, 9 e cd44 no câncer de mama

    Directory of Open Access Journals (Sweden)

    Fábio Bagnoli

    2010-01-01

    Full Text Available OBJECTIVE: This study intends to verify the expression levels and correlation of aromatase, matrix metalloproteinase 2 (MMP-2, matrix metalloproteinase 9 (MMP-9 and CD44 in ductal carcinoma in situ (DCIS and infiltrating ductal carcinoma (IDC when both are found in the same breast. METHODS: One hundred and ten cases were evaluated by tissue microarray (TMA and immunohistochemically screened with anti-aromatase polyclonal antibodies, anti-MMP-2 monoclonal antibodies, anti-MMP-9 policlonal antibodies and anti-CD44 monoclonal antibodies. RESULTS: Aromatase was expressed in IDC and DCIS in 63 (57.3% and 60 (67% of the cases respectively; MMP-2 was similarly expressed in IDC and DCIS in 15 (13.60% cases; MMP-9 was positively expressed in IDC and DCIS in 83 (75.50% and 82 (74.50% cases, respectively; CD44 was positively expressed in IDC and DCIS in 49 (44.50% and 48 (42.60% of the cases, respectively; all of them were highly correlated (pOBJETIVO: O objetivo desse estudo é verificar as expressões e correlações da aromatase, metalloproteinase 2 da matriz (MMP2, metalloproteinase 9 da matriz (MMP-9 e CD44 no carcinoma ductal in situ (CDIS e carcinoma ductal infiltrativo (CDI quando ambos estão presentes simultaneamente na mesma mama. MÉTODOS: Foram avaliados 110 casos pelo método de tissue microarray (TMA e através da utilização de anticorpos policlonais antiaromatase, anticorpos monoclonais anti-MMP-2, anticorpos policlonais anti-MMP-9 e anticorpos monoclonais anti-CD44. RESULTADOS: A aromatase estava expressa de forma positiva no CDI e CDIS em 63 (57,3% e 60 (67% casos, respectivamente. A expressão de MMP-2 estava expressa de forma positiva em 15 (13,6% casos tanto no CDI, quanto no CDIS. A expressão da MMP-9 estava expressa de forma positiva em 83 (75,5% e 82 (74,5% casos de CDI e CDIS, respectivamente. A expressão de CD44 estava expressa de forma positiva em 49 (44,5% e 48 (42,6% casos de CDI e CDIS, respectivamente. Todos eles

  13. Mouse population-guided resequencing reveals that variants in CD44 contribute to acetaminophen-induced liver injury in humans

    OpenAIRE

    Harrill, Alison H.; Watkins, Paul B; Su, Stephen; Ross, Pamela K.; Harbourt, David E; Stylianou, Ioannis M; Boorman, Gary A.; Russo, Mark W.; Sackler, Richard S.; Harris, Stephen C.; Smith, Philip C.; Tennant, Raymond; Bogue, Molly; Paigen, Kenneth; Harris, Christopher

    2009-01-01

    Interindividual variability in response to chemicals and drugs is a common regulatory concern. It is assumed that xenobiotic-induced adverse reactions have a strong genetic basis, but many mechanism-based investigations have not been successful in identifying susceptible individuals. While recent advances in pharmacogenetics of adverse drug reactions show promise, the small size of the populations susceptible to important adverse events limits the utility of whole-genome association studies c...

  14. Identification of the Molecular Mechanisms Responsible for the Inhibition of Homing of AML Cells Triggered by CD44-Ligation

    KAUST Repository

    Al-Jifri, Ablah

    2011-08-03

    Acute Myeloid Leukemia (AML) is a cancerous disease that is defined by the inability to produce functional and mature blood cells, as well as the uncontrolled proliferation due to failure to undergo apoptosis of abnormal cells. The most common therapy for Leukemia, chemotherapy, has proven only to be partially efficient since it does not target the leukemic stem cells (LSCs) that have a high self-renewal and repopulation capacity and result in remission of the disease. Therefore targeting LSCs will provide more efficient therapy. One way to achieve this would be to inhibit their homing capability to the bone marrow. It has recently been shown that CD44, an adhesive molecule, plays a crucial role in cell trafficking and lodgement of both normal and leukemic stem cells. More importantly anti-CD44 monoclonal antibodies, along with its ability to induce differentiation of leukemic blasts, it inhibits specifically the homing capacity of LSCs to their micro-environmental niches. However, these molecular mechanisms that underlie the inhibition of homing have yet to be determined. To address these questions we conducted in vitro adhesion and blot-rolling assays to analyze the adherence and rolling capacity of these LSCs before and after treatment with anti-CD44 monoclonal antibody (mAb). Since glycosyltransferases play a crucial role in post translational carbohydrate decoration on adhesion molecules, we analyzed the expression (using quantitative PCR) of the different glycosyltransferases expressed in LSC\\'s before and after CD44 ligation (mAb treatment). Furthermore, we analyzed differentiation by flow cytometric analysis of treated and non-treated LSC\\'s. We anticipate that our results will set forth new insights into targeted therapies for AML.

  15. Assembly of pericellular matrices by COS-7 cells transfected with CD44 lymphocyte-homing receptor genes.

    OpenAIRE

    Knudson, W.; Bartnik, E; Knudson, C B

    1993-01-01

    The capacity to assemble and retain a pericellular matrix is correlated with the expression of the cell surface binding sites specific for the extracellular matrix macromolecule hyaluronan. These binding proteins have been termed hyaluronan receptors. The lymphocyte-homing receptor CD44 may have identity with these hyaluronan receptors. To determine whether hyaluronan receptors function independently in this capacity for matrix assembly, mammalian cells were transfected with cDNA encoding the...

  16. Abrogating Drug Resistance in Malignant Peripheral Nerve Sheath Tumors by Disrupting Hyaluronan-CD44 Interactions with Small Hyaluronan Oligosaccharides

    OpenAIRE

    Slomiany, Mark G.; Dai, Lu; Bomar, Paul A.; Knackstedt, Thomas J.; Kranc, D. Alex; Tolliver, Lauren; Maria, Bernard L.; Toole, Bryan P.

    2009-01-01

    Malignant peripheral nerve sheath tumors (MPNST) develop in ~10% of neurofibromatosis type-1 patients and are a major contributing factor to neurofibromatosis-1 patient mortality and morbidity. MPNSTs are multidrug resistant, and thus long-term patient survival rates are poor after standard doxorubicin or multiagent chemotherapies. We show that the hyaluronan receptor CD44 forms complexes with multidrug transporters, BCRP (ABCG2) and P-glycoprotein (ABCB1), in the plasma membrane of human MPN...

  17. Proteoglycan from salmon nasal cartridge promotes in vitro wound healing of fibroblast monolayers via the CD44 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Ito, Gen; Kobayashi, Takeshi; Takeda, Yoshie [Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550 (Japan); Sokabe, Masahiro, E-mail: msokabe@med.nagoya-u.ac.jp [Department of Physiology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550 (Japan); Mechanobiology Laboratory, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550 (Japan); Mechanobiology Institute Singapore, National University of Singapore, Singapore 117411 (Singapore)

    2015-01-16

    Highlights: • Proteoglycan from salmon nasal cartridge (SNC-PG) promoted wound healing in fibroblast monolayers. • SNC-PG stimulated both cell proliferation and cell migration. • Interaction between chondroitin sulfate-units and CD44 is responsible for the effect. - Abstract: Proteoglycans (PGs) are involved in various cellular functions including cell growth, adhesion, and differentiation; however, their physiological roles are not fully understood. In this study, we examined the effect of PG purified from salmon nasal cartilage (SNC-PG) on wound closure using tissue-cultured cell monolayers, an in vitro wound-healing assay. The results indicated that SNC-PG significantly promoted wound closure in NIH/3T3 cell monolayers by stimulating both cell proliferation and cell migration. SNC-PG was effective in concentrations from 0.1 to 10 μg/ml, but showed much less effect at higher concentrations (100–1000 μg/ml). The effect of SNC-PG was abolished by chondroitinase ABC, indicating that chondroitin sulfates (CSs), a major component of glycosaminoglycans (GAGs) in SNC-PG, are crucial for the SNC-PG effect. Furthermore, chondroitin 6-sulfate (C-6-S), a major CS of SNC-PG GAGs, could partially reproduce the SNC-PG effect and partially inhibit the binding of SNC-PG to cells, suggesting that SNC-PG exerts its effect through an interaction between the GAGs in SNC-PG and the cell surface. Neutralization by anti-CD44 antibodies or CD44 knockdown abolished SNC-PG binding to the cells and the SNC-PG effect on wound closure. These results suggest that interactions between CS-rich GAG-chains of SNC-PG and CD44 on the cell surface are responsible for the SNC-PG effect on wound closure.

  18. Clustering High-Dimensional Data: The Expression of E-cadherin, CD44 and p53 Molecules in Lip Cancer

    OpenAIRE

    KITIKIDOU, Kyriaki; Aris NTOMOUCHTSIS; Chrisoula TSOMPANIDOU; Konstantinos VAHTSEVANOS

    2010-01-01

    Objective: Clustering techniques can determine which expression patterns are important and which genes contribute to such patterns. We evaluate performance on data from a lip carcinoma study in Greece. Lip carcinoma is one of the most common malignant oral and maxillofacial tumours and in advanced clinical stages has a poor prognosis. E-cadherin, CD44 and p53 molecules are associated with cellular adhesion. Material and Methods: To prepare for clustering, we divided each of the median normali...

  19. Over expression of hyaluronan promotes progression of HCC via CD44-mediated pyruvate kinase M2 nuclear translocation

    Science.gov (United States)

    Li, Jing-Huan; Wang, Ying-Cong; Qin, Cheng-Dong; Yao, Rong-Rong; Zhang, Rui; Wang, Yan; Xie, Xiao-Ying; Zhang, Lan; Wang, Yan-Hong; Ren, Zheng-Gang

    2016-01-01

    Hyaluronan is expressed in hepatocellular carcinoma (HCC) as HCC generally arises from a cirrhotic liver in which excessive production and accumulation of HA leads to developing cirrhosis. Though it has been suggested HA is involved in progression of HCC, the mechanisms underlying the connection between HA and HCC progression are unclear. Since increased aerobic glycolysis is a metabolic trait of malignant cells and HA-CD44 can modulate glucose metabolism, we aim to investigate the roles of PKM2, a key enzyme in glucose metabolism, in the HA-CD44 axis facilitated the progress of HCC. We shown PKM2 was required for HA-promoted HCC progression, which was not modulated by PKM2 kinase activity but by nuclear translocation of PKM2. PKM2 translocation was Erk (Thr202/Tyr204) phosphorylation dependent, which functioned at the downstream of HA-CD44 binding. Furthermore, elevated HA expression significantly correlated with PKM2 nuclear location and was an independent factors predicting poor HCC prognosis. In conclusions PKM2 nuclear translocation is required for mediating the described HA biological effects on HCC progression and our results imply that inhibition of HA may have therapeutic value in treating HCC. PMID:27186420

  20. Activation of c-Met and upregulation of CD44 expression are associated with the metastatic phenotype in the colorectal cancer liver metastasis model.

    Directory of Open Access Journals (Sweden)

    Victoria A Elliott

    Full Text Available BACKGROUND: Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized. METHODS: HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice. RESULTS: CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance. CONCLUSIONS: Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.

  1. Immunoselection of breast and ovarian cancer cells with trastuzumab and natural killer cells: selective escape of CD44high/CD24low/HER2low breast cancer stem cells.

    Science.gov (United States)

    Reim, Florian; Dombrowski, Yvonne; Ritter, Cathrin; Buttmann, Mathias; Häusler, Sebastian; Ossadnik, Monika; Krockenberger, Mathias; Beier, Dagmar; Beier, Christoph P; Dietl, Johannes; Becker, Jürgen C; Hönig, Arnd; Wischhusen, Jörg

    2009-10-15

    Although trastuzumab (Herceptin) has substantially improved the overall survival of patients with mammary carcinomas, even initially well-responding tumors often become resistant. Because natural killer (NK) cell-mediated antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to contribute to the therapeutic effects of trastuzumab, we have established a cell culture system to select for ADCC-resistant SK-OV-3 ovarian cancer and MCF7 mammary carcinoma cells. Ovarian cancer cells down-regulated HER2 expression, resulting in a more resistant phenotype. MCF7 breast cancer cells, however, failed to develop resistance in vitro. Instead, treatment with trastuzumab and polyclonal NK cells resulted in the preferential survival of individual sphere-forming cells that displayed a CD44(high)CD24(low) "cancer stem cell-like" phenotype and expressed significantly less HER2 compared with non-stem cells. Likewise, the CD44(high)CD24(low) population was also found to be more immunoresistant in SK-BR3, MDA-MB231, and BT474 breast cancer cell lines. When immunoselected MCF7 cells were then re-expanded, they mostly lost the observed phenotype to regenerate a tumor cell culture that displayed the initial HER2 surface expression and ADCC-susceptibility, but was enriched in CD44(high)CD24(low) cancer stem cells. This translated into increased clonogenicity in vitro and tumorigenicity in vivo. Thus, we provide evidence that the induction of ADCC by trastuzumab and NK cells may spare the actual tumor-initiating cells, which could explain clinical relapse and progress. Moreover, our observation that the "relapsed" in vitro cultures show practically identical HER2 surface expression and susceptibility toward ADCC suggests that the administration of trastuzumab beyond relapse might be considered, especially when combined with an immune-stimulatory treatment that targets the escape variants. PMID:19826050

  2. Lubricin/Proteoglycan 4 Binding to CD44 Receptor: A Mechanism of Lubricin’s suppression of Pro-inflammatory Cytokine Induced Synoviocyte Proliferation

    Science.gov (United States)

    Al-Sharif, Afnan; Jamal, Maha; Zhang, Ling; Larson, Katherine; Schmidt, Tannin; Jay, Gregory; Elsaid, Khaled

    2015-01-01

    Objective To evaluate recombinant human proteoglycan 4 (rhPRG4) binding to CD44 receptor and its consequence on cytokine induced synoviocyte proliferation. Methods rhPRG4 binding to CD44 and competition with high molecular weight hyaluronic acid (HMW HA) was evaluated using a direct enzyme linked immunosorbent assay (ELISA) and surface plasmon resonance. Sialidase-A and O-glycosidase digestion of rhPRG4 was performed and CD44 binding was evaluated using ELISA. Rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were stimulated with interleukin-1 beta (IL-1β) or tumor necrosis factor alpha (TNF-α) for 48 hours in the presence or absence of rhPRG4 or HMW HA at 20, 40 and 80μg/ml and cell proliferation was measured. CD44 contribution was assessed by co-incubation with a CD44 antibody (IM7). The anti-proliferative effect of rhPRG4 was investigated following treatment of Prg4−/− synoviocytes with IL-1β or TNF-α in the presence or absence of IM7. Results rhPRG4 binds CD44 and interferes with HMW HA CD44 binding. Removal of sialic acid and O-glycosylations significantly increased CD44 binding by rhPRG4 (p<0.001). rhPRG4 and HMW HA at 40 and 80μg/ml significantly suppressed IL-1β induced RA-FLS proliferation (p<0.05). rhPRG4 at 20, 40 and 80μg/ml significantly suppressed TNF-α induced RA-FLS proliferation (p<0.05). CD44 neutralization reversed the effect of rhPRG4 on IL-1β and TNF-α stimulated RA-FLS and the effect of HMW HA on IL-1β stimulated RA-FLS. rhPRG4 inhibited cytokine-induced proliferation of Prg4−/− synoviocytes which could be prevented by blocking CD44. Conclusion Lubricin is a novel putative ligand for CD44 and may control synoviocyte overgrowth in inflammatory arthropathies via a CD44-mediated mechanism. PMID:25708025

  3. Hyaluronan-decorated polymer nanoparticles targeting the CD44 receptor for the combined photo/chemo-therapy of cancer

    Science.gov (United States)

    Maiolino, Sara; Moret, Francesca; Conte, Claudia; Fraix, Aurore; Tirino, Pasquale; Ungaro, Francesca; Sortino, Salvatore; Reddi, Elena; Quaglia, Fabiana

    2015-03-01

    In the attempt to develop novel concepts in designing targeted nanoparticles for combination therapy of cancer, we propose here CD44-targeted hyaluronan-decorated double-coated nanoparticles (dcNPs) delivering the lipophilic chemotherapeutic docetaxel (DTX) and an anionic porphyrin (TPPS4). dcNPs are based on electrostatic interactions between a negative DTX-loaded nanoscaffold of poly(lactide-co-glycolide), a polycationic shell of polyethyleneimine entangling negatively-charged TPPS4 and finally decorated with hyaluronan (HA) to promote internalization through CD44 receptor-mediated endocytosis. DTX/TPPS4-dcNPs, prepared through layer-by-layer deposition, showed a hydrodynamic diameter of around 180 nm, negative zeta potential and efficient loading of both DTX and TPPS4. DTX/TPPS4-dcNPs were freeze-dried with trehalose giving a powder that could be easily dispersed in different media. Excellent stability of dcNPs in specific salt- and protein-containing media was found. Spectroscopic behavior of DTX/TPPS4-dcNPs demonstrated a face-to-face arrangement of the TPPS4 units in non-photoresponsive H-type aggregates accounting for an extensive aggregation of the porphyrin embedded in the shell. Experiments in MDA-MB-231 cells overexpressing the CD44 receptor demonstrated a 9.4-fold increase in the intracellular level of TPPS4 delivered from dcNPs as compared to free TPPS4. Light-induced death increased tremendously in cells that had been treated with a combination of TPPS4 and DTX delivered through dcNPs as compared with free drugs, presumably due to efficient uptake and co-localization inside the cells. In perspective, the strategy proposed here to target synergistic drug combinations through HA-decorated nanoparticles seems very attractive to improve the specificity and efficacy of cancer treatment.In the attempt to develop novel concepts in designing targeted nanoparticles for combination therapy of cancer, we propose here CD44-targeted hyaluronan-decorated double

  4. Effect of CD44 binding peptide conjugated to an engineered inert matrix on maintenance of breast cancer stem cells and tumorsphere formation.

    Directory of Open Access Journals (Sweden)

    Xiaoming Yang

    Full Text Available INTRODUCTION: As cancer cells are affected by many factors in their microenvironment, a major challenge is to isolate the effect of a specific factor on cancer stem cells (CSCs while keeping other factors unchanged. We have developed a synthetic inert 3D polyethylene glycol diacrylate (PEGDA gel culture system as a unique tool to study the effect of microenvironmental factors on CSCs response. We have reported that CSCs formed in the inert PEGDA gel by encapsulation of breast cancer cells maintain their stemness within a certain range of gel stiffness. The objective was to investigate the effect of CD44 binding peptide (CD44BP conjugated to the gel on the maintenance of breast CSCs. METHODS: 4T1 or MCF7 breast cancer cells were encapsulated in PEGDA gel with CD44BP conjugation. Control groups included dissolved CD44BP and the gel with mutant CD44BP conjugation. Tumorsphere size and density, and expression of CSC markers were determined after 9 days. For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined after 4 weeks. Effect of CD44BP conjugation on breast CSC maintenance was compared with integrin binding RGD peptide (IBP and fibronectin-derived heparin binding peptide (FHBP. RESULTS: Conjugation of CD44BP to the gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. CONCLUSION: CD44BP and IBP conjugated to the gel abolished tumorsphere formation by encapsulated 4T1 cells while FHBP enhanced tumorsphere formation compared to cells in the gel without peptide. The PEGDA hydrogel culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors.

  5. Clustering High-Dimensional Data: The Expression of E-cadherin, CD44 and p53 Molecules in Lip Cancer

    Directory of Open Access Journals (Sweden)

    Kyriaki KITIKIDOU

    2010-01-01

    Full Text Available Objective: Clustering techniques can determine which expression patterns are important and which genes contribute to such patterns. We evaluate performance on data from a lip carcinoma study in Greece. Lip carcinoma is one of the most common malignant oral and maxillofacial tumours and in advanced clinical stages has a poor prognosis. E-cadherin, CD44 and p53 molecules are associated with cellular adhesion. Material and Methods: To prepare for clustering, we divided each of the median normalized gene expression values by the range of that gene. Next, we set our prior parameters and we performed the final inference using pooled sets of Markov chain Monte Carlo (MCMC runs. After pooling the chains, we grouped the data into clusters and selected E-cadherin, CD44 and p53 molecules using the marginal median model as cut off. The selection of a small set of genes is advantageous here. A small number of selected genes is appealing to biologists because they constitute a manageable set of candidates on which further studies can be performed. Results: E-cadherin, CD44 and p53 molecules were selected as discriminatory. Results highlight the fact that clustering method has successfully selected genes that are biologically consistent with current research and that provide strong biological validation of the cluster configuration suggested. Conclusion: A clustering method that takes advantage of known substructure in the data when simultaneously clustering high-dimensional data with an unknown number of clusters, and selecting the best discriminating variables for those clusters implies the opportunity to handle bigger datasets. When analyzing real data, clustering has found three genes that agree with current biological research and literature and that provide biological validation of the cluster configuration. Overall, clustering can provide biologists with both useful and manageable information for further experimental research.

  6. Expression of PCNA and CD44mRNA in colorectal cancer with venous invasion and its relationship to liver metastasis

    Institute of Scientific and Technical Information of China (English)

    Shu-Qiang Yue; Yan-Ling Yang; Ke-Feng Dou; Kai-Zong Li

    2003-01-01

    AIM: To investigate the expression of proliferating cell nuclear antigen (PCNA) and CD44mRNA in colorectal cancer with venous invasion and its relationship with liver metastasis.METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of PCNA and CD44mRNA in 31 cases of colorectal cancer with venous invasion.RESULTS: Positive expression rates of PCNA and CD44mRNA in colorectal cancer were higher than those without liver metastasis (P<0.05 and P<0.01). In case of colorectal cancer with liver metastasis, strongly positive rates of PCNA and CD44mRNA were 94.1% and 70.6 %,respectively, significantly higher than those without liver metastasis. There was a positive relationship between the expressions of PCNA and CD44mRNA (r=0.67, P<0.05).CONCLUSION: Detection of PCNA and CD44mRNA expression in colorectal cancer may be useful for evaluating liver metastasis of cancer cells.

  7. STUDY ON THE EFFECT OF T-2 TOXIN AND SELENIUM ON CD44 EXPRESSION IN THE CULTURED HUMAN FETAL CHONDROCYTES IN VITRO

    Institute of Scientific and Technical Information of China (English)

    谢龙; 曹峻岭; 岳燕; 朱建宏; 张增铁; 张富军; 李思远

    2003-01-01

    Objective To investigate the effect on the structure of reestablished cartilage in vitro and CD44 expression on chondrocytes and compare the inducing effect on the reestablished cartilage in vitro between cortical bone matrix gelatin and cancellous bone matrix gelatin. Methods To plant human fetal chondrocytes on the BMG, the damage of the cultured chondrocytes was observed by the optical microscope (HE staining). The immunohistochemistry of CD44 was quantitative analysis by the image collection and analysis system. Results With the increasing concentration of T-2 toxin, the damage of chondroytes was more and more evident and CD44 expression was lowered. After adding selenium, the damage was relieved and CD44 expression increased. The density of chondrocytes on the cortical bone matrix gelatin was much higher than that on the cancellous bone matrix gelatin. Conclusion T-2 toxin can lower the CD44 expression on the chondrocytes and adding selenium can relieve the damage caused by T-2toxin and increased CD44 expression. The inducing effect on reestablished cartilage in vitro of cortical bone matrix gelatin was much higher than that of cancellous bone matrix gelatin.

  8. Activation of VCAM-1 and Its Associated Molecule CD44 Leads to Increased Malignant Potential of Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Pei-Chen Wang

    2014-02-01

    Full Text Available VCAM-1 (CD106, a transmembrane glycoprotein, was first reported to play an important role in leukocyte adhesion, leukocyte transendothelial migration and cell activation by binding to integrin VLA-1 (α4β1. In the present study, we observed that VCAM-1 expression can be induced in many breast cancer epithelial cells by cytokine stimulation in vitro and its up-regulation directly correlated with advanced clinical breast cancer stage. We found that VCAM-1 over-expression in the NMuMG breast epithelial cells controls the epithelial and mesenchymal transition (EMT program to increase cell motility rates and promote chemoresistance to doxorubicin and cisplatin in vitro. Conversely, in the established MDAMB231 metastatic breast cancer cell line, we confirmed that knockdown of endogenous VCAM-1 expression reduced cell proliferation and inhibited TGFβ1 or IL-6 mediated cell migration, and increased chemosensitivity. Furthermore, we demonstrated that knockdown of endogenous VCAM-1 expression in MDAMB231 cells reduced tumor formation in a SCID xenograft mouse model. Signaling studies showed that VCAM-1 physically associates with CD44 and enhances CD44 and ABCG2 expression. Our findings uncover the possible mechanism of VCAM-1 activation facilitating breast cancer progression, and suggest that targeting VCAM-1 is an attractive strategy for therapeutic intervention.

  9. The anti-migratory effects of FKBPL and its peptide derivative, AD-01: regulation of CD44 and the cytoskeletal pathway.

    Directory of Open Access Journals (Sweden)

    Anita Yakkundi

    Full Text Available FK506 binding protein-like (FKBPL and its peptide derivatives exert potent anti-angiogenic activity in vitro and in vivo and control tumour growth in xenograft models, when administered exogenously. However, the role of endogenous FKBPL in angiogenesis is not well characterised. Here we investigated the molecular effects of the endogenous protein and its peptide derivative, AD-01, leading to their anti-migratory activity. Inhibition of secreted FKBPL using a blocking antibody or siRNA-mediated knockdown of FKBPL accelerated the migration of human microvascular endothelial cells (HMEC-1. Furthermore, MDA-MB-231 tumour cells stably overexpressing FKBPL inhibited tumour vascular development in vivo suggesting that FKBPL secreted from tumour cells could inhibit angiogenesis. Whilst FKBPL and AD-01 target CD44, the nature of this interaction is not known and here we have further interrogated this aspect. We have demonstrated that FKBPL and AD-01 bind to the CD44 receptor and inhibit tumour cell migration in a CD44 dependant manner; CD44 knockdown abrogated AD-01 binding as well as its anti-migratory activity. Interestingly, FKBPL overexpression and knockdown or treatment with AD-01, regulated CD44 expression, suggesting a co-regulatory pathway for these two proteins. Downstream of CD44, alterations in the actin cytoskeleton, indicated by intense cortical actin staining and a lack of cell spreading and communication were observed following treatment with AD-01, explaining the anti-migratory phenotype. Concomitantly, AD-01 inhibited Rac-1 activity, up-regulated RhoA and the actin binding proteins, profilin and vinculin. Thus the anti-angiogenic protein, FKBPL, and AD-01, offer a promising and alternative approach for targeting both CD44 positive tumours and vasculature networks.

  10. ETS-1-mediated transcriptional up-regulation of CD44 is required for sphingosine-1-phosphate receptor subtype 3-stimulated chemotaxis.

    Science.gov (United States)

    Zhang, Wenliang; Zhao, Jiawei; Lee, Jen-Fu; Gartung, Allison; Jawadi, Hiba; Lambiv, Wanyu Louis; Honn, Kenneth V; Lee, Menq-Jer

    2013-11-01

    Sphingosine-1-phosphate (S1P)-regulated chemotaxis plays critical roles in various physiological and pathophysiological conditions. S1P-regulated chemotaxis is mediated by the S1P family of G-protein-coupled receptors. However, molecular details of the S1P-regulated chemotaxis are incompletely understood. Cultured human lung adenocarcinoma cell lines abundantly express S1P receptor subtype 3 (S1P3), thus providing a tractable in vitro system to characterize molecular mechanism(s) underlying the S1P3 receptor-regulated chemotactic response. S1P treatment enhances CD44 expression and induces membrane localization of CD44 polypeptides via the S1P3/Rho kinase (ROCK) signaling pathway. Knockdown of CD44 completely diminishes the S1P-stimulated chemotaxis. Promoter analysis suggests that the CD44 promoter contains binding sites of the ETS-1 (v-ets erythroblastosis virus E26 oncogene homolog 1) transcriptional factor. ChIP assay confirms that S1P treatment stimulates the binding of ETS-1 to the CD44 promoter region. Moreover, S1P induces the expression and nuclear translocation of ETS-1. Knockdown of S1P3 or inhibition of ROCK abrogates the S1P-induced ETS-1 expression. Furthermore, knockdown of ETS-1 inhibits the S1P-induced CD44 expression and cell migration. In addition, we showed that S1P3/ROCK signaling up-regulates ETS-1 via the activity of JNK. Collectively, we characterized a novel signaling axis, i.e., ROCK-JNK-ETS-1-CD44 pathway, which plays an essential role in the S1P3-regulated chemotactic response.

  11. Selective regain of egfr gene copies in CD44+/CD24-/low breast cancer cellular model MDA-MB-468

    Directory of Open Access Journals (Sweden)

    Andreas Antje

    2010-03-01

    Full Text Available Abstract Background Increased transcription of oncogenes like the epidermal growth factor receptor (EGFR is frequently caused by amplification of the whole gene or at least of regulatory sequences. Aim of this study was to pinpoint mechanistic parameters occurring during egfr copy number gains leading to a stable EGFR overexpression and high sensitivity to extracellular signalling. A deeper understanding of those marker events might improve early diagnosis of cancer in suspect lesions, early detection of cancer progression and the prediction of egfr targeted therapies. Methods The basal-like/stemness type breast cancer cell line subpopulation MDA-MB-468 CD44high/CD24-/low, carrying high egfr amplifications, was chosen as a model system in this study. Subclones of the heterogeneous cell line expressing low and high EGF receptor densities were isolated by cell sorting. Genomic profiling was carried out for these by means of SNP array profiling, qPCR and FISH. Cell cycle analysis was performed using the BrdU quenching technique. Results Low and high EGFR expressing MDA-MB-468 CD44+/CD24-/low subpopulations separated by cell sorting showed intermediate and high copy numbers of egfr, respectively. However, during cell culture an increase solely for egfr gene copy numbers in the intermediate subpopulation occurred. This shift was based on the formation of new cells which regained egfr gene copies. By two parametric cell cycle analysis clonal effects mediated through growth advantage of cells bearing higher egfr gene copy numbers could most likely be excluded for being the driving force. Subsequently, the detection of a fragile site distal to the egfr gene, sustaining uncapped telomere-less chromosomal ends, the ladder-like structure of the intrachromosomal egfr amplification and a broader range of egfr copy numbers support the assumption that dynamic chromosomal rearrangements, like breakage-fusion-bridge-cycles other than proliferation drive the gain

  12. CD44-Targeted Hyaluronic Acid-Coated Redox-Responsive Hyperbranched Poly(amido amine)/Plasmid DNA Ternary Nanoassemblies for Efficient Gene Delivery.

    Science.gov (United States)

    Gu, Jijin; Chen, Xinyi; Ren, Xiaoqing; Zhang, Xiulei; Fang, Xiaoling; Sha, Xianyi

    2016-07-20

    Hyaluronic acid (HA), which can specifically bind to CD44 receptor, is a specific ligand for targeting to CD44-overexpressing cancer cells. The current study aimed to develop ternary nanoassemblies based on HA-coating for targeted gene delivery to CD44-positive tumors. A novel reducible hyperbranched poly(amido amine) (RHB) was assembled with plasmid DNA (pDNA) to form RHB/pDNA nanoassemblies. HA/RHB/pDNA nanoassemblies were fabricated by coating HA on the surface of the RHB/pDNA nanoassembly core through electrostatic interaction. After optimization, HA/RHB/pDNA nanoassemblies were spherical, core-shell nanoparticles with nanosize (187.6 ± 11.4 nm) and negative charge (-9.1 ± 0.3 mV). The ternary nanoassemblies could efficiently protect the condensed pDNA from enzymatic degradation by DNase I, and HA could significantly improve the stability of nanoassemblies in the sodium heparin solution or serum in vitro. As expected, HA significantly decreased the cytotoxicity of RHB/pDNA nanoassemblies due to the negative surface charges. Moreover, it revealed that HA/RHB/pDNA nanoassemblies showed higher transfection activity than RHB/pDNA nanoassemblies in B16F10 cells, especially in the presence of serum in vitro. Because of the active recognition between HA and CD44 receptor, there was significantly different transfection efficiency between B16F10 (CD44+) and NIH3T3 (CD44-) cells after treatment with HA/RHB/pDNA nanoassemblies. In addition, the cellular targeting and transfection activity of HA/RHB/pDNA nanoassemblies were further evaluated in vivo. The results indicated that the interaction between HA and CD44 receptor dramatically improved the accumulation of HA/RHB/pDNA nanoassemblies in CD44-positive tumor, leading to higher gene expression than RHB/pDNA nanoassemblies. Therefore, HA/RHB/pDNA ternary nanoassemblies may be a potential gene vector for delivery of therapeutic genes to treat CD44-overexpressing tumors in vivo. PMID:27311558

  13. Targeting CD44 by Hyaluronic Acid-Based Nano Drug Delivery Systems May Eradicate Cancer Stem Cells in Human Breast Cancer

    Directory of Open Access Journals (Sweden)

    Rassoul Dinarvand

    2011-01-01

    Full Text Available Despite the significant progress in cancer diagnosis and therapy, still invasion and metastasis of cancer cells, development of drug resistance and cancer recurrence are the main causes of mortality in cancer patients. Recent researches on cancer stem cells (CSCs along with the role of CD44 marker in drug resistance and as the main marker of breast CSCs, highlight the importance of CD44 in cancer targeted therapy. Additionally, co-localization of MDR1 and CD44 in cancer cell population showed that one protein directly influences the expression of the other and disruption of interaction has significant effects on drug resistance, cell migration and in vitro invasion. Based on the above information, using nanotechnology-derived CD44 targeted drug delivery systems will be able to address recurrence of the disease and other major obstacles in cancer chemotherapy. Therefore, we hypotheses that using combination of cytotoxic agents and CSC specific agents anchored in hyaluronic acid (as the endogenous substrate of CD44, have the potential to develop novel drug delivery systems to eradicate breast cancer.

  14. Efficient CD44-targeted magnetic resonance imaging (MRI) of breast cancer cells using hyaluronic acid (HA)-modified MnFe2O4 nanocrystals

    Science.gov (United States)

    Lee, Taeksu; Lim, Eun-Kyung; Lee, Jaemin; Kang, Byunghoon; Choi, Jihye; Park, Hyo Seon; Suh, Jin-Suck; Huh, Yong-Min; Haam, Seungjoo

    2013-04-01

    Targeted molecular imaging with hyaluronic acid (HA) has been highlighted in the diagnosis and treatment of CD44-overexpressing cancer. CD44, a receptor for HA, is closely related to the growth of cancer including proliferation, metastasis, invasion, and angiogenesis. For the efficient detection of CD44, we fabricated a few kinds of HA-modified MnFe2O4 nanocrystals (MNCs) to serve as specific magnetic resonance (MR) contrast agents (HA-MRCAs) and compared physicochemical properties, biocompatibility, and the CD44 targeting efficiency. Hydrophobic MNCs were efficiently phase-transferred using aminated polysorbate 80 (P80) synthesized by introducing spermine molecules on the hydroxyl groups of P80. Subsequently, a few kinds of HA-MRCAs were fabricated, conjugating different ratios of HA on the equal amount of phase-transferred MNCs. The optimized conjugation ratio of HA against magnetic content was identified to exhibit not only effective CD44 finding ability but also high cell viability through in vitro experiments. The results of this study demonstrate that the suggested HA-MRCA shows strong potential to be used for accurate tumor diagnosis.

  15. Structure-Function Analysis of the Non-Muscle Myosin Light Chain Kinase (nmMLCK) Isoform by NMR Spectroscopy and Molecular Modeling: Influence of MYLK Variants.

    Science.gov (United States)

    Shen, Kui; Ramirez, Benjamin; Mapes, Brandon; Shen, Grace R; Gokhale, Vijay; Brown, Mary E; Santarsiero, Bernard; Ishii, Yoshitaka; Dudek, Steven M; Wang, Ting; Garcia, Joe G N

    2015-01-01

    The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

  16. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels

    DEFF Research Database (Denmark)

    Hviid, Thomas Vauvert F; Hylenius, Sine; Rørbye, Christina;

    2003-01-01

    During pregnancy, the human extra-villous trophoblast in the contact zone between maternal and fetal tissue in the placenta does not express the classical MHC class I and II molecules. Instead, HLA-G and -C, and possibly HLA-E, are expressed. HLA-G may modulate the immunological relationship...... between mother and fetus in several ways. Finally, the expression of membrane-bound HLA-G and soluble HLA-G has been proposed to influence the outcome of pregnancy, and an aberrant HLA-G expression in pre-eclamptic placentas and spontaneous abortions has been reported. Here, an association between certain...... HLA-G polymorphisms and the mRNA levels of the different alternatively spliced HLA-G isoforms in first trimester trophoblast cell populations is reported. Several alternatively spliced HLA-G mRNA isoforms, including a 14-bp polymorphism in the 3'UTR end (exon 8) of the HLA-G gene, are expressed at a...

  17. Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

    Directory of Open Access Journals (Sweden)

    Olgun eGuvench

    2015-06-01

    Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

  18. Role of the Phosphorylation of mTOR in the Differentiation of AML Cells Triggered with CD44 Antigen

    KAUST Repository

    Darwish, Manar M

    2013-05-01

    Acute myeloid leukemia (AML) is a hematological disorder characterized by blockage of differentiation of myeloblasts. To date, the main therapy for AML is chemotherapy. Yet, studies are seeking a better treatment to enhance the survival rate of patients and minimize the relapsing of the disease. Since the major problem in these cells is that they are arrested in cellular differentiation, drugs that could induce their differentiation have proven to be efficient and of major interest for AML therapy. CD44 triggering appeared as a promising target for AML therapy as it has been shown that specific monoclonal antibodies, such as A3D8 and H90, reversed the blockage of differentiation, inhibited the proliferation of all AML subtypes, and in some cases, induced cell apoptosis. Studies conducted in our laboratory have added strength to these antibodies as potential treatment for AML. Indeed, our laboratory found that treating HL60 cells with A3D8 shows a decrease in the phosphorylation of the mammalian target of Rapamycin (mTOR) kinase correlated with the inhibition of proliferation/induction of differentiation of AML cells.The relationship between the induction of differentiation and the inhibition of proliferation and the decrease of mTOR phosphorylation remains to be clarified. To study the importance of the de-phosphorylation of mTOR and the observed effect of CD44 triggering on differentiation and/or proliferation, we sought to prepare phospho-mimic mutants of the mTOR kinase that will code for a constitutively phosphorylated form of mTOR and used two main methods to express this mutant in HL60 cells: lentiviral and simple transfection (cationic-liposomal transfection).

  19. CD44+/CD24-细胞在乳腺癌组织及细胞系中的数量与分布%Quantity and distribution of CD44+/CD24-cells in breast cancer tissue and the cell lines

    Institute of Scientific and Technical Information of China (English)

    吕新全; Zhenhe Suo; 马长路; 徐珂佳; 刘羿杉; 李惠翔

    2009-01-01

    Objective To study the distribution and quantity of CD44VCD24- cells in breast cancer tissue and the cell lines,and as well as its correlation with the expression of various breast cancer markers and molecular subtyping of breast carcinoma.Methods The expression of CD44 / CD24,estrogen receptor,progesterone receptor,HER2,human estrogen-induced protein PS2,bcl-2 and nm23 in 60 cases of invasive ductal carcinoma of breast were studied by either single or double immunohistochemical staining.The co-expression of CD44 and CD24 in 3 breast cancer cell lines (MCF-7,MDA-MB-468,and MDA-MB- 231) was also examined.Results The quantity and distribution of CD44 + /CD24- cells varied greatly and no specific patterns were identified.The percentage of CD44 + /CD24- in breast cancer was 65%.The amount of CD44+/CD24- cells did not correlate with the age of patients,lymph node metastasis,tumor  size,molecular subtypes and expression of various breast cancer markers in breast carcinoma.The proportion of CD44+/CD24- cells in MCF-7,MDA-MB-468,and MDA-MB-231 cell lines was 80% ,respectively.Conclusions CD44+ /CD24- cells are demonstrated in certain breast cancer tissues and cell lines.However,there is no relationship obtained between the quantity or the distribution of these cells and the molecular subtyping or the clinicopathologic parameters in breast cancer.%目的 检测CD44+/CIY24-细胞在乳腺癌组织及细胞系中的分布及数量,探讨其与乳腺癌常用标志物表达和乳腺癌分子亚型的关系.方法 采用免疫组织化学SP双染及单染法,分别检测了60例乳腺浸润性导管癌中的CD44及C1724的共表达情况和ER、PR、HER2、人雌激素诱导蛋白PS2、bcl-2、nm23的单独表达情况,同时检测了三种乳腺痛细胞系(MCF-7、MDA-MB-468及MDA-MB-231)中CD44及CD24的表达情况.结果 不同病例标本中CD44+/C1724-细胞的数量差异较大,分布无明显规律,总阳性率为65.0%;CD44+/CD24-细胞数量与患者年龄、肿瘤

  20. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

    Science.gov (United States)

    Maisel, Daniela; Birzele, Fabian; Voss, Edgar; Nopora, Adam; Bader, Sabine; Friess, Thomas; Goller, Bernhard; Laifenfeld, Daphna; Weigand, Stefan; Runza, Valeria

    2016-01-01

    CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages) to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP) of the malignant cells by macrophages. PMID:27463372

  1. Targeting Tumor Cells with Anti-CD44 Antibody Triggers Macrophage-Mediated Immune Modulatory Effects in a Cancer Xenograft Model.

    Directory of Open Access Journals (Sweden)

    Daniela Maisel

    Full Text Available CD44, a transmembrane receptor reported to be involved in various cellular functions, is overexpressed in several cancer types and supposed to be involved in the initiation, progression and prognosis of these cancers. Since the sequence of events following the blockage of the CD44-HA interaction has not yet been studied in detail, we profiled xenograft tumors by RNA Sequencing to elucidate the mode of action of the anti-CD44 antibody RG7356. Analysis of tumor and host gene-expression profiles led us to the hypothesis that treatment with RG7356 antibody leads to an activation of the immune system. Using cytokine measurements we further show that this activation involves the secretion of chemo-attractants necessary for the recruitment of immune cells (i.e. macrophages to the tumor site. We finally provide evidence for antibody-dependent cellular phagocytosis (ADCP of the malignant cells by macrophages.

  2. HER2阳性乳腺癌中CD44v6的表达与曲妥珠单抗耐药的关系%Relationship between Expression of CD44v6 and Resistance of Trastuzumab Chemotheraphy in Metastatic Breast Cancer Patients with HER2 Positive

    Institute of Scientific and Technical Information of China (English)

    王延桂; 高美华

    2011-01-01

    目的:探讨曲妥珠单抗在治疗HER2阳性的转移性乳腺癌时,产生耐药性与CD44v6表达的相关性.方法:共66例HER2阳性转移性乳腺癌患者入组.接受曲妥珠单抗治疗的患者37例,其中18例获得了治疗前和治疗后转移性癌组织.采用免疫组化S-P法对治疗前、治疗后的不同乳腺组织进行CD44v6表达的研究.结果:CD44v6在经曲妥珠单抗治疗产生耐药的活检组织中阳性表达程度明显高于治疗前.结论:CD44v6的表达与曲妥珠单抗耐药相关.%Objective: To explore the correlation between the expression of CD44v6 and resistance of trastuzumab chemotheraphy for metastatic breast cancer patient with HER2 positive.Methods: 66 metastatic breast cancer patients with HER2 positive were eligible.Among 66 patients, 37 were received trastuzumab, and there were 18 patients who had tumor tissues before and after therapy.All species were analyzed by S-P immunohistochemical method using the monoclonal antibody CD44 v6.Results: CD44v6 in breast cancer tissues of resistance to traszuzumab were signifcantly higher than before the treatment.Conclusion: There may be correlation between the expression of CD44v6 and resistance to trastuzumab.

  3. 粘附因子CD44v3在正常绒毛和葡萄胎中的表达及相关性研究%Expression of CD44 v3 and Their Correlation in Normal Placetal Villi and Hydatidiform Mole

    Institute of Scientific and Technical Information of China (English)

    白兴武

    2011-01-01

    目的:探讨CD44v3在正常绒毛和葡萄胎中的表达及相关性.方法:采用免疫组化SABC法检测CD44v3在50例正常绒毛,50例良性葡萄胎,40例恶性葡萄胎的表达情况.结果:CD44v3在正常绒毛,良性葡萄胎,恶性葡萄胎中阳性表达率分别是:8.0%,30.6%,90.0%,组间比较差异有统计学意义(P<0.05).结论:CD44v3的过表达促进葡萄胎的浸润、转移,可作为葡萄胎的浸润、转移及评价预后生物学指标.%Objective: To investigate the expression of CD44 v3 and their correlation in normal placetal villi and hydatidiform mole. Method: The expression of CD44 v3 protein was detected in 50 specimens nomal placetal villi ,50 specimens benign hydatidiform mole and 40 specimens of malignant hydatidiform mole by immunohistochemistry (SABC method ). Result: In normal placetal villi, benign hydatidiform mole, malignant hydatidiform mole ,the expression rate of CD44 v3 were 8.0%, 30.6% and 90.0% respectively . Conclusion:The expressions of CD44v3 are correlated to invasion and metastasis of hydatidiform mole, thus can be as markers to predict metastasis and invasion of hydatidiform mole and the prognosis of these patients as well.

  4. CD44 targeted chemotherapy for co-eradication of breast cancer stem cells and cancer cells using polymeric nanoparticles of salinomycin and paclitaxel.

    Science.gov (United States)

    Muntimadugu, Eameema; Kumar, Rajendra; Saladi, Shantikumar; Rafeeqi, Towseef Amin; Khan, Wahid

    2016-07-01

    This combinational therapy is mainly aimed for complete eradication of tumor by killing both cancer cells and cancer stem cells. Salinomycin (SLM) was targeted towards cancer stem cells whereas paclitaxel (PTX) was used to kill cancer cells. Drug loaded poly (lactic-co-glycolic acid) nanoparticles were prepared by emulsion solvent diffusion method using cationic stabilizer. Size of the nanoparticles (below 150nm) was determined by dynamic light scattering technique and transmission electron microscopy. In vitro release study confirmed the sustained release pattern of SLM and PTX from nanoparticles more than a month. Cytotoxicity studies on MCF-7 cells revealed the toxicity potential of nanoparticles over drug solutions. Hyaluronic acid (HA) was coated onto the surface of SLM nanoparticles for targeting CD44 receptors over expressed on cancer stem cells and they showed the highest cytotoxicity with minimum IC50 on breast cancer cells. Synergistic cytotoxic effect was also observed with combination of nanoparticles. Cell uptake studies were carried out using FITC loaded nanoparticles. These particles showed improved cellular uptake over FITC solution and HA coating further enhanced the effect by 1.5 folds. CD44 binding efficiency of nanoparticles was studied by staining MDA-MB-231 cells with anti CD44 human antibody and CD44(+) cells were enumerated using flow cytometry. CD44(+) cell count was drastically decreased when treated with HA coated SLM nanoparticles indicating their efficiency towards cancer stem cells. Combination of HA coated SLM nanoparticles and PTX nanoparticles showed the highest cytotoxicity against CD44(+) cells. Hence combinational therapy using conventional chemotherapeutic drug and cancer stem cell inhibitor could be a promising approach in overcoming cancer recurrence due to resistant cell population. PMID:27045981

  5. Osteopontin and the C-terminal peptide of thrombospondin-4 compete for CD44 binding and have opposite effects on CD133+ cell colony formation

    Directory of Open Access Journals (Sweden)

    Dobocan Monica C

    2009-10-01

    Full Text Available Abstract Background C21, the C-terminal peptide of thrombospondin-4, has growth promoting activity and was discovered as one of several erythropoietin-dependent endothelial proteins. C21 stimulates red cell formation in anemic mice and is a growth factor for CD34+ and CD36+ hematopoietic cells, skin fibroblasts and kidney epithelial cells. ROD1 has been identified as an intracellular mediator. Nothing is known about the existence of putative C21 receptors on plasma membranes of target cells. Findings We analyzed the nature of C21-binding proteins in cell lysates of skin fibroblasts using C21 affinity columns. The membrane receptor CD44 was identified as C21-binding protein by mass spectrometry. We were unable to demonstrate any direct involvement of CD44 on cell growth or the effect of C21 on cell proliferation. A soluble form of CD44 was synthesized in insect cells and purified from culture supernatants with a combination of PVDF filtration in the presence of ammonium sulphate and HPLC. Both osteopontin and hyaluronic acid competitively displaced Biotin-C21 binding to CD44. In a colony-forming assay using primitive CD133+ hematopoietic stem cells from cord blood, osteopontin and C21 had opposite effects and C21 reduced the inhibitory action of osteopontin. Conclusion CD44 is a C21-binding membrane protein. We could not demonstrate an involvement of CD44 in the proliferative action of C21. Nevertheless, based on the antagonism of C21 and osteopontin in hematopoietic precursors, we speculate that C21 could indirectly have a major impact on hematopoietic stem cell proliferation, by hindering osteopontin membrane binding at the level of the bone marrow niche.

  6. Impact of CD44v6 overexpression on invasion and metastasis of colon cancer SW480 cells%过表达CD44v6基因对人大肠癌SW480细胞侵袭迁移能力的影响

    Institute of Scientific and Technical Information of China (English)

    吕林; 刘海光; 张筱骅

    2016-01-01

    目的:研究CD44v6基因过表达对SW480细胞侵袭和迁移能力的影响.方法:慢病毒介导的CD44v6过表达细胞(CD44v6组)和空载体对照细胞(NC组)由前期实验构建,采用荧光显微镜观察增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)表达、实时荧光定量PCR检测CD44v6 mRNA表达水平、免疫荧光检测Flag标签蛋白三种方法重新鉴定过表达细胞模型;CCK-8法检测细胞增殖活性;划痕试验和Transwell试验检测细胞侵袭和迁移能力.结果:绿色荧光蛋白观察显示细胞转染效率近100%;实时荧光定量PCR显示CD44v6组细胞CD44v6 mRNA表达水平较对照组显著升高(P<0.001);Flag标签蛋白免疫荧光染色显示过表达C D44v6蛋白主要定位于细胞膜.CCK-8结果显示2组细胞增殖无明显差异;划痕试验结果显示CD44v6组细胞划痕愈合指数较对照组显著增高(P<0.05);Transwell试验结果显示CD44v6组细胞迁移和侵袭相关指数均较对照组显著增高(均P<0.05),且CD44v6抗体处理后,CD44v6组细胞迁移和侵袭相关指数均较前显著减低(均P<0.05).结论:CD44v基因过表达能显著增强SW480细胞侵袭和迁移能力.%AIM:To investigate the impact of CD44v6 overexpression on the invasion and metastasis of human colon cancer SW480 cells.METHODS:SW480 cells stably overexpressing CD44v6 (CD44v6 group) and negative control cells (NC group) were developed through lentivirus infection.Transfection efficiency was evaluated by detecting the expression of enhanced green fluorescent protein (EGFP).CD44v6 mRNA levels were determined using quantitative real-time PCR.Localization of the overexpressed protein was observed by immunofluorescence staining of Flag protein.Cell proliferation was determined by cell counting kit (CCK)-8 assay.Cell invasion and metastasis were examined by scratch assay and transwell assay.RESULTS:EGFP detection indicated that transfection efficiency was close to 100% in both groups.CD

  7. Induced growth inhibition, cell cycle arrest and apoptosis in CD133+/CD44+ prostate cancer stem cells by flavopiridol

    Science.gov (United States)

    SONER, BURAK CEM; AKTUG, HUSEYIN; ACIKGOZ, EDA; DUZAGAC, FAHRIYE; GUVEN, UMMU; AYLA, SULE; CAL, CAG; OKTEM, GULPERI

    2014-01-01

    Flavopiridol is a flavone that inhibits several cyclin-dependent kinases and exhibits potent growth-inhibitory activity, apoptosis and G1-phase arrest in a number of human tumor cell lines. Flavopiridol is currently undergoing investigation in human clinical trials. The present study focused on the effect of flavopiridol in cell proliferation, cell cycle progression and apoptosis in prostate cancer stem cells (CSCs). Therefore, cluster of differentiation 133 (CD133)+high/CD44+high prostate CSCs were isolated from the DU145 human prostate cancer cell line. The cells were treated with flavopiridol in a dose- and time-dependent manner to determine the inhibitory effect. Cell viability and proliferation were analyzed and the efficiency of flavopiridol was assessed using the sphere-forming assay. Flavopiridol was applied to monolayer cultures of CD133high/CD44high human prostate CSCs at the following final concentrations: 100, 300, 500 and 1000 nM. The cultures were incubated for 24, 48 and 72 h. The half maximal inhibitory concentration (IC50) value of the drug was determined as 500 nM for monolayer cells. Dead cells were analyzed prior and subsequent to exposure to increasing flavopiridol doses. Annexin-V and immunofluorescence analyses were performed for the evaluation of apoptotic pathways. According to the results, flavopiridol treatment caused significant growth inhibition at 500 and 1000 nM when compared to the control at 24 h. G0/G1 analysis showed a statistically significant difference between 100 and 500 nM (P<0.005), 100 and 1000 nM (P<0.001), 300 and 1000 nM (P<0.001), and 500 and 1000 nM (P<0.001). Flavopiridol also significantly influenced the cells in the G2/M phase, particularly at high-dose treatments. Flavopiridol induced growth inhibition and apoptosis at the IC50 dose (500 nM), resulting in a significant increase in immunofluorescence staining of caspase-3, caspase-8 and p53. In conclusion, the present results indicated that flavopiridol could be a

  8. Epithelial mesenchymal transition and pancreatic tumor initiating CD44+/EpCAM+ cells are inhibited by γ-secretase inhibitor IX.

    Directory of Open Access Journals (Sweden)

    Vindhya Palagani

    Full Text Available Pancreatic ductal adenocarcinoma (PDAC is an aggressive disease with a high rate of metastasis. Recent studies have indicated that the Notch signalling pathway is important in PDAC initiation and maintenance, although the specific cell biological roles of the pathway remain to be established. Here we sought to examine this question in established pancreatic cancer cell lines using the γ-secretase inhibitor IX (GSI IX to inactivate Notch. Based on the known roles of Notch in development and stem cell biology, we focused on effects on epithelial mesenchymal transition (EMT and on pancreatic tumor initiating CD44+/EpCAM+ cells. We analyzed the effect of the GSI IX on growth and epithelial plasticity of human pancreatic cancer cell lines, and on the tumorigenicity of pancreatic tumor initiating CD44+/EpCAM+ cells. Notably, apoptosis was induced after GSI IX treatment and EMT markers were selectively targeted. Furthermore, under GSI IX treatment, decline in the growth of pancreatic tumor initiating CD44+/EpCAM+ cells was observed in vitro and in a xenograft mouse model. This study demonstrates a central role of Notch signalling pathway in pancreatic cancer pathogenesis and identifies an effective approach to inhibit selectively EMT and suppress tumorigenesis by eliminating pancreatic tumor initiating CD44+/EpCAM+ cells.

  9. CD44v6 Monoclonal Antibody-Conjugated Gold Nanostars for Targeted Photoacoustic Imaging and Plasmonic Photothermal Therapy of Gastric Cancer Stem-like Cells

    Science.gov (United States)

    Liang, Shujing; Li, Chao; Zhang, Chunlei; Chen, Yunsheng; Xu, Liang; Bao, Chenchen; Wang, Xiaoyong; liu, Gang; zhang, Fengchun; Cui, Daxiang

    2015-01-01

    Developing safe and effective nanoprobes for targeted imaging and selective therapy of gastric cancer stem cells (GCSCs) has become one of the most promising anticancer strategies. Herein, gold nanostars-based PEGylated multifunctional nanoprobes were prepared with conjugated CD44v6 monoclonal antibodies (CD44v6-GNS) as the targeting ligands. It was observed that the prepared nanoprobes had high affinity towards GCSC spheroid colonies and destroyed them completely with a low power density upon near-infrared (NIR) laser treatment (790 nm, 1.5 W/cm2, 5 min) in vitro experiment. Orthotopic and subcutaneous xenografted nude mice models of human gastric cancer were established. Subsequently, biodistribution and photothermal therapeutic effects after being intravenously injected with the prepared nanoprobes were assessed. Photoacoustic imaging revealed that CD44v6-GNS nanoprobes could target the gastric cancer vascular system actively at 4 h post-injection, while the probes inhibited tumor growth remarkably upon NIR laser irradiation, and even extended survivability of the gastric cancer-bearing mice. The CD44v6-GNS nanoprobes exhibited great potential for applications of gastric cancer targeted imaging and photothermal therapy in the near future. PMID:26155313

  10. Relationship between expression of CD44v6 and nm23-H1 and tumor invasion and metastasis in hepatocellular carcinoma

    OpenAIRE

    Xiao, Cheng-Zhi; Dai, Yi-Min; Yu, Hong-Yu; Wang, Jian-Jun; Ni, Can-Rong

    1998-01-01

    AIM: To detect the expression of CD44v6 mRNA and nm23-H1 mRNA in hepatocellular carcinoma (HCC) by in situ hybridization, and to evaluate the relationship between their expression and also relationship between their expressions and tumor invasion and metastasis.

  11. Intragraft Tubular Vimentin and CD44 Expression Correlate With Long-Term Renal Allograft Function and Interstitial Fibrosis and Tubular Atrophy

    NARCIS (Netherlands)

    J. Kers; Y.C. Xu-Dubois; E. Rondeau; N. Claessen; M.M. Idu; J.J.T.H. Roelofs; F.J. Bemelman; R.J.M. ten Berge; S. Florquin

    2010-01-01

    Background. Development of interstitial fibrosis and tubular atrophy (IF/TA) is the main histologic feature involved in renal allograft deterioration. The aim of this study was to validate whether de novo tubular expression of CD44 (transmembrane glycoprotein) and vimentin (mesenchymal cell marker),

  12. ICAM-1、CD44V6与食管鳞癌淋巴结转移关系的研究

    Institute of Scientific and Technical Information of China (English)

    王永霞

    2007-01-01

    目的探讨黏附分子ICAM-1、CD44V6在食管鳞癌淋巴结转移中的作用。方法采用S—P免疫组化染色法检测食管鳞癌组织中ICAM-1、CD44V6的表达情况。结果在发生淋巴结转移的食管鳞癌组织中ICAM-1、CD44V6的阳性表达率分别为69.2%(18/26)、76.9%(20/26),且两者具有明显的一致性(P〈0.05)。结论ICAM-1、CD44V6与食管鳞癌的淋巴结转移情况密切相关,食管鳞癌淋巴结的转移是一个涉及多种黏附分子参与的复杂过程。

  13. BACE1 gene variants do not influence BACE1 activity, levels of APP or Aβ isoforms in CSF in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Minthon Lennart

    2010-09-01

    Full Text Available Abstract The BACE1 gene encodes the beta-site APP-cleaving enzyme 1 and has been associated with Alzheimer's disease (AD. BACE1 is the most important β-secretase responsible for the generation of Alzheimer-associated amyloid β-proteins (Aβ and may play a role in the amyloidogenic process in AD. We hypothesized that BACE1 gene variants might influence BACE1 activity or other markers for APP metabolism in the cerebrospinal fluid (CSF and thereby contribute to the development of AD. We genotyped a Swedish sample of 269 AD patients for the rs638405 single nucleotide polymorphism (SNP in the BACE1 gene and correlated genotype data to a broad range of amyloid-related biomarkers in CSF, including BACE1 activity, levels of Aβ40, Aβ42, α- and β-cleaved soluble APP (α-sAPP and β-sAPP, as well as markers for Alzheimer-type axonal degeneration, i.e., total-tau and phospho-tau181. Gene variants of BACE1 were neither associated with amyloid-related biomarkers, nor with markers for axonal degeneration in AD.

  14. Binding of human leukocytes to fibronectin is augmented by an anti-CD44 mAb (TL-1) and blocked by another anti-CD44 mAb (Hermes-3) but not by anti-VLA-4/VLA-5 mAbs.

    Science.gov (United States)

    Cao, L; Yoshino, T; Kawasaki, N; Yanai, H; Kawahara, K; Kondo, E; Omonishi, K; Takahashi, K; Akagi, T

    Fibronectin (FN) forms meshworks in extracellular spaces, and it plays an important role in cellular trafficking. Lymphoid cells are activated by binding to FN of the VLA-4 and VLA-5 receptors. CD44 also acts as a receptor of FN, but the mechanism and physiologic regulation of their binding are poorly understood. We have developed an anti-CD44 monoclonal antibody (mAb) (TL-1) in which lymphoid cells are activated and form homotypic cell aggregation. In this study, we found that the adhesion of CEM, HSB2, and LAD lymphoid cells to FN was augmented by TL-1 treatment and was apparently blocked by another anti-CD44 mAb (Hermes-3), but TL-1 Fab' fragments treatment did not induce FN-binding. A similar phenomenon is reported in the binding of the CD44 molecule to HA. This augmentation was not inhibited by the CS1 and RGD peptides of FN or by anti-VLA-4 and -VLA-5 mAbs; it was energy-dependent and associated with cytoplasmic actin filaments. Tl-1 treatment did not alter the cell surface expression of CD44 molecules. These findings above suggested that activated and/or altered cell surface distribution of CD44 molecules via a conformational change augmented the avidity of its binding to FN, which may be similar to lymphocyte-hyaluronate and lymphocyte-endothelial cell binding. As the Hermes-3 binding site is also involved in the interaction between lymphocytes and endothelial cells, activation of lymphocytes via CD44 molecules may facilitate the binding of lymphocytes to endothelial cells, extravasation, and migration to inflammatory sites rich in FN. PMID:9145328

  15. Nuclear β-catenin and CD44 upregulation characterize invasive cell populations in non-aggressive MCF-7 breast cancer cells

    International Nuclear Information System (INIS)

    In breast cancer cells, the metastatic cell state is strongly correlated to epithelial-to-mesenchymal transition (EMT) and the CD44+/CD24- stem cell phenotype. However, the MCF-7 cell line, which has a luminal epithelial-like phenotype and lacks a CD44+/CD24- subpopulation, has rare cell populations with higher Matrigel invasive ability. Thus, what are the potentially important differences between invasive and non-invasive breast cancer cells, and are the differences related to EMT or CD44/CD24 expression? Throughout the sequential selection process using Matrigel, we obtained MCF-7-14 cells of opposite migratory and invasive capabilities from MCF-7 cells. Comparative analysis of epithelial and mesenchymal marker expression was performed between parental MCF-7, selected MCF-7-14, and aggressive mesenchymal MDA-MB-231 cells. Furthermore, using microarray expression profiles of these cells, we selected differentially expressed genes for their invasive potential, and performed pathway and network analysis to identify a set of interesting genes, which were evaluated by RT-PCR, flow cytometry or function-blocking antibody treatment. MCF-7-14 cells had enhanced migratory and invasive ability compared with MCF-7 cells. Although MCF-7-14 cells, similar to MCF-7 cells, expressed E-cadherin but neither vimentin nor fibronectin, β-catenin was expressed not only on the cell membrane but also in the nucleus. Furthermore, using gene expression profiles of MCF-7, MCF-7-14 and MDA-MB-231 cells, we demonstrated that MCF-7-14 cells have alterations in signaling pathways regulating cell migration and identified a set of genes (PIK3R1, SOCS2, BMP7, CD44 and CD24). Interestingly, MCF-7-14 and its invasive clone CL6 cells displayed increased CD44 expression and downregulated CD24 expression compared with MCF-7 cells. Anti-CD44 antibody treatment significantly decreased cell migration and invasion in both MCF-7-14 and MCF-7-14 CL6 cells as well as MDA-MB-231 cells. MCF-7-14 cells are a

  16. Dual-targeting hybrid nanoparticles for the delivery of SN38 to Her2 and CD44 overexpressed human gastric cancer

    Science.gov (United States)

    Yang, Zhe; Luo, Huiyan; Cao, Zhong; Chen, Ya; Gao, Jinbiao; Li, Yingqin; Jiang, Qing; Xu, Ruihua; Liu, Jie

    2016-06-01

    Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor 2 (Her2) and cluster determinant 44 (CD44), is one of the most malignant human tumors which causes a high mortality rate due to rapid tumor growth and metastasis. To develop effective therapeutic treatments, a dual-targeting hybrid nanoparticle (NP) system was designed and constructed to deliver the SN38 agent specifically to human solid gastric tumors bearing excessive Her2 and CD44. The hybrid NPs consist of a particle core made of the biodegradable polymer PLGA and a lipoid shell prepared by conjugating the AHNP peptides and n-hexadecylamine (HDA) to the carboxyl groups of hyaluronic acid (HA). Upon encapsulation of the SN38 agent in the NPs, the AHNP peptides and HA on the NP surface allow preferential delivery of the drug to gastric cancer cells (e.g., HGC27 cells) by targeting Her2 and CD44. Cellular uptake and in vivo biodistribution experiments verified the active targeting and prolonged in vivo circulation properties of the dual-targeting hybrid NPs, leading to enhanced accumulation of the drug in tumors. Furthermore, the anti-proliferation mechanism studies revealed that the inhibition of the growth and invasive activity of HGC27 cells was not only attributed to the enhanced cellular uptake of dual-targeting NPs, but also benefited from the suppression of CD44 and Her2 expression by HA and AHNP moieties. Finally, intravenous administration of the SN38-loaded dual-targeting hybrid NPs induced significant growth inhibition of HGC27 tumor xenografted in nude mice compared with a clinical antitumor agent, Irinotecan (CPT-11), and the other NP formulations. These results demonstrate that the designed dual-targeting hybrid NPs are promising for targeted anti-cancer drug delivery to treat human gastric tumors over-expressing Her2 and CD44.Gastric cancer (GC), particularly of the type with high expression of both human epidermal growth factor receptor

  17. Transforming growth factor-β1 induces EMT by the transactivation of epidermal growth factor signaling through HA/CD44 in lung and breast cancer cells.

    Science.gov (United States)

    Li, Lingmei; Qi, Lisha; Liang, Zhijie; Song, Wangzhao; Liu, Yanxue; Wang, Yalei; Sun, Baocun; Zhang, Bin; Cao, Wenfeng

    2015-07-01

    Epithelial-mesenchymal transition (EMT), a process closely related to tumor development, is regulated by a variety of signaling pathways and growth factors, such as transforming growth factor-β1 (TGF-β1) and epidermal growth factor (EGF). Hyaluronan (HA) has been shown to induce EMT through either TGF-β1 or EGF signaling and to be a regulator of the crosstalk between these two pathways in fibroblasts. In this study, in order to clarify whether HA has the same effect in tumor cells, we utilized the lung cancer cell line, A549, and the breast cancer cell line, MCF-7, and found that the effects of stimulation with TGF-β1 were more potent than those of EGF in regulating the expression of EMT-associated proteins and in enhancing cell migration and invasion. In addition, we observed that TGF-β1 activated EGF receptor (EGFR) and its downstream AKT and extracellular signal-regulated kinase (ERK) pathways. Furthermore, we found that TGF-β1 upregulated the expression of hyaluronan synthases (HAS1, HAS2 and HAS3) and promoted the expression of CD44, a cell surface receptor for HA, which interacts with EGFR, resulting in the activation of the downstream AKT and ERK pathways. Conversely, treatment with 4-methylumbelliferone (4-MU; an inhibitor of HAS) prior to stimulation with TGF-β1, inhibited the expression of CD44 and EGFR, abolished the interaction between CD44 and EGFR. Furthermore, the use of shRNA targeting CD44 impaired the expression of EGFR, deactivated the AKT and ERK pathways, reversed EMT and decreased the migration and invasion ability of cells. In conclusion, our data demonstrate that TGF-β1 induces EMT by the transactivation of EGF signaling through HA/CD44 in lung and breast cancer cells.

  18. Inference of Isoforms from Short Sequence Reads

    Science.gov (United States)

    Feng, Jianxing; Li, Wei; Jiang, Tao

    Due to alternative splicing events in eukaryotic species, the identification of mRNA isoforms (or splicing variants) is a difficult problem. Traditional experimental methods for this purpose are time consuming and cost ineffective. The emerging RNA-Seq technology provides a possible effective method to address this problem. Although the advantages of RNA-Seq over traditional methods in transcriptome analysis have been confirmed by many studies, the inference of isoforms from millions of short sequence reads (e.g., Illumina/Solexa reads) has remained computationally challenging. In this work, we propose a method to calculate the expression levels of isoforms and infer isoforms from short RNA-Seq reads using exon-intron boundary, transcription start site (TSS) and poly-A site (PAS) information. We first formulate the relationship among exons, isoforms, and single-end reads as a convex quadratic program, and then use an efficient algorithm (called IsoInfer) to search for isoforms. IsoInfer can calculate the expression levels of isoforms accurately if all the isoforms are known and infer novel isoforms from scratch. Our experimental tests on known mouse isoforms with both simulated expression levels and reads demonstrate that IsoInfer is able to calculate the expression levels of isoforms with an accuracy comparable to the state-of-the-art statistical method and a 60 times faster speed. Moreover, our tests on both simulated and real reads show that it achieves a good precision and sensitivity in inferring isoforms when given accurate exon-intron boundary, TSS and PAS information, especially for isoforms whose expression levels are significantly high.

  19. Correlation of Pokemon, E-cadherin and CD44v6 expression with the ultrasonic appearance in breast carcinoma%Pokemon、E-cadherin和CD44v6蛋白在乳腺癌组织中的表达及其与影像学特征的关系

    Institute of Scientific and Technical Information of China (English)

    鲁豫; 李文涛; 张斌; 吴刚; 于洋

    2010-01-01

    目的 检测Pokemon、E-cadherin和CD44v6蛋白在乳腺癌组织及癌旁组织中的表达,探讨其与影像学表现的关系.方法 60例新鲜乳腺癌标本,所有标本均含有乳腺癌组织及癌旁组织,采用免疫组织化学法分别检测Pokemon、E-cadherin和CD44v6蛋白的表达.用B超观察乳腺癌的影像学特征.结果 乳腺癌组织中Pokemon蛋白表达率为75.0%,高于癌旁乳腺组织中Pokemon蛋白表达率31.7%(P<0.05),乳腺癌组织中E-cadherin蛋白的阳性表达率(55.0%)明显低于癌旁乳腺组织中E-cadherin蛋白的阳性表达率(100.0%)(P<0.05),乳腺癌组织中CD44v6蛋白的表达率(60.0%)显著高于癌旁乳腺组织CD44v6蛋白的表达率(5.0%)(P<0.05).超声观察,Pokemon、E-cadherin和CD44v6蛋白的表达与乳腺癌的浸润、转移明显相关.结论 Pokemon、E-cadherin和CD44v6蛋白可能在乳腺癌发生、发展及转移中发挥作用,Pokemon、E-cadherin和CD44v6蛋白与超声影像结合可能与乳腺癌的预后有关.%Objective To investigate the correlations of Pokemon, E-caderin and CD44v6 expression with the ultrasonic appearance in breast carcinoma. Methods All sixty breast carcinoma samples included both breast carcinoma and surrounding non-tumour breast tissues. Expression of Pokemon, E-caderin and CD44v6 were examined using immunohistochemistry technique. The results were compared with the ultrasonic features of breast carcinoma. Results The frequency of Pokemon protein expression was 31.7% for morphological normal breast tissue and 75.0% for breast cancer. The frequency of E-caderin protein expression was 100.0% for morphological normal breast tissue and 55.0% for breast cancer. The frequency of CD44v6 protein expression was 5.0% for morphological normal breast tissue and 60.0% for breast cancer. Pokemon, E-cadherin and CD44v6 protein expression correlated with breast cancer invasion and metastasis in ultrasonography. Conclusion Expression of Pokemon, E-caderin and CD44v6

  20. 粘附分子CD62P和CD44在毛细支气管炎患儿外周血中的表达及意义%Expression and significance of adhesion molecules CD62P and CD44 in peripheral blood of infants with bronchiolitis

    Institute of Scientific and Technical Information of China (English)

    邹丽萍; 王伟; 张艳丽; 张艳; 王莉

    2015-01-01

    目的:探讨黏附分子CD62P和CD44在毛细支气管炎(简称毛支炎)患儿外周血中的表达及意义。方法选取2014年11月至2015年5月住院治疗的毛支炎发病期患儿33例和恢复期患儿19例为研究对象,同期选取支气管肺炎患儿30例为支气管肺炎组,非感染患儿26例为对照组。采用流式细胞术检测各组患儿外周血CD62P的表达百分比,ELISA法测定血清CD44的水平。结果毛支炎发病期组CD62P和CD44水平显著高于毛支炎恢复期组、支气管肺炎组及对照组(P<0.05);毛支炎恢复期组CD62P和CD44的水平仍高于对照组(P<0.05)。毛支炎发病期患儿外周血中黏附分子CD62P百分比与血清CD44的水平呈正相关(r=0.91, P<0.05)。结论黏附分子CD62P、CD44参与了毛细支气管炎的发病过程,其水平高低可反映毛细支气管炎炎症反应的严重程度。%ObjectiveTo explore the expression and signiifcance of the adhesion molecules CD62P and CD44 in the peripheral blood of infants with bronchiolitis.MethodsThirty-three infants with bronchiolitis in the acute phase and 19 infants with bronchiolitis in the recovery phase, who were hospitalized between November 2014 and May 2015, were enrolled. Thirty infants with bronchopneumonia and 26 infants without infection were enrolled as the bronchopneumonia group and the control group, respectively. The CD62P expression in the peripheral blood of each group was measured by lfow cytometry, and the CD44 level in serum was determined using ELISA.ResultsThe levels of the adhesion molecules CD62P and CD44 in the bronchiolitis group in the acute phase were signiifcantly higher than those in the bronchiolitis group in the recovery phase, the bronchopneumonia group, and the control group (P<0.05). The levels of the adhesion molecules CD62P and CD44 in the bronchiolitis group in the recovery phase were also signiifcantly higher than those in the control group (P<0.05). In the

  1. Expression of CCR7, L-selectin, CD44v6 and MMP9 in colorectal cancer and their relative with lymphatic metastatic%大肠癌中CCR7、L-selectin、CD44v6和MMP9表达及其与淋巴转移的关系

    Institute of Scientific and Technical Information of China (English)

    武欣; 李坤; 张凡; 刘军超; 林媛媛; 金春亭

    2013-01-01

    目的 探讨CC趋化因子受体7(CCR7)、L-selectin、CD44v6和MMP9在大肠癌组织中的表达及其与大肠癌淋巴转移的关系.方法 采用免疫组化PV 9000两步法检测104例大肠癌组织(大肠癌组)、55例癌旁正常组织(癌旁组)和34例转移灶组织(转移组)中CCR7和L-selectin、CD44v6和MMP9的表达.结果 大肠癌组、转移组中CCR7、L-selectin、CD44v6和MMP9阳性率明显高于癌旁组(P<0.05),有淋巴结转移者明显高于无转移者(P<0.05);CCR7与L-selectin、MMP9表达呈正相关(P<0.05);CD44v6与L-selectin、MMP9表达相关;L-selectin与MMP9的表达呈正相关.结论 CCR7、L-selectin、CD44v6和MMP9在大肠癌中的表达与大肠癌的发生、淋巴转移有关,它们可能共同参与了大肠癌发生及淋巴结转移过程.%Purpose To investigate the expression of chemokine receptor 7 ( CCR7 ), adhesion molecule L-selectin, CD44v6 and MMP9 in human colorectal carcinoma, and analyze their correlation with lymph node metastasis. Methods The expression of CCR7, L-selectin, CD44v6 and MMP9 were tested by immunohistochemical method in 104 cases of colorectal carcinoma specimens ( colorectal carcinoma group ), 55 cases of normal intestinal mucosa adjacent to carcinoma ( adjacent group ) and 34 cases of metastatic tumor tissue ( metastasis group). Results The positive rate of CCR7, L-selectin, CD44v6 and MMP9 expression in the colorectal carcinoma group and metastatic tumor tissue were significantly higher than that in adjacent group and metastasis group ( P < 0. 05 ). The expression of CCR7, L-selectin, CD44v6 and MMP9 in the tissue with lymph node metastasis was significantly higher than that in the tissue without lymph node metastasis ( P <0. 05 ). There was significantly positive correlation between expression of CCR7 and L-selectin, CCR7 and MMP9, CD44v6 and L-selectin, CD44v6 and MMP9, L-selectin and MMP9 ( P < 0. 05 ). Conclusions The expression of CCR7, L-selectin, CD44v6 and MMP9 are closely

  2. Correlation between Abnormal Expressions of CD44 Protein and Gene and Lymph Node Metastasis for Human Lung Carcinoma%人肺癌CD44蛋白和基因的异常表达与淋巴结转移的相关性

    Institute of Scientific and Technical Information of China (English)

    张连斌; 孙玉鹗

    2001-01-01

    目的:研究 CD44蛋白和基因在人肺癌组织中的异常表达,以及这种异常表达与淋巴结转移的相关性。方法:应用 SP免疫组织化学染色方法和 RT PCR/Southern方法,研究人肺癌组织中 CD44蛋白和基因的表达,并应用 QTM970计算机图像分析仪,对免疫组织化学染色结果进行定量分析。结果: SP免疫组织化学染色和计算机图像定量分析结果显示,非小细胞肺癌( non small cell lung carcinomas, NSCLCs)组织比癌旁肺组织,有淋巴结转移比无淋巴结转移的非小细胞肺癌组织染色强度深,差异有极显著意义( F=16.67, P< 0.01); RT PCR/Southern杂交结果显示,有淋巴结转移较无淋巴结转移的非小细胞肺癌表达更多的 CD44 mRNA异常转录子(χ 2=12.13, P< 0.01)。结论: CD44蛋白和基因在人非小细胞肺癌中出现异常表达,这种异常表达在人非小细胞肺癌的发展和淋巴结转移中起重要作用。检测 CD44蛋白和基因的表达,可能对判断非小细胞肺癌病人的预后、预测淋巴结的转移趋势 ,具有重要的临床意义。%Objective:The aim of this study was to investigate the abnormal expressions of CD44 protein and gene, relationship between the abnormal expressions of CD44 protein and gene and lymph node metastasis of human lung carcinomas. Methods: SP immunohistochemistry and RT PCR/Southern methods were used to investigate the expressions of CD44 protein and gene of human lung carcinomas. The immunohistochemistry results were quantitatively analyzed with QTM970 computed image analyzer. Results: SP immunohistochemistry results and quantitative analysis revealed that stronger expression of CD44 protein was observed in non small cell lung carcinomas than in para carcinomatous pulmonary tissues, in non small cell lung carcinomas with lymph node metastasis than in non small cell lung carcinomas without lymph node metastasis

  3. Expression of cancer stem cell markers, CD133, CD44 and OCT-4, in small cell lung cancer and their clinical significances%肿瘤干细胞标志物CD133、CD44、OCT-4在小细胞肺癌中的表达及其临床意义

    Institute of Scientific and Technical Information of China (English)

    吉亚君; 白玲; 王鑫; 刘政宏

    2015-01-01

    Objective To determine the expression of cancer stem cell markers, CD133, CD44 and OCT-4, in small cell lung cancer cell line NCI-H82 and confirm the specific markers. Methods NCI-H82 cells were cultured, and the expressions of markers (CD133, CD44 and OCT-4) were detected by immunofluorescence staining. The immunohistochemistry was performed to detect the expressions of CD133, CD44 and OCT-4 in 79 tissues with small cell lung cancer. Results In NCI-H82 cells, the florescence was positive in CD133 and CD44 and negative in OCT-4. In the tissues, the expression of CD133 and CD 44 was related with tumor size, lymph node metastasis and clinical stage (P< 0.05), while OCT-4 was negative expression. Conclusion CD133 and CD44 might be the cancer stem cell markers of small cell lung cancer, which maybe have clinical significance on diagnosis and treatment.%目的:探讨肿瘤干细胞标志物CD133、CD44、OCT-4与小细胞肺癌临床病理特征之间的相关性及临床意义。方法应用免疫荧光技术检测小细胞肺癌细胞株NCI-H82中肿瘤干细胞标志物CD133、CD44、OCT-4的表达;同时应用免疫组织化学法检测79例小细胞肺癌组织中CD133、CD44、OCT-4的表达。结果在小细胞肺癌细胞株NCI-H82中,CD133和CD 44的荧光信号为阳性表达,OCT-4的荧光信号为阴性表达。小细胞肺癌组织中CD133和CD44表达与肿瘤直径、淋巴结转移和临床分期相关,差异具有统计学意义(P<0.05)。小细胞肺癌组织中OCT-4为阴性表达。结论 CD133和CD44可能是小细胞肺癌肿瘤干细胞的标志物,对小细胞肺癌的诊断和治疗有一定的临床意义。

  4. 肺癌原发灶及转移淋巴结肿瘤干细胞标记物CD133和CD44的表达情况分析%Expression of CD133 and CD44 in Tumor Stem Cells of Primary Lung Cancer and Metastatic Lymph Nodes

    Institute of Scientific and Technical Information of China (English)

    迟艳飞

    2015-01-01

    Objective To study the analysis of the primary tumors and lung cancer metastasis lymph node cancer stem cell marker CD133 and the expression of CD44. Methods 40 cases of our hospital patients with lung cancer as research object, using immunohisto chemical staining detection CD133 and CD44 in patients with primary tumors and tumor metastasis lymph nodes in the expression, analyze the tumor size, lymph node metastasis, the relationship between the degree of cell differentiation and its expression. Results 40 cases of lung cancer patients, lymph node metastasis 25 cases, 62.5%(25/40). CD133 and positive expression of CD44 in lymph node metastasis rate was 92% (23/25), 72% (18/25), CD44 and CD133 positive expression rate in the original lesions were 72.5%(29/40), 57.5%(23/40), CD44 and CD133 positive group and negative group comparison between primary focal tumor size had no signiifcant difference (P>0.05). Conclusion Lung cancer primary tumors and the metastasis lymph nodes in the cancer stem cell marker CD133 and the expression of CD44 and tumor biology has a direct relationship, has a direct inlfuence on the prognosis of patients.%目的:探讨分析肺癌原发灶及转移淋巴结肿瘤干细胞标记物CD133和CD44的表达情况。方法选择我院收治的40例肺癌患者作为观察对象,采用免疫组化染色检测CD133和CD44在患者的肿瘤原发灶以及转移淋巴结中的表达情况,观察分析肿瘤大小、淋巴结转移情况、细胞分化程度与其表达情况的关系。结果40例肺癌患者中,淋巴结转移25例,占62.5%(25/40)。CD133和CD44在淋巴结转移中的阳性表达率分别为92%(23/25)、72%(18/25);CD133和CD44在原病灶中的阳性表达率分别为72.5%(29/40)、57.5%(23/40);CD133和CD44阳性组与阴性组原发灶肿瘤大小之间的比较均无显著差异(P>0.05)。结论肺癌原发灶及转移淋巴结中肿瘤干细胞标记物CD133和CD44的表达情况与肿瘤

  5. A novel mouse model of human breast cancer stem-like cells with high CD44+CD24-/lower phenotype metastasis to human bone

    Institute of Scientific and Technical Information of China (English)

    LING Li-jun; WANG Feng; WANG Shui; LIU Xiao-an; SHEN En-chao; DING Qiang; LU Chao; XU Jian; CAO Qin-hong; ZHU Hai-qing

    2008-01-01

    Background A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel "human-source" model of human breast cancer skeletal metastasis.Methods Human breast cancer stem-like cells, the CD44+/CD24-/lower subpopulation, was separated and cultured. Before injection with the stem-like cells, mice were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1x105, 1x106 human breast cancer stem-like cells, and 1x106 parental MDA-MB-231 cells, respectively. A positive control group (D) without implantation of human bone was also injected with 1x106 MDA-MB-231 cells. Immunohistochemistry was performed for determination of CD34, CD105, smooth muscle antibody, CD44, CD24, cytokine, CXC chemokine receptor-4 (CXCR4), and osteopontin (OPN). mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction (PCR). Results Our results demonstrated that cells in implanted human bones of group B, which received 1x106 cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas those of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis (77.8%, P=0.0230) and no accompaniment of other tissue metastasis. The real-time PCR showed an increase of CD44, CXCR4, and OPN mRNA in metastatic bone tissues in group B compared with those of groups C and D, however the expression of CD24 mRNA in group B were the lowest. Conclusions In the novel "human source" model of breast cancer, breast cancer stem-like cells demonstrated a higher human bone-seeking ability. Its mechanism might be related to the higher expressions of CD44, CXCR4, and OPN, and the lower expression of CD24 in breast cancer stem-like cells.

  6. Expression of Osteopontin and CD44v6 and their significance in keratocystic odontogenic tumor before and after decompression%牙源性角化囊性瘤开窗减压前后骨桥蛋白及 CD44 v6的表达及意义

    Institute of Scientific and Technical Information of China (English)

    朱江; 武和明; 王婧; 丁旭; 李怀奇; 邢树忠; 吴煜农

    2014-01-01

    Objective:To investigate the expression of osteopontin (OPN) and CD44v6 and their significance in keratocystic odonto-genic tumor ( KCOT) before and after decompression .Methods:All 16 pairs of paraffin-embedded tissue specimens of KCOT before and after decompression were collected and then used for immunohistochemical detection of OPN and CD 44v6.The results were statistically analyzed.Results:The expression of OPN in KCOT was down-regulated after decompression (P<0.05).And CD44v6 expression in post-operative KCOT epithelium was significantly less than that in preoperative KCOT epithelium (P<0.05).Conclusions:The local aggres-sive behavior of KCOT epithelium after decompression was decreased , which may be part of the mechanisms of decompression therapy .%目的:观察骨桥蛋白(osteopontin,OPN)及细胞粘附分子CD44v6在牙源性角化囊性瘤(keratocystic odontogenic tumor , KCOT)中开窗减压前后表达水平的变化。方法:收集16例经病理证实的KCOT患者开窗减压前后的病理标本,应用免疫组织化学染色法检测OPN及CD44v6的表达,并进行统计分析。结果:开窗减压前,OPN及CD44v6在KCOT中均呈高表达。减压后,16例患者OPN均呈阴性表达,统计学分析其表达在开窗后显著下降(P<0.05);CD44v6在开窗减压后标本中,10例(63%)表达下降,5例(31%)表达未发生变化,1例(6%)表达升高,统计学分析其表达在开窗后显著下降(P<0.05)。结论:OPN及CD44v6在KCOT中呈高表达,开窗减压后二者的表达水平明显降低,这可能是开窗减压术治疗KCOT的部分机制。

  7. Relationship between CD44,CD133 + cells expression and clinical characteristics in breast cancer%乳腺癌中 CD44、CD133+细胞表达及其与临床特征相关性研究

    Institute of Scientific and Technical Information of China (English)

    张子杰

    2014-01-01

    Objective To investigate the relationship between CD44,CD133 + cells expression and clinical characteristics in breast canc-er,which would provide basis for clinical diagnosis and treatment,prognosis. Methods The pathology specimens of 200 cases of breast(25 cases of breast hyperplasia,22 cases of atypical hyperplasia,153 cases of breast cancer)and 30 cases of normal breast tissue were included as the study subject. The CD44,CD133 + expression in different breast lesions were detected by immunohistochemical staining. The expression of CD44, CD133 + in different clinical and pathological characteristics of breast cancer were analyzed. Results The positive expression rate of CD44、CD133 + in breast cancer were both significantly higher than those of breast hyperplasia,atypical hyperplasia( P ﹤ 0. 05). The dual expression rate of CD133 + ,CD44 in breast cancer,breast Hyperplasia,atypical hyperplasia was 58. 2% ,32. 0% ,18. 2%( P = 0. 00),respectively. The expression level of CD44,CD133 + increased with histological grade,TNM staging increasing( P ﹤ 0. 05). The expression level of CD44 CD133 + in recurrent breast cancer was significantly higher than that in non recurrent breast cancer( P ﹤ 0. 05). Conclusion There was some correlation between expression levels of CD44,CD133 + and clinical pathological characteristics of breast cancer. Therefore,a combined analysis of CD44,CD133 + expression is useful for evaluation of malignant degree and prognosis of breast cancer,which can be a direction in study of breast cancer therapy.%目的:探讨乳腺癌中 CD44、CD133+细胞表达与临床特征的相关性,为临床诊治、预后评估提供依据。方法收集乳腺病理标本200例(乳腺增生25例,不典型增生22例,乳腺癌153例)及正常乳腺组织30例。采用免疫组织化学染色测定乳腺不同病变组织中的 CD44、CD133+表达情况,分析乳腺癌不同临床病理特征的 CD44、CD133+表达水平。结果 CD44、CD133+

  8. Experimental research of immunophenotype CD133,CD34,CD44 in human lung adenocarcinoma%人肺腺癌肿瘤细胞免疫表型CD133、CD34、CD44的实验研究

    Institute of Scientific and Technical Information of China (English)

    谭思创; 王若星; 谭斯品; 胡文; 马宇超; 喻风雷

    2011-01-01

    Objective To validate the possibility of CD133 CD34 CD44 be served as biomarkers in cancer stem cell of human lung adenocarcinoma. Methods Two kinds of culturing methods were performed to generate adhesive tumor cells and floating aggregates, and the differences of expression of CD133 CD34 CD44 between 2 kinds of cultured cells were observed by immunofluorescence. Results Floating aggregates grew more slowly, kept activity for longer period than adhesive cells (72.5% vs 47.5%,P<0.05). Floating aggregates expressed higher level of CD133, CD34 and CD44 than adhesive cells (68.97%,82.76%,93.10% vs 5.26%,15.79%,5.26%,P<0.01). Conclusions The combination of CD133, CD34 and CD44 probably can be used as surface markers of cancer stem cells for human lung adenocarcinoma.%目的 拟验证CD133、CD34、CD44作为人肺腺癌肿瘤干细胞表面标记物的合理性.方法 采集新鲜肺腺癌组织标本,利用两种体外培养方法扩增出贴壁细胞和悬浮细胞球两种肿瘤细胞,采用免疫荧光检测比较CD133、CD34和CD44在两种培养细胞中表达的差异.结果 悬浮球肿瘤细胞培养较贴壁肿瘤细胞生长速度慢、维持时间长且成功率高(72.5% vs 47.5%,P<0.05).CD133、CD34和CD44在悬浮细胞球中表达率和表达量明显高于贴壁肿瘤细胞(68.97%,82.76%,93.10% vs 5.26%,15.79%,5.26%,P<0.01).结论 CD133、CD34和CD44可能作为分离人肺腺癌肿瘤干细胞的表面蛋白表标记组合.

  9. Functional heterogeneity within the CD44 high human breast cancer stem cell-like compartment reveals a gene signature predictive of distant metastasis

    DEFF Research Database (Denmark)

    Leth-Larsen, Rikke; Terp, Mikkel Green; Christensen, Anne G;

    2012-01-01

    The CD44(hi) compartment in human breast cancer is enriched in tumor-initiating cells; however the functional heterogeneity within this subpopulation remains poorly defined. We used a triple-negative breast cancer cell line with a known bi-lineage phenotype to isolate and clone CD44(hi) single...... performed comparative quantitative proteomic and gene array analyses of these cells and identified potential novel markers of breast cancer cells with tumor-initiating features, such as LSR, RAB25, S100A14 and MUC1, as well as a novel 31-gene signature capable of predicting distant metastasis in cohorts...... of estrogen receptor-negative human breast cancers. These findings strongly favor functional heterogeneity in the breast cancer cell compartment and hold promise for further refinements of prognostic marker profiling. Our work confirms that, in addition to cancer stem cells with mesenchymal-like morphology...

  10. Aplicación terapéutica de agentes inhibidores de CD44 frente a la leucemia linfoblástica aguda (ALL) humana

    OpenAIRE

    Toribio, María Luisa; García-Peydró, Marina; Sánchez Madrid, Francisco

    2012-01-01

    [ES] La presente invención describe la aplicación terapéutica de agentes inhibidores de CD44 frente a la leucemia linfoblástica aguda (ALL) humana. En concreto, la presente invención hace referencia a un agente inhibidor de la función de CD44 para su uso en medicina, preferentemente para la prevención y/o tratamiento de la T-ALL humana, así como a una composición farmacéutica y un kit. La presente invención también hace referencia aun método de prevención y/o tratamiento de la T-ALL humana, a...

  11. Aplicación terapéutica de agentes inhibidores de CD44 frente a la leucemia linfoblástica aguda (ALL) humana

    OpenAIRE

    Toribio, María Luisa; García-Peydró, Marina; Sánchez Madrid, Francisco

    2012-01-01

    La presente invención describe la aplicación terapéutica de agentes inhibidores de CD44 frente a la leucemia linfoblástica aguda (ALL) humana. En concreto, la presente invención hace referencia a un agente inhibidor de la función de CD44 para su uso en medicina, preferentemente para la prevención y/o tratamiento de la T-ALL humana, así como a una composición farmacéutica y un kit. La presente invención también hace referencia a un método de prevención y/o tratamient...

  12. 结肠癌细胞源exosomes的分离鉴定及其表面蛋白CD44v6的表达

    Institute of Scientific and Technical Information of China (English)

    刘朋飞; 朱磊; 李响; 孙一夫; 石毅; 丛登立; 王浩天; 周余来

    2010-01-01

    目的 探索结肠癌colo205细胞系对exosomes的分泌功能,并分析热休克作用对表面蛋白CD44v6表达量的影响.方法 采用超速离心法分离colo205细胞分泌的exosomes和热休克处理后colo205细胞分泌的exosomes(Heat shocked exosomes,HS-Exo),经220 nm微孔滤膜过滤纯化后,在透射电镜下观察其形态,并用SDS-PAGE方法分析细胞与exosomes的蛋白组成,Western Blot检测表面CD44v6的表达情况.结果 经透射电镜观察,正常exosomes与HS-Exo形态基本相似,均为圆形或椭圆形膜性囊泡,直径大多在30~100 nm之间,且经热休克处理的colo205细胞及其分泌的HS-Exo,在CD44v6表达量上较正常colo205细胞及exosomes显著上调(P<0.05).结论 结肠癌colo205细胞系可分泌exosomes,且超速离心结合滤膜过滤的分离纯化方法切实可行;热休克作用可使CD44v6表达上调,说明HS-Exo较exosomes可能在肿瘤免疫治疗方面有更重要的应用价值.

  13. Overexpression of CD44 accompanies acquired tamoxifen resistance in MCF7 cells and augments their sensitivity to the stromal factors, heregulin and hyaluronan

    OpenAIRE

    Hiscox Stephen; Baruha Bedanta; Smith Chris; Bellerby Rebecca; Goddard Lindy; Jordan Nicola; Poghosyan Zaruhi; Nicholson Robert I; Barrett-Lee Peter; Gee Julia

    2012-01-01

    Abstract Background Acquired resistance to endocrine therapy in breast cancer is a significant problem with relapse being associated with local and/or regional recurrence and frequent distant metastases. Breast cancer cell models reveal that endocrine resistance is accompanied by a gain in aggressive behaviour driven in part through altered growth factor receptor signalling, particularly involving erbB family receptors. Recently we identified that CD44, a transmembrane cell adhesion receptor ...

  14. The microRNA miR-34a inhibits non-small cell lung cancer (NSCLC) growth and the CD44hi stem-like NSCLC cells.

    Science.gov (United States)

    Shi, Yang; Liu, Can; Liu, Xin; Tang, Dean G; Wang, Junchen

    2014-01-01

    Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate, which is probably due to the existence of lung cancer stem cells (CSCs). CSCs in many tumors including non-small cell lung cancer (NSCLC) have been identified using adhesion molecular CD44, either individually or in combination with other marker(s). MicroRNAs (miRNAs) regulate both normal stem cells and CSCs and dysregulation of miRNAs has been implicated in tumorigenesis. Recently, miR-34a was found to be downregulated in NSCLC cells but the biological functions of miR-34a in regulating NSCLC cell behavior have not been extensively studied. Here we show that transfection of synthetic miR-34a, but not the negative control (NC) miRNA oligonucleotides (oligos) in three NSCLC cell lines, i.e., A549, H460, and H1299, inhibited their holoclone formation, clonogenic expansion, and tumor regeneration in vivo. Furthermore, the lentiviral vector-mediated overexpression of miR-34a in purified CD44hi H460 cells also inhibited tumor outgrowth. In contrast, expression of miR-34a antagomirs (i.e., antisense oligos) in the CD44lo H460 cells promoted tumor development. Our study shows that miR-34a is a negative regulator of the tumorigenic properties of NSCLC cells and CD44hi lung CSCs, and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against NSCLC. PMID:24595209

  15. The microRNA miR-34a inhibits non-small cell lung cancer (NSCLC growth and the CD44hi stem-like NSCLC cells.

    Directory of Open Access Journals (Sweden)

    Yang Shi

    Full Text Available Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate, which is probably due to the existence of lung cancer stem cells (CSCs. CSCs in many tumors including non-small cell lung cancer (NSCLC have been identified using adhesion molecular CD44, either individually or in combination with other marker(s. MicroRNAs (miRNAs regulate both normal stem cells and CSCs and dysregulation of miRNAs has been implicated in tumorigenesis. Recently, miR-34a was found to be downregulated in NSCLC cells but the biological functions of miR-34a in regulating NSCLC cell behavior have not been extensively studied. Here we show that transfection of synthetic miR-34a, but not the negative control (NC miRNA oligonucleotides (oligos in three NSCLC cell lines, i.e., A549, H460, and H1299, inhibited their holoclone formation, clonogenic expansion, and tumor regeneration in vivo. Furthermore, the lentiviral vector-mediated overexpression of miR-34a in purified CD44hi H460 cells also inhibited tumor outgrowth. In contrast, expression of miR-34a antagomirs (i.e., antisense oligos in the CD44lo H460 cells promoted tumor development. Our study shows that miR-34a is a negative regulator of the tumorigenic properties of NSCLC cells and CD44hi lung CSCs, and establishes a strong rationale for developing miR-34a as a novel therapeutic agent against NSCLC.

  16. Human breast cancer stem cell markers CD44 and CD24: enriching for cells with functional properties in mice or in man?

    OpenAIRE

    Fillmore, Christine; Kuperwasser, Charlotte

    2007-01-01

    Identification of breast cancer stem cells as the cells within breast tumors that have the ability to give rise to cells that make up the bulk of the tumor mass has shifted the focus of cancer research. However, there is still much debate concerning the unique nature of the markers that distinguish cancer stem cells in the breast. As such, understanding whether CD44+/CD24- breast cancer cells are merely more successful in overcoming an engraftment incompatibility that exists when injecting hu...

  17. The expression and clinical significance ofβ-catenin and colorectal cancer stem cells marker EpCAMhigh/CD44+in colorectal cancer

    Institute of Scientific and Technical Information of China (English)

    Dan Liu; Jinghua Sun; Jinming Zhu; Huan Zhou; Yang Zhang

    2013-01-01

    Objective:The aim of the study was to explore the role of Wnt/β-catenin signal ing pathway in the maintenance, invasion and metastasis of colorectal cancer stem cells. Methods:Double immunohistochemical staining was used to detect the expression of EpCAMhigh/CD44+which is regarded as the marker of colorectal cancer stem cells in 80 cases of colorectal cancer and their corresponding liver metastases. The SP method of immunohistochemistry was used to detect the expression of the key proteinβ-catenin in the Wnt pathway in these tissue. The expression and correlation ofβ-catenin and EpCAMhigh/CD44+in colorectal cancer were analyzed and their role on the biological behavior of colorectal cancer was explored. Results:The abnormal expression ofβ-catenin was significantly higher in colorectal cancer than in the paraneoplastic normal intes-tinal mucosa [55%(44/80) vs 10%(2/20), P0.05), but it was significantly correlated with degree of dif erentiation (P=0.001), depth of invasion (P=0.001), clinical stage (P=0.000) and metastasis (P=0.000). In the colorectal cancer, the expression of EpCAMhigh/CD44+cells has no correlation with gender (P=0.934) and the magnitude (P=0.160) of the tumor (P>0.05), but was significantly correlated with age (P=0.021), degree of dif erentiation (P=0.013), depth of invasion (P=0.000), clinical stage (P=0.000) and metastasis (P=0.000). In the corresponding liver metastases, we could also detecte EpCAMhigh/CD44+cells. In cases with abnormal expression ofβ-catenin, the positive expression rate of EpCAMhigh/CD44+was significantly higher than those with normal expression ofβ-catenin (84.1%vs 44.4%), and the dif erence was statistical y significant (P<0.05). Conclusion:The abnormal activation of Wnt/β-catenin signal ing pathway may prompt the abnormal proliferation of the colorectal cancer stem cells, which leads to the recurrence and metastasis of the cancer.

  18. Relationship between the expressions of CD44H, p-selectin and VEGF and the metastasis with lymph node in esophageal squamous cell carcinoma%食管鳞癌中CD44H、P-选择素和血管内皮生长因子的表达与淋巴结转移的关系

    Institute of Scientific and Technical Information of China (English)

    李娜; 李继煜; 张景航; 王洪兴; 焦云娟

    2006-01-01

    目的 探讨CD44H、P-选择素(P-selectin)和血管内皮生长因子(VEGF)的表达与食管鳞癌淋巴结转移的关系.方法 采用免疫组织化学SP法检测60例食管鳞癌组织中CD44H、P-selectin和VEGF的表达.结果 淋巴结转移组CD44H、P-selectin和VEGF的表达增强,与无淋巴结转移组的表达差异有显著性意义(P<0.05),P-selectin和VEGF在淋巴结转移组的表达呈正相关(P<0.05).结论 CD44H、P-selectin和VEGF的表达与食管鳞癌淋巴结的转移密切相关,P-selectin和VEGF存在协同关系,可作为评估食管鳞癌预后的生物学标志.

  19. CORRELATION BETWEEN CELL ADHESION MOLLECULES CD44v6 EXPRESSION, DNA CONTENT AND INVASIVE BREAST CARCINOMA%黏附分子CD44v6蛋白表达及DNA含量与乳腺浸润性癌的相关性研究

    Institute of Scientific and Technical Information of China (English)

    吴岳; 王登文; 王成

    2005-01-01

    目的研究CD44v6蛋白表达及DNA含量在浸润性乳腺癌侵袭、转移和预后中的作用.方法应用免疫组化S-P法检测103例浸润性乳腺癌石蜡切片中肿瘤细胞CD44v6的表达,并用图像分析法进行DNA含量分析.结果CD44v6主要定位于细胞质,与乳腺浸润性癌组织学的类型无关(P>0.05),而与组织学的浸润程度有关.CD44v6蛋白在组织学分级:Ⅰ级阳性表达率为57.9%,Ⅱ级为87.0%,Ⅲ级为88.1%(P<0.01).乳腺浸润性癌DNA异倍体情况:采用Feulgen染色示细胞核呈紫红色,胞浆不着色.DNA图像分析结果显示CD44v6蛋白表达阳性者肿瘤细胞异倍体率为82.3%(65/79),表达阴性者肿瘤异倍体为45.9%(11/24),经x2检验有统计学意义(P<0.05).各组织学类型之间异倍体比较,无显著性差异(P>0.05).结论CD44v6蛋白表达与DNA异倍体水平呈平行关系.CD44v6、DNA倍体的联合检测对于指导临床医生的诊断具有重要的意义.因此,结合DNA含量和CD44v6蛋白表达的联合检测,对乳腺浸润性癌的诊断、判断预后价值更大.

  20. Immunohistochemical study using T-cell lymphoma antibody 1 and CD44 in diagnosis of Burkitt's lymphoma%T细胞淋巴瘤1和CD44蛋白在Burkitt淋巴瘤中的表达及其诊断价值

    Institute of Scientific and Technical Information of China (English)

    水若鸿; 陆洪芬; 朱雄增

    2009-01-01

    Objective To study the values of immunohistochemistry using T-cell lymphoma antibody (TCL) 1 and CD44 in the diagnosis of Burkitt's lymphoma. Methods Immunohistochemical study for TCL1, CD44, CD10, bcl-2, bcl-6, c-myc and Ki-67 was performed on paraffin-embedded sections of lymphoma cases, including 25 cases of Burkitt's lymphoma and 25 cases of diffuse large B-cell lymphoma. Results Burkitt's lymphoma commonly expressed TCL1 (96% ,24 cases), CDI0 (88% ,22 cases), bcl-6 and c-myc (92% ,23 cases). Only 1 case(4%) expressed CD44 and bcl-2. The Ki-67 proliferation index ranged from 95% to 100%. On the other hand, diffuse large B-cell lymphoma expressed CD44(84% ,21 cases), CD10(32% ,8 cases), bcl-6 (72%, 18 cases) and bcl-2(72%, 18 cases). Four cases(16%) were weakly positive for TCL1. The staining for c-myc was all negative. The Ki-67 proliferation index ranged from 40% to 90%. Conclusion Immunohistochemical staining for TCL1 and CD44 is a useful ancillary tool in the pathologic diagnosis of Burkitt's lymphoma which is also helpful for the differential diagnosis from diffuse large B-cell lymphoma.%目的 探讨T细胞淋巴瘤1(TCL1)和CD44蛋白在Burkitt淋巴瘤中的表达及其诊断价值.方法 在石蜡包埋的实验组25例Burkitt淋巴瘤和对照组25例非特指弥漫性大B细胞淋巴瘤(DLBCL)中采用免疫组织化学EnVision法检测CD44、TCLl以及CD10、bel-2、bcl-6、c-myc、Ki-67等常用抗体表达情况.结果 Burkitt淋巴瘤中瘤细胞96%(24例)呈TCL1阳性,仅4%(1例)CD44阳性;88%(22例)CD10阳性、92%(23例)bcl-6和c-myc阳性,仅4%(1例)bcl-2阳性;Ki-67增殖指数为95%~100%.非特指DLBCL中仅16%(4例)TCL1弱阳性,84%(21例)CD44阳性、32%(8例)CD10阳性、72%(18例)bcl-6和bcl-2阳性、c-myc均阴性,Ki-67增殖指数40%~90%.结论 当形态和免疫表型不典型时,TCL1和CD44两种蛋白的检测有助于提高Burkitt淋巴瘤的确诊率及其与DLBCL的鉴别诊断.

  1. Relationship between the expressions of lymph homing recptors P-selectin, CD44 and α6β1 and lymph metastasis in cervical carcinoma%淋巴归巢受体P选择素、CD44、α6β1在宫颈癌中表达与淋巴转移关系研究

    Institute of Scientific and Technical Information of China (English)

    张素贞; 张凡; 常永霞; 张九鸿; 赵秀芳; 成日青

    2010-01-01

    目的 探讨P选择素(P-selectin)、CD44、整合素α6β1对宫颈癌淋巴转移的作用.方法 应用免疫组织化学方法对比检测35例宫颈鳞状细胞癌原发灶及其淋巴转移灶中P-selectin、CD44、α6β1的表达.结果 与原发灶相比,淋巴转移灶中P-selectin阳性率显著降低(P<0.05),CD44阳性率无显著变化(P=1.230 5),ICAM-1阳性率则显著增高(P<0.01).原发灶中P-selectin与CD44、P-selectin与ICAM-1表达水平均存在显著正相关性(r=0.873 7,P<0.01;r=0.795 7,P<0.01),而CD44与α6β1则呈明显负相关(r=-0.658 3,P<0.01);淋巴转移灶中P-selectin、CD44、α6β1表达水平之间均不存在相关关系;P-selectin原发灶与淋巴转移中表达水平存在明显正相关(r=0.753 4,P<0.01),CD44原发灶与淋巴转移中表达水平之间无明显相关,α6β1原发灶与淋巴转移中表达水平之间存在明显负相关(r=-0.536 1,P<0.01).原发灶中P-selectin表达与肿瘤分化、淋巴结转移存在正相关(r=0.842 0,P<0.01和r=0.768 9,P<0.01);CD44表达与肿瘤侵犯深度、淋巴结转移之间存在正相关(r=0.678 2,P<0.01;r=0.863 4,P<0.01);α6β1表达与淋巴结转移存在正相关(r=0.654 8,P<0.01).结论 P-selectin、CD44、α6β1都与宫颈癌淋巴转移相关,其中P-selectin、CD44在淋巴转移的始动阶段发挥重要作用.

  2. Bioinformatics research of CD44 and epithelial cell adhesion molecule related genes and pathways in colorectal cancer%结直肠癌中CD44和上皮细胞黏附分子相关基因与通路的生物信息学研究

    Institute of Scientific and Technical Information of China (English)

    马敏星; 周瑞; 张嘉刚; 马洪卫; 陶文惠; 赵秋; 李瑾

    2016-01-01

    Objective To investigate genes and involved biological processes closely associated with stem cell markers of colorectal cancer-epithelial cell adhesion molecule (EpCAM) + and CD44+.Methods By the bioinformatics method,with microarray data of colorectal cancer from gene expression omnibus (GEO) database and R2 platform,the genes significantly related with CD44 and EpCAM expression were screened out.The differences in expression of related genes were analyzed on the basis of gender,family history of cancer,alcohol and Dukes stage.The expression of related genes in colorectal cancer was compared with that of other tumors and healthy subjects.At same time,the pathways of the genes and Kyoto encyclopedia of genes and genomes (KEGG) of CD44 and EpCAM significantly related genes were analyzed with gene ontology (GO) and KEGG method.Single factor analysis of variance and Chi-square test of four-fold table with correction for continuity were used for statistical analysis by R2 platform embedded statistical tools.Results The expressions of CD44 and EpCAM were detected in all 315 colorectal cancer samples.A total of 888 and 6 316 genes were screened out which were significantly associated with CD44 and EpCAM expression.CD44 was positively correlated with EpCAM.There was no obvious correlation between the expression of five genes which expressed in all 315 tissues and gender family history of cancer,alcohol and Dukes stage (all P>0.05).By further compared with the expression in other tumors and tissues,the expressions of two genes solute carrier family 12,member 2 (SLC12A2) and proteome of centriole 1 centriolar protein B (POC1B) in colorectal tumor were significantly higher than that in other tumors (F=289.422、128.456,all P<0.01),and its expression in colorectal cancer was obviously higher than that in tissues of health subjects (F=349.519、128.456,all P<0.01).GO analysis indicated there were 15 GO semantics related with both CD44 and EpCAM.The genes related with CD

  3. Disturbance of nivalenol on expression of adhesion molecule CD44 on surface of cultured chondrocytes%雪腐镰刀菌烯醇能干扰培养软骨细胞表面黏附分子CD44的表达

    Institute of Scientific and Technical Information of China (English)

    孙健; 曹峻岭; 杨旭东; 孟列素

    2006-01-01

    BACKGROUND: Deficit of nivalenol (NIV) and selenium (Se) is related with kashin-beck disease (KBD) to certain extent. Hyaluronic acid (HA) metabolism affects directly the polymerization of proteoglycans (PG) and normal structure and function of cartilage. The integration with HA receptor on surface of cartilage tissue is the key link in HA metabolism. Being the main receptor of HA on chondrocytic membrane, CD44 expression impacts directly HA metabolism, further affects cartilage matrix metabolism, which is extremely important to maintaining the structure and function of cartilage matrix.OBJECTIVE: To probe into the injury and protection of related etiology of KBD to target tissue cells and the mechanism on degenerative necrosis of chondrocytes.DESIGN: Randomized controlled observation was designed.SETTING: Department of Genetics and Molecular-Biology of Medical College of Xi' an Jiaotong University.MATERIALS: The experiment was performed in Key Laboratory Room on Ministry of Education associated with Environment and Disease of Xian Jiaotong University from October 2002 to July 2004. One New Zealand pedigree young rabbit aged 30 days was employed and its humerus, femurs and tibia were cut out in surgery.METHODS: With cell culture, the model of bone tissue was reconstructed in vitro, in which, NIV of various concentrations, KBD suspicious infectious agent and Se, the protective factor were added. HA receptor CD44 on chondrocytic membrane and soluble CD44 in cell culture solution were determined.MAIN OUTCOME MEASURES: ① Microscopic observation of adhesion molecule CD44 on chondrocytic surface. ② Soluble CD44 in chongrocytic culture solution.RESULTS: ① Microscopic observation of adhesion molecule CD44 on chondrocytic surface: CD expression in chondrocytic membrane was decreased with increasing of NIV concentration and it was in tendency of increasing with Se added. ② Soluble CD44 in chongrocytic culture solution:The concentration of soluble CD44 in cell culture

  4. Effect of soluble CD44 molecule on the expression of apoptosis regulatory protein bcl-2 associated death factor bad in human trabecular meshwork cell%可溶性CD44分子对人眼小梁网细胞凋亡调节蛋白bcl-2相关死亡因子bad表达的影响

    Institute of Scientific and Technical Information of China (English)

    梁宗宝; 吴瑜瑜; 郭茂生

    2012-01-01

    背景 研究表明可溶性CD44分子(sCD44)在原发性开角型青光眼(POAG)患者眼中的质量浓度高于正常人,POAG患者房水中sCD44水平与小梁网细胞凋亡相关蛋白的表达是否有关及其对POAG的共同作用机制至今尚不明确. 目的 探讨不同质量浓度的sCD44对POAG患者小梁网细胞凋亡调节蛋白bcl-2相关死亡因子bad表达的影响.方法 收集POAG患者行小梁切除术中切除的小梁网组织,采用组织块培养法原代培养小梁网细胞,并用免疫细胞化学法对培养细胞进行鉴定,将第3代的小梁网细胞按随机数字表法随机分为6个组,分别在无血清培养基中加入含终质量浓度为0(对照组)、1、5、10、25、50 μg/L的sCD44,培养细胞48 h.采用细胞计数试剂8(CCK-8)法和ELISA法检测小梁网细胞中凋亡调节蛋白bc1-2相关死亡因子bad蛋白的表达.结果 培养的细胞经层黏连蛋白(LM)、神经元特异性烯醇化酶(NSE)单克隆抗体、纤维连接蛋白(FN)免疫组织化学染色呈阳性反应.CCK-8法检测0、l、5、10、25、50μg/L sCD44作用48 h后小梁网细胞吸光度(A450)值分别为:0.2460±0.0019、0.1874±0.0015、0.1570±0.0016、0.1302±0.0019、0.1084±0.0018、0.0940±0.0020,差异有统计学意义(F=14.922,P=0.000),各实验组A450值均低于对照组,差异均有统计学意义(P=0.013、0.008、0.011、0.005、0.004).ELISA法检测表明,0、l、5、10、25、50 μg/LsCD44作用48 h后小梁网细胞中bad蛋白质量浓度分别为(114.8461±2.9560)、(137.8270±2.4259)、(161.4194±3.7381)、(170.9453±3.2006)、(221.2252±4.3738)、(324.6167±4.4220) ng/L,差异有统计学意义(F=16.610,P=0.000),各实验组小梁网细胞中bad蛋白质量浓度均明显高于对照组,差异均有统计学意义(P=0.017、0.013、0.008、0.007、0.006).结论 sCD44在一定质量浓度范围内可促进POAG小梁网细胞的凋亡,并且能上调凋亡调节蛋白bcl-2相关

  5. Expression of CD44 in Liver Tissues of Rats with Severe Acute Pancreatitis and Protective Effect of Qingyi Ⅱ Granules%CD44在重症急性胰腺炎大鼠肝脏表达及清胰Ⅱ号颗粒剂保护作用的实验研究

    Institute of Scientific and Technical Information of China (English)

    李鹏; 王玲君; 兑丹华

    2011-01-01

    Objective: To explore the role of CD44 in pancreas and liver damage in severe acute pan-creastitis (SAP) , and to investigate the effects of Chinese medicine Qingyi Ⅱ granules on the expression of CD44 and its mechanism. Methods: A total of 36 healthy SD rats were randomly divided into three groups: sham-operated group ( group A) , SAP model group (group B) and treatment group ( group C). SAP model was established by retrograde injection of 30 g/L sodium taurocholate into bile-pancreas duct. Qingyi Ⅱ Granules 250 g/L was administered by gavage every 6 hours in group C . While the other two groups were infused with same volume of saline instead. All animals were sacrificed after modeling for 24 h. The CD44 expressions in pancreas and liver tissues were detected with immunohistochemistry method. Serum amylase (AMY) , aspartate aminotransferasea (AST) and ala-nine aminotransferase (ALT) were tested. Pathologic changes of pancreas and liver tissues were observed under microscope. Results: Pancreas and liver tissues were damaged seriously in group B, and the expression of CD44 was positive, the serum levels of AMY, AST and ALT were significantly higher than those of groups A and C ( P<0.05 ). The expression level of CD44 was lower and activities of serum AMY, AST and ALT were higher significantly in group C than those in group B. (P<0. 05). Conclusions: The expression of CD44 may play an important role in pancreas and liver injury in SAP rats. Chinese medicine Qingyi II granules could depress the expressions of CD44 in pancreas and liver tissues, and alleviate the pathologic damage of pancreas and liver in SAP rats.%目的:探讨CD44在重症急性胰腺炎(severe acute pancreastitis,SAP)时胰腺及肝损害机制中的作用,并观察中药清胰Ⅱ号颗粒剂对CD44的影响及机理.方法:将36只SD大鼠随机等分为假手术组(A组)、SAP组(B组)、治疗组(C组),各组以30 g/L牛磺胆酸钠逆行注入胰胆管制作大鼠SAP模型,A组大鼠开

  6. Human breast cancer stem cell maker CD44+CD24-/low enriched in basal-like breast carcinoma%CD44+CD24-/low在基底样乳腺癌中过表达与其恶性预后的相关性研究

    Institute of Scientific and Technical Information of China (English)

    于晓宇; 耿敬姝; 宋鸿涛; 冯迪; 蒋旸

    2013-01-01

    Objective To study the expression of breast cancer stem cell - like markers CD44+ CD24 -/low in basal - like breast carcinoma( BLBC )and their relation with the prognosis of BLBC. Methods We classified breast carcinomas into 4 groups: luminal A, luminal B/C , Her - 2 over - expressing and triple - negative subtype with the expressions of ER,PR and Her - 2, respectively. We classified triple - negative subtype into 2 groups:BLBC and normal breast - like group with the expression of CK5/6 and EGFR, respectively. Immunohisto-chemical( Envision method )was performed to all of the 5 groups, with the selection of antibodies for CD44 and CD24. Results ( 1 ) triple - negative group were 60 cases in all, expressed CK5/6 and EGFR in 41cases ( 68. 3% ),which was identified as BLBC;low expressed CK5/6 and EGFR in 19 cases( 31.7% ),which identified as the normal breast - like group. ( 2 )32 cases expressed CD44+ CD24-/low phenotype in 41 cases of BLBC78.0%( 32/41 ),compared with luminal A group of 37.9%( 11/29 ),luminal B of25.9%( 7/27 ),Her-2 over - expression group of 17. 2%( 5/29 )and normal breast - like group of 26. 3%( 5/21 )in BLBC( P < 0. 05 ). ( 3 )ln all cases of breast cancer which can be evaluated in 145 cases with CD44 + CD24 -/low immun - phenotype, Ki - 67 index was higher and statistically significant! P < 0. 05 ). Conclusion The expression of human breast cancer stem cell markers CD44 + CD24-/low in BLBC was significantly higher than other types of breast cancer. Then, we can suppose an association between the prognosis of BLBC and the presence of CD44+ CD24 -/low cell.%目的 探讨乳腺癌干细胞样标志物CD44+CD24-/low在基底样乳腺癌(basal-like breast carcinoma,BLBC)中过表达与BLBC恶性预后的相关性.方法 在乳腺癌基因表达分型的基础上,根据雌激素受体(ER)、孕激素受体(PR)、人类表皮生长因子受体2(Her-2)免疫表型的表达选取乳腺癌组织四组:管腔A组、管腔B/C组、Her-2高表达组

  7. Synergistic effects of CD44 and TGF-β1 through AKT/GSK-3β/β-catenin signaling during epithelial-mesenchymal transition in liver cancer cells.

    Science.gov (United States)

    Park, Na Ri; Cha, Jung Hoon; Jang, Jeong Won; Bae, Si Hyun; Jang, Bohyun; Kim, Jung-Hee; Hur, Wonhee; Choi, Jong Young; Yoon, Seung Kew

    2016-09-01

    Cancer metastasis is strongly correlated with epithelial-mesenchymal transition (EMT), in which transforming growth factor-β (TGF-β) signaling plays a central role. CD44 has emerged as a cancer stem cell (CSC) marker that strongly induces EMT together with TGF-β1. This study aimed to investigate the link between high CD44 and TGF-β1 levels during EMT in HCC cell lines. FACS analysis showed high expression of CD44 in TGF-β1-positive SNU-368 cells and TGF-β1-negative SNU-354 cells. SNU-368 CD44(+) cells showed EMT through up-regulation of the AKT/GSK-3β/β-catenin pathway. By comparison, SNU-354 CD44(+) cells showed only increased N-cadherin expression, which was not accompanied by a decrease in E-cadherin expression, and also down-regulated the AKT/GSK-3β/β-catenin pathway. However, TGF-β1-stimulated SNU-354 cells (CD44/TGF-β1(+)) exhibited lower E-cadherin and higher N-cadherin expression with increased AKT/GSK-3β/β-catenin pathway activity. CD44/TGF-β1(+) SNU-354 cells also showed enhanced migration and formed larger spheres, while the TGF-β1-induced stem cell properties returned to their original state with the TGF-β1 inhibitor SB431542. SB431542-treated SNU-368 (CD44/TGF-β1(-)) cells also showed diminished N-cadherin and AKT/GSK-3β/β-catenin pathway activity and further decreased cell motility in a wound healing assay. However, CD44 knockdown in SNU-354 cells did not induce EMT even after treatment with TGF-β1. Finally, double inhibition of both CD44 and TGF-β1 further decreased migration and sphere formation more strongly than a single inhibition in SNU-368 cells. In conclusion, the current study demonstrated the synergistic interactions between CD44 and TGF-β1 in EMT induction and CSC properties through the AKT/GSK-3β/β-catenin pathway in HCC cells.

  8. Synergistic active targeting of dually integrin αvβ3/CD44-targeted nanoparticles to B16F10 tumors located at different sites of mouse bodies.

    Science.gov (United States)

    Shi, Sanjun; Zhou, Min; Li, Xin; Hu, Min; Li, Chenwen; Li, Min; Sheng, Fangfang; Li, Zhuoheng; Wu, Guolin; Luo, Minghe; Cui, Huanhuan; Li, Ziwei; Fu, Ruoqiu; Xiang, Mingfeng; Xu, Jing; Zhang, Qian; Lu, Laichun

    2016-08-10

    Conventional enhanced permeation and retention (EPR) mediates the effects of many drugs, including the accumulation of nanocarriers at tumor sites, but its efficiency remains low. In this study, this limitation was overcome by developing a dual-targeting delivery system based on hyaluronan (HA, a major ligand of CD44) and tetraiodothyroacetic acid (tetrac, a specific ligand of αvβ3), which was exploited to carry docetaxel (DTX) for the synergistic active targeting to tumors. First, a tetrac-HA (TeHA) conjugate was synthesized and grafted onto the surfaces of solid lipid nanoparticles (SLNs) (TeHA-SLNs/DTX), with a high encapsulation efficiency of >91.6%. The resulting SLNs exhibited an approximately toroid morphology revealed using TEM. The cellular uptake and cytotoxicity of various formulations on CD44/αvβ3-enriched B16F10 cells were then assessed, and both results confirmed the selective uptake and high cytotoxicity of the TeHA-SLNs/DTX in a TeHA-dependent manner. In vivo imaging and vessel distribution tests revealed the efficiency of synergistic active targeting was higher than that of EPR-mediated passive targeting by the TeHA-SLNs to αvβ3-expressing tumor blood vessels and CD44-expressing tumor cells via selective targeting. Finally, in both xenograft tumor mice and in situ lung metastasis tumor mice, tumor growth was significantly inhibited by TeHA-SLNs/DTX. Therefore, TeHA-SLNs are an efficient system for the dual-targeted delivery of drugs to treat cancer in vivo. PMID:27235150

  9. The microRNA miR-34a Inhibits Non-Small Cell Lung Cancer (NSCLC) Growth and the CD44hi Stem-Like NSCLC Cells

    OpenAIRE

    Shi, Yang; Liu, Can; Liu, Xin; Tang, Dean G.; Wang, Junchen

    2014-01-01

    Lung cancer is among the most lethal malignancies with a high metastasis and recurrence rate, which is probably due to the existence of lung cancer stem cells (CSCs). CSCs in many tumors including non-small cell lung cancer (NSCLC) have been identified using adhesion molecular CD44, either individually or in combination with other marker(s). MicroRNAs (miRNAs) regulate both normal stem cells and CSCs and dysregulation of miRNAs has been implicated in tumorigenesis. Recently, miR-34a was found...

  10. CD44+/CD24- breast cancer cells exhibit phenotypic reversion in three-dimensional self-assembling peptide RADA16 nanofiber scaffold

    Directory of Open Access Journals (Sweden)

    Mi K

    2015-04-01

    Full Text Available Kun Mi,1 Zhihua Xing2 1Department of Biochemistry and Molecular Biology, Sichuan Cancer Hospital and Institute, 2Laboratory of Ethnopharmacology, Institute for Nanobiomedical Technology and Membrane Biology, West China Hospital, Sichuan University, Chengdu, People’s Republic of China Background: Self-assembling peptide nanofiber scaffolds have been shown to be a ­permissive biological material for tissue repair, cell proliferation, differentiation, etc. Recently, a subpopulation (CD44+/CD24- of breast cancer cells has been reported to have stem/progenitor cell properties. The aim of this study was to investigate whether this subpopulation of cancer cells have different phenotypes in self-assembling COCH3-RADARADARADARADA-CONH2 (RADA16 peptide nanofiber scaffold compared with Matrigel® (BD Biosciences, Two Oak Park, Bedford, MA, USA and collagen I.Methods: CD44 and CD24 expression was determined by flow cytometry. Cell proliferation was measured by 5-bromo-2'-deoxyuridine assay and DNA content measurement. Immunostaining was used to indicate the morphologies of cells in three-dimensional (3D cultures of different scaffolds and the localization of β-catenin in the colonies. Western blot was used to determine the expression of signaling proteins. In vitro migration assay and inoculation into nude mice were used to evaluate invasion and tumorigenesis in vivo.Results: The breast cancer cell line MDA-MB-435S contained a high percentage (>99% of CD44+/CD24- cells, which exhibited phenotypic reversion in 3D RADA16 nanofiber scaffold compared with collagen I and Matrigel. The newly formed reverted acini-like colonies reassembled a basement membrane and reorganized their cytoskeletons. At the same time, cells cultured and embedded in RADA16 peptide scaffold exhibited growth arrest. Also, they exhibited different migration potential, which links their migration ability with their cellular morphology. Consistent with studies in vitro, the in vivo tumor

  11. Observation the inhibitory effect and expression of MMP -2, CD44v6 of common turmeric among tumor-bearing nude mice%温郁金对荷肿瘤裸鼠抑瘤作用和MMP -2、CD44v6表达影响的观察

    Institute of Scientific and Technical Information of China (English)

    王光亮; 张俊会

    2012-01-01

    Objective To study the inhibitory effect of common turmeric and the expression of MMP - 2 and CD44v6 proteins in human gastric SGC -7901 cell, explore the possible mechanisms on gastric cancer metastasis. Method Established nude mouse orthotopic transplantation mode of SGC - 7901 and then randomly divided the nude mouse into control group and common turmeric group. The tumor growth and metastasis were observed, the expression of MMP - 2 and CD44v6 proteins in the tumor tissue were detected by immunohisto-chemistry. Results The rate of successfully orthotopic transplantation was 100%. The weight of the tumors in common turmeric group was (2.73 ±0.92) g, in control group was (4. 09 ± 1.17) g, there was statistical significance between the two group (P <0.05) , The inhibitory rate of common turmeric group was 33. 25%. The metastasis of cavitas peritonealis, liver and lymph node in common turmeric group were significantly lower than those of control group (P < 0. 05) . Meanwhile, we found that the positive rates of MMP - 2 and CD44v6 expression in the common turmeric group were obviously lower than that in control group (P<0.05) . Conclusions Common turmeric can inhibit gastric cancer growth and metastasis in orthotopic transplantation model of nude mice, the mechanism might be related to down - regulation of MMP - 2 and CD44v6 expression.%目的 观察温郁金对胃癌细胞抑制作用和MMP-2、CD44v6蛋白表达的影响,探讨其抗胃癌细胞转移的作用机制.方法 以SGC - 7901胃癌细胞株建立胃癌裸鼠原位移植瘤模型,将裸鼠随机分为对照组(0.9%氯化钠溶液)及实验组(温郁金水煎剂).观察裸小鼠胃癌种植后肿瘤生长及转移灶情况,用免疫组化法检测2组肿瘤组织中MMP-2和CD44v6蛋白的表达.结果 2组荷瘤鼠胃壁均有肿瘤生长,荷瘤率100%,对照组瘤重(4.09±1.17) g,实验组瘤重(2.73±0.92) g(与对照组比较P<0.05),抑瘤率为33.25%;实验组肝、腹腔和淋巴结转

  12. Research on the enrichment of CD44+CD24-cancer stem cells in breast cancer multi-drug resistant cell lines MCF-7/PTX%MCF-7/PTX多药耐药细胞株中CD44+CD24-乳腺癌干细胞富集的研究

    Institute of Scientific and Technical Information of China (English)

    韩娜娜; 孙长岗; 庄静; 杨静

    2013-01-01

    Objective To establish human breast cancer multi-drug resistant cell lines MCF-7/PTX in order to study the enrichment of CD+44CD-24 breast cancer cells. Methods We established the multidrug resistant cell line MCF-7/PTX by method of intermittently induction with high dose of PTX. The resistance and cross-resistance of MCF-7 and MCF-7/PTX on PTX were identified by MTT method. The proportion of CD+44CD-24 cells was detected by method of flow cytometry. Results The resistance indices of MCF-7/PTX on paclitaxel, doxorubicin, docetaxel, epirubicin, vincristine, methotrexate, cis-platin were 53, 45.72, 38.30,41.26, 23.75, 17.62 and 35. 80, respectively. The proportion of CD+44CD-24 cells in MCF-7 and MCF-7/PTX were 8.60% , 73. 20% , respectively. Conclusion The MCF-7/PTX cell line established in this study was a kind of multi-drug resistant cell line. The CD+44CD-24 cells in MCF-7/PTX were enriched.%目的 建立人乳腺癌紫杉醇(PTX)多药耐药细胞株MCF-7/PTX,观察MCF-7、MCF-7/PTX细胞株中CD44+CD24-乳腺癌干细胞的富集情况.方法 以MCF-7为亲本细胞,以PTX为诱导药物,高浓度间歇诱导法建立多药耐药细胞株MCF-7/PTX;MTT法检测MCF-7、MCF-7/PTX对PTX等多种化疗药物的耐药性及交叉耐药性;流式细胞术检测细胞株CD44+CD24-乳腺癌干细胞的含量.结果 ①MCF-7/PTX对PTX、阿霉素、多西紫杉醇、表阿霉素、长春新碱、甲氨蝶呤、顺铂的耐药指数分别为53.00、45.72、38.30、41.26、23.75、17.62、35.80.②MCF-7、MCF-7/PTX细胞株中CD44+CD24-细胞的比例分别为8.60%、73.20%,P<0.01.结论 本实验所建立的MCF-7/PTX为多药耐药细胞株;MCF-7/PTX存在着CD44+CD24-乳腺癌干细胞的富集.

  13. 宫颈上皮内瘤样病变和宫颈癌中CD44v6和P63蛋白表达及其意义%Expression and significance of CD44v6 and P63 protein in cervical intraepithclial neoplasia and cervical cercinoma

    Institute of Scientific and Technical Information of China (English)

    谷化平

    2008-01-01

    目的 探讨CD44v6和p63蛋白在宫颈上皮内瘤样病变(CIN)和浸润宫颈鳞癌(ICC)中的表达及其临床意义.方法 应用免疫组织化学方法,检测30例ICC、20例CIN和10例正常宫颈组织(NCE)中CD44v6和p63蛋白的表达水平,并结合临床病理特征进行分析.结果 (1)在ICC组织中CD44v6蛋白表达阳性率为80.0%(24/30),明显高于CIN(35.0%,7/20,P<0.05)和NCE(30.0%,3/10,P<0.05).(2)全部30例ICC和10例CIN中p63染色均为阳性.ICC分化程度越低,阳性细胞数越高、细胞的着色越深.NCE中仅基底层及上基层细胞阳性.(3)p63表达与ICC组织学分级和淋巴结转移相关(P<0.05).结论 CD44v6和p63可能参与了调控ICC的发生、发展过程,可作为ICC早期诊断指标,其高表达预示ICC预后不良.

  14. Mechanistic Insight into Receptor-Mediated Delivery of Cationic-β-Cyclodextrin:Hyaluronic Acid-Adamantamethamidyl Host:Guest pDNA Nanoparticles to CD44(+) Cells.

    Science.gov (United States)

    Badwaik, Vivek; Liu, Linjia; Gunasekera, Dinara; Kulkarni, Aditya; Thompson, David H

    2016-03-01

    Targeted delivery is a key element for improving the efficiency and safety of nonviral vectors for gene therapy. We have recently developed a CD44 receptor targeted, hyaluronic acid-adamantamethamidyl based pendant polymer system (HA-Ad), capable of forming complexes with cationic β-cyclodextrins (CD-PEI(+)) and pDNA. Complexes formed using these compounds (HA-Ad:CD-PEI(+):pDNA) display high water solubility, good transfection efficiency, and low cytotoxicity. Spatial and dynamic tracking of the transfection complexes by confocal microscopy and multicolor flow cytometry techniques was used to evaluate the target specificity, subcellular localization, and endosomal escape process. Our data shows that cells expressing the CD44 receptor undergo enhanced cellular uptake and transfection efficiency with HA-Ad:CD-PEI(+):pDNA complexes. This transfection system, comprised noncovalent assembly of cyclodextrin:adamantamethamidyl-modified hyaluronic acid via host:guest interactions to condense pDNA, is a potentially useful tool for targeted delivery of nucleic acid therapeutics. PMID:26900622

  15. Human Umbilical Cord Mesenchymal Stem Cells Promote Breast Cancer Metastasis by Interleukin-8- and Interleukin-6-Dependent Induction of CD44(+)/CD24(-) Cells.

    Science.gov (United States)

    Ma, Fengxia; Chen, Dandan; Chen, Fang; Chi, Ying; Han, Zhibo; Feng, Xiaoming; Li, Xue; Han, Zhongchao

    2015-01-01

    Although emerging evidence links mesenchymal stem cells (MSCs) with cancer metastasis, the underlying mechanisms are poorly understood. In the present study, we found that human umbilical cord-derived MSCs (UC-MSCs) promoted MCF-7 cell migration in vitro and metastasis in vivo. To explore the mechanisms, the characteristics of MCF-7 cells cocultured with UC-MSCs were assessed. The expression and secretion of interleukin-8 (IL-8) and IL-6 were induced in MCF-7 cells cocultured with UC-MSCs. However, neutralization of IL-8 or IL-6 secreted by UC-MSCs could attenuate the enhanced expression of IL-8 and IL-6 in MCF-7 cells cocultured with UC-MSCs, which subsequently alleviated the enhanced migration. Similar to UC-MSCs, exogenous human recombinant IL-8 or IL-6 also promoted IL-8 and IL-6 expression and MCF-7 cell migration. In addition to enhanced IL-8 and IL-6 expression, MCF-7 cells cocultured with UC-MSCs displayed enhanced mammosphere-forming ability and increased percentage of CD44(+)/CD24(-) cells. However, epithelial-to-mesenchymal transition (EMT) was not observed in MCF-7 cells cocultured with UC-MSCs. Taken together, these results suggested that IL-8 and IL-6 secreted by UC-MSCs activated the autocrine IL-8 and IL-6 signaling in MCF-7 cells and induced CD44(+)/CD24(-) cells, which subsequently promoted MCF-7 cell migration in vitro and metastasis in vivo.

  16. Salinomycin Promotes Anoikis and Decreases the CD44+/CD24- Stem-Like Population via Inhibition of STAT3 Activation in MDA-MB-231 Cells.

    Directory of Open Access Journals (Sweden)

    Hyunsook An

    Full Text Available Triple-negative breast cancer (TNBC is an aggressive tumor subtype with an enriched CD44+/CD24- stem-like population. Salinomycin is an antibiotic that has been shown to target cancer stem cells (CSC; however, the mechanisms of action involved have not been well characterized. The objective of the present study was to investigate the effect of salinomycin on cell death, migration, and invasion, as well as CSC-like properties in MDA-MB-231 breast cancer cells. Salinomycin significantly induced anoikis-sensitivity, accompanied by caspase-3 and caspase-8 activation and PARP cleavage, during anchorage-independent growth. Salinomycin treatment also caused a marked suppression of cell migration and invasion with concomitant downregulation of MMP-9 and MMP-2 mRNA levels. Notably, salinomycin inhibited the formation of mammospheres and effectively reduced the CD44+/CD24- stem-like population during anchorage-independent growth. These observations were associated with the inhibition of STAT3 phosphorylation (Tyr705. Furthermore, interleukin-6 (IL-6-induced STAT3 activation was strongly suppressed by salinomycin challenge. These findings support the notion that salinomycin may be potentially efficacious for targeting breast cancer stem-like cells through the inhibition of STAT3 activation.

  17. MDR1 siRNA loaded hyaluronic acid-based CD44 targeted nanoparticle systems circumvent paclitaxel resistance in ovarian cancer

    Science.gov (United States)

    Yang, Xiaoqian; Lyer, Arun K.; Singh, Amit; Choy, Edwin; Hornicek, Francis J.; Amiji, Mansoor M.; Duan, Zhenfeng

    2015-02-01

    Development of multidrug resistance (MDR) is an almost universal phenomenon in patients with ovarian cancer, and this severely limits the ultimate success of chemotherapy in the clinic. Overexpression of the MDR1 gene and corresponding P-glycoprotein (Pgp) is one of the best known MDR mechanisms. MDR1 siRNA based strategies were proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Cluster of differentiation 44 (CD44) targeted hyaluronic acid (HA) based nanoparticle has been shown to successfully deliver chemotherapy agents or siRNAs into tumor cells. The goal of this study is to evaluate the ability of HA-PEI/HA-PEG to deliver MDR1 siRNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA with paclitaxel to suppress growth of ovarian cancer. We observed that HA-PEI/HA-PEG nanoparticles can efficiently deliver MDR1 siRNA into MDR ovarian cancer cells, resulting in down-regulation of MDR1 and Pgp expression. Administration of HA-PEI/HA-PEG/MDR1 siRNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Our findings suggest that CD44 targeted HA-PEI/HA-PEG/MDR1 siRNA nanoparticles can serve as a therapeutic tool with great potentials to circumvent MDR in ovarian cancer.

  18. 透明质酸对人骨关节炎软骨细胞骨桥蛋白和CD44表达的影响%Effects of hyaluronic acid on osteopontin mRNA and CD44 mRNA expression in human osteoarthritic chondrocytes

    Institute of Scientific and Technical Information of China (English)

    周斌; 张方杰; 罗伟; 高曙光; 曾超; 熊依林; 李宇晟; 雷光华

    2014-01-01

    背景:软骨进行性破坏为晚期骨关节炎的特征性病变,骨关节炎相关因子透明质酸、骨桥蛋白、CD44在骨关节炎软骨中表达增加。  目的:通过透明质酸干预体外培养的人膝骨关节炎软骨细胞,探讨透明质酸对人膝骨关节炎软骨细胞 CD44与骨桥蛋白表达的影响。  方法:将软骨标本进行体外培养获取纯化的软骨细胞,分为3组:空白对照组、透明质酸干预组(100 mg/L)和透明质酸酶干预组(200 mg/L)。培养48 h后,采用Real-time Q PCR检测软骨细胞骨桥蛋白mRNA,CD44 mRNA表达水平。用SPSS 17.0统计软件包分析骨桥蛋白mRNA和CD44 mRNA经透明质酸干预前后表达的差异。  结果与结论:透明质酸组的骨桥蛋白 mRNA表达水平较空白组高,透明质酸酶组的骨桥蛋白 mRNA表达水平较空白组低;透明质酸组及透明质酸酶组的CD44 mRNA表达水平均较空白组低。结果提示透明质酸可以上调骨关节炎软骨细胞骨桥蛋白的表达;透明质酸在骨关节炎软骨细胞内对 CD44表达的影响具有双相性,其影响结果可能与透明质酸的相对分子质量有关。%BACKGROUND:Progressive fracture of the cartilage is considered the characteristic lesion in later osteoarthritis, the expression of osteoarthritis-related factors such as hyaluronic acid, osteopontin and CD44 in osteoarthritic cartilage is increased. OBJECTIVE:To investigate the effect of hyaluronic acid on the expression of osteopontin mRNA and CD44 mRNA of chondrocytes in the in vitro cultured chondrocytes of patients with knee osteoarthritis. METHODThe cartilage samples obtained from osteoarthritic patients were cultured and purified into acquire chondrocytes in vitro, and the cells were divided into three groupblank control group, hyaluronic acid (100 mg/mL) group and hyaluronidase (200 mg/mL) group. After 48 hours of cellculture, real-time quantitative polymerase chain reaction

  19. Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model

    Energy Technology Data Exchange (ETDEWEB)

    Spiegelberg, Diana; Mortensen, Anja C.; Stenerloew, Bo [Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala (Sweden); Selvaraju, Ram K.; Eriksson, Olof [Uppsala University, Preclinical PET Platform, Uppsala (Sweden); Nestor, Marika [Uppsala University, Department of Immunology, Genetics and Pathology, Uppsala (Sweden); Uppsala University, Unit of Otolaryngology and Head and Neck Surgery, Department of Surgical Sciences, Uppsala (Sweden)

    2016-05-15

    Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition. Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 x 50 mg/kg), and were imaged with PET using either {sup 18}F-FDG or {sup 124}I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining. AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC{sub 50} values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with {sup 124}I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with {sup 124}I-AbD19384 as well as {sup 18}F-FDG uptake, were not significantly altered by AT13387 treatment. We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of

  20. Molecular imaging of EGFR and CD44v6 for prediction and response monitoring of HSP90 inhibition in an in vivo squamous cell carcinoma model

    International Nuclear Information System (INIS)

    Heat shock protein 90 (HSP90) is essential for the activation and stabilization of numerous oncogenic client proteins. AT13387 is a novel HSP90 inhibitor promoting degradation of oncogenic proteins upon binding, and may also act as a radiosensitizer. For optimal treatment there is, however, the need for identification of biomarkers for patient stratification and therapeutic response monitoring, and to find suitable targets for combination treatments. The aim of this study was to assess the response of surface antigens commonly expressed in squamous cell carcinoma to AT13387 treatment, and to find suitable biomarkers for molecular imaging and radioimmunotherapy in combination with HSP90 inhibition. Cancer cell proliferation and radioimmunoassays were used to evaluate the effect of AT13387 on target antigen expression in vitro. Inhibitor effects were then assessed in vivo in mice-xenografts. Animals were treated with AT13387 (5 x 50 mg/kg), and were imaged with PET using either 18F-FDG or 124I-labelled tracers for EGFR and CD44v6, and this was followed by ex-vivo biodistribution analysis and immunohistochemical staining. AT13387 exposure resulted in high cytotoxicity and possible radiosensitization with IC50 values below 4 nM. Both in vitro and in vivo AT13387 effectively downregulated HSP90 client proteins. PET imaging with 124I-cetuximab showed a significant decrease of EGFR in AT13387-treated animals compared with untreated animals. In contrast, the squamous cell carcinoma-associated biomarker CD44v6, visualized with 124I-AbD19384 as well as 18F-FDG uptake, were not significantly altered by AT13387 treatment. We conclude that AT13387 downregulates HSP90 client proteins, and that molecular imaging of these proteins may be a suitable approach for assessing treatment response. Furthermore, radioimmunotherapy targeting CD44v6 in combination with AT13387 may potentiate the radioimmunotherapy outcome due to radiosensitizing effects of the drug, and could potentially

  1. Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

    Directory of Open Access Journals (Sweden)

    Schwarzenbach Heidi

    2010-06-01

    Full Text Available Abstract Background The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation. Methods In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies. Results Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3 at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR. Conclusions This study is one

  2. Biomarker screening of oral cancer cell lines revealed sub-populations of CD133-, CD44-, CD24- and ALDH1- positive cancer stem cells

    Directory of Open Access Journals (Sweden)

    Kendall K

    2013-05-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC ranks sixth worldwide for cancer-related mortality. For the past several decades the mainstay of treatment for HNSCC has been surgery and external beam radiation, although more recent trials combining chemotherapy and radiation have demonstrated improvements. However, cancer recurrence and treatment failures continue to occur in a significant percentage of patients. Recent advances in tumor biology have led to the discovery that many cancers, including HNSCC, may contain subpopulations of cells with stem cell-like properties that may explain relapse and recurrence. The objective of this study was to screen existing oral cancer cell lines for biomarkers specific for cells with stem cell-like properties. RNA was isolated for RT-PCR screening using primers for specific mRNA of the biomarkers: CD44, CD24, CD133, NANOG, Nestin, ALDH1, and ABCG2 in CAL27, SCC25 and SCC15 cells. This analysis revealed that some oral cancer cell lines (CAL27 and SCC25 may contain small subpopulations of adhesion- and contact-independent cells (AiDC that also express tumor stem cell markers, including CD44, CD133, and CD24. In addition, CAL27 cells also expressed the intracellular tumor stem cell markers, ALDH1 and ABCG2. Isolation and culture of the adhesion- and contact-independent cells from CAL27 and SCC25 populations revealed differential proliferation rates and more robust inhibition by the MEK inhibitor PD98059, as well as the chemotherapeutic agents Cisplatin and Paclitaxel, within the AiDC CAL27 cells. At least one oral cancer cell line (CAL27 contained subpopulations of cells that express specific biomarkers associated with tumor stem cells which were morphologically and phenotypically distinct from other cells within this cell line.

  3. CD44+/CD105+ Human Amniotic Fluid Mesenchymal Stem Cells Survive and Proliferate in the Ovary Long-Term in a Mouse Model of Chemotherapy-Induced Premature Ovarian Failure

    Directory of Open Access Journals (Sweden)

    Te Liu, Yongyi Huang, Lihe Guo, Weiwei Cheng, Gang Zou

    2012-01-01

    Full Text Available Objectives: Stem cell transplantation has been reported to rescue ovarian function in a preclinical mouse model of chemotherapy-induced premature ovarian failure (POF; however, maintaining the survival and self-renewal of transplanted seed cells in ovarian tissues over the long-term remains a troublesome issue. In this study we aimed to determine whether the CD44+/CD105+ human amniotic fluid cell (HuAFCs subpopulation represent potential seed cells for stem cell transplantation treatments in POF. Materials and methods: The CD44+/CD105+ subpopulation were isolated from HuAFCs, cultured in vitro, and injected into a cyclophosphamide-induced mouse model of POF. Results: Under continuous subculture in vitro, CD44+/CD105+ cells proliferated rapidly and expressed high levels of the proliferative markers Ki67 and survivin, as well as high levels of a number of mesenchymal stem cell biomarkers. Moreover, when red fluorescence protein (RFP-transduced CD44+/CD105+ HuAFCs were transplanted into the ovaries of POF mice, the cells could be detected by fluorescence microscopy up to three weeks after injection. Furthermore, the BrdUrd incorporation assay and immunofluorescent staining demonstrated that CD44+/CD105+ HuAFCs underwent normal cycles of cell proliferation and self-renewal in the ovarian tissues of POF mice over the long-term. Conclusions: The mesenchymal stem cell properties and long-term in vivo survival of CD44+/CD105+ HuAFCs make them ideal seed cells for stem cell transplantation to treat POF.

  4. MMP-9、CD44、TGF-β1在脑星形细胞瘤中应用研究%The Brain Star Polygon Cell Lump Immunity Set Turns a Clinical Application Research

    Institute of Scientific and Technical Information of China (English)

    王连军; 汪刚; 张发顺; 王丽华; 刘燕伟; 梁辉; 李惠翔

    2009-01-01

    Objective MMP-9 and CD44 together position in the cell surface,and this kind of together positions to help the CD44 vto settle anchor roans MMP-9s to also decline a solution to the gum original IV at the cell surface and promote tumor cell gradually. Stopping the empress of the function of CD44 with the dissolubility CD44 can immediately make it lose the function that the anchor man series MMP-9. But is settle by the CD44 anchor manses superficial at the cell of the MMP-9 can also make TGF-β1 of have no activity-TGF-β1 ex- bodies activate and activate of TGF-β1 new-born blood vessel growth is the essential growth factor. This text passes the MMP-9 and TGF-β1 which settles to the CD44 anchor manses-TGF-β1 the expression circumstance carries on analysis in the brain star polygon ceil lump,as to it's express meaning to carry on a research,to the CD44,MMP-9,TGF-β1 gradually transfer amedium function to carry on a first step study in the brain star polygon cell lump,in the meantime to the CD44,MMP-9,TGF-β1 relativities carry on an analytical research. Methods Totally collect 2006 subsidiary hospital of Zheng4 Zhou's university first and Xu3 Chang hospital surgical operation in the city center excision of the brain star polygon coil lump the specimen be 98 and apply an immunity set to turn the SP method examination tumor the CD44,MMP-9,TGF-β1 in the organization-TGF-β1 expression,analytical CD44, MMP-9,TGF-β1 with and CD44, MMP-9,TGF-β1 of the brain star polygon cell lump ratings-TGF- 131. The relation of one its. Results (1)CD44 the expression rote is 77.55%(76/98) in the star polygon cell lump organization and show Zhao Gao to organize 0 at the normal brain (P<0.05). Pass the related examination of the Spearman grade,different Class CD44 expression r=0.95,P=0.046, have covariance to learn meaning,the of the star polygon ceil lump Class and the CD44 male degree-present just related. Use the Fishers precision examination method to carry on an examination to

  5. Expression of bax、bcl-2、CD44v6、nm23 protein in transitional cell carcinoma of the bladder%膀胱移行细胞癌中bax、bcl-2、CD44v6、nm23表达

    Institute of Scientific and Technical Information of China (English)

    张玉华; 陈萍; 朴颖实; 韩影; 李弘; 张伟东; 刘宪军; 巩雷; 谢江

    2004-01-01

    目的探讨bax、bcl-2、CD44、nm23基因蛋白在膀胱移行细胞癌(TCC)中的表达.方法应用免疫组化S-P法对64例膀胱TCC及20例正常膀胱黏膜组织中bax、bcl-2、CD44v6、nm23进行检测.结果 64例膀胱TCC中bax阳性率为17.2%(11/64),正常膀胱黏膜组织为90.0%(18/20)(P《0.05). 膀胱TCC中bcl-2阳性表达率为82.8%(53/64),正常膀胱黏膜为20.0%(4/20)(P《0.05).bax 、bcl-2阳性率随组织学分级的提高有逐渐增强的趋势,但无统计学意义(P》0.05);在无浸润及有深层浸润的膀胱TCC中,bax的阳性表达率分别为16.3%、20.0%(P》0.05),bcl-2则分别为83.7%、80.0%(P》0.05);在无复发转移及有复发转移的膀胱TCC中,bax阳性率分别为16.1%、25.0%(P》0.05),bcl-2分别为82.1%、87.5%(P》0.05).64例膀胱TCC中CD44v6阳性表达率为50.0%(32/64),正常膀胱黏膜组织为5.0%(1/20)(P《0.05).nm23在膀胱TCC中的阳性率为76.6%(49/64),正常膀胱黏膜组织为20.0%(4/20)(P《0.05).CD44v6阳性率Ⅰ、Ⅱ级与Ⅲ级相比差异有显著性(P《0.05),nm23阳性率Ⅰ级与Ⅱ、Ⅲ级相比差异有显著性(P《0.05);在有深层浸润的膀胱TCC中CD44v6、nm 23阳性表达率均明显高于无浸润的病例(P《0.05);有复发转移的膀胱TCC CD44v6阳性率高于无复发转移的病例(P《0.05),而nm23在膀胱TCC中的阳性表达率与有无复发转移无明显相关性(P》0.05).结论 bax表达水平下降及bcl-2的表达增强在膀胱TCC的发生中起到重要作用,CD44v6、nm23也参与促进了膀胱TCC的发生.bax、bcl-2与肿瘤的组织学分级、浸润深度、复发、转移等无关,但CD44v6与之呈正相关.nm23与组织学分级、浸润深度呈正相关,而与复发或转移无相关性.

  6. 乳腺癌外周血微转移hSBEM mRNA和CD44V6 mRNA的检测%The detection of micrometastases in the peripheral blood of patients with breast cancer for hSBEM mRNA and CD44V6 mRNA

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective:Successful treatment of breast cancer greatly depends on the early detection of its metastasis, therefore a sensitive and specific biomarker for detecting dissemination of the cancer cells will help to achieve this goal. This study was to evaluate the prognostic significance of human small breast epithelial mucin (hSBEM) and CD44V6 in breast cancer. Methods: The expressions of hSBEM mRNA and CD44V6 mRNA were detected with nested reverse transcription polymerase chain reaction (nested RT-PCR) in 67 samples of breast cancer and adjacent normal breast tissue, 16 samples of breast benign lesions tissue, and 67 specimens of peripheral blood from patients with breast cancer, 16 specimens of benign breast lesions, 20 specimens of healthy volunteers, and 25 (each five cases) other carcinomas tissue samples, including those of gastric carcinoma, colorectal carcinoma, esophageal carcinoma, lung carcinoma, and ovary carcinoma, were analyzed for hSBEM mRNA expression by nested RT-PCR. Results: hSBEM mRNA expression was observed in 62/67 (92.54%)of breast cancer, 14/16 (87.50%) of breast benign lesions and 59/67 (88.05%) of normal breast tissue, with no significant differences between them (P>0.05). None of the samples from other cancer tissues were positive. In peripheral blood the expression of hSBEM mRNA was detected in 34/67 (50.75%) from patients with breast cancer, with significant increasing (P< 0.05) in the cases of metastatic disease (stage Ⅳ) and those with lymph node metastasis compared with localized disease (stage Ⅰ, Ⅱ and Ⅲ) and without lymph node metastasis, but its expression was not found in peripheral blood of patients with benign breast lesions or healthy volunteers. Although CD44V6 mRNA was significantly higher in breast cancer than in benign breast lesions tissue and normal breast tissue, its expression in peripheral blood show no significant difference (P>0.05) in the patients with breast cancer (82.09%), benign breast lesion (75

  7. 宫颈癌及其淋巴转移灶淋巴归巢受体L-selectin、CD44、ICAM-1表达的对比研究

    Institute of Scientific and Technical Information of China (English)

    陈江平; 张凡; 常永霞; 张九鸿; 赵秀芳; 成日青; 舒丽莎

    2009-01-01

    目的 探讨L-selectin、CD44、ICAM-1对宫颈癌淋巴转移的作用.方法 应用免疫组织化学方法 对比检测35例宫颈鳞状细胞癌原发灶及其淋巴转移灶中L-selectin、CD44、ICAM-1的表达.结果 L-selectin淋巴转移灶中阳性率明显低于原发灶(P<0.05);CD44淋巴转移灶中阳性率与原发灶无明显区别(P>0.05);ICAM-1淋巴转移灶中阳性率明显高于原发灶(P<0.01);L-selectin与CD44、L-selectin与ICAM-1在原发灶中表达水平之间存在明显相关(r=0.873 7,P<0.01;r=0.795,P<0.01),CD44、ICAM-1在原发灶中表达水平呈明显负相关(r=-0.658 3,P<0.01);淋巴转移灶中L-selectin、CD44、ICAM-1表达水平之间均不存在相关;L-selectin原发灶与淋巴转移中表达水平之间存在明显相关(r=0.753 4,P<0.01),CD44原发灶与淋巴转移中表达水平之间无明显相关,ICAM-1原发灶与淋巴转移中表达水平之间存在明显负相关(r=-0.536 1,P<0.01).原发灶中L-selectin表达与肿瘤分化、淋巴结转移存在相关(r=0.842 0,P<0.01;r=0.768 9,P<0.01);CD44表达与肿瘤侵犯深度、淋巴结转移之间存在相关(r=0.678 2,P<0.01;r=0.863 4,P<0.01);ICAM-1表达与淋巴结转移存在相关(r=0.654 8,P<0.01).结论 L-selectin、CD44、ICAM-1都与宫颈癌淋巴转移相关,其中L-selectin、CD44在淋巴转移的始动阶段发挥重要作用.

  8. LNA aptamer based multi-modal, Fe3O4-saturated lactoferrin (Fe3O4-bLf) nanocarriers for triple positive (EpCAM, CD133, CD44) colon tumor targeting and NIR, MRI and CT imaging.

    Science.gov (United States)

    Roy, Kislay; Kanwar, Rupinder K; Kanwar, Jagat R

    2015-12-01

    This is the first ever attempt to combine anti-cancer therapeutic effects of emerging anticancer biodrug bovine lactoferrin (bLf), and multimodal imaging efficacy of Fe3O4 nanoparticles (NPs) together, as a saturated Fe3O4-bLf. For cancer stem cell specific uptake of nanocapsules/nanocarriers (NCs), Fe3O4-bLf was encapsulated in alginate enclosed chitosan coated calcium phosphate (AEC-CP) NCs targeted (Tar) with locked nucleic acid (LNA) modified aptamers against epithelial cell adhesion molecule (EpCAM) and nucleolin markers. The nanoformulation was fed orally to mice injected with triple positive (EpCAM, CD133, CD44) sorted colon cancer stem cells in the xenograft cancer stem cell mice model. The complete regression of tumor was observed in 70% of mice fed on non-targeted (NT) NCs, with 30% mice showing tumor recurrence after 30 days, while only 10% mice fed with Tar NCs showed tumor recurrence indicating a significantly higher survival rate. From tumor tissue analyses of 35 apoptotic markers, 55 angiogenesis markers, 40 cytokines, 15 stem cell markers and gene expression studies of important signaling molecules, it was revealed that the anti-cancer mechanism of Fe3O4-bLf was intervened through TRAIL, Fas, Fas-associated protein with death domain (FADD) mediated phosphorylation of p53, to induce activation of second mitochondria-derived activator of caspases (SMAC)/DIABLO (inhibiting survivin) and mitochondrial depolarization leading to release of cytochrome C. Induction of apoptosis was observed by inhibition of the Akt pathway and activation of cytokines released from monocytes/macrophages and dendritic cells (interleukin (IL) 27, keratinocyte chemoattractant (KC)). On the other hand, the recurrence of tumor in AEC-CP-Fe3O4-bLf NCs fed mice mainly occurred due to activation of alternative pathways such as mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK) and Wnt signaling leading to an increase in expression of survivin

  9. High levels of the adhesion molecule CD44 on leukemic cells generate acute myeloid leukemia relapse after withdrawal of the initial transforming event.

    Science.gov (United States)

    Quéré, R; Andradottir, S; Brun, A C M; Zubarev, R A; Karlsson, G; Olsson, K; Magnusson, M; Cammenga, J; Karlsson, S

    2011-03-01

    Multiple genetic hits are detected in patients with acute myeloid leukemia (AML). To investigate this further, we developed a tetracycline-inducible mouse model of AML, in which the initial transforming event, overexpression of HOXA10, can be eliminated. Continuous overexpression of HOXA10 is required to generate AML in primary recipient mice, but is not essential for maintenance of the leukemia. Transplantation of AML to secondary recipients showed that in established leukemias, ∼80% of the leukemia-initiating cells (LICs) in bone marrow stopped proliferating upon withdrawal of HOXA10 overexpression. However, the population of LICs in primary recipients is heterogeneous, as ∼20% of the LICs induce leukemia in secondary recipients despite elimination of HOXA10-induced overexpression. Intrinsic genetic activation of several proto-oncogenes was observed in leukemic cells resistant to inactivation of the initial transformation event. Interestingly, high levels of the adhesion molecule CD44 on leukemic cells are essential to generate leukemia after removal of the primary event. This suggests that extrinsic niche-dependent factors are also involved in the host-dependent outgrowth of leukemias after withdrawal of HOXA10 overexpression event that initiates the leukemia.

  10. The coordinate alteration of actin cytoskeleton, CD44 and matrix metalloproteinase-2 in the metastasis of breast cancer cells%转移相关分子链Actin-CD44-MMP-2在乳腺癌转移实验中的改变

    Institute of Scientific and Technical Information of China (English)

    赵威; 韩海勃; 林仲翔; 张志谦

    2011-01-01

    Objective To study the roles of actin and associated molecules in the control of human breast cancer cell malignant behaviors in vitro and in vivo.Methods A highly metastatic human breast cancer cell line BICR-H1 was compared with another breast cancer cell line MCF-7, which was well differentiated and non-metastatic.Western blot, immunofluorescence, gelatin zymography analysis and a chick embryonic chorioallantoic membrane (CAM) assay were used in this research.5~30 μg cisplatin or MMP-2 C terminal PEX domain were injected i.v.in CAM.Results BICR - H 1 expressed high level of CD44, which was closely associated with actin aggregates at the bottom side of attached cells.It was also shown with MMP-2 activity.On the contrary, MCF-7 cells showed weak disruption of actin cytoskeleton structures and a few actin aggregates.It expressed low or minimal level of CD44 and MMP-2.The expression of CD44 was down-regulated in cisplatin-treated BICR-H1 cells, and the activity of MMP-2 was also decreased upon PEX treatment.Both cell lines could form tumors in CAM, but only BICR-H1 cells could metastasize to distant tissues.Cisplatin inhibited the growth of BICR-H1 and MCF-7 cells in a time and dose dependent manner in CAM.The lung metastatic foci of BICR-H1 cells treated with 30 μg cisplatin were reduced from 30 ± 15/embryo (PBS group) to 8 ± 6/embryo, and the same dose of PEX could completely inhibit BICR-H1 metastasis.Conclusion It is concluded that actin cytoskeleton, CD44 and MMP-2 (ACM) molecular linkage is associated with breast cancer metastatic phenotypes, and both cisplatin and PEX can interfere with the ACM molecular linkage, resulting in the suppression of both tumor growth and metastasis.%目的 研究乳腺癌转移相关的分子机制及抑制体内外转移的作用和机制.方法 选择高、低转移性乳腺癌细胞系BICR-H1和MCF-7,用明胶底物非变性电泳分析法、Western blot和免疫荧光染色等方法,观察肌动蛋白、CD44

  11. 二甲双胍联合奥沙利铂对肺癌裸鼠移植瘤 VEGF-C、VEGFR-3、CD44 v6和β-catenin 表达的影响%Effects of metformin combined with oxaliplatin on VEGF-C, VEGFR-3, CD44v6 andβ-catenin expression of lung cancer xenograft in nude mice

    Institute of Scientific and Technical Information of China (English)

    陈玉琴; 陈刚

    2015-01-01

    目的:探讨二甲双胍联合奥沙利铂对肺癌裸鼠移植瘤血管内皮生长因子-C( VEGF-C)、血管内皮生长因子受体-3(VEGFR-3)、CD44变异体蛋白6(CD44v6)和β-链蛋白(β-catenin)分子表达水平的影响。方法建立肺癌裸鼠移植瘤模型,将裸鼠随机分为二甲双胍组、奥沙利铂组、联合用药组及对照组,每组分别给予二甲双胍、奥沙利铂、二甲双胍+奥沙利铂及灭菌蒸馏水等干预,42 d后处死动物,留取肿瘤组织,分别采用免疫组织化学法及实时荧光定量PCR法检测肿瘤组织中VEGF-C、VEGFR-3、CD44v6和β-catenin蛋白及mRNA的表达。结果二甲双胍组、奥沙利铂组和联合用药组的抑瘤率分别为28.97%、34.37%和50.27%。与对照组相比,奥沙利铂组、联合用药组VEGF-C、VEGFR-3、CD44v6和β-catenin蛋白及mRNA表达水平均降低(P均<0.05);与二甲双胍组、奥沙利铂组比较,联合用药组VEGF-C、VEGFR-3、CD44v6和β-catenin 蛋白及mRNA表达水平均降低(P 均<0.05), VEGF-C、VEGFR-3、CD44v6和 β-catenin 蛋白组间比较有统计学差异( F 值分别为79.281、151.275、171.294、164.149,P均<0.01);VEGF-C、VEGFR-3、CD44v6和β-catenin mRNA组间比较差异均有统计学意义(F值分别为78.897、81.029、64.100、83.892,P均<0.01)。结论奥沙利铂及二甲双胍均可抑制VEGF-C、VEGFR-3、CD44v6和β-catenin的表达,联合应用抑制效果更强。%Objective To investigate the effects of metformin combined with oxaliplatin on the expression levels of vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3), CD44v6 andβ-catenin of lung cancer xenograft in nude mice .Methods We established human lung cancer xenograft models in the nude mice.The nude mice were randomly divided into metformin-treated, oxaliplatin-treated, metformin combined with ox

  12. CD44+/CD105+ Human Amniotic Fluid Mesenchymal Stem Cells Survive and Proliferate in the Ovary Long-Term in a Mouse Model of Chemotherapy-Induced Premature Ovarian Failure

    OpenAIRE

    LIU, TE; HUANG, YONGYI; Guo, Lihe; Cheng, Weiwei; Zou, Gang

    2012-01-01

    Objectives: Stem cell transplantation has been reported to rescue ovarian function in a preclinical mouse model of chemotherapy-induced premature ovarian failure (POF); however, maintaining the survival and self-renewal of transplanted seed cells in ovarian tissues over the long-term remains a troublesome issue. In this study we aimed to determine whether the CD44+/CD105+ human amniotic fluid cell (HuAFCs) subpopulation represent potential seed cells for stem cell transplantation treatments i...

  13. Inhibition of Sonic Hedgehog Signaling Pathway by Thiazole Antibiotic Thiostrepton Attenuates the CD44+/CD24-Stem-Like Population and Sphere-Forming Capacity in Triple-Negative Breast Cancer

    Directory of Open Access Journals (Sweden)

    Na Yang

    2016-03-01

    Full Text Available Background/Aim: Triple-negative breast cancer (TNBC represents a particular clinical challenge because these cancers do not respond to endocrine therapy or other available targeted agents. The lack of effective agents and obvious targets are major challenges in treating TNBC. In this study we explored the cytostatic effect of thiazole ring containing antibiotic drug thiostrepton on TNBC cell lines and investigated the molecular mechanism. Methods: Cell viability was measured by MTT assay. Cell surface marker was monitored by FCM. Western blot was applied to assess the protein expression levels of target genes. Results: We found that thiostrepton remarkably suppressed the CD44+/CD24- stem-like population and sphere forming capacity of TNBC cell lines. Notably, we showed for the first time that thiostrepton exerted its pharmacological action by targeting sonic hedgehog (SHH signaling pathway. Thiostrepton repressed SHH ligand expression and reduced Gli-1 nuclear localization in TNBC cell line. Furthermore, the downstream target of SHH signaling undergone dose-dependent, rapid, and sustained loss of mRNA transcript level after thiostrepton treatment. Finally, we showed that SHH ligand was essential for maintaining CD44+/CD24- stem-like population in TNBC cell line. Conclusion: We conclude that thiostrepton suppresses the CD44+/CD24- stem-like population through inhibition of SHH signaling pathway. Our results give a new insight into the mechanism of thiostrepton anti-tumor activity and suggest thiostrepton as a promising agent that targets hedgehog signaling pathway in TNBC.

  14. Expression and Significance of Cancer Stem Cell Markers CD133, CD44, SOX2, OCT4 and ALDH1 in Non-small Cell Lung Cancer%肿瘤干细胞标志物CD133、CD44、SOX2、OCT4、ALDH1在非小细胞肺癌组织中的表达及临床意义

    Institute of Scientific and Technical Information of China (English)

    梁洪享; 钟竑; 罗勇; 黄燕; 丁昭珩; 丁罡

    2013-01-01

    目的 研究肿瘤干细胞标记物CD133、CD44、SOX2、OCT4、ALDH1在非小细胞肺癌(NSCLC)组织中的表达及临床意义,为探索非小细胞肺癌肿瘤干细胞提供参考.方法 采用免疫组织化学方法检测70例NSCLC组织、14例非癌组织中的CD133、CD44、SOX2、OCT4、ALDH1蛋白的表达并对结果进行分析. 结果 (1) CD133、CD44、SOX2、OCT4、ALDH1在70例NSCLC组织中的阳性表达率分别为88.57%、98.57%、100%、100%、100%,强阳性表达率分别为48.57%、67.14%、67.14%、31.43%、50%; CD133、CCD44在NSCLC与非癌组织中的表达差异存在统计学意义(P均<0.0001),SOX2、OCT4、ALDH1在NSCLC与非癌组织中的表达差异无统计学意义(P均>0.05) (2)随着病理级别的升高,CD133、CD44、SOX2、OCT4及ALDH1的表达呈上升趋势,分化越低的NSCLC表达上述指标的概率越高,其中CD133、SOX2、OCT4的表达在高、中、低分化组织中差异存在统计学意义(P值分别为0.001、0.040、<0.0001);CD133的表达在吸烟史、分化程度、淋巴结转移、肿瘤分期四个因素上差异存在统计学意义(P值分别为0.033、0.001、0.033、0.046);CD44与SOX2的表达在年龄上的差异存在统计学意义(P值分别为0.001、0.040) 结论 NSCLC组织中CD133、CD44、SOX2、OCT4、ALDH1阳性率高,CD133、CD44的表达明显高于非癌组织;CD133、SOX2、OCT4与NSCLC的恶性程度有关;CD44与SOX2与年龄因素有关.

  15. Utility of a triple antibody cocktail intraurothelial neoplasm-3 (IUN-3-CK20/CD44s/p53) and α-methylacyl-CoA racemase (AMACR) in the distinction of urothelial carcinoma in situ (CIS) and reactive urothelial atypia.

    Science.gov (United States)

    Aron, Manju; Luthringer, Daniel J; McKenney, Jesse K; Hansel, Donna E; Westfall, Danielle E; Parakh, Rugvedita; Mohanty, Sambit K; Balzer, Bonnie; Amin, Mahul B

    2013-12-01

    Urothelial carcinoma in situ (CIS) is a prognostically and therapeutically significant lesion with considerable morphologic overlap with reactive conditions especially in the setting of prior therapy. Various markers including CK20, CD44s, and p53 have been used as an adjunct in making this distinction; however, the utility of these markers in the posttreatment scenario is not fully established. α-Methylacyl-CoA racemase (AMACR) is a tumor-associated marker that is expressed in a subset of high-grade urothelial carcinomas but has not been studied in CIS. This study was undertaken to evaluate the immunoreactivity of CK20, CD44s, and p53 as a triple antibody cocktail intraurothelial neoplasm-3 (IUN-3) in distinguishing CIS from its mimics and to compare its utility with AMACR in the diagnosis of CIS. A total of 135 specimens (7 benign ureters and 128 bladder biopsies-28 reactive, 33 posttherapy reactive, 43 CIS, 24 CIS posttherapy) were included in this study. Immunostaining for p53 (brown, nuclear), CD44s (brown, membranous), and CK20 (red, cytoplasmic and membranous) was performed as a cocktail, and the staining pattern was further classified as: malignant (full-thickness CK20 and/or full-thickness p53 with CD44s negativity), reactive/benign (CK20 limited to the umbrella cell layer, p53 negative, and CD44s positivity ranging from basal to full thickness), and indeterminate (CK20 and p53 positive but not full thickness and/or CD44s positive). AMACR staining was performed in 50 cases. Cytoplasmic staining for AMACR was graded as negative (absent to weak focal staining [<5% cells]) and positive (≥5%). The "IUN-3 malignant" pattern was observed in 84% of cases of CIS without a history of prior therapy and in 71% of the cases of CIS with a history of prior therapy. Cases with posttherapy reactive atypia showed an "IUN-3 reactive" pattern in 84% cases and "IUN-3 indeterminate" pattern in 16% of the cases; the IUN-3 malignant pattern was not identified in any of the

  16. Cell-specific expression of TLR9 isoforms in inflammation.

    Science.gov (United States)

    McKelvey, Kelly J; Highton, John; Hessian, Paul A

    2011-02-01

    Toll-like receptors (TLRs) are key pattern recognition receptors during an immune response. With five isoforms of human TLR9 described, we hypothesised that differential expression of TLR9 isoforms in different cell types would result in variable contributions to the overall input from TLR9 during inflammation. We assessed the molecular expression of the TLR9 isoforms, TLR9-A, -C and -D. In normal peripheral blood mononuclear cells, B-lymphocytes express ∼100-fold more TLR9-A transcript than monocytes or T-lymphocytes, which predominantly express the TLR9-C transcript. Switches in isoform predominance accompany B-lymphocyte development. TLR9 protein expression in rheumatoid inflammatory lesions reflected the TLR9 isoform expression by immune cells. Herein we suggest that B-lymphocytes and plasmacytoid dendritic cells contribute the ∼3-fold higher TLR9-A transcript levels observed in inflamed synovium when compared to subcutaneous rheumatoid nodules. In contrast, macrophages and T-lymphocytes contribute the ∼4-fold higher TLR9-C transcript levels seen in nodules, compared to synovia. From protein sequence, predictions of subcellular localisation suggest TLR9-B may locate to the mitochondria, whereas TLR9-D adopts an opposing orientation in the endoplasmic reticulum. Consistent with this, structure models raise the possibility of alternative ligands for the TLR9-B and TLR9-D variants. Our results highlight differences in the expression of human TLR9 isoforms in normal and inflamed tissues, with differing contributions to inflammation.

  17. Molecular basis for Duarte and Los Angeles variant galactosemia.

    OpenAIRE

    Langley, S D; Lai, K.; Dembure, P P; Hjelm, L. N.; Elsas, L. J.

    1997-01-01

    Human orythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the "Los Angeles (LA) phenotype," which has increased GALT enzyme activity. We evaluated GALT enzyme activity and scre...

  18. Thermochemotherapy effect of nanosized As2O3/Fe3O4 complex on experimental mouse tumors and its influence on the expression of CD44v6, VEGF-C and MMP-9

    Directory of Open Access Journals (Sweden)

    Zhang Dongsheng

    2009-10-01

    Full Text Available Abstract Background Both thermotherapy and arsenic have been shown to be active against a broad spectrum of cancers. To reduce the limitations of conventional thermotherapy, improve therapeutic anticancer activity, reduce the toxicity of arsenic on normal tissue, and increase tissue-specific delivery, we prepared a nanosized As2O3/Fe3O4 complex (Fe3O4 magnetic nanoparticles encapsulated in As2O3. We assessed the thermodynamic characteristics of this complex and validated the hyperthermia effect, when combined with magnetic fluid hyperthermia (MFH, on xenograft HeLa cells (human cervical cancer cell line in nude mice. We also measured the effect on the expression of CD44v6, VEGF-C, and MMP-9 which were related to cancer and/or metastasis. Results The nanosized As2O3/Fe3O4 particles were approximately spherical, had good dispersibility as evidenced by TEM, and an average diameter of about 50 nm. With different concentrations of the nanosized As2O3/Fe3O4 complex, the correspondingsuspension of magnetic particles could attain a steady temperature ranging from 42°C to 65°C when placed in AMF for 40 min. Thermochemotherapy with the nanosized As2O3/Fe3O4 complex showed a significant inhibitory effect on the mass (88.21% and volume (91.57% of xenograft cervical tumors (p 2O3/Fe3O4 complex significantly inhibited the expression of CD44v6, VEGF-C, and MMP-9 mRNA (p Conclusion As2O3/Fe3O4 complex combined with MFH had is a promising technique for the minimally invasive elimination of solid tumors and may be have anticancerometastasic effect by inhibiting the expression of CD44v6, VEGF-C, and MMP-9.

  19. Blockade of Notch3 inhibits the stem-like property and is associated with ALDH1A1 and CD44 via autophagy in non-small lung cancer.

    Science.gov (United States)

    Ma, Yuanyuan; Li, Mingzhen; Si, Jiahui; Xiong, Ying; Lu, Fangliang; Zhang, Jianzhi; Zhang, Liyi; Zhang, Panpan; Yang, Yue

    2016-06-01

    Acquired resistance to standard chemotherapy causes treatment failure in patients with local advanced and advanced non-small lung cancer (NSCLC). Cancer stem cells (CSCs) are a small subpopulation within cancer that is thought to be resistant to conventional chemotherapy. The Notch pathway is one of the most intensively studied for putative therapeutic targets of CSCs in solid tumors. In our study, suppression of Notch3 decreased colony and sphere formation of stem-like property in lung cancer cells. In addition, Notch3 expression was demonstrated to be upregulated in the patients with chemoresistance and related to poor prognosis of NSCLC patients. Our results also showed that CSC markers ALDH1A1 and CD44 were highly expressed in NSCLC patients with chemoresistance and these two markers were positively correlated with Notch3 expression in lung cancer specimens from TCGA database. Furthermore, the lung cancer cells with drug resistance were shown to be associated with activation of autophagy. All the data support a crucial role of Notch3 in the increase of stem-like property in NSCLC cells that might be associated with upregulation of ALDH1A1 and CD44 and activation of autophagy. PMID:27035162

  20. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism.

    Science.gov (United States)

    Corominas, Roser; Yang, Xinping; Lin, Guan Ning; Kang, Shuli; Shen, Yun; Ghamsari, Lila; Broly, Martin; Rodriguez, Maria; Tam, Stanley; Trigg, Shelly A; Fan, Changyu; Yi, Song; Tasan, Murat; Lemmens, Irma; Kuang, Xingyan; Zhao, Nan; Malhotra, Dheeraj; Michaelson, Jacob J; Vacic, Vladimir; Calderwood, Michael A; Roth, Frederick P; Tavernier, Jan; Horvath, Steve; Salehi-Ashtiani, Kourosh; Korkin, Dmitry; Sebat, Jonathan; Hill, David E; Hao, Tong; Vidal, Marc; Iakoucheva, Lilia M

    2014-04-11

    Increased risk for autism spectrum disorders (ASD) is attributed to hundreds of genetic loci. The convergence of ASD variants have been investigated using various approaches, including protein interactions extracted from the published literature. However, these datasets are frequently incomplete, carry biases and are limited to interactions of a single splicing isoform, which may not be expressed in the disease-relevant tissue. Here we introduce a new interactome mapping approach by experimentally identifying interactions between brain-expressed alternatively spliced variants of ASD risk factors. The Autism Spliceform Interaction Network reveals that almost half of the detected interactions and about 30% of the newly identified interacting partners represent contribution from splicing variants, emphasizing the importance of isoform networks. Isoform interactions greatly contribute to establishing direct physical connections between proteins from the de novo autism CNVs. Our findings demonstrate the critical role of spliceform networks for translating genetic knowledge into a better understanding of human diseases.

  1. Cloning, expression, purification and characterization of tryptophan hydroxylase variants

    DEFF Research Database (Denmark)

    Boesen, Jane

    such as depression and obsessive-compulsive disorder (OCD). Characterization of TPH and elucidation of the enzymes regulation and catalytic mechanism is therefore vital to our understanding of the serotonin balance. This study concerns variants of both human TPH isoform 1 (hTPH1) and human TPH isoform 2 (h PH2...... the variant. The GST-rhTPH1 variant could be purified using affinity chromatography followed by gel filtration with high purity and a yield of 40 mg/l culture. The purified GST-rhTPH1 exists as a dimer in solution due to dimerization of GST. The GST could be cleaved successfully from the fusion protein using...

  2. HIV-1 p17 matrix protein interacts with heparan sulfate side chain of CD44v3, syndecan-2, and syndecan-4 proteoglycans expressed on human activated CD4+ T cells affecting tumor necrosis factor alpha and interleukin 2 production.

    Science.gov (United States)

    De Francesco, Maria A; Baronio, Manuela; Poiesi, Claudio

    2011-06-01

    HIV-1 p17 contains C- and N-terminal sequences with positively charged residues and a consensus cluster for heparin binding. We have previously demonstrated by affinity chromatography that HIV-1 p17 binds strongly to heparin-agarose at physiological pH and to human activated CD4(+) T cells. In this study we demonstrated that the viral protein binds to heparan sulfate side chains of syndecan-2, syndecan-4, and CD44v3 purified from HeLa cells and that these heparan sulfate proteoglycans (HSPGs) co-localize with HIV-1 p17 on activated human CD4(+) T cells by confocal fluorescence analysis. Moreover, we observed a stimulatory or inhibitory activity when CD4(+) T cells were activated with mitogens together with nanomolar or micromolar concentrations of the matrix protein.

  3. Two Domains of Vimentin Are Expressed on the Surface of Lymph Node, Bone and Brain Metastatic Prostate Cancer Lines along with the Putative Stem Cell Marker Proteins CD44 and CD133

    International Nuclear Information System (INIS)

    Vimentin was originally identified as an intermediate filament protein present only as an intracellular component in many cell types. However, this protein has now been detected on the surface of a number of different cancer cell types in a punctate distribution pattern. Increased vimentin expression has been indicated as an important step in epithelial-mesenchymal transition (EMT) required for the metastasis of prostate cancer. Here, using two vimentin-specific monoclonal antibodies (SC5 and V9 directed against the coil one rod domain and the C-terminus of the vimentin protein, respectively), we examined whether either of these domains would be displayed on the surface of three commonly studied prostate cancer cell lines isolated from different sites of metastases. Confocal analysis of LNCaP, PC3 and DU145 prostate cancer cell lines (derived from lymph node, bone or brain prostate metastases, respectively) demonstrated that both domains of vimentin are present on the surface of these metastatic cancer cell types. In addition, flow cytometric analysis revealed that vimentin expression was readily detected along with CD44 expression but only a small subpopulation of prostate cancer cells expressed vimentin and the putative stem cell marker CD133 along with CD44. Finally, Cowpea mosaic virus (CPMV) nanoparticles that target vimentin could bind and internalize into tested prostate cancer cell lines. These results demonstrate that at least two domains of vimentin are present on the surface of metastatic prostate cancer cells and suggest that vimentin could provide a useful target for nanoparticle- or antibody- cancer therapeutic agents directed against highly invasive cancer and/or stem cells

  4. Cellulase variants

    Energy Technology Data Exchange (ETDEWEB)

    Blazej, Robert; Toriello, Nicholas; Emrich, Charles; Cohen, Richard N.; Koppel, Nitzan

    2015-07-14

    This invention provides novel variant cellulolytic enzymes having improved activity and/or stability. In certain embodiments the variant cellulotyic enzymes comprise a glycoside hydrolase with or comprising a substitution at one or more positions corresponding to one or more of residues F64, A226, and/or E246 in Thermobifida fusca Cel9A enzyme. In certain embodiments the glycoside hydrolase is a variant of a family 9 glycoside hydrolase. In certain embodiments the glycoside hydrolase is a variant of a theme B family 9 glycoside hydrolase.

  5. Learning-dependent gene expression of CREB1 isoforms in the molluscan brain

    Directory of Open Access Journals (Sweden)

    Hisayo Sadamoto

    2010-05-01

    Full Text Available Cyclic AMP-responsive element binding protein1 (CREB1 has multiple functions in gene regulation. Various studies have reported that CREB1-dependent gene induction is necessary for memory formation and long-lasting behavioral changes in both vertebrates and invertebrates. In the present study, we characterized Lymnaea CREB1 (LymCREB1 mRNA isoforms of spliced variants in the central nervous system (CNS of the pond snail Lymnaea stagnalis. Among these spliced variants, the three isoforms that code a whole LymCREB1 protein are considered to be the activators for gene regulation. The other four isoforms, which code truncated LymCREB1 proteins with no kinase inducible domain, are the repressors. For a better understanding of the possible roles of different LymCREB1 isoforms, the expression level of these isoform mRNAs was investigated by a real-time quantitative RT-PCR method. Further, we examined the changes in gene expression for all the isoforms in the CNS after conditioned taste aversion (CTA learning or backward conditioning as a control. The results showed that CTA learning increased LymCREB1 gene expression, but it did not change the activator/repressor ratio. Our findings showed that the repressor isoforms, as well as the activator ones, are expressed in large amounts in the CNS, and the gene expression of CREB1 isoforms appeared to be specific for the given stimulus. This was the first quantitative analysis of the expression patterns of CREB1 isoforms at the mRNA level and their association with learning behavior.

  6. Two-dimensional zymography differentiates gelatinase isoforms in stimulated microglial cells and in brain tissues of acute brain injuries.

    Science.gov (United States)

    Chen, Shanyan; Meng, Fanjun; Chen, Zhenzhou; Tomlinson, Brittany N; Wesley, Jennifer M; Sun, Grace Y; Whaley-Connell, Adam T; Sowers, James R; Cui, Jiankun; Gu, Zezong

    2015-01-01

    Excessive activation of gelatinases (MMP-2/-9) is a key cause of detrimental outcomes in neurodegenerative diseases. A single-dimension zymography has been widely used to determine gelatinase expression and activity, but this method is inadequate in resolving complex enzyme isoforms, because gelatinase expression and activity could be modified at transcriptional and posttranslational levels. In this study, we investigated gelatinase isoforms under in vitro and in vivo conditions using two-dimensional (2D) gelatin zymography electrophoresis, a protocol allowing separation of proteins based on isoelectric points (pI) and molecular weights. We observed organomercuric chemical 4-aminophenylmercuric acetate-induced activation of MMP-2 isoforms with variant pI values in the conditioned medium of human fibrosarcoma HT1080 cells. Studies with murine BV-2 microglial cells indicated a series of proform MMP-9 spots separated by variant pI values due to stimulation with lipopolysaccharide (LPS). The MMP-9 pI values were shifted after treatment with alkaline phosphatase, suggesting presence of phosphorylated isoforms due to the proinflammatory stimulation. Similar MMP-9 isoforms with variant pI values in the same molecular weight were also found in mouse brains after ischemic and traumatic brain injuries. In contrast, there was no detectable pI differentiation of MMP-9 in the brains of chronic Zucker obese rats. These results demonstrated effective use of 2D zymography to separate modified MMP isoforms with variant pI values and to detect posttranslational modifications under different pathological conditions.

  7. Entropy-based model for miRNA isoform analysis.

    Directory of Open Access Journals (Sweden)

    Shengqin Wang

    Full Text Available MiRNAs have been widely studied due to their important post-transcriptional regulatory roles in gene expression. Many reports have demonstrated the evidence of miRNA isoform products (isomiRs in high-throughput small RNA sequencing data. However, the biological function involved in these molecules is still not well investigated. Here, we developed a Shannon entropy-based model to estimate isomiR expression profiles of high-throughput small RNA sequencing data extracted from miRBase webserver. By using the Kolmogorov-Smirnov statistical test (KS test, we demonstrated that the 5p and 3p miRNAs present more variants than the single arm miRNAs. We also found that the isomiR variant, except the 3' isomiR variant, is strongly correlated with Minimum Free Energy (MFE of pre-miRNA, suggesting the intrinsic feature of pre-miRNA should be one of the important factors for the miRNA regulation. The functional enrichment analysis showed that the miRNAs with high variation, particularly the 5' end variation, are enriched in a set of critical functions, supporting these molecules should not be randomly produced. Our results provide a probabilistic framework for miRNA isoforms analysis, and give functional insights into pre-miRNA processing.

  8. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Science.gov (United States)

    Guillaudeau, Angélique; Durand, Karine; Bessette, Barbara; Chaunavel, Alain; Pommepuy, Isabelle; Projetti, Fabrice; Robert, Sandrine; Caire, François; Rabinovitch-Chable, Hélène; Labrousse, François

    2012-01-01

    The EGFR (epidermal growth factor receptor) is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a), normal and tumor cells produce soluble EGFR isoforms (sEGFR) that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2), 3 (v3) and 4 (v4) mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab) and intracellular domain targeted antibody (ICD-Ab). EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade), histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS). PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types. PMID:22623992

  9. EGFR soluble isoforms and their transcripts are expressed in meningiomas.

    Directory of Open Access Journals (Sweden)

    Angélique Guillaudeau

    Full Text Available The EGFR (epidermal growth factor receptor is involved in the oncogenesis of many tumors. In addition to the full-length EGFR (isoform a, normal and tumor cells produce soluble EGFR isoforms (sEGFR that lack the intracellular domain. sEGFR isoforms b, c and d are encoded by EGFR variants 2 (v2, 3 (v3 and 4 (v4 mRNA resulting from gene alternative splicing. Accordingly, the results of EGFR protein expression analysis depend on the domain targeted by the antibodies. In meningiomas, EGFR expression investigations mainly focused on EGFR isoform a. sEGFR and EGFRvIII mutant, that encodes a constitutively active truncated receptor, have not been studied. In a 69 meningiomas series, protein expression was analyzed by immunohistochemistry using extracellular domain targeted antibody (ECD-Ab and intracellular domain targeted antibody (ICD-Ab. EGFRv1 to v4 and EGFRvIII mRNAs were quantified by RT-PCR and EGFR amplification revealed by MLPA. Results were analyzed with respect to clinical data, tumor resection (Simpson grade, histological type, tumor grade, and patient outcome.Immunochemical staining was stronger with ECD-Ab than with ICD-Ab. Meningiomas expressed EGFRv1 to -v4 mRNAs but not EGFRvIII mutant. Intermediate or high ECD-Ab staining and high EGFRv1 to v4 mRNA levels were associated to a better progression free survival (PFS. PFS was also improved in women, when tumor resection was evaluated as Simpson 1 or 2, in grade I vs. grade II and III meningiomas and when Ki67 labeling index was lower than 10%. Our results suggest that, EGFR protein isoforms without ICD and their corresponding mRNA variants are expressed in meningiomas in addition to the whole isoform a. EGFRvIII was not expressed. High expression levels seem to be related to a better prognosis. These results indicate that the oncogenetic mechanisms involving the EGFR pathway in meningiomas could be different from other tumor types.

  10. Genomic organization and the tissue distribution of alternatively spliced isoforms of the mouse Spatial gene

    Directory of Open Access Journals (Sweden)

    Mattei Marie-Geneviève

    2004-07-01

    Full Text Available Abstract Background The stromal component of the thymic microenvironment is critical for T lymphocyte generation. Thymocyte differentiation involves a cascade of coordinated stromal genes controlling thymocyte survival, lineage commitment and selection. The "Stromal Protein Associated with Thymii And Lymph-node" (Spatial gene encodes a putative transcription factor which may be involved in T-cell development. In the testis, the Spatial gene is also expressed by round spermatids during spermatogenesis. Results The Spatial gene maps to the B3-B4 region of murine chromosome 10 corresponding to the human syntenic region 10q22.1. The mouse Spatial genomic DNA is organised into 10 exons and is alternatively spliced to generate two short isoforms (Spatial-α and -γ and two other long isoforms (Spatial-δ and -ε comprising 5 additional exons on the 3' site. Here, we report the cloning of a new short isoform, Spatial-β, which differs from other isoforms by an additional alternative exon of 69 bases. This new exon encodes an interesting proline-rich signature that could confer to the 34 kDa Spatial-β protein a particular function. By quantitative TaqMan RT-PCR, we have shown that the short isoforms are highly expressed in the thymus while the long isoforms are highly expressed in the testis. We further examined the inter-species conservation of Spatial between several mammals and identified that the protein which is rich in proline and positive amino acids, is highly conserved. Conclusions The Spatial gene generates at least five alternative spliced variants: three short isoforms (Spatial-α, -β and -γ highly expressed in the thymus and two long isoforms (Spatial-δ and -ε highly expressed in the testis. These alternative spliced variants could have a tissue specific function.

  11. Novel CD44 receptor targeting multifunctional “nano-eggs” based on double pH-sensitive nanoparticles for co-delivery of curcumin and paclitaxel to cancer cells and cancer stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Daquan, E-mail: cdq1981@126.com [Peking University, School of Pharmaceutical Sciences, Health Science Center (China); Wang, Guohua [China Academy of Chinese Medical Sciences, Institute of Chinese Materia Madica (China); Song, Weiguo [Shouguang Fukang Pharmceutial Co., Ltd. (China); Zhang, Qiang, E-mail: zqdodo@bjmu.edu.cn [Peking University, School of Pharmaceutical Sciences, Health Science Center (China)

    2015-10-15

    Most anticancer drugs cannot kill cancer stem cells (CSCs) effectively, which lead to the failure of anticancer chemotherapy, such as relapse and metastasis. In this study, we prepared a multifunctional oligosaccharides of hyaluronan (oHA) conjugates, oHA-histidine-menthone 1,2-glycerol ketal (oHM). The oHM conjugates possess pH-sensitive menthone 1,2-glycerol ketal (MGK) as hydrophobic moieties and oHA as the target of CD44 receptor. Anticancer drugs, curcumin(Cur) and paclitaxel(PTX), were loaded into oHM micelles via self-assembly. Then, oHM micelles were mineralized through controlled deposition of inorganic calcium and phosphate ions on the nanoparticular shell via a sequential addition method to fabricate the “nano-eggs.” The formed nano-eggs had a smaller size (120.6 ± 4.5 nm) than oHM micelles (158.6 ± 6.4 nm), indicating that mineralization made the appearance of compact nanoparticles. Interestingly, when the nano-eggs were put into the acidic conditions (pH 6.5), their outer shell(inorganic minerals) will be destroyed with the larger size, while the “nano-eggs” were stable under pH 7.4. For both nano-eggs and oHM micelles, the Cur and PTX were released in a sustained manner depending on the pH of the solution. However, the nano-eggs showed much lower released than the oHM micelles due to the dissolution of the inorganic minerals and pH-sensitive ketal at mildly acidic environments (pH 6.5). In vivo study, the nano-eggs could get to the tumor site more effectively than oHM micelles. CSCs were sorted by a side population assay from MDA-MB-231 breast cancer cell lines over-expressing CD44 receptors. Antitumor activity was also evaluated on MDA-MB-231 xenografts in nude mice. The antitumor efficacy indicated that nano-eggs with co-delivery of Cur and PTX produced the strongest antitumor efficacy, and nano-eggs showed strong activity against cancer stem cells. These double pH-sensitive nano-eggs may provide a promising strategy for drug

  12. Novel CD44 receptor targeting multifunctional “nano-eggs” based on double pH-sensitive nanoparticles for co-delivery of curcumin and paclitaxel to cancer cells and cancer stem cells

    International Nuclear Information System (INIS)

    Most anticancer drugs cannot kill cancer stem cells (CSCs) effectively, which lead to the failure of anticancer chemotherapy, such as relapse and metastasis. In this study, we prepared a multifunctional oligosaccharides of hyaluronan (oHA) conjugates, oHA-histidine-menthone 1,2-glycerol ketal (oHM). The oHM conjugates possess pH-sensitive menthone 1,2-glycerol ketal (MGK) as hydrophobic moieties and oHA as the target of CD44 receptor. Anticancer drugs, curcumin(Cur) and paclitaxel(PTX), were loaded into oHM micelles via self-assembly. Then, oHM micelles were mineralized through controlled deposition of inorganic calcium and phosphate ions on the nanoparticular shell via a sequential addition method to fabricate the “nano-eggs.” The formed nano-eggs had a smaller size (120.6 ± 4.5 nm) than oHM micelles (158.6 ± 6.4 nm), indicating that mineralization made the appearance of compact nanoparticles. Interestingly, when the nano-eggs were put into the acidic conditions (pH 6.5), their outer shell(inorganic minerals) will be destroyed with the larger size, while the “nano-eggs” were stable under pH 7.4. For both nano-eggs and oHM micelles, the Cur and PTX were released in a sustained manner depending on the pH of the solution. However, the nano-eggs showed much lower released than the oHM micelles due to the dissolution of the inorganic minerals and pH-sensitive ketal at mildly acidic environments (pH 6.5). In vivo study, the nano-eggs could get to the tumor site more effectively than oHM micelles. CSCs were sorted by a side population assay from MDA-MB-231 breast cancer cell lines over-expressing CD44 receptors. Antitumor activity was also evaluated on MDA-MB-231 xenografts in nude mice. The antitumor efficacy indicated that nano-eggs with co-delivery of Cur and PTX produced the strongest antitumor efficacy, and nano-eggs showed strong activity against cancer stem cells. These double pH-sensitive nano-eggs may provide a promising strategy for drug

  13. The inhibition of autoreactive T cell functions by a peptide based on the CDR1 of an anti-DNA autoantibody is via TGF-beta-mediated suppression of LFA-1 and CD44 expression and function.

    Science.gov (United States)

    Sela, Uri; Mauermann, Nora; Hershkoviz, Rami; Zinger, Heidy; Dayan, Molly; Cahalon, Liora; Liu, Jian Ping; Mozes, Edna; Lider, Ofer

    2005-12-01

    Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-beta secretion 10 days later. Addition of TGF-beta suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFbeta-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 (alphaLbeta2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-beta-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1alpha and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-beta on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation. PMID:16301630

  14. Jeep variants

    CERN Document Server

    de Bondt, Michiel

    2010-01-01

    The jeep problem was first solved by O. Helmer and N.J. Fine. But not much later, C.G. Phipps formulated a more general solution. He formulated a so-called convoy or caravan variant of the jeep problem and reduced the original problem to it. The convoy idea of Phipps was refined in [3]. Here we will apply this refined idea to several variants of the jeep problem.

  15. Distinct expression and ligand-binding profiles of two constitutively active GPR17 splice variants

    DEFF Research Database (Denmark)

    Benned-Jensen, Tau; Rosenkilde, M M

    2010-01-01

    In humans and non-human primates, the 7TM receptor GPR17 exists in two isoforms differing only by the length of the N-terminus. Of these, only the short isoform has previously been characterized. Hence, we investigated gene expression and ligand-binding profiles of both splice variants and furthe...

  16. Quantitative Characterization of E-selectin Interaction with Native CD44 and P-selectin Glycoprotein Ligand-1 (PSGL-1) Using a Real Time Immunoprecipitation-based Binding Assay

    KAUST Repository

    Abu Samra, Dina Bashir Kamil

    2015-06-29

    Selectins (E-, P-, and L-selectins) interact with glycoprotein ligands to mediate the essential tethering/rolling step in cell transport and delivery that captures migrating cells from the circulating flow. In this work, we developed a real time immunoprecipitation assay on a surface plasmon resonance chip that captures native glycoforms of two well known E-selectin ligands (CD44/hematopoietic cell E-/L-selectin ligand and P-selectin glycoprotein ligand-1) from hematopoietic cell extracts. Here we present a comprehensive characterization of their binding to E-selectin. We show that both ligands bind recombinant monomeric E-selectin transiently with fast on- and fast off-rates, whereas they bind dimeric E-selectin with remarkably slow onand off-rates. This binding requires the sialyl Lewis x sugar moiety to be placed on both O- and N-glycans, and its association, but not dissociation, is sensitive to the salt concentration. Our results suggest a mechanism through which monomeric selectins mediate initial fast on and fast off kinetics to help capture cells out of the circulating shear flow; subsequently, tight binding by dimeric/oligomeric selectins is enabled to significantly slow rolling. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Splicing variants of porcine synphilin-1

    DEFF Research Database (Denmark)

    Larsen, Knud Erik; Madsen, Lone Bruhn; Farajzadeh, Leila;

    2015-01-01

    %) and to mouse (84%) synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel......RNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa) synphilin-1 cDNA (SNCAIP) and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1) of 919 amino acids which shows a high similarity to human (90...... splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation....

  18. Identification of novel chicken estrogen receptor-alpha messenger ribonucleic acid isoforms generated by alternative splicing and promoter usage.

    Science.gov (United States)

    Griffin, C; Flouriot, G; Sonntag-Buck, V; Nestor, P; Gannon, F

    1998-11-01

    Using the rapid amplification of complementary DNA ends (RACE) methodology we have identified three new chicken estrogen receptor-alpha (cER alpha) messenger RNA (mRNA) variants in addition to the previously described form (isoform A). Whereas one of the new variants (isoform B) presents a 5'-extremity contiguous to the 5'-end of isoform A, the two other forms (isoforms C and D) are generated by alternative splicing of upstream exons (C and D) to a common site situated 70 nucleotides upstream of the translation start site in the previously assigned exon 1 (A). The 3'-end of exon 1C has been located at position -1334 upstream of the transcription start site of the A isoform (+1). Whereas the genomic location of exon 1D is unknown, 700 bp 5' to this exon were isolated by genomic walking, and their sequence was determined. The transcription start sites of the cER alpha mRNA isoforms were defined. In transfection experiments, the regions immediately upstream of the A-D cER alpha mRNA isoforms were shown to possess cell-specific promoter activities. Three of these promoters were down-regulated in the presence of estradiol and ER alpha protein. It is concluded, therefore, that the expression of the four different cER alpha mRNA isoforms is under the control of four different promoters. Finally, RT-PCR, S1 nuclease mapping, and primer extension analysis of these different cER alpha mRNA isoforms revealed a differential pattern of expression of the cER alpha gene in chicken tissues. Together, the results suggest that alternative 5'-splicing and promoter usage may be mechanisms used to modulate the levels of expression of the chicken ER alpha gene in a tissue-specific and/or developmental stage-specific manner. PMID:9794473

  19. Altered Alpha-Synuclein, Parkin, and Synphilin Isoform Levels in Multiple System Atrophy Brains

    DEFF Research Database (Denmark)

    Brudek, Tomasz; Winge, Kristian; Bredo Rasmussen, Nadja;

    2016-01-01

    Together with Parkinson's disease (PD) and dementia with Lewy bodies (DLB), Multiple System Atrophy (MSA) is a member of a diverse group of neurodegenerative disorders termed α-synucleinopathies. Previously, it has been shown that alpha-synuclein, parkin and synphilin-1 display disease specific...... controls using isoform-specific primers and exon specific antibodies in substantia nigra, striatum, cerebellar cortex, and nucleus dentatus. These regions are severely affected by alpha-synuclein pathology and neurodegeneration. Further, we have also investigated transcript levels for parkin and synphilin...... increased levels of parkin isoforms lacking the N-terminal ubiquitin-like domain and an aggregation-prone synphiln-1A isoform that causes neuronal toxicity in MSA. In PD brains, Parkin transcript variant 3, 7 and 11 were significantly and specifically overexpressed in the striatum and cerebellar cortex...

  20. Splicing variants of porcine synphilin-1

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    Knud Larsen

    2015-09-01

    Full Text Available Parkinson's disease (PD, idiopathic and familial, is characterized by degradation of dopaminergic neurons and the presence of Lewy bodies (LB in the substantia nigra. LBs contain aggregated proteins of which α-synuclein is the major component. The protein synphilin-1 interacts and colocalizes with α-synuclein in LBs. The aim of this study was to isolate and characterize porcine synphilin-1 and isoforms hereof with the future perspective to use the pig as a model for Parkinson's disease. The porcine SNCAIP cDNA was cloned by reverse transcriptase PCR. The spatial expression of SNCAIP mRNA was investigated by RNAseq. The presented work reports the molecular cloning and characterization of the porcine (Sus scrofa synphilin-1 cDNA (SNCAIP and three splice variants hereof. The porcine SNCAIP cDNA codes for a protein (synphilin-1 of 919 amino acids which shows a high similarity to human (90% and to mouse (84% synphilin-1. Three shorter transcript variants of the synphilin-1 gene were identified, all lacking one or more exons. SNCAIP transcripts were detected in most examined organs and tissues and the highest expression was found in brain tissues and lung. Conserved splicing variants and a novel splice form of synhilin-1 were found in this study. All synphilin-1 isoforms encoded by the identified transcript variants lack functional domains important for protein degradation.

  1. Isolation and characterization of patatin isoforms

    NARCIS (Netherlands)

    Pots, A.M.; Gruppen, H.; Hessing, M.; Boekel, M.A.J.S. van; Voragen, A.G.J.

    1999-01-01

    Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented.

  2. Silencing GFAP isoforms in astrocytoma cells disturbs laminin-dependent motility and cell adhesion.

    Science.gov (United States)

    Moeton, Martina; Kanski, Regina; Stassen, Oscar M J A; Sluijs, Jacqueline A; Geerts, Dirk; van Tijn, Paula; Wiche, Gerhard; van Strien, Miriam E; Hol, Elly M

    2014-07-01

    Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in astrocytes and neural stem cells. The GFAP gene is alternatively spliced, and expression of GFAP is highly regulated during development, on brain damage, and in neurodegenerative diseases. GFAPα is the canonical splice variant and is expressed in all GFAP-positive cells. In the human brain, the alternatively spliced transcript GFAPδ marks specialized astrocyte populations, such as subpial astrocytes and the neurogenic astrocytes in the human subventricular zone. We here show that shifting the GFAP isoform ratio in favor of GFAPδ in astrocytoma cells, by selectively silencing the canonical isoform GFAPα with short hairpin RNAs, induced a change in integrins, a decrease in plectin, and an increase in expression of the extracellular matrix component laminin. Together, this did not affect cell proliferation but resulted in a significantly decreased motility of astrocytoma cells. In contrast, a down-regulation of all GFAP isoforms led to less cell spreading, increased integrin expression, and a >100-fold difference in the adhesion of astrocytoma cells to laminin. In summary, isoform-specific silencing of GFAP revealed distinct roles of a specialized GFAP network in regulating the interaction of astrocytoma cells with the extracellular matrix through laminin.-Moeton, M., Kanski, R., Stassen, O. M. J. A., Sluijs, J. A., Geerts, D., van Tijn, P., Wiche, G., van Strien, M. E., Hol, E. M. Silencing GFAP isoforms in astrocytoma cells disturbs laminin dependent motility and cell adhesion.

  3. Functional studies of sodium pump isoforms

    DEFF Research Database (Denmark)

    Clausen, Michael Jakob

    unique expression profiles and specialized functional features. We use a Two Electrode Voltage Clamp setup to determine pre-steady-state and steady-state characteristics of each isoform and design chimeras to pin-point the structural elements responsible for observed differences. With this strategy we...... and glial cells express multiple isoforms of the Na+,K+-ATPase that are sorted to different specialized subcellular compartments. We are setting up a novel assay to study the details of Na+,K+-ATPase trafficking in polarized cells. With SNAP and CLIP tagged Na+,K+-ATPase isoforms we can track newly...

  4. FSH isoform pattern in classic galactosemia

    OpenAIRE

    Gubbels, Cynthia S.; Thomas, Chris M.G.; Wodzig, Will K. W. H.; Olthaar, André J.; Jaeken, Jaak; Sweep, Fred C. G. J.; Rubio-Gozalbo, M. Estela

    2010-01-01

    Female classic galactosemia patients suffer from primary ovarian insufficiency (POI). The cause for this long-term complication is not fully understood. One of the proposed mechanisms is that hypoglycosylation of complex molecules, a known secondary phenomenon of galactosemia, leads to FSH dysfunction. An earlier study showed less acidic isoforms of FSH in serum samples of two classic galactosemia patients compared to controls, indicating hypoglycosylation. In this study, FSH isoform patterns...

  5. PKC Isoform Expression in Modeled Microgravity

    Science.gov (United States)

    Risin, Diana; Sundaresan, Alamelu; Pellis, Neal R.; Dawson, David L. (Technical Monitor)

    1999-01-01

    Our previous studies showed that modeled (MMG) and true (USA Space Shuttle Missions STS-54 and STS-56) microgravity (MG) inhibit human lymphocyte locomotion, Modeled MG also suppressed polyclonal and antigen-specific lymphocyte activation. Activation of PKC by phorbol myristate acetate (PMA) restored the microgravity-inhibited lymphocyte locomotion as well as activation by phytohaemagglutinin (PHA), whereas calcium ionophore (ionomycin) was unable to restore these functions. Based on these results we hypothesized that MG-induced changes in lymphocyte functions are caused by a fundamental defect in signal transduction mechanism. This defect may be localized either at the PKC level or upstream of PKC, most likely, at the cell membrane level. In this study we examined the expression of PKC isoforms alpha, epsilon and delta in PBMC cultured in rotating wall vessel bioreactor, developed at NASA JSC, which models microgravity by sustaining cells in continuous free fall. The assessment of the isoforms was performed by FACS analysis following cell permeabilization. A decrease in the expression of isoforms epsilon and delta, but not isoform a, was observed in PBMC cultured in microgravity conditions. These data suggest that MMG might selectively affect the expression of Ca2+ independent isoforms of PKC Molecular analysis confirm selective suppression of Ca2+ independent isoforms of PKC.

  6. Isoform Specificity of Protein Kinase Cs in Synaptic Plasticity

    Science.gov (United States)

    Sossin, Wayne S.

    2007-01-01

    Protein kinase Cs (PKCs) are implicated in many forms of synaptic plasticity. However, the specific isoform(s) of PKC that underlie(s) these events are often not known. We have used "Aplysia" as a model system in order to investigate the isoform specificity of PKC actions due to the presence of fewer isoforms and a large number of documented…

  7. A unified phylogeny-based nomenclature for histone variants

    Directory of Open Access Journals (Sweden)

    Talbert Paul B

    2012-06-01

    Full Text Available Abstract Histone variants are non-allelic protein isoforms that play key roles in diversifying chromatin structure. The known number of such variants has greatly increased in recent years, but the lack of naming conventions for them has led to a variety of naming styles, multiple synonyms and misleading homographs that obscure variant relationships and complicate database searches. We propose here a unified nomenclature for variants of all five classes of histones that uses consistent but flexible naming conventions to produce names that are informative and readily searchable. The nomenclature builds on historical usage and incorporates phylogenetic relationships, which are strong predictors of structure and function. A key feature is the consistent use of punctuation to represent phylogenetic divergence, making explicit the relationships among variant subtypes that have previously been implicit or unclear. We recommend that by default new histone variants be named with organism-specific paralog-number suffixes that lack phylogenetic implication, while letter suffixes be reserved for structurally distinct clades of variants. For clarity and searchability, we encourage the use of descriptors that are separate from the phylogeny-based variant name to indicate developmental and other properties of variants that may be independent of structure.

  8. Antiangiogenic VEGF Isoform in Inflammatory Myopathies

    Directory of Open Access Journals (Sweden)

    Nila Volpi

    2013-01-01

    Full Text Available Objective. To investigate expression of vascular endothelial growth factor (VEGF antiangiogenic isoform A-165b on human muscle in idiopathic inflammatory myopathies (IIM and to compare distribution of angiogenic/antiangiogenic VEGFs, as isoforms shifts are described in other autoimmune disorders. Subjects and Methods. We analyzed VEGF-A165b and VEGF-A by western blot and immunohistochemistry on skeletal muscle biopsies from 21 patients affected with IIM (polymyositis, dermatomyositis, and inclusion body myositis and 6 control muscle samples. TGF-β, a prominent VEGF inductor, was analogously evaluated. Intergroup differences of western blot bands density were statistically examined. Endomysial vascularization, inflammatory score, and muscle regeneration, as pathological parameters of IIM, were quantitatively determined and their levels were confronted with VEGF expression. Results. VEGF-A165b was significantly upregulated in IIM, as well as TGF-β. VEGF-A was diffusely expressed on unaffected myofibers, whereas regenerating/atrophic myofibres strongly reacted for both VEGF-A isoforms. Most inflammatory cells and endomysial vessels expressed both isoforms. VEGF-A165b levels were in positive correlation to inflammatory score, endomysial vascularization, and TGF-β. Conclusions. Our findings indicate skeletal muscle expression of antiangiogenic VEGF-A165b and preferential upregulation in IIM, suggesting that modulation of VEGF-A isoforms may occur in myositides.

  9. A novel MCPH1 isoform complements the defective chromosome condensation of human MCPH1-deficient cells.

    Directory of Open Access Journals (Sweden)

    Ioannis Gavvovidis

    Full Text Available Biallelic mutations in MCPH1 cause primary microcephaly (MCPH with the cellular phenotype of defective chromosome condensation. MCPH1 encodes a multifunctional protein that notably is involved in brain development, regulation of chromosome condensation, and DNA damage response. In the present studies, we detected that MCPH1 encodes several distinct transcripts, including two major forms: full-length MCPH1 (MCPH1-FL and a second transcript lacking the six 3' exons (MCPH1Δe9-14. Both variants show comparable tissue-specific expression patterns, demonstrate nuclear localization that is mediated independently via separate NLS motifs, and are more abundant in certain fetal than adult organs. In addition, the expression of either isoform complements the chromosome condensation defect found in genetically MCPH1-deficient or MCPH1 siRNA-depleted cells, demonstrating a redundancy of both MCPH1 isoforms for the regulation of chromosome condensation. Strikingly however, both transcripts are regulated antagonistically during cell-cycle progression and there are functional differences between the isoforms with regard to the DNA damage response; MCPH1-FL localizes to phosphorylated H2AX repair foci following ionizing irradiation, while MCPH1Δe9-14 was evenly distributed in the nucleus. In summary, our results demonstrate here that MCPH1 encodes different isoforms that are differentially regulated at the transcript level and have different functions at the protein level.

  10. Novel Kidins220/ARMS Splice Isoforms: Potential Specific Regulators of Neuronal and Cardiovascular Development.

    Directory of Open Access Journals (Sweden)

    Nathalie Schmieg

    Full Text Available Kidins220/ARMS is a transmembrane protein playing a crucial role in neuronal and cardiovascular development. Kidins220/ARMS is a downstream target of neurotrophin receptors and interacts with several signalling and trafficking factors. Through computational modelling, we found two potential sites for alternative splicing of Kidins220/ARMS. The first is located between exon 24 and exon 29, while the second site replaces exon 32 by a short alternative terminal exon 33. Here we describe the conserved occurrence of several Kidins220/ARMS splice isoforms at RNA and protein levels. Kidins220/ARMS splice isoforms display spatio-temporal regulation during development with distinct patterns in different neuronal populations. Neurotrophin receptor stimulation in cortical and hippocampal neurons and neuroendocrine cells induces specific Kidins220/ARMS splice isoforms and alters the appearance kinetics of the full-length transcript. Remarkably, alternative terminal exon splicing generates Kidins220/ARMS variants with distinct cellular localisation: Kidins220/ARMS containing exon 32 is targeted to the plasma membrane and neurite tips, whereas Kidins220/ARMS without exon 33 mainly clusters the full-length protein in a perinuclear intracellular compartment in PC12 cells and primary neurons, leading to a change in neurotrophin receptor expression. Overall, this study demonstrates the existence of novel Kidins220/ARMS splice isoforms with unique properties, revealing additional complexity in the functional regulation of neurotrophin receptors, and potentially other signalling pathways involved in neuronal and cardiovascular development.

  11. Ikaros isoforms:The saga continues

    Institute of Scientific and Technical Information of China (English)

    Laura; A; Perez-Casellas; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    Through alternate splicing,the Ikaros gene produces multiple proteins.Ikaros is essential for normal hematopoiesis and possesses tumor suppressor activity.Ikaros isoforms interact to form dimers and potentially multimeric complexes.Diverse Ikaros complexes produced by the presence of different Ikaros isoforms are hypothesized to confer distinct functions.Small dominantnegative Ikaros isoforms have been shown to inhibit the tumor suppressor activity of full-length Ikaros.Here,we describe how Ikaros activity is regulated by the coordinated expression of the largest Ikaros isoforms IK-1 and IK-H.Although IK-1 is described as full-length Ikaros,IK-H is the longest Ikaros isoform.IK-H,which includes residues coded by exon 3B (60 bp that lie between exons 3 and 4),is abundant in human but not murine hematopoietic cells.Specific residues that lie within the 20 amino acids encoded by exon 3B give IK-H DNA-binding characteristics that are distinct from those of IK-1.Moreover,IK-H can potentiate or inhibit the ability of IK-1 to bind DNA.IK-H binds to the regulatory regions of genes that are upregulated by Ikaros,but not genes that are repressed by Ikaros.Although IK-1 localizes to pericentromeric heterochromatin,IK-H can be found in both pericentromeric and non-pericentromeric locations.Anti-silencing activity of gamma satellite DNA has been shown to depend on the binding of IK-H,but not other Ikaros isoforms.The unique features of IK-H,its influence on Ikaros activity,and the lack of IK-H expression in mice suggest that Ikaros function in humans may be more complex and possibly distinct from that in mice.

  12. Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

    Science.gov (United States)

    Carthew, James; Karakesisoglou, Iakowos

    2016-01-01

    Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins. PMID:27147045

  13. Troponin T isoform expression is modulated during Atlantic Halibut metamorphosis

    Directory of Open Access Journals (Sweden)

    Llewellyn Lynda

    2007-06-01

    Full Text Available Abstract Background Flatfish metamorphosis is a thyroid hormone (TH driven process which leads to a dramatic change from a symmetrical larva to an asymmetrical juvenile. The effect of THs on muscle and in particular muscle sarcomer protein genes is largely unexplored in fish. The change in Troponin T (TnT, a pivotal protein in the assembly of skeletal muscles sarcomeres and a modulator of calcium driven muscle contraction, during flatfish metamophosis is studied. Results In the present study five cDNAs for halibut TnT genes were cloned; three were splice variants arising from a single fast TnT (fTnT gene; a fourth encoded a novel teleost specific fTnT-like cDNA (AfTnT expressed exclusively in slow muscle and the fifth encoded the teleost specific sTnT2. THs modified the expression of halibut fTnT isoforms which changed from predominantly basic to acidic isoforms during natural and T4 induced metamorphosis. In contrast, expression of red muscle specific genes, AfTnT and sTnT2, did not change during natural metamorphosis or after T4 treatment. Prior to and after metamorphosis no change in the dorso-ventral symmetry or temporal-spatial expression pattern of TnT genes and muscle fibre organization occurred in halibut musculature. Conclusion Muscle organisation in halibut remains symmetrical even after metamorphosis suggesting TH driven changes are associated with molecular adaptations. We hypothesize that species specific differences in TnT gene expression in teleosts underlies different larval muscle developmental programs which better adapts them to the specific ecological constraints.

  14. A human polymorphism affects NEDD4L subcellular targeting by leading to two isoforms that contain or lack a C2 domain

    Directory of Open Access Journals (Sweden)

    Lalouel Jean-Marc

    2009-04-01

    Full Text Available Abstract Background Ubiquitination serves multiple cellular functions, including proteasomal degradation and the control of stability, function, and intracellular localization of a wide variety of proteins. NEDD4L is a member of the HECT class of E3 ubiquitin ligases. A defining feature of NEDD4L protein isoforms is the presence or absence of an amino-terminal C2 domain, a class of subcellular, calcium-dependent targeting domains. We previously identified a common variant in human NEDD4L that generates isoforms that contain or lack a C2 domain. Results To address the potential functional significance of the NEDD4L common variant on NEDD4L subcellular localization, NEDD4L isoforms that either contained or lacked a C2 domain were tagged with enhanced green fluorescent protein, transfected into Xenopus laevis kidney epithelial cells, and imaged by performing confocal microscopy on live cells. We report that the presence or absence of this C2 domain exerts differential effects on the subcellular distribution of NEDD4L, the ability of C2 containing and lacking NEDD4L isoforms to mobilize in response to a calcium stimulus, and the intracellular transport of subunits of the NEDD4L substrate, ENaC. Furthermore, the ability of the C2-containing isoform to influence β-ENaC mobilization from intracellular pools involves the NEDD4L active site for ubiquitination. We propose a model to account for the potential impact of this common genetic variant on protein function at the cellular level. Conclusion NEDD4L isoforms that contain or lack a C2 domain target different intracellular locations. Additionally, whereas the C2-containing NEDD4L isoform is capable of shuttling between the plasma membrane and intracellular compartments in response to calcium stimulus the C2-lacking isoform can not. The C2-containing isoform differentially affects the mobilization of ENaC subunits from intracellular pools and this trafficking step requires NEDD4L ubiquitin ligase

  15. New isoforms of rat Aquaporin-4

    DEFF Research Database (Denmark)

    Moe, Svein Erik; Sorbo, Jan Gunnar; Søgaard, Rikke;

    2008-01-01

    an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus...

  16. p53 isoforms change p53 paradigm

    OpenAIRE

    Bourdon, JC

    2014-01-01

    Although p53 defines cellular responses to cancer treatment it is not clear how p53 can be used to control cell fate outcome. Data demonstrate that so-called p53 does not exist as a single protein, but is in fact a group of p53 protein isoforms whose expression can be manipulated to control the cellular response to treatment.

  17. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

    Science.gov (United States)

    Pavlidou, Anastasia; Dalamaga, Maria; Kroupis, Christos; Konstantoudakis, George; Belimezi, Maria; Athanasas, George; Dimas, Kleanthi

    2011-01-01

    AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas. METHODS: We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler-Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. RESULTS: Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-ΔΕx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-ΔΕx3, survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin (P < 0.001). The mRNA expression of wild-survivin and survivin-ΔΕx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-ΔEx3/wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. CONCLUSION: Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer. PMID:21472129

  18. Survivin isoforms and clinicopathological characteristics in colorectal adenocarcinomas using real-time qPCR

    Institute of Scientific and Technical Information of China (English)

    Anastasia Pavlidou; Maria Dalamaga; Christos Kroupis; George Konstantoudakis; Maria Belimezi; George Athanasas; Kleanthi Dimas

    2011-01-01

    AIM: To investigate three isoforms of survivin in colorectal adenocarcinomas. METHODS: We used the LightCycler Technology (Roche), along with a common forward primer and reverse primers specific for the splice variants and two common hybridization probes labeled with fluorescein and LightCycler- Red fluorophore (LC-Red 640). Real time quantitative polymerase chain reaction (PCR) was performed on cDNAs from 52 tumor specimens from colorectal cancer patients and 10 unrelated normal colorectal tissues. In the patients group, carcinoembryonic antigen (CEA) and CA19-9 tumor markers were also measured immunochemically. RESULTS: Wild type survivin mRNA isoform was expressed in 48% of the 52 tumor samples, survivin-2b in 38% and survivin-ΔΕx3 in 29%, while no expression was found in normal tissues. The mRNA expression of wild type survivin presented a significant correlation with the expression of the ratio of survivin-2b, survivin-ΔΕx3, survivin-2b/wild type survivin and survivin-ΔΕx3/wild type survivin (P < 0.001). The mRNA expression of wildsurvivin and survivin-ΔΕx3 was related with tumor size and invasion (P = 0.006 and P < 0.005, respectively). A significant difference was found between survivin-2b and morphologic cancer type. Also, the ratio of survivin-ΔEx3/ wild-survivin was significantly associated with prognosis. No association was observed between the three isoforms and grade, metastasis, Dukes stage and gender. The three isoforms were not correlated with CEA and CA19-9. CONCLUSION: Survivin isoforms may play a role in cell apoptosis and their quantification could provide information about clinical management of patients suffering from colorectal cancer.

  19. Stability Mechanisms of Laccase Isoforms using a Modified FoldX Protocol Applicable to Widely Different Proteins

    DEFF Research Database (Denmark)

    Christensen, Niels J.; Kepp, Kasper P.

    2013-01-01

    A recent computational protocol that accurately predicts and rationalizes protein multisite mutant stabilities has been extended to handle widely different isoforms of laccases. We apply the protocol to four isoenzymes of Trametes versicolor laccase (TvL) with variable lengths (498–503 residues) ......, and 245, or near substrate, mainly 265, are identified that contribute to stability-function trade-offs, of relevance to the search for new proficient and stable variants of these important industrial enzymes....

  20. First Trimester Pregnancy Loss and the Expression of alternatively spliced NKp30 isoforms in Maternal Blood and Placental Tissue

    Directory of Open Access Journals (Sweden)

    Avishai eShemesh

    2015-06-01

    Full Text Available In this study, we aimed to investigate whether first trimester pregnancy loss is associated with differences in expression of NKp30 splice variants (isoforms in maternal peripheral blood or placental tissue. We conducted a prospective case-control study; a total of 33 women undergoing dilation and curettage due to first trimester pregnancy loss were further subdivided into groups with sporadic or recurrent pregnancy loss. The control group was comprised of women undergoing elective termination of pregnancy. The qPCR approach was employed to assess the relative expression of NKp30 isoforms as well as the total expression of NKp30 and NKp46 receptors between the selected groups. Results show that in both PBMC and placental tissue, NKp46 and NKp30 expression was mildly elevated in the pregnancy loss groups compared with the elective group. In particular, NKp46 elevation was significant. Moreover, expression analysis of NKp30 isoforms manifested a different profile between PBMC and the placenta. NKp30-a and NKp30-b isoforms in the placental tissue, but not in PBMC, showed a significant increase in the pregnancy loss groups compared with the elective group. Placental expression of NKp30 activating isoforms -a and -b in the pregnancy loss groups was negatively correlated with PLGF expression. In contrast, placental expression of these isoforms in the elective group was positively correlated with TNFα, IL-10 and VEGF-A expression. The altered expression of NKp30 activating isoforms in placental tissue from patients with pregnancy loss compared to the elective group and the different correlations with cytokine expression point to the involvement of NKp30-mediated function in pregnancy loss.

  1. Isoforms of murine and human serum amyloid P component

    DEFF Research Database (Denmark)

    Nybo, Mads; Hackler, R; Kold, B;

    1998-01-01

    Isoelectric focusing (IEF) and immunofixation of murine serum amyloid P component (SAP), purified and in serum, showed a distinct and strain-dependent isoform pattern with up to seven bands (pI 5.1-5.7). Neuraminidase treatment caused a shift of the isoforms to more basic pI values, but did...... of isoforms of human SAP required the presence of urea and higher SAP concentrations. TEF and immunofixation of SAP monomers showed five to eight isoforms, ranging from pI 4.7-5.7. IEF of SAP in human serum resulted in a less distinct pattern and more acidic isoforms. As with murine SAP, neuraminidase...

  2. EASI—enrichment of alternatively spliced isoforms

    OpenAIRE

    Julian P Venables; Burn, John

    2006-01-01

    Alternative splicing produces more than one protein from the majority of genes and the rarer forms can have dominant functions. Instability of alternative transcripts can also hinder the study of regulation of gene expression by alternative splicing. To investigate the true extent of alternative splicing we have developed a simple method of enriching alternatively spliced isoforms (EASI) from PCRs using beads charged with Thermus aquaticus single-stranded DNA-binding protein (T.Aq ssb). This ...

  3. Differential water permeability and regulation of three aquaporin 4 isoforms

    DEFF Research Database (Denmark)

    Fenton, Robert A.; Moeller, Hanne B; Zelenina, Marina;

    2010-01-01

    Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed...... in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 > Mz > M23. None of the three isoforms were permeable to small osmolytes nor were they affected...... by changes in external K(+) concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms...

  4. Alternative Splicing Generates Different Parkin Protein Isoforms: Evidences in Human, Rat, and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Soraya Scuderi

    2014-01-01

    Full Text Available Parkinson protein 2, E3 ubiquitin protein ligase (PARK2 gene mutations are the most frequent causes of autosomal recessive early onset Parkinson’s disease and juvenile Parkinson disease. Parkin deficiency has also been linked to other human pathologies, for example, sporadic Parkinson disease, Alzheimer disease, autism, and cancer. PARK2 primary transcript undergoes an extensive alternative splicing, which enhances transcriptomic diversification. To date several PARK2 splice variants have been identified; however, the expression and distribution of parkin isoforms have not been deeply investigated yet. Here, the currently known PARK2 gene transcripts and relative predicted encoded proteins in human, rat, and mouse are reviewed. By analyzing the literature, we highlight the existing data showing the presence of multiple parkin isoforms in the brain. Their expression emerges from conflicting results regarding the electrophoretic mobility of the protein, but it is also assumed from discrepant observations on the cellular and tissue distribution of parkin. Although the characterization of each predicted isoforms is complex, since they often diverge only for few amino acids, analysis of their expression patterns in the brain might account for the different pathogenetic effects linked to PARK2 gene mutations.

  5. Developmentally regulated switch in alternatively spliced SNAP-25 isoforms alters facilitation of synaptic transmission.

    Science.gov (United States)

    Bark, Christina; Bellinger, Frederick P; Kaushal, Ashutosh; Mathews, James R; Partridge, L Donald; Wilson, Michael C

    2004-10-01

    Although the basic molecular components that promote regulated neurotransmitter release are well established, the contribution of these proteins as regulators of the plasticity of neurotransmission and refinement of synaptic connectivity during development is elaborated less fully. For example, during the period of synaptic growth and maturation in brain, the expression of synaptosomal protein 25 kDa (SNAP-25), a neuronal t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) essential for action potential-dependent neuroexocytosis, is altered through alternative splicing of pre-mRNA transcripts. We addressed the role of the two splice-variant isoforms of SNAP-25 with a targeted mouse mutation that impairs the shift from SNAP-25a to SNAP-25b. Most of these mutant mice die between 3 and 5 weeks of age, which coincides with the time when SNAP-25b expression normally reaches mature levels in brain and synapse formation is essentially completed. The altered expression of these SNAP-25 isoforms influences short-term synaptic function by affecting facilitation but not the initial probability of release. This suggests that mechanisms controlling alternative splicing between SNAP-25 isoforms contribute to a molecular switch important for survival that helps to guide the transition from immature to mature synaptic connections, as well as synapse regrowth and remodeling after neural injury.

  6. Splice isoforms of the polyglutamine disease protein ataxin-3 exhibit similar enzymatic yet different aggregation properties.

    Directory of Open Access Journals (Sweden)

    Ginny Marie Harris

    Full Text Available Protein context clearly influences neurotoxicity in polyglutamine diseases, but the contribution of alternative splicing to this phenomenon has rarely been investigated. Ataxin-3, a deubiquitinating enzyme and the disease protein in SCA3, is alternatively spliced to encode either a C-terminal hydrophobic stretch or a third ubiquitin interacting motif (termed 2UIM and 3UIM isoforms, respectively. In light of emerging insights into ataxin-3 function, we examined the significance of this splice variation. We confirmed neural expression of several minor 5' variants and both of the known 3' ataxin-3 splice variants. Regardless of polyglutamine expansion, 3UIM ataxin-3 is the predominant isoform in brain. Although 2UIM and 3UIM ataxin-3 display similar in vitro deubiquitinating activity, 2UIM ataxin-3 is more prone to aggregate and more rapidly degraded by the proteasome. Our data demonstrate how alternative splicing of sequences distinct from the trinucleotide repeat can alter properties of the encoded polyglutamine disease protein and thereby perhaps contribute to selective neurotoxicity.

  7. High-throughput proteomics detection of novel splice isoforms in human platelets.

    LENUS (Irish Health Repository)

    Power, Karen A

    2009-01-01

    Alternative splicing (AS) is an intrinsic regulatory mechanism of all metazoans. Recent findings suggest that 100% of multiexonic human genes give rise to splice isoforms. AS can be specific to tissue type, environment or developmentally regulated. Splice variants have also been implicated in various diseases including cancer. Detection of these variants will enhance our understanding of the complexity of the human genome and provide disease-specific and prognostic biomarkers. We adopted a proteomics approach to identify exon skip events - the most common form of AS. We constructed a database harboring the peptide sequences derived from all hypothetical exon skip junctions in the human genome. Searching tandem mass spectrometry (MS\\/MS) data against the database allows the detection of exon skip events, directly at the protein level. Here we describe the application of this approach to human platelets, including the mRNA-based verification of novel splice isoforms of ITGA2, NPEPPS and FH. This methodology is applicable to all new or existing MS\\/MS datasets.

  8. Hemoglobin variants in Cyprus.

    Science.gov (United States)

    Kyrri, Andreani R; Felekis, Xenia; Kalogerou, Eleni; Wild, Barbara J; Kythreotis, Loukas; Phylactides, Marios; Kleanthous, Marina

    2009-01-01

    Cyprus, located at the eastern end of the Mediterranean region, has been a place of eastern and western civilizations, and the presence of various hemoglobin (Hb) variants can be considered a testimony to past colonizations of the island. In this study, we report the structural Hb variants identified in the Cypriot population (Greek Cypriots, Maronites, Armenians, and Latinos) during the thalassemia screening of 248,000 subjects carried out at the Thalassaemia Centre, Nicosia, Cyprus, over a period of 26 years. A sample population of 65,668 people was used to determine the frequency and localization of several of the variants identified in Cyprus. The localization of some of the variants in regions where the presence of foreign people was most prevalent provides important clues to the origin of the variants. Twelve structural variants have been identified by DNA sequencing, nine concerning the beta-globin gene and three concerning the alpha-globin gene. The most common beta-globin variants identified were Hb S (0.2%), Hb D-Punjab (0.02%), and Hb Lepore-Washington-Boston (Hb Lepore-WB) (0.03%); the most common alpha-globin variant was Hb Setif (0.1%). The presence of some of these variants is likely to be directly linked to the history of Cyprus, as archeological monuments have been found throughout the island which signify the presence for many years of the Greeks, Syrians, Persians, Arabs, Byzantines, Franks, Venetians, and Turks. PMID:19373583

  9. Molecular Analysis of Collagen XVIII Reveals Novel Mutations, Presence of a Third Isoform, and Possible Genetic Heterogeneity in Knobloch Syndrome

    Science.gov (United States)

    Suzuki, O. T.; Sertié, A. L.; Der Kaloustian, V. M.; Kok, F.; Carpenter, M.; Murray, J.; Czeizel, A. E.; Kliemann, S. E.; Rosemberg, S.; Monteiro, M.; Olsen, B. R.; Passos-Bueno, M. R.

    2002-01-01

    Knobloch syndrome (KS) is a rare disease characterized by severe ocular alterations, including vitreoretinal degeneration associated with retinal detachment and occipital scalp defect. The responsible gene, COL18A1, has been mapped to 21q22.3, and, on the basis of the analysis of one family, we have demonstrated that a mutation affecting only one of the three COL18A1 isoforms causes this phenotype. We report here the results of the screening of both the entire coding region and the exon-intron boundaries of the COL18A1 gene (which includes 43 exons), in eight unrelated patients with KS. Besides 20 polymorphic changes, we identified 6 different pathogenic changes in both alleles of five unrelated patients with KS (three compound heterozygotes and two homozygotes). All are truncating mutations leading to deficiency of one or all collagen XVIII isoforms and endostatin. We have verified that, in exon 41, the deletion c3514-3515delCT, found in three unrelated alleles, is embedded in different haplotypes, suggesting that this mutation has occurred more than once. In addition, our results provide evidence of nonallelic genetic heterogeneity in KS. We also show that the longest human isoform (NC11-728) is expressed in several tissues (including the human eye) and that lack of either the short variant or all of the collagen XVIII isoforms causes similar phenotypes but that those patients who lack all forms present more-severe ocular alterations. Despite the small sample size, we found low endostatin plasma levels in those patients with mutations leading to deficiency of all isoforms; in addition, it seems that absence of all collagen XVIII isoforms causes predisposition to epilepsy. PMID:12415512

  10. A rare sequence variant in intron 1 of THAP1 is associated with primary dystonia.

    Science.gov (United States)

    Vemula, Satya R; Xiao, Jianfeng; Zhao, Yu; Bastian, Robert W; Perlmutter, Joel S; Racette, Brad A; Paniello, Randal C; Wszolek, Zbigniew K; Uitti, Ryan J; Van Gerpen, Jay A; Hedera, Peter; Truong, Daniel D; Blitzer, Andrew; Rudzińska, Monika; Momčilović, Dragana; Jinnah, Hyder A; Frei, Karen; Pfeiffer, Ronald F; LeDoux, Mark S

    2014-05-01

    Although coding variants in THAP1 have been causally associated with primary dystonia, the contribution of noncoding variants remains uncertain. Herein, we examine a previously identified Intron 1 variant (c.71+9C>A, rs200209986). Among 1672 subjects with mainly adult-onset primary dystonia, 12 harbored the variant in contrast to 1/1574 controls (P A variant was associated with an elevated ratio of Isoform 1 (NM_018105) to Isoform 2 (NM_199003) in leukocytes. In silico and minigene analyses indicated that c.71+9C>A alters THAP1 splicing. Lymphoblastoid cells harboring the c.71+9C>A variant showed extensive apoptosis with relatively fewer cells in the G2 phase of the cell cycle. Differentially expressed genes from lymphoblastoid cells revealed that the c.71+9C>A variant exerts effects on DNA synthesis, cell growth and proliferation, cell survival, and cytotoxicity. In aggregate, these data indicate that THAP1 c.71+9C>A is a risk factor for adult-onset primary dystonia. PMID:24936516

  11. Tumorigenic properties of alternative osteopontin isoforms in mesothelioma

    Energy Technology Data Exchange (ETDEWEB)

    Ivanov, Sergey V., E-mail: Sergey.Ivanov@med.nyu.edu [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States); Ivanova, Alla V.; Goparaju, Chandra M.V.; Chen, Yuanbin; Beck, Amanda; Pass, Harvey I. [Thoracic Surgery Laboratory, Cardiothoracic Surgery Department, NYU Langone Medical Center, 462 First Ave., Bellevue Hospital, Room 15N20, NY 10016 (United States)

    2009-05-08

    Osteopontin (SPP1) is an inflammatory cytokine that we previously characterized as a diagnostic marker in patients with asbestos-induced malignant mesothelioma (MM). While SPP1 shows both pro- and anti-tumorigenic biological effects, little is known about the molecular basis of these activities. In this study, we demonstrate that while healthy pleura possesses all three differentially spliced SPP1 isoforms (A-C), in clinical MM specimens isoform A is markedly up-regulated and predominant. To provide a clue to possible functions of the SPP1 isoforms we next performed their functional evaluation via transient expression in MM cell lines. As a result, we report that isoforms A-C demonstrate different activities in cell proliferation, wound closure, and invasion assays. These findings suggest different functions for SPP1 isoforms and underline pro-tumorigenic properties of isoforms A and B.

  12. Developmental expression of two Haliotis asinina hemocyanin isoforms.

    Science.gov (United States)

    Streit, Klaus; Jackson, Daniel; Degnan, Bernard M; Lieb, Bernhard

    2005-09-01

    Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

  13. Androgen receptor isoforms in human and rat prostate

    Institute of Scientific and Technical Information of China (English)

    Shu-JieXIA; Gang-YaoHAO; Xiao-DaTANG

    2000-01-01

    Aim: To investigate the androgen receptor (AR) isoforms and its variability of expression in human and rat prostatic tissues. Methods: Human benign prostatic hyperplasia (BPH) and prostatic cancer tissues were obtained from patients undergoing prostatectomy, and rat ventral prostate was incised 3 days after castration. Forty-one AR-positive BPH specimens, 3 prostatic cancer specimens, and 6 rat prostates were used. After processing at 4℃, the tissues were examined by means of high resolution isoelectric focusing (IEF) technique to determine their AR isoforms. Results:From the prostatic specimens, 3 types of AR isoforms were detected with pI values at 6.5, 6.0, and 5.3. In human BPH tissues, 15/41 (36.6%) specimens showed all the three types of isoforms, while 19/41 (46.3%) showed 2 isoforms at various combinations and 7/41(17.1%), 1 isoform. For the 3 prostatic cancer specimens, one showed 3 isoforms, one, 2 isoforms, and the other failed to show any isoform. All rat prostatic tissues showed 2 isoforms at different combinations. Binding of 3H-dihydrotestosterone (DHT) to the isoforms was inhibited by the addition of 100-fold excess of DHT or testosterone, but not progesterone, oestradiol or diethylstilboestrol. Conclusion: AR isoforms are different in different patients. Although their genesis is not clear, the therapeutic implication of the present observation appears to be interesting, that may help clarifying the individual differences in the response to hormonal therapy.(Asian J Androl 2000 Dec;2:307-310)

  14. Two new isoforms of the human hepatoma-derived growth factor interact with components of the cytoskeleton.

    Science.gov (United States)

    Nüße, Jessica; Mirastschijski, Ursula; Waespy, Mario; Oetjen, Janina; Brandes, Nadine; Rebello, Osmond; Paroni, Federico; Kelm, Sørge; Dietz, Frank

    2016-05-01

    Hepatoma-derived growth factor (HDGF) is involved in diverse, apparently unrelated processes, such as cell proliferation, apoptosis, DNA-repair, transcriptional control, ribosome biogenesis and cell migration. Most of the interactions of HDGF with diverse molecules has been assigned to the hath region of HDGF. In this study we describe two previously unknown HDGF isoforms, HDGF-B and HDGF-C, generated via alternative splicing with structurally unrelated N-terminal regions of their hath region, which is clearly different from the well described isoform, HDGF-A. In silico modeling revealed striking differences near the PHWP motif, an essential part of the binding site for glycosaminoglycans and DNA/RNA. This observation prompted the hypothesis that these isoforms would have distinct interaction patterns with correspondingly diverse roles on cellular processes. Indeed, we discovered specific associations of HDGF-B and HDGF-C with cytoskeleton elements, such as tubulin and dynein, suggesting previously unknown functions of HDGF in retrograde transport, site directed localization and/or cytoskeleton organization. In contrast, the main isoform HDGF-A does not interact directly with the cytoskeleton, but via RNA with messenger ribonucleoprotein (mRNP) complexes. In summary, the discovery of HDGF splice variants with their discrete binding activities and subcellular distributions opened new avenues for understanding its biological function and importance. PMID:26845719

  15. Naturally Occurring Variants of Human A9 Nicotinic Receptor Differentially Affect Bronchial Cell Proliferation and Transformation

    OpenAIRE

    Chikova, Anna; Grando, Sergei A.

    2011-01-01

    Isolation of polyadenilated mRNA from human immortalized bronchial epithelial cell line BEP2D revealed the presence of multiple isoforms of RNA coded by the CHRNA9 gene for α9 nicotinic acetylcholine receptor (nAChR). BEP2D cells were homozygous for the rs10009228 polymorphism encoding for N442S amino acid substitution, and also contained mRNA coding for several truncated isoforms of α9 protein. To elucidate the biologic significance of the naturally occurring variants of α9 nAChR, we compare...

  16. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer

    Energy Technology Data Exchange (ETDEWEB)

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Sun, Shiqin [College of Pharmacy, Harbin Medical University-Daqing, Daqing, Heilongjiang 163319 (China); Chen, Xiangmei, E-mail: xm_chen6176@bjmu.edu.cn [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China); Lu, Fengmin [Department of Microbiology and Infectious Disease Center, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191 (China)

    2014-04-25

    Highlights: • The expression of Fbx4 was significantly lower in HCC tissues. • Novel splicing variants of Fbx4 were identified. • These novel variants are much more abundant in human cancer tissues and cells. • The novel Fbx4 isoforms could promote cell proliferation and migration in vitro. • These isoforms showed less capability for cyclin D1 binding and degradation. - Abstract: Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168–245nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant

  17. Mucopolysaccharidosis: A New Variant?

    Science.gov (United States)

    Primrose, D. A.

    1972-01-01

    Described is a possibly new variant of mucopolysaccharidosis characterized by progressive mental and motor deficiency, bone abnormalities, a generalized skin lesion, and abnormal mucopolysaccharides in the urine as seen in a 20-year-old female. (DB)

  18. Evidence for leptin receptor isoforms heteromerization at the cell surface.

    Science.gov (United States)

    Bacart, Johan; Leloire, Audrey; Levoye, Angélique; Froguel, Philippe; Jockers, Ralf; Couturier, Cyril

    2010-06-01

    Leptin mediates its metabolic effects through several leptin receptor (LEP-R) isoforms. In humans, long (LEPRb) and short (LEPRa,c,d) isoforms are generated by alternative splicing. Most of leptin's effects are believed to be mediated by the OB-Rb isoform. However, the role of short LEPR isoforms and the possible existence of heteromers between different isoforms are poorly understood. Using BRET1 and optimized co-immunoprecipitation, we observed LEPRa/b and LEPRb/c heteromers located at the plasma membrane and stabilized by leptin. Given the widespread coexpression of LEPRa and LEPRb, our results suggest that LEPRa/b heteromers may represent a major receptor species in most tissues.

  19. Transmembrane and truncated (SEC isoforms of MUC1 in the human endometrium and Fallopian tube

    Directory of Open Access Journals (Sweden)

    Wreschner Daniel H

    2003-01-01

    Full Text Available Abstract The cell surface mucin MUC1 is expressed by endometrial epithelial cells with increased abundance in the secretory phase of the menstrual cycle, when it is found both at the apical cell surface and in secretions. This suggests the presence of a maternal cell surface glycoprotein barrier to embryo implantation, arising from the anti-adhesive property of MUC1. In previous work, we demonstrated alternatively spliced MUC1 variant forms in tumour cells. The variant MUC1/SEC lacks the transmembrane and cytoplasmic sequences found in the full-length variant. We now show that MUC1/SEC mRNA is present in endometrial carcinoma cell lines, endometrial tissue and primary cultured endometrial epithelial cells. The protein can be detected using isoform-specific antibodies in uterine flushings, suggesting release from endometrium in vivo. However, on the basis of immunolocalisation studies, MUC1/SEC also remains associated with the apical epithelial surface both in tissue and in cultured cells. Transmembrane MUC1 and MUC1/SEC are both strikingly localised to the apical surface of tubal epithelium. Thus MUC1 may contribute to the anti-adhesive character of the tubal surface, inhibiting ectopic implantation. The mechanism by which this barrier is overcome in endometrium at implantation is the subject of ongoing investigation.

  20. Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.

    Science.gov (United States)

    Harauz, George; Boggs, Joan M

    2013-05-01

    The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5-kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two-dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3-domains, participation in Fyn-mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post-translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5-kDa MBP's protein-membrane and protein-protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5-kDa MBP has significant roles beyond membrane adhesion. The full-length, early-developmental 21.5-kDa splice isoform is predominantly karyophilic due to a non-traditional P-Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.

  1. A novel alternatively spliced isoform of the mu-opioid receptor: functional antagonism

    Directory of Open Access Journals (Sweden)

    Wentworth Sean

    2010-06-01

    Full Text Available Abstract Background Opioids are the most widely used analgesics for the treatment of clinical pain. They produce their therapeutic effects by binding to μ-opioid receptors (MORs, which are 7 transmembrane domain (7TM G-protein-coupled receptors (GPCRs, and inhibiting cellular activity. However, the analgesic efficacy of opioids is compromised by side-effects such as analgesic tolerance, dependence and opioid-induced hyperalgesia (OIH. In contrast to opioid analgesia these side effects are associated with cellular excitation. Several hypotheses have been advanced to explain these phenomena, yet the molecular mechanisms underlying tolerance and OIH remain poorly understood. Results We recently discovered a new human alternatively spliced isoform of MOR (MOR1K that is missing the N-terminal extracellular and first transmembrane domains, resulting in a 6TM GPCR variant. To characterize the pattern of cellular transduction pathways activated by this human MOR1K isoform, we conducted a series of pharmacological and molecular experiments. Results show that stimulation of MOR1K with morphine leads to excitatory cellular effects. In contrast to stimulation of MOR1, stimulation of MOR1K leads to increased Ca2+ levels as well as increased nitric oxide (NO release. Immunoprecipitation experiments further reveal that unlike MOR1, which couples to the inhibitory Gαi/o complex, MOR1K couples to the stimulatory Gαs complex. Conclusion The major MOR1 and the alternative MOR1K isoforms mediate opposite cellular effects in response to morphine, with MOR1K driving excitatory processes. These findings warrant further investigations that examine animal and human MORK1 expression and function following chronic exposure to opioids, which may identify MOR1K as a novel target for the development of new clinically effective classes of opioids that have high analgesic efficacy with diminished ability to produce tolerance, OIH, and other unwanted side-effects.

  2. Functional refolding and characterization of two Tom40 isoforms from human mitochondria.

    Science.gov (United States)

    Mager, Frauke; Gessmann, Dennis; Nussberger, Stephan; Zeth, Kornelius

    2011-07-01

    Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.

  3. HER4 and its cytoplasmic isoforms are associated with progression-free survival of malignant melanoma

    DEFF Research Database (Denmark)

    Nielsen, Trine O; Poulsen, Steen Seier; Journe, Fabrice;

    2014-01-01

    RNA quantification was carried out of all four EGF receptors (EGFR, HER2, HER3, and HER4) and the HER4 cytoplasmic isoforms in lymph node metastases from patients with malignant melanoma. We related their expression to progression of the disease. HER4 expression was found to be an indicator of short progression......-free survival (P=0.0340). Interestingly, of the two cytoplasmic splice variants of HER4, the association of CYT1 (P=0.0176) with progression-free survival was more pronounced than that for CYT2 (P=0.0458). Also, HER3 was associated with progression-free survival (P=0.0169), whereas no association was found for...

  4. Existence of multiple isoforms of HS1-associated protein X-1 in murine and human tissues.

    Science.gov (United States)

    Lees, Delphine M; Hart, Ian R; Marshall, John F

    2008-06-13

    To date, the literature concerning the HS1 (haematopoietic cell-specific protein 1)-associated protein X-1 (HAX1) protein has reported considerable variation regarding its function in mammalian cells, subcellular localisation and binding partners. We show here that HAX1 comprises a family of proteins. Murine tissues express three mRNA variants, encoded by two genes on chromosomes 2 and 3. The chromosome 2 gene is intronless and would encode a protein 100% identical with that encoded by chromosome 3. In humans, alternative splice variants, encoded by the chromosome 1 gene, produce a family of transcripts composed of up to eight members. Based on the sequences published in GenBank and Ensembl, we designed specific primers and detected by PCR three mRNA species in murine tissues and eight variants in human cells. We screened a panel of 19 human cell lines as well as primary fibroblasts, oral keratinocytes and freshly isolated peripheral blood mononuclear cells. All human cells studied expressed at least six of the possible HAX1 mRNA variants. In silico analysis of the variants revealed an open reading frame in all of them, suggesting that murine and human tissues can express two and eight HAX1 proteins, respectively. Analysis of human protein lysates by Western blotting with the use of a monoclonal anti-HAX1 antibody revealed multiple bands. These bands were decreased after treatment of cells with a single small interfering RNA duplex targeting a region common to six of the variants, confirming their identity as HAX1 proteins. Comparison of the human variants with the six HAX1 homologues described to date in the chimpanzee (Pan troglodytes) and the four homologues described in macaque (Macaca mulatta) revealed very high conservation with only one amino acid substitution between human and chimpanzee homologues. Moreover, a number of additional products were amplified and sequenced, which indicated that further human isoforms are likely to exist. These findings are

  5. Alternative splicing variants of human Fbx4 disturb cyclin D1 proteolysis in human cancer.

    Science.gov (United States)

    Chu, Xiufeng; Zhang, Ting; Wang, Jie; Li, Meng; Zhang, Xiaolei; Tu, Jing; Sun, Shiqin; Chen, Xiangmei; Lu, Fengmin

    2014-04-25

    Fbx4 is a specific substrate recognition component of SCF ubiquitin ligases that catalyzes the ubiquitination and subsequent degradation of cyclin D1 and Trx1. Two isoforms of human Fbx4 protein, the full length Fbx4α and the C-terminal truncated Fbx4β have been identified, but their functions remain elusive. In this study, we demonstrated that the mRNA level of Fbx4 was significantly lower in hepatocellular carcinoma tissues than that in the corresponding non-tumor tissues. More importantly, we identified three novel splicing variants of Fbx4: Fbx4γ (missing 168-245 nt of exon1), Fbx4δ (missing exon6) and a N-terminal reading frame shift variant (missing exon2). Using cloning sequencing and RT-PCR, we demonstrated these novel splice variants are much more abundant in human cancer tissues and cell lines than that in normal tissues. When expressed in Sk-Hep1 and NIH3T3 cell lines, Fbx4β, Fbx4γ and Fbx4δ could promote cell proliferation and migration in vitro. Concordantly, these isoforms could disrupt cyclin D1 degradation and therefore increase cyclin D1 expression. Moreover, unlike the full-length isoform Fbx4α that mainly exists in cytoplasm, Fbx4β, Fbx4γ, and Fbx4δ locate in both cytoplasm and nucleus. Since cyclin D1 degradation takes place in cytoplasm, the nuclear distribution of these Fbx4 isoforms may not be involved in the down-regulation of cytoplasmic cyclin D1. These results define the impact of alternative splicing on Fbx4 function, and suggest that the attenuated cyclin D1 degradation by these novel Fbx4 isoforms provides a new insight for aberrant cyclin D1 expression in human cancers. PMID:24704453

  6. Molecular basis for Duarte and Los Angeles variant galactosemia

    Energy Technology Data Exchange (ETDEWEB)

    Langley, S.D.; Lai, K.; Dembure, P.P. [Emory Univ. School of Medicine, Atlanta, GA (United States)] [and others

    1997-02-01

    Human erythrocytes that are homozygous for the Duarte enzyme variant of galactosemia (D/D) have a characteristic isoform on isoelectric focusing and 50% reduction in galactose-1-phosphate uridyltransferase (GALT) enzyme activity. The Duarte biochemical phenotype has a molecular genotype of N314D/N314D. The characteristic Duarte isoform is also associated with a variant called the {open_quotes}Los Angeles (LA) phenotype,{close_quotes} which has increased GALT enzyme activity. We evaluated GALT enzyme activity and screened the GALT genes of 145 patients with one or more N314D-containing alleles. We found seven with the LA biochemical phenotype, and all had a 1721C{r_arrow}T transition in exon 7 in cis with the N314D missense mutation. The 1721C{r_arrow}T transition is a neutral polymorphism for leucine at amino acid 218 (L218L). In pedigree analyses, this 1721C{r_arrow}T transition segregated with the LA phenotype of increased GALT activity in three different biochemical phenotypes (LA/N, LA/G, and LA/D). To determine the mechanism for increased activity of the LA variant, we compared GALT mRNA, protein abundance, and enzyme thermal stability in lymphoblast cell lines of D and LA phenotypes with comparable genotypes. GALT protein abundance was increased in LA compared to D alleles, but mRNA was similar among all genotypes. We conclude that the codon change N314D in cis with the base-pair transition 1721C{r_arrow}T produces the LA variant of galactosemia and that this nucleotide change increases GALT activity by increasing GALT protein abundance without increasing transcription or decreasing thermal lability. A favorable codon bias for the mutated codon with consequently increased translation rates is postulated as the mechanism. 23 refs., 3 figs., 4 tabs.

  7. Hemoglobin Variants in Mice

    Energy Technology Data Exchange (ETDEWEB)

    Popp, Raymond A.

    1965-04-22

    Variability among mammalian hemoglobins was observed many years ago (35). The chemical basis for differences among hemoglobins from different species of mammals has been studied by several investigators (5, 11, 18, 48). As well as interspecies differences, hemoglobin variants are frequently found within a species of mammals (2, 3, 7, 16) The inheritance of these intraspecies variants can be studied, and pedigrees indicate that the type of hemoglobin synthesized in an individual is genetically controlled (20). Several of the variant human hemoglobins are f'unctionally deficient (7, 16). Such hemoglobin anomalies are of basic interest to man because of the vital role of hemoglobin for transporting oxygen to all tissues of the body.

  8. p53 Family: Role of Protein Isoforms in Human Cancer

    Directory of Open Access Journals (Sweden)

    Jinxiong Wei

    2012-01-01

    Full Text Available TP53, TP63, and TP73 genes comprise the p53 family. Each gene produces protein isoforms through multiple mechanisms including extensive alternative mRNA splicing. Accumulating evidence shows that these isoforms play a critical role in the regulation of many biological processes in normal cells. Their abnormal expression contributes to tumorigenesis and has a profound effect on tumor response to curative therapy. This paper is an overview of isoform diversity in the p53 family and its role in cancer.

  9. A reliable method for quantification of splice variants using RT-qPCR

    OpenAIRE

    Camacho Londoño, Julia; Philipp, Stephan E.

    2016-01-01

    Background The majority of protein isoforms arise from alternative splicing of the encoding primary RNA transcripts. To understand the significance of single splicing events, reliable techniques are needed to determine their incidence. However, existing methods are labour-intensive, error-prone or of limited use. Results Here, we present an improved method to determine the relative incidence of transcripts that arise from alternative splicing at a single site. Splice variants were quantified ...

  10. Structural and Functional Characterization of Two Alternative Splicing Variants of Mouse Endothelial Cell-Specific Chemotaxis Regulator (ECSCR

    Directory of Open Access Journals (Sweden)

    Yongchang Chang

    2012-04-01

    Full Text Available Endothelial cells (ECs that line the lumen of blood vessels are important players in blood vessel formation, and EC migration is a key component of the angiogenic process. Thus, identification of genes that are specifically or preferentially expressed in vascular ECs and in-depth understanding of their biological functions may lead to discovery of new therapeutic targets. We have previously reported molecular characterization of human endothelial cell-specific molecule 2 (ECSM2/endothelial cell-specific chemotaxis regulator (ECSCR. In the present study, we cloned two mouse full-length cDNAs by RT-PCR, which encode two putative ECSCR isoform precursors with considerable homology to the human ECSCR. Nucleotide sequence and exon-intron junction analyses suggested that they are alternative splicing variants (ECSCR isoform-1 and -2, differing from each other in the first and second exons. Quantitative RT-PCR results revealed that isoform-2 is the predominant form, which was most abundant in heart, lung, and muscles, and moderately abundant in uterus and testis. In contrast, the expression of isoform-1 seemed to be more enriched in testis. To further explore their potential cellular functions, we expressed GFP- and FLAG-tagged ECSCR isoforms, respectively, in an ECSCR deficient cell line (HEK293. Interestingly, the actual sizes of either ECSCR-GFP or -FLAG fusion proteins detected by immunoblotting are much larger than their predicted sizes, suggesting that both isoforms are glycoproteins. Fluorescence microscopy revealed that both ECSCR isoforms are localized at the cell surface, which is consistent with the structural prediction. Finally, we performed cell migration assays using mouse endothelial MS1 cells overexpressing GFP alone, isoform-1-GFP, and isoform-2-GFP, respectively. Our results showed that both isoforms significantly inhibited vascular epidermal growth factor (VEGF-induced cell migration. Taken together, we have provided several lines

  11. Structural diversity and evolution of the N-terminal isoform-specific region of ecdysone receptor-A and -B1 isoforms in insects

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    Kubo Takeo

    2010-02-01

    Full Text Available Abstract Background The ecdysone receptor (EcR regulates various cellular responses to ecdysteroids during insect development. Insects have multiple EcR isoforms with different N-terminal A/B domains that contain the isoform-specific activation function (AF-1 region. Although distinct physiologic functions of the EcR isoforms have been characterized in higher holometabolous insects, they remain unclear in basal direct-developing insects, in which only A isoform has been identified. To examine the structural basis of the EcR isoform-specific AF-1 regions, we performed a comprehensive structural comparison of the isoform-specific region of the EcR-A and -B1 isoforms in insects. Results The EcR isoforms were newly identified in 51 species of insects and non-insect arthropods, including direct-developing ametabolous and hemimetabolous insects. The comprehensive structural comparison revealed that the isoform-specific region of each EcR isoform contained evolutionally conserved microdomain structures and insect subgroup-specific structural modifications. The A isoform-specific region generally contained four conserved microdomains, including the SUMOylation motif and the nuclear localization signal, whereas the B1 isoform-specific region contained three conserved microdomains, including an acidic activator domain-like motif. In addition, the EcR-B1 isoform of holometabolous insects had a novel microdomain at the N-terminal end. Conclusions Given that the nuclear receptor AF-1 is involved in cofactor recruitment and transcriptional regulation, the microdomain structures identified in the isoform-specific A/B domains might function as signature motifs and/or as targets for cofactor proteins that play essential roles in the EcR isoform-specific AF-1 regions. Moreover, the novel microdomain in the isoform-specific region of the holometabolous insect EcR-B1 isoform suggests that the holometabolous insect EcR-B1 acquired additional transcriptional

  12. Allele-Selective Transcriptome Recruitment to Polysomes Primed for Translation: Protein-Coding and Noncoding RNAs, and RNA Isoforms.

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    Roshan Mascarenhas

    Full Text Available mRNA translation into proteins is highly regulated, but the role of mRNA isoforms, noncoding RNAs (ncRNAs, and genetic variants remains poorly understood. mRNA levels on polysomes have been shown to correlate well with expressed protein levels, pointing to polysomal loading as a critical factor. To study regulation and genetic factors of protein translation we measured levels and allelic ratios of mRNAs and ncRNAs (including microRNAs in lymphoblast cell lines (LCL and in polysomal fractions. We first used targeted assays to measure polysomal loading of mRNA alleles, confirming reported genetic effects on translation of OPRM1 and NAT1, and detecting no effect of rs1045642 (3435C>T in ABCB1 (MDR1 on polysomal loading while supporting previous results showing increased mRNA turnover of the 3435T allele. Use of high-throughput sequencing of complete transcript profiles (RNA-Seq in three LCLs revealed significant differences in polysomal loading of individual RNA classes and isoforms. Correlated polysomal distribution between protein-coding and non-coding RNAs suggests interactions between them. Allele-selective polysome recruitment revealed strong genetic influence for multiple RNAs, attributable either to differential expression of RNA isoforms or to differential loading onto polysomes, the latter defining a direct genetic effect on translation. Genes identified by different allelic RNA ratios between cytosol and polysomes were enriched with published expression quantitative trait loci (eQTLs affecting RNA functions, and associations with clinical phenotypes. Polysomal RNA-Seq combined with allelic ratio analysis provides a powerful approach to study polysomal RNA recruitment and regulatory variants affecting protein translation.

  13. Expression of Ik6 and Ik8 Isoforms and Their Association with Relapse and Death in Mexican Children with Acute Lymphoblastic Leukemia.

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    Adriana Reyes-León

    Full Text Available Expression of the 6 and 8 dominant-negative Ikaros isoforms in pediatric patients with acute lymphoblastic leukemia has been associated with a high risk of relapse and death; due to these isoforms disrupting the differentiation and proliferation of lymphoid cells. The aim of this study was to know the frequency of Ik6 and Ik8 in 113 Mexican ALL-children treated within the National Popular Medical Insurance Program to determine whether there was an association with relapse-free survival, event-free survival and overall survival, and to assess its usefulness in the initial stratification of patients. The expression of these isoforms was analyzed using specific primer sets and nested RT-PCR. The detected transcripts were classified according to the isoforms's sizes reported. A non-expected band of 300 bp from one patient was analyzed by sequencing. Twenty-six patients expressed Ik6 and/or Ik8 and one of them expressed a variant of Ik8 denominated Ik8-deleted. Although the presence of them was not statistically associated with lower relapse free survival (p = 0.432, event free survival (p = 0.667 or overall survival (p = 0.531, inferior overall survival was observed in patients that expressed these isoforms and showed high or standard risk by age and white blood-cell count at diagnosis. Of the 26 patients Ik6+ and/or Ik8+, 14 did not present adverse events; from them 6 were exclusively Ik6+ and/or Ik8+, and 8 were positive for the other Ikaros isoforms (Ik1, Ik2, Ik5, Ik3A, Ik4, Ik4A, Ik7. In the patients studied, the expression of Ik6 and Ik8 did not constitute an independent prognostic factor for relapse or death related to disease; therefore, they could not be used in the initial risk stratification.

  14. Regulation of estrogen receptor (ER) isoform messenger RNA expression by different ER ligands in female rat pituitary.

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    Tena-Sempere, M; Navarro, V M; Mayen, A; Bellido, C; Sánchez-Criado, J E

    2004-03-01

    Net estrogen sensitivity in target tissues critically depends on the regulated expression of full-length and alternately processed estrogen receptor (ER) isoforms. However, the molecular mechanisms for the control of pituitary responsiveness to estrogen remain partially unknown. In the present communication, we report the ability of different ligands, with distinct agonistic or antagonistic properties at the ER, to modulate the expression of the transcripts encoding ERalpha and ERbeta isoforms, as well as those for the truncated ERalpha product (TERP), and the variant ERbeta2, in pituitaries from ovariectomized rats, i.e., a background devoid of endogenous estrogen. Compared with expression levels at the morning of proestrus, ovariectomy (OVX) resulted in increased pituitary expression of ERbeta and ERbeta2 mRNAs, whereas it decreased TERP-1 and -2 levels without affecting those of ERalpha. Administration of estradiol benzoate (as potent agonist for alpha and beta forms of ER) or the selective ERalpha agonist, propyl pyrazole triol, fully reversed the responses to OVX, while the ERbeta ligand, diarylpropionitrile, failed to induce any significant effect except for a partial stimulation of TERP-1 and -2 mRNA expression levels. To note, the ERbeta agonist was also ineffective in altering pituitary expression of progesterone receptor-B mRNA, i.e., a major estrogen-responsive target. In all parameters tested, tamoxifen, a selective ER modulator with mixed agonist/antagonist activity, behaved as ERalpha agonist, although the magnitude of tamoxifen effects was significantly lower than those of the ERalpha ligand, except for TERP induction. In contrast, the pure antiestrogen RU-58668 did not modify the expression of any of the targets under analysis. Overall, our results indicate that endogenous estrogen differentially regulates pituitary expression of the mRNAs encoding several ER isoforms with distinct functional properties, by a mechanism that is mostly conducted

  15. Alternative splicing of the human gene SYBL1 modulates protein domain architecture of longin VAMP7/TI-VAMP, showing both non-SNARE and synaptobrevin-like isoforms

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    De Franceschi Nicola

    2011-05-01

    Full Text Available Abstract Background The control of intracellular vesicle trafficking is an ideal target to weigh the role of alternative splicing in shaping genomes to make cells. Alternative splicing has been reported for several Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptors of the vesicle (v-SNAREs or of the target membrane (t-SNARES, which are crucial to intracellular membrane fusion and protein and lipid traffic in Eukaryotes. However, splicing has not yet been investigated in Longins, i.e. the most widespread v-SNAREs. Longins are essential in Eukaryotes and prototyped by VAMP7, Sec22b and Ykt6, sharing a conserved N-terminal Longin domain which regulates membrane fusion and subcellular targeting. Human VAMP7/TI-VAMP, encoded by gene SYBL1, is involved in multiple cell pathways, including control of neurite outgrowth. Results Alternative splicing of SYBL1 by exon skipping events results in the production of a number of VAMP7 isoforms. In-frame or frameshift coding sequence modifications modulate domain architecture of VAMP7 isoforms, which can lack whole domains or domain fragments and show variant or extra domains. Intriguingly, two main types of VAMP7 isoforms either share the inhibitory Longin domain and lack the fusion-promoting SNARE motif, or vice versa. Expression analysis in different tissues and cell lines, quantitative real time RT-PCR and confocal microscopy analysis of fluorescent protein-tagged isoforms demonstrate that VAMP7 variants have different tissue specificities and subcellular localizations. Moreover, design and use of isoform-specific antibodies provided preliminary evidence for the existence of splice variants at the protein level. Conclusions Previous evidence on VAMP7 suggests inhibitory functions for the Longin domain and fusion/growth promoting activity for the Δ-longin molecule. Thus, non-SNARE isoforms with Longin domain and non-longin SNARE isoforms might have somehow opposite regulatory functions

  16. Glycolipid binding preferences of Shiga toxin variants.

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    Sayali S Karve

    Full Text Available The major virulence factor of Shiga toxin producing E. coli, is Shiga toxin (Stx, an AB5 toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. The two major isoforms, Stx1 and Stx2, and Stx2 variants (Stx2a-h significantly differ in toxicity. The exact reason for this toxicity difference is unknown, however different receptor binding preferences are speculated to play a role. Previous studies used enzyme linked immunosorbent assay (ELISA to study binding of Stx1 and Stx2a toxoids to glycolipid receptors. Here, we studied binding of holotoxin and B-subunits of Stx1, Stx2a, Stx2b, Stx2c and Stx2d to glycolipid receptors globotriaosylceramide (Gb3 and globotetraosylceramide (Gb4 in the presence of cell membrane components such as phosphatidylcholine (PC, cholesterol (Ch and other neutral glycolipids. In the absence of PC and Ch, holotoxins of Stx2 variants bound to mixtures of Gb3 with other glycolipids but not to Gb3 or Gb4 alone. Binding of all Stx holotoxins significantly increased in the presence of PC and Ch. Previously, Stx2a has been shown to form a less stable B-pentamer compared to Stx1. However, its effect on glycolipid receptor binding is unknown. In this study, we showed that even in the absence of the A-subunit, the B-subunits of both Stx1 and Stx2a were able to bind to the glycolipids and the more stable B-pentamer formed by Stx1 bound better than the less stable pentamer of Stx2a. B-subunit mutant of Stx1 L41Q, which shows similar stability as Stx2a B-subunits, lacked glycolipid binding, suggesting that pentamerization is more critical for binding of Stx1 than Stx2a.

  17. Distinct functional interactions between actin isoforms and nonsarcomeric myosins.

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    Mirco Müller

    Full Text Available Despite their near sequence identity, actin isoforms cannot completely replace each other in vivo and show marked differences in their tissue-specific and subcellular localization. Little is known about isoform-specific differences in their interactions with myosin motors and other actin-binding proteins. Mammalian cytoplasmic β- and γ-actin interact with nonsarcomeric conventional myosins such as the members of the nonmuscle myosin-2 family and myosin-7A. These interactions support a wide range of cellular processes including cytokinesis, maintenance of cell polarity, cell adhesion, migration, and mechano-electrical transduction. To elucidate differences in the ability of isoactins to bind and stimulate the enzymatic activity of individual myosin isoforms, we characterized the interactions of human skeletal muscle α-actin, cytoplasmic β-actin, and cytoplasmic γ-actin with human myosin-7A and nonmuscle myosins-2A, -2B and -2C1. In the case of nonmuscle myosins-2A and -2B, the interaction with either cytoplasmic actin isoform results in 4-fold greater stimulation of myosin ATPase activity than was observed in the presence of α-skeletal muscle actin. Nonmuscle myosin-2C1 is most potently activated by β-actin and myosin-7A by γ-actin. Our results indicate that β- and γ-actin isoforms contribute to the modulation of nonmuscle myosin-2 and myosin-7A activity and thereby to the spatial and temporal regulation of cytoskeletal dynamics. FRET-based analyses show efficient copolymerization abilities for the actin isoforms in vitro. Experiments with hybrid actin filaments show that the extent of actomyosin coupling efficiency can be regulated by the isoform composition of actin filaments.

  18. Assessment of orthologous splicing isoforms in human and mouse orthologous genes

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    Horner David S

    2010-10-01

    Full Text Available Abstract Background Recent discoveries have highlighted the fact that alternative splicing and alternative transcripts are the rule, rather than the exception, in metazoan genes. Since multiple transcript and protein variants expressed by the same gene are, by definition, structurally distinct and need not to be functionally equivalent, the concept of gene orthology should be extended to the transcript level in order to describe evolutionary relationships between structurally similar transcript variants. In other words, the identification of true orthology relationships between gene products now should progress beyond primary sequence and "splicing orthology", consisting in ancestrally shared exon-intron structures, is required to define orthologous isoforms at transcript level. Results As a starting step in this direction, in this work we performed a large scale human- mouse gene comparison with a twofold goal: first, to assess if and to which extent traditional gene annotations such as RefSeq capture genuine splicing orthology; second, to provide a more detailed annotation and quantification of true human-mouse orthologous transcripts defined as transcripts of orthologous genes exhibiting the same splicing patterns. Conclusions We observed an identical exon/intron structure for 32% of human and mouse orthologous genes. This figure increases to 87% using less stringent criteria for gene structure similarity, thus implying that for about 13% of the human RefSeq annotated genes (and about 25% of the corresponding transcripts we could not identify any mouse transcript showing sufficient similarity to be confidently assigned as a splicing ortholog. Our data suggest that current gene and transcript data may still be rather incomplete - with several splicing variants still unknown. The observation that alternative splicing produces large numbers of alternative transcripts and proteins, some of them conserved across species and others truly species

  19. Differential regulation of renal phospholipase C isoforms by catecholamines.

    Science.gov (United States)

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam or pramipexole) or antinatriuresis (NE) occurred, the kidneys were removed for analysis of PLC isoform protein expression activity. Western blot analysis revealed that in renal cortical membranes, fenoldopam and pramipexole increased expression of PLC beta 1 and decreased expression of PLC gamma 1; PLC delta was unchanged. In the cytosol, pramipexole and fenoldopam increased expression of both PLC beta 1 and PLC gamma 1. No effects were noted in the medulla. A preferential D1 antagonist, SKF 83742, which by itself had no effect, blocked the effects of pramipexole, thus confirming the involvement of the D1 receptor. In contrast, NE also increased PLC beta 1 but did not affect PLC gamma 1 protein expression in membranes. The changes in PLC isoform expression were accompanied by similar changes in PLC isoform activity. These studies demonstrate for the first time differential regulation of PLC isoforms by catecholamines. PMID:7814630

  20. SURVIV for survival analysis of mRNA isoform variation.

    Science.gov (United States)

    Shen, Shihao; Wang, Yuanyuan; Wang, Chengyang; Wu, Ying Nian; Xing, Yi

    2016-01-01

    The rapid accumulation of clinical RNA-seq data sets has provided the opportunity to associate mRNA isoform variations to clinical outcomes. Here we report a statistical method SURVIV (Survival analysis of mRNA Isoform Variation), designed for identifying mRNA isoform variation associated with patient survival time. A unique feature and major strength of SURVIV is that it models the measurement uncertainty of mRNA isoform ratio in RNA-seq data. Simulation studies suggest that SURVIV outperforms the conventional Cox regression survival analysis, especially for data sets with modest sequencing depth. We applied SURVIV to TCGA RNA-seq data of invasive ductal carcinoma as well as five additional cancer types. Alternative splicing-based survival predictors consistently outperform gene expression-based survival predictors, and the integration of clinical, gene expression and alternative splicing profiles leads to the best survival prediction. We anticipate that SURVIV will have broad utilities for analysing diverse types of mRNA isoform variation in large-scale clinical RNA-seq projects. PMID:27279334

  1. Estrogens induce expression of membrane-associated estrogen receptor α isoforms in lactotropes.

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    Sandra Zárate

    Full Text Available Estrogens are key to anterior pituitary function, stimulating hormone release and controlling cell fate to achieve pituitary dynamic adaptation to changing physiological conditions. In addition to their classical mechanism of action through intracellular estrogen receptors (ERs, estrogens exert rapid actions via cell membrane-localized ERs (mERs. We previously showed that E2 exerts a rapid pro-apoptotic action in anterior pituitary cells, especially in lactotropes and somatotropes, through activation of mERs. In the present study, we examined the involvement of mERα in the rapid pro-apoptotic action of estradiol by TUNEL in primary cultures of anterior pituitary cells from ovariectomized rats using a cell-impermeable E2 conjugate (E2-BSA and an ERα selective antagonist (MPP dihydrochloride. We studied mERα expression during the estrous cycle and its regulation by gonadal steroids in vivo by flow cytometry. We identified ERα variants in the plasma membrane of anterior pituitary cells during the estrous cycle and studied E2 regulation of these mERα variants in vitro by surface biotinylation and Western Blot. E2-BSA-induced apoptosis was abrogated by MPP in total anterior pituitary cells and lactotropes. In cycling rats, we detected a higher number of lactotropes and a lower number of somatotropes expressing mERα at proestrus than at diestrus. Acute E2 treatment increased the percentage of mERα-expressing lactotropes whereas it decreased the percentage of mERα-expressing somatotropes. We detected three mERα isoforms of 66, 39 and 22 kDa. Expression of mERα66 and mERα39 was higher at proestrus than at diestrus, and short-term E2 incubation increased expression of these two mERα variants. Our results indicate that the rapid apoptotic action exerted by E2 in lactotropes depends on mERα, probably full-length ERα and/or a 39 kDa ERα variant. Expression and activation of mERα variants in lactotropes could be one of the mechanisms through

  2. Intron 4 containing novel GABAB1 isoforms impair GABAB receptor function.

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    Changhoon Lee

    Full Text Available BACKGROUND: Gamma-aminobutyric acid type B (GABAB receptors decrease neural activity through G protein signaling. There are two subunits, GABAB1 and GABAB2. Alternative splicing provides GABAB1 with structural and functional diversity. cDNA microarrays showed strong signals from human brain RNA using GABAB1 intron 4 region probes. Therefore, we predicted the existence of novel splice variants. METHODOLOGY/PRINCIPAL FINDINGS: Based on the probe sequence analysis, we proposed two possible splice variants, GABAB1j and GABAB1k. The existence of human GABAB1j was verified by quantitative real-time PCR, and mouse GABAB1j was found from a microarray probe set based on human GABAB1j sequence. GABAB1j open reading frames (ORF and expression patterns are not conserved across species, and they do not have any important functional domains except sushi domains. Thus, we focused on another possible splice variant, GABAB1k. After obtaining PCR evidence for GABAB1k existence from human, mouse, and rat, it was cloned from human and mouse by PCR along with three additional isoforms, GABAB1l, GABAB1m, and GABAB1n. Their expression levels by quantitative real-time PCR are relatively low in brain although they may be expressed in specific cell types. GABAB1l and GABAB1m inhibit GABAB receptor-induced G protein-activated inwardly rectifying K(+ channel (GIRK currents at Xenopus oocyte two-electrode voltage clamp system. CONCLUSIONS/SIGNIFICANCE: This study supports previous suggestions that intron 4 of GABAB1 gene is a frequent splicing spot across species. Like GABAB1e, GABAB1l and GABAB1m do not have transmembrane domains but have a dimerization motif. So, they also could be secreted and bind GABAB2 dominantly instead of GABAB1a. However, only GABAB1l and GABAB1m are N- and C-terminal truncated splicing variants and impair receptor function. This suggests that the intron 4 containing N-terminal truncation is necessary for the inhibitory action of the new splice

  3. A novel CDX2 isoform regulates alternative splicing.

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    Matthew E Witek

    Full Text Available Gene expression is a dynamic and coordinated process coupling transcription with pre-mRNA processing. This regulation enables tissue-specific transcription factors to induce expression of specific transcripts that are subsequently amplified by alternative splicing allowing for increased proteome complexity and functional diversity. The intestine-specific transcription factor CDX2 regulates development and maintenance of the intestinal epithelium by inducing expression of genes characteristic of the mature enterocyte phenotype. Here, sequence analysis of CDX2 mRNA from colonic mucosa-derived tissues revealed an alternatively spliced transcript (CDX2/AS that encodes a protein with a truncated homeodomain and a novel carboxy-terminal domain enriched in serine and arginine residues (RS domain. CDX2 and CDX2/AS exhibited distinct nuclear expression patterns with minimal areas of co-localization. CDX2/AS did not activate the CDX2-dependent promoter of guanylyl cyclase C nor inhibit transcriptional activity of CDX2. Unlike CDX2, CDX2/AS co-localized with the putative splicing factors ASF/SF2 and SC35. CDX2/AS altered splicing patterns of CD44v5 and Tra2-β1 minigenes in Lovo colon cancer cells independent of CDX2 expression. These data demonstrate unique dual functions of the CDX2 gene enabling it to regulate gene expression through both transcription (CDX2 and pre-mRNA processing (CDX2/AS.

  4. Association of cancer stem cell markers genetic variants with gallbladder cancer susceptibility, prognosis, and survival.

    Science.gov (United States)

    Yadav, Anu; Gupta, Annapurna; Rastogi, Neeraj; Agrawal, Sushma; Kumar, Ashok; Kumar, Vijay; Mittal, Balraj

    2016-02-01

    Genes important to stem cell progression have been involved in the genetics and clinical outcome of cancers. We investigated germ line variants in cancer stem cell (CSC) genes to predict susceptibility and efficacy of chemoradiotherapy treatment in gallbladder cancer (GBC) patients. In this study, we assessed the effect of SNPs in CSC genes (surface markers CD44, ALCAM, EpCAM, CD133) and (molecular markers NANOG, SOX-2, LIN-28A, ALDH1A1, OCT-4) with GBC susceptibility and prognosis. Total 610 GBC patients and 250 controls were genotyped by using PCR-RFLP, ARMS-PCR, and TaqMan allelic discrimination assays. Chemotoxicity graded 2-4 in 200 patients and tumor response was recorded in 140 patients undergoing neoadjuvant chemotherapy (NACT). Differences in genotype and haplotype frequency distributions were calculated by binary logistic regression. Gene-gene interaction model was analyzed by generalized multifactor dimensionality reduction (GMDR). Overall survival was assessed by Kaplan-Meier survival curve and multivariate Cox-proportional methods. ALCAM Ars1157Crs10511244 (P = 0.0035) haplotype was significantly associated with GBC susceptibility. In GMDR analysis, ALCAM rs1157G>A, EpCAM rs1126497T>C emerged as best significant interaction model with GBC susceptibility and ALDH1A1 rs13959T>G with increased risk of grade 3-4 hematological toxicity. SOX-2 rs11915160A>C, OCT-4 rs3130932T>G, and NANOG rs11055786T>C were found best gene-gene interaction model for predicting response to NACT. In both Cox-proportional and recursive partitioning ALCAM rs1157GA+AA genotype showed higher mortality and hazard ratio. ALCAM gene polymorphisms associated with GBC susceptibility and survival while OCT-4, SOX-2, and NANOG variants showed an interactive role with treatment response. PMID:26318430

  5. Proteogenomic Analysis Identifies a Novel Human SHANK3 Isoform

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    Fahad Benthani

    2015-05-01

    Full Text Available Mutations of the SHANK3 gene have been associated with autism spectrum disorder. Individuals harboring different SHANK3 mutations display considerable heterogeneity in their cognitive impairment, likely due to the high SHANK3 transcriptional diversity. In this study, we report a novel interaction between the Mutated in colorectal cancer (MCC protein and a newly identified SHANK3 protein isoform in human colon cancer cells and mouse brain tissue. Hence, our proteogenomic analysis identifies a new human long isoform of the key synaptic protein SHANK3 that was not predicted by the human reference genome. Taken together, our findings describe a potential new role for MCC in neurons, a new human SHANK3 long isoform and, importantly, highlight the use of proteomic data towards the re-annotation of GC-rich genomic regions.

  6. Laminin isoforms in endothelial and perivascular basement membranes

    Science.gov (United States)

    Yousif, Lema F.; Di Russo, Jacopo; Sorokin, Lydia

    2013-01-01

    Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis. PMID:23263631

  7. Oxygenation properties and isoform diversity of snake hemoglobins

    DEFF Research Database (Denmark)

    Storz, Jay F.; Natarajan, Chandrasekhar; Moriyama, Hideaki;

    2015-01-01

    Available data suggest that snake hemoglobins (Hbs) are characterized by a combination of unusual structural and functional properties relative to the Hbs of other amniote vertebrates, including oxygenation-linked tetramer- dimer dissociation. However, standardized comparative data are lacking...... for snake Hbs, and the Hb isoform composition of snake red blood cells has not been systematically characterized. Here we present the results of an integrated analysis of snake Hbs and the underlying - and -type globin genes to characterize 1) Hb isoform composition of definitive erythrocytes, and 2......) the oxygenation properties of isolated isoforms as well as composite hemolysates. We used species from three families as subjects for experimental studies of Hb function: South American rattlesnake, Crotalus durissus (Viperidae); Indian python, Python molurus (Pythonidae); and yellow-bellied sea snake, Pelamis...

  8. Identification and characterization of novel NuMA isoforms

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Jin, E-mail: petersdu2112@hotmail.com [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Xu, Zhe [Department of Clinical Laboratory Diagnosis, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); Core Laboratory for Clinical Medical Research, Beijing Tiantan Hospital, Capital Medical University, Beijing (China); He, Dacheng [Key Laboratory for Cell Proliferation and Regulation of the Ministry of Education, Beijing Normal University, Beijing (China); Lu, Guanting, E-mail: guantlv@126.com [Beijing DnaLead Science and Technology Co., LTD, Beijing (China)

    2014-11-21

    Highlights: • Seven NuMA isoforms generated by alternative splicing were categorized into 3 groups: long, middle and short. • Both exons 15 and 16 in long NuMA were “hotspot” for alternative splicing. • Lower expression of short NuMA was observed in cancer cells compared with nonneoplastic controls. • Distinct localization pattern of short isoforms indicated different function from that of long and middle NuMA. - Abstract: The large nuclear mitotic apparatus (NuMA) has been investigated for over 30 years with functions related to the formation and maintenance of mitotic spindle poles during mitosis. However, the existence and functions of NuMA isoforms generated by alternative splicing remains unclear. In the present work, we show that at least seven NuMA isoforms (categorized into long, middle and short groups) generated by alternative splicing from a common NuMA mRNA precursor were discovered in HeLa cells and these isoforms differ mainly at the carboxyl terminus and the coiled-coil domains. Two “hotspot” exons with molecular mass of 3366-nt and 42-nt tend to be spliced during alternative splicing in long and middle groups. Furthermore, full-length coding sequences of long and middle NuMA obtained by using fusion PCR were constructed into GFP-tagged vector to illustrate their cellular localization. Long NuMA mainly localized in the nucleus with absence from nucleoli during interphase and translocated to the spindle poles in mitosis. Middle NuMA displayed the similar cell cycle-dependent distribution pattern as long NuMA. However, expression of NuMA short isoforms revealed a distinct subcellular localization. Short NuMA were present in the cytosol during the whole cycle, without colocalization with mitotic apparatus. These results have allowed us tentatively to explore a new research direction for NuMA’s various functions.

  9. A Review of Metallothionein Isoforms and their Role in Pathophysiology

    OpenAIRE

    Senthil kumar M; Manisenthil Kumar KT; Shyam Sunder A; Thirumoorthy N; Ganesh GNK; Chatterjee Malay

    2011-01-01

    Abstract The Metallothionein (MT) is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological s...

  10. Expression of CD150 in tumors of the central nervous system: identification of a novel isoform.

    Directory of Open Access Journals (Sweden)

    Olga Romanets-Korbut

    Full Text Available CD150 (IPO3/SLAM belongs to the SLAM family of receptors and serves as a major entry receptor for measles virus. CD150 is expressed on normal and malignant cells of the immune system. However, little is known about its expression outside the hematopoietic system, especially tumors of the central nervous system (CNS. Although CD150 was not found in different regions of normal brain tissues, our immunohistochemical study revealed its expression in 77.6% of human CNS tumors, including glioblastoma, anaplastic astrocytoma, diffuse astrocytoma, ependymoma, and others. CD150 was detected in the cytoplasm, but not on the cell surface of glioma cell lines, and it was colocalized with the endoplasmic reticulum and Golgi complex markers. In addition to the full length mRNA of the mCD150 splice isoform, in glioma cells we found a highly expressed novel CD150 transcript (nCD150, containing an 83 bp insert. The insert is derived from a previously unrecognized exon designated Cyt-new, which is located 510 bp downstream of the transmembrane region exon, and is a specific feature of primate SLAMF1. Both mCD150 and nCD150 cDNA variants did not contain any mutations and had the leader sequence. The nCD150 transcript was also detected in normal and malignant B lymphocytes, primary T cells, dendritic cells and macrophages; however, in glioma cells nCD150 was found to be the predominant CD150 isoform. Similarly to mCD150, cell surface expression of nCD150 allows wild type measles virus entry to the cell. Our data indicate that CD150 expression in CNS tumors can be considered a new diagnostic marker and potential target for novel therapeutic approaches.

  11. XBAT35, a Novel Arabidopsis RING E3 Ligase Exhibiting Dual Targeting of Its Splice Isoforms,Is Involved in Ethylene-Mediated Regulation of Apical Hook Curvature

    Institute of Scientific and Technical Information of China (English)

    Sofia D.Carvalho; Rita Saraiva; Teresa M.Maia; Isabel A.Abreu; Paula Duque

    2012-01-01

    The Arabidopsis XBAT35 is one of five structurally related ankyrin repeat-containing Really interesting New Gene (RING) E3 ligases involved in ubiquitin-mediated protein degradation,which plays key roles in a wide range of cellular processes.Here,we show that the XBAT35 gene undergoes alternative splicing,generating two transcripts that are constitutively expressed in all plant tissues.The two splice variants derive from an exon skipping event that excludes an in-frame segment from the XBAT35 precursor mRNA,giving rise to two protein isoforms that differ solely in the presence of a nuclear localization signal (NLS).Transient expression assays indicate that the isoform lacking the NLS localizes in the cytoplasm of plant cells,whereas the other is targeted to the nucleus,accumulating in nuclear speckles.Both isoforms are functional E3 ligases,as assessed by in vitro ubiquitination assays.Two insertion mutant alleles and RNA-interference (RNAi) silencing lines for XBAT35 display no evident phenotypes under normal growth conditions,but exhibit hypersensitivity to the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) during apical hook exaggeration in the dark,which is rescued by an inhibitor of ethylene perception.Independent expression of each XBAT35 splice variant in the mutant background indicates that the two isoforms may differentially contribute to apical hook formation but are both functional in this ethylene-mediated response.Thus,XBAT35 defines a novel player in ethylene signaling involved in negatively regulating apical hook curvature,with alternative splicing controlling dual targeting of this E3 ubiquitin ligase to the nuclear and cytoplasmic compartments.

  12. ApoE isoform-dependent changes in hippocampal synaptic function

    Directory of Open Access Journals (Sweden)

    Sullivan Patrick M

    2009-05-01

    Full Text Available Abstract The lipoprotein receptor system in the hippocampus is intimately involved in the modulation of synaptic transmission and plasticity. The association of specific apoE isoform expression with human neurodegenerative disorders has focused attention on the role of these apoE isoforms in lipoprotein receptor-dependent synaptic modulation. In the present study, we used the apoE2, apoE3 and apoE4 targeted replacement (TR mice along with recombinant human apoE isoforms to determine the role of apoE isoforms in hippocampus area CA1 synaptic function. While synaptic transmission is unaffected by apoE isoform, long-term potentiation (LTP is significantly enhanced in apoE4 TR mice versus apoE2 TR mice. ApoE isoform-dependent differences in LTP induction require NMDA-receptor function, and apoE isoform expression alters activation of both ERK and JNK signal transduction. Acute application of specific apoE isoforms also alters LTP induction while decreasing NMDA-receptor mediated field potentials. Furthermore, acute apoE isoform application does not have the same effects on ERK and JNK activation. These findings demonstrate specific, isoform-dependent effects of human apoE isoforms on adult hippocampus synaptic plasticity and highlight mechanistic differences between chronic apoE isoform expression and acute apoE isoform exposure.

  13. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    International Nuclear Information System (INIS)

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  14. Pairwise comparisons of ten porcine tissues identify differential transcriptional regulation at the gene, isoform, promoter and transcription start site level

    Energy Technology Data Exchange (ETDEWEB)

    Farajzadeh, Leila; Hornshøj, Henrik; Momeni, Jamal; Thomsen, Bo; Larsen, Knud; Hedegaard, Jakob; Bendixen, Christian; Madsen, Lone Bruhn, E-mail: LoneB.Madsen@agrsci.dk

    2013-08-23

    Highlights: •Transcriptome sequencing yielded 223 mill porcine RNA-seq reads, and 59,000 transcribed locations. •Establishment of unique transcription profiles for ten porcine tissues including four brain tissues. •Comparison of transcription profiles at gene, isoform, promoter and transcription start site level. •Highlights a high level of regulation of neuro-related genes at both gene, isoform, and TSS level. •Our results emphasize the pig as a valuable animal model with respect to human biological issues. -- Abstract: The transcriptome is the absolute set of transcripts in a tissue or cell at the time of sampling. In this study RNA-Seq is employed to enable the differential analysis of the transcriptome profile for ten porcine tissues in order to evaluate differences between the tissues at the gene and isoform expression level, together with an analysis of variation in transcription start sites, promoter usage, and splicing. Totally, 223 million RNA fragments were sequenced leading to the identification of 59,930 transcribed gene locations and 290,936 transcript variants using Cufflinks with similarity to approximately 13,899 annotated human genes. Pairwise analysis of tissues for differential expression at the gene level showed that the smallest differences were between tissues originating from the porcine brain. Interestingly, the relative level of differential expression at the isoform level did generally not vary between tissue contrasts. Furthermore, analysis of differential promoter usage between tissues, revealed a proportionally higher variation between cerebellum (CBE) versus frontal cortex and cerebellum versus hypothalamus (HYP) than in the remaining comparisons. In addition, the comparison of differential transcription start sites showed that the number of these sites is generally increased in comparisons including hypothalamus in contrast to other pairwise assessments. A comprehensive analysis of one of the tissue contrasts, i

  15. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jianmin, E-mail: jmhuang@partners.org [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Levitsky, Lynne L. [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States); Rhoads, David B., E-mail: rhoads@helix.mgh.harvard.edu [Pediatric Endocrine Unit, MassGeneral Hospital for Children and Harvard Medical School, Boston, Massachusetts, 02114-2696 (United States)

    2009-04-15

    Hepatocyte nuclear factor 4{alpha} (HNF4{alpha}) is a critical transcription factor for pancreas and liver development and functions in islet {beta} cells to maintain glucose homeostasis. Mutations in the human HNF4A gene lead to maturity onset diabetes of the young (MODY1) and polymorphisms are associated with increased risk for type 2 diabetes mellitus (T2DM). Expression of six HNF4{alpha} variants, three each from two developmentally regulated promoters, has been firmly established. We have now detected a new set of HNF4{alpha} variants designated HNF4{alpha}10-12 expressed from distal promoter P2. These variants, generated by inclusion of previously undetected exon 1E (human = 222 nt, rodent = 136 nt) following exon 1D have an altered N-terminus but identical remaining reading frame. HNF4{alpha}10-{alpha}12 are expressed in pancreatic islets (and liver) and exhibit transactivation potentials similar to the corresponding {alpha}7-{alpha}9 isoforms. DNA-binding analyses implied much higher protein levels of HNF4{alpha}10-{alpha}12 in liver than expected from the RT-PCR data. Our results provide evidence for a more complex expression pattern of HNF4{alpha} than previously appreciated. We recommend inclusion of exon 1E and nearby DNA sequences in screening for HNF4{alpha} mutations and polymorphisms in genetic analyses of MODY1 and T2DM.

  16. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    Science.gov (United States)

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities.

  17. Bacteria-Induced Dscam Isoforms of the Crustacean, Pacifastacus leniusculus.

    Directory of Open Access Journals (Sweden)

    Apiruck Watthanasurorot

    2011-06-01

    Full Text Available The Down syndrome cell adhesion molecule, also known as Dscam, is a member of the immunoglobulin super family. Dscam plays an essential function in neuronal wiring and appears to be involved in innate immune reactions in insects. The deduced amino acid sequence of Dscam in the crustacean Pacifastacus leniusculus (PlDscam, encodes 9(Ig-4(FNIII-(Ig-2(FNIII-TM and it has variable regions in the N-terminal half of Ig2 and Ig3 and the complete Ig7 and in the transmembrane domain. The cytoplasmic tail can generate multiple isoforms. PlDscam can generate more than 22,000 different unique isoforms. Bacteria and LPS injection enhanced the expression of PlDscam, but no response in expression occurred after a white spot syndrome virus (WSSV infection or injection with peptidoglycans. Furthermore, PlDscam silencing did not have any effect on the replication of the WSSV. Bacterial specific isoforms of PlDscam were shown to have a specific binding property to each tested bacteria, E. coli or S. aureus. The bacteria specific isoforms of PlDscam were shown to be associated with bacterial clearance and phagocytosis in crayfish.

  18. Role of p53 isoforms and aggregations in cancer.

    Science.gov (United States)

    Kim, SeJin; An, Seong Soo A

    2016-06-01

    p53 is a master regulatory protein that is involved in diverse cellular metabolic processes such as apoptosis, DNA repair, and cell cycle arrest. The protective function of p53 (in its homotetrameric form) as a tumor suppressor is lost in more than 50% of human cancers.Despite considerable experimental evidence suggesting the presence of multiple p53 states, it has been difficult to correlate the status of p53 with cancer response to treatments and clinical outcomes, which suggest the importance of complex but essential p53 regulatory pathways.Recent studies have indicated that the expression pattern of p53 isoforms may play a crucial role in regulating normal and cancer cell fates in response to diverse stresses. The human TP53 gene encodes at least 12 p53 isoforms, which are produced in normal tissue through alternative initiation of translation, usage of alternative promoters, and alternative splicing. Furthermore, some researchers have suggested that the formation of mutant p53 aggregates may be associated with cancer pathogenesis due to loss-of function (LoF), dominant-negative (DN), and gain-of function (GoF) effects.As different isoforms or the aggregation state of p53 may influence tumorigenesis, this review aims to examine the correlation of p53 isoforms and aggregation with cancer. PMID:27368003

  19. Identification and characterization of a novel isoform of hepatopoietin

    Institute of Scientific and Technical Information of China (English)

    Jun Lu; Wang-Xiang Xu; Yi-Qun Zhan; Xiao-Lin Cui; Wei-Min Cai; Fu-Chu He; Xiao-Ming Yang

    2002-01-01

    AIM: To isolate a novel isoform of human HPO (HPO-205)human fetal liver Marathon-reedy cDNA andcharacterize its primary biological function.METHODS: 5'-RACE (rapid amplification of cDNA 5' ends)was used to isolate a novel isoform of hHPO in this paperThe constructed pcDNAHPO-205, pcDNAHPO and pcDNA eukaryotic expression vectors were respectively transfectedby lipofectamine method and the stimulation of DNAsynthesis was observed by 3H-TdR incorporation assay.Proteins extracted from different cells were analyzed byWestern blot.RESULTS: A novel isoform of hHPO (HPO-205) encoding a205 amino acid ORF corresponding to a translatedproduction of 23 kDa was isolated and distinguished fromthe previous HPO that lacked the N-terminal 80 amino acids.The dnse-dspendent stimulation of DNA synthesis of HepG2hepatoma cells by HPO-205 demonstrated its similarbiological activity with HPO in vitro. The level of MAPK(Mitogen-activated protein kinase) phnsphorylarion byWestern blot analysis revealed that HPO-205 might have thestronger activity of stimulating hepatic cell proliferation thanthat of HPO.CONCLUSION: A novel isoform of hHPO (HPO-205) wasisolated from hepatic-derived cells. The comparison of HPO-205 and HPO will lead to a new insight into the structure andfunction of hHPO, and provide the new way of thinking todeeply elucidate the biological roles of HPO/ALR.

  20. Distinct Functions of Endophilin Isoforms in Synaptic Vesicle Endocytosis

    Directory of Open Access Journals (Sweden)

    Jifeng Zhang

    2015-01-01

    Full Text Available Endophilin isoforms perform distinct characteristics in their interactions with N-type Ca2+ channels and dynamin. However, precise functional differences for the endophilin isoforms on synaptic vesicle (SV endocytosis remain unknown. By coupling RNA interference and electrophysiological recording techniques in cultured rat hippocampal neurons, we investigated the functional differences of three isoforms of endophilin in SV endocytosis. The results showed that the amplitude of normalized evoked excitatory postsynaptic currents in endophilin1 knockdown neurons decreased significantly for both single train and multiple train stimulations. Similar results were found using endophilin2 knockdown neurons, whereas endophilin3 siRNA exhibited no change compared with control neurons. Endophilin1 and endophilin2 affected SV endocytosis, but the effect of endophilin1 and endophilin2 double knockdown was not different from that of either knockdown alone. This result suggested that endophilin1 and endophilin2 functioned together but not independently during SV endocytosis. Taken together, our results indicate that SV endocytosis is sustained by endophilin1 and endophilin2 isoforms, but not by endophilin3, in primary cultured hippocampal neurons.

  1. Isoforms of transferrin in psoriasis patients abusing alcohol

    NARCIS (Netherlands)

    P. Hoefkens (Peter); E.M. Higgins; R.J. Ward (Roberta); H.G. van Eijk (Henk)

    1997-01-01

    textabstractThe different isoforms of transferrin have been quantified by isoelectric focusing in the sera of psoriasis patients with and without a history of abusing alcohol. In both male and female psoriasis subjects abusing alcohol, there were significant increases in the 2-sial

  2. Tropomyosin-binding properties modulate competition between tropomodulin isoforms.

    Science.gov (United States)

    Colpan, Mert; Moroz, Natalia A; Gray, Kevin T; Cooper, Dillon A; Diaz, Christian A; Kostyukova, Alla S

    2016-06-15

    The formation and fine-tuning of cytoskeleton in cells are governed by proteins that influence actin filament dynamics. Tropomodulin (Tmod) regulates the length of actin filaments by capping the pointed ends in a tropomyosin (TM)-dependent manner. Tmod1, Tmod2 and Tmod3 are associated with the cytoskeleton of non-muscle cells and their expression has distinct consequences on cell morphology. To understand the molecular basis of differences in the function and localization of Tmod isoforms in a cell, we compared the actin filament-binding abilities of Tmod1, Tmod2 and Tmod3 in the presence of Tpm3.1, a non-muscle TM isoform. Tmod3 displayed preferential binding to actin filaments when competing with other isoforms. Mutating the second or both TM-binding sites of Tmod3 destroyed its preferential binding. Our findings clarify how Tmod1, Tmod2 and Tmod3 compete for binding actin filaments. Different binding mechanisms and strengths of Tmod isoforms for Tpm3.1 contribute to their divergent functional capabilities. PMID:27091317

  3. Variants of windmill nystagmus.

    Science.gov (United States)

    Choi, Kwang-Dong; Shin, Hae Kyung; Kim, Ji-Soo; Kim, Sung-Hee; Choi, Jae-Hwan; Kim, Hyo-Jung; Zee, David S

    2016-07-01

    Windmill nystagmus is characterized by a clock-like rotation of the beating direction of a jerk nystagmus suggesting separate horizontal and vertical oscillators, usually 90° out of phase. We report oculographic characteristics in three patients with variants of windmill nystagmus in whom the common denominator was profound visual loss due to retinal diseases. Two patients showed a clock-like pattern, while in the third, the nystagmus was largely diagonal (in phase or 180° out of phase) but also periodically changed direction by 180°. We hypothesize that windmill nystagmus is a unique manifestation of "eye movements of the blind." It emerges when the central structures, including the cerebellum, that normally keep eye movements calibrated and gaze steady can no longer perform their task, because they are deprived of the retinal image motion that signals a need for adaptive recalibration. PMID:27159990

  4. Expression of Ik6 and Ik8 Isoforms and Their Association with Relapse and Death in Mexican Children with Acute Lymphoblastic Leukemia

    Science.gov (United States)

    Reyes-León, Adriana; Juárez-Velázquez, Rocío; Medrano-Hernández, Alma; Cuenca-Roldán, Teresa; Salas-Labadía, Consuelo; del Pilar Navarrete-Meneses, María; Rivera-Luna, Roberto; López-Hernández, Gerardo; Paredes-Aguilera, Rogelio; Pérez-Vera, Patricia

    2015-01-01

    Expression of the 6 and 8 dominant-negative Ikaros isoforms in pediatric patients with acute lymphoblastic leukemia has been associated with a high risk of relapse and death; due to these isoforms disrupting the differentiation and proliferation of lymphoid cells. The aim of this study was to know the frequency of Ik6 and Ik8 in 113 Mexican ALL-children treated within the National Popular Medical Insurance Program to determine whether there was an association with relapse-free survival, event-free survival and overall survival, and to assess its usefulness in the initial stratification of patients. The expression of these isoforms was analyzed using specific primer sets and nested RT-PCR. The detected transcripts were classified according to the isoforms’s sizes reported. A non-expected band of 300 bp from one patient was analyzed by sequencing. Twenty-six patients expressed Ik6 and/or Ik8 and one of them expressed a variant of Ik8 denominated Ik8-deleted. Although the presence of them was not statistically associated with lower relapse free survival (p = 0.432), event free survival (p = 0.667) or overall survival (p = 0.531), inferior overall survival was observed in patients that expressed these isoforms and showed high or standard risk by age and white blood-cell count at diagnosis. Of the 26 patients Ik6+ and/or Ik8+, 14 did not present adverse events; from them 6 were exclusively Ik6+ and/or Ik8+, and 8 were positive for the other Ikaros isoforms (Ik1, Ik2, Ik5, Ik3A, Ik4, Ik4A, Ik7). In the patients studied, the expression of Ik6 and Ik8 did not constitute an independent prognostic factor for relapse or death related to disease; therefore, they could not be used in the initial risk stratification. PMID:26131904

  5. Cloning, expression and alternative splicing of the novel isoform of hTCP11 gene

    DEFF Research Database (Denmark)

    Ma, Yong-xin; Zhang, Si-zhong; Wu, Qia-qing;

    2003-01-01

    To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing.......To identify a novel isoform of hTCP11 gene and investigate its expression and alternative splicing....

  6. Alternative splice variants of the human PD-1 gene

    DEFF Research Database (Denmark)

    Nielsen, Christian; Ohm-Laursen, Line; Barington, Torben;

    2005-01-01

    PD-1 is an immunoregulatory receptor expressed on the surface of activated T cells, B cells, and monocytes. We describe four alternatively spliced PD-1 mRNA transcripts (PD-1Deltaex2, PD-1Deltaex3, PD-1Deltaex2,3, and PD-1Deltaex2,3,4) in addition to the full length isoform. PD-1Deltaex2 and PD-1......Deltaex3 are generated by alternative splicing where exon 2 (extracellular IgV-like domain) and exon 3 (transmembrane domain) respectively are spliced out. PD-1Deltaex3 is therefore likely to encode a soluble form of PD-1. PD-1Deltaex2,3 lacks exon 2 and 3. These three variants have unaffected open...

  7. Growth hormone isoforms in a girl with gigantism.

    Science.gov (United States)

    Ng, L L; Chasalow, F I; Escobar, O; Blethen, S L

    1999-01-01

    Several previous investigations have suggested that there may be different growth hormone isoforms in patients with acromegaly. We used three different site-specific monoclonal antibodies (MAbs) to investigate growth hormone (GH) isoforms in serum from an 8 year-old girl with a GH and prolactin secreting adenoma. The pattern of GH-immunoreactivity was dependent on the circumstances of collection. Serum obtained after oral glucose had very little cross reactivity with MAb 352 although concentrations of up to 15 micrograms/l were found with two other MAbs, 033 and 665. MAb 352 does not recognize the 20,000 dalton isoform of GH (20K) while both MAb 033 and 665 do. The same pattern of GH immunoreactivity (low MAb 352, equal and higher MAb 033 and 665) was seen in other baseline samples. In contrast, samples obtained after TRH/GnRH showed immunoreactivity patterns expected for a mixture of 22,000 dalton isoform of GH (22K) with only a small amount of 20K. GH samples obtained during sleep showed both patterns with episodic peaks with equal immunoreactivity superimposed on the basal pattern (decreased activity with MAb 352). Affinity chromatography of basal samples showed that a portion of the GH immunoreactivity was neither 22K nor 20K, although in stimulated samples, over 70% of GH was 22K or 20K GH. In conclusion, the nature of GH isoforms present in serum varies with GH concentration. These differences may contribute to the known difficulty in correlating disease activity and random GH measurements in patients with GH secreting adenomas. PMID:10392356

  8. Histone variants and lipid metabolism

    NARCIS (Netherlands)

    Borghesan, Michela; Mazzoccoli, Gianluigi; Sheedfar, Fareeba; Oben, Jude; Pazienza, Valerio; Vinciguerra, Manlio

    2014-01-01

    Within nucleosomes, canonical histones package the genome, but they can be opportunely replaced with histone variants. The incorporation of histone variants into the nucleosome is a chief cellular strategy to regulate transcription and cellular metabolism. In pathological terms, cellular steatosis i

  9. Effects of Δ40p53, an isoform of p53 lacking the N-terminus, on transactivation capacity of the tumor suppressor protein p53

    OpenAIRE

    Hafsi, Hind; Santos-Silva, Daniela; Courtois-Cox, Stéphanie; Hainaut, Pierre

    2013-01-01

    Background The p53 protein is expressed as multiple isoforms that differ in their N- and C-terminus due to alternative splicing, promoter or codon initiation usage. Δ40p53 lacks the first 39 residues containing the main transcriptional activation domain, resulting from initiation of translation at AUG +40 in fully spliced p53 mRNA or in a specific variant mRNA retaining intron 2. Overexpression of Δ40p53 antagonizes wild-type p53 in vitro. However, animal models of Δ40p53 in mouse or Zebrafis...

  10. Determination of the class and isoform selectivity of small-molecule histone deacetylase inhibitors

    DEFF Research Database (Denmark)

    Khan, N.; Jeffers, M.; Kumar, S.;

    2008-01-01

    ) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rh...

  11. Expression, purification and enzymatic characterization of the catalytic domains of human tryptophan hydroxylase isoforms

    DEFF Research Database (Denmark)

    Windahl, Michael Skovbo; Boesen, Jane; Karlsen, Pernille Efferbach;

    2009-01-01

    Tryptophan hydroxylase exists in two isoforms: Isoform 1 catalyses the first and rate-limiting step in the synthesis of serotonin in the peripheral parts of the body while isoform 2 catalyses this step in the brain. The catalytic domains of human tryptophan hydroxylase 1 and 2 have been expressed...

  12. A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

    Science.gov (United States)

    Isenmann, Sandra; Khew-Goodall, Yeesim; Gamble, Jennifer; Vadas, Mathew; Wattenberg, Binks W.

    1998-01-01

    Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell. PMID:9658161

  13. One isoform of Arg/Abl2 tyrosine kinase is nuclear and the other seven cytosolic isoforms differently modulate cell morphology, motility and the cytoskeleton

    Energy Technology Data Exchange (ETDEWEB)

    Bianchi, Cristina; Torsello, Barbara; Di Stefano, Vitalba; Zipeto, Maria A.; Facchetti, Rita; Bombelli, Silvia; Perego, Roberto A., E-mail: roberto.perego@unimib.it

    2013-08-01

    The non-receptor tyrosine kinase Abelson related gene (Arg/Abl2) regulates cell migration and morphogenesis by modulating the cytoskeleton. Arg promotes actin-based cell protrusions and spreading, and inhibits cell migration by attenuating stress fiber formation and contractility via activation of the RhoA inhibitor, p190RhoGAP, and by regulating focal adhesion dynamics also via CrkII phosphorylation. Eight full-length Arg isoforms with different N- and C-termini are endogenously expressed in human cells. In this paper, the eight Arg isoforms, subcloned in the pFLAG-CMV2 vector, were transfected in COS-7 cells in order to study their subcellular distribution and role in cell morphology, migration and cytoskeletal modulation. The transfected 1BSCTS Arg isoform has a nuclear distribution and phosphorylates CrkII in the nucleus, whilst the other isoforms are detected in the cytoplasm. The 1BLCTL, 1BSCTL, 1ASCTS isoforms were able to significantly decrease stress fibers, induce cell shrinkage and filopodia-like protrusions with a significant increase in p190RhoGAP phosphorylation. In contrast, 1ALCTL, 1ALCTS, 1ASCTL and 1BLCTS isoforms do not significantly decrease stress fibers and induce the formation of retraction tail-like protrusions. The 1BLCTL and 1ALCTL isoforms have different effects on cell migration and focal adhesions. All these data may open new perspectives to study the mechanisms of cell invasiveness. -Highlights: • Each of the eight Arg isoforms was transfected in COS-7 cells. • Only the 1BSCTS Arg isoform has a nuclear distribution in transfected cells. • The cytoplasmic isoforms and F-actin colocalize cortically and in cell protrusions. • Arg isoforms differently phosphorylate p190RhoGAP and CrkII. • Arg isoforms differently modulate stress fibers, cell protrusions and motility.

  14. Treponema pallidum Lipoprotein TP0435 Expressed in Borrelia burgdorferi Produces Multiple Surface/Periplasmic Isoforms and mediates Adherence.

    Science.gov (United States)

    Chan, Kamfai; Nasereddin, Thayer; Alter, Laura; Centurion-Lara, Arturo; Giacani, Lorenzo; Parveen, Nikhat

    2016-01-01

    The ability of Treponema pallidum, the syphilis spirochete to colonize various tissues requires the presence of surface-exposed adhesins that have been difficult to identify due to the inability to culture and genetically manipulate T. pallidum. Using a Borrelia burgdorferi-based heterologous system and gain-in-function approach, we show for the first time that a highly immunogenic lipoprotein TP0435 can be differentially processed into multiple isoforms with one variant stochastically displayed on the spirochete surface. TP0435 was previously believed to be exclusively located in T. pallidum periplasm. Furthermore, non-adherent B. burgdorferi strain expressing TP0435 acquires the ability to bind to a variety of host cells including placental cells and exhibits slow opsonophagocytosis in vitro similar to poor ex vivo phagocytosis of T. pallidum by host macrophages reported previously. This phenomenon of production of both surface and periplasmic immunogenic lipoprotein isoforms has possible implications in immune evasion of the obligate pathogen T. pallidum during infection. PMID:27161310

  15. Differential regulation of macropinocytosis by Abi1/Hssh3bp1 isoforms.

    Directory of Open Access Journals (Sweden)

    Patrycja M Dubielecka

    Full Text Available BACKGROUND: Macropinocytosis, which is a constitutive cellular process of fluid and macromolecule uptake, is regulated by actin cytoskeleton rearrangements near the plasma membrane. Activation of Rac1, which is proposed to act upstream of the actin polymerization regulatory Wave 2 complex, has been found to correlate with enhanced macropinocytosis. One of the components of the Wave 2 complex is Abi1. Multiple, alternatively spliced isoforms of Abi1 are expressed in mammalian cells, but the functional significance of the various isoforms is unknown. PRINCIPAL FINDINGS: Here, using flow cytometric assay analysis for Alexa Fluor 647, we demonstrate that Abi1 isoforms 2 and 3 differentially regulate macropinocytosis. LNCaP cells expressing isoform 3 had increased macropinocytic uptake that correlated with enhanced cell spreading and higher Rac1 activation in comparison to cells expressing isoform 2. Isoform 2 expressing cells had decreased macropinocytic uptake, but demonstrated greater sensitivity to Rac1 activation. Moreover, more isoform 2 was localized within the cytoplasm in comparison to isoform 3, which was more associated with the plasma membrane. Activated Rac1 was found to specifically bind to a site in exon 10 of isoform 2 in vitro. Because of alternative mRNA splicing, exon 10 is absent from isoform 3, precluding similar binding of activated Rac1. Both isoforms, however, bound to inactive Rac1 through the same non-exon 10 site. Thus, Abi1 isoform 3-containing Wave 2 complex exhibited a differential binding to activated vs. inactive Rac1, whereas isoform 2-containing Wave 2 complex bound activated or inactive Rac1 comparably. CONCLUSION: Based on these observations, we postulate that Abi1 isoforms differentially regulate macropinocytosis as a consequence of their different relative affinities for activated Rac1 in Wave 2 complex. These findings also raise the possibility that isoform-specific roles occur in other Abi1 functions.

  16. Laminin isoforms differentially regulate adhesion, spreading, proliferation, and ERK activation of β1 integrin-null cells

    International Nuclear Information System (INIS)

    The presence of many laminin receptors of the β1 integrin family on most cells makes it difficult to define the biological functions of other major laminin receptors such as integrin α6β4 and dystroglycan. We therefore tested the binding of a β1 integrin-null cell line GD25 to four different laminin variants. The cells were shown to produce dystroglycan, which based on affinity chromatography bound to laminin-1, -2/4, and -10/11, but not to laminin-5. The cells also expressed the integrin α6Aβ4A variant. GD25 β1 integrin-null cells are known to bind poorly to laminin-1, but we demonstrate here that these cells bind avidly to laminin-2/4, -5, and -10/11. The initial binding at 20 min to each of these laminins could be inhibited by an integrin α6 antibody, but not by a dystroglycan antibody. Hence, integrin α6Aβ4A of GD25 cells was identified as a major receptor for initial GD25 cell adhesion to three out of four tested laminin isoforms. Remarkably, cell adhesion to laminin-5 failed to promote cell spreading, proliferation, and extracellular signal-regulated kinase (ERK) activation, whereas all these responses occurred in response to adhesion to laminin-2/4 or -10/11. The data establish GD25 cells as useful tools to define the role integrin α6Aβ4A and suggest that laminin isoforms have distinctly different capacities to promote cell adhesion and signaling via integrin α6Aβ4A

  17. A short caspase-3 isoform inhibits chemotherapy-induced apoptosis by blocking apoptosome assembly.

    Directory of Open Access Journals (Sweden)

    Frédérique Végran

    Full Text Available Alternative splicing of caspase-3 produces a short isoform caspase-3s that antagonizes caspase-3 apoptotic activity. However, the mechanism of apoptosis inhibition by caspase-3s remains unknown. Here we show that exogenous caspase-3 sensitizes MCF-7 and HBL100 breast cancers cells to chemotherapeutic treatments such as etoposide and methotrexate whereas co-transfection with caspase-3s strongly inhibits etoposide and methotrexate-induced apoptosis underlying thus the anti-apoptotic role of caspase-3s. In caspase-3 transfected cells, lamin-A and α-fodrin were cleaved when caspase-3 was activated by etoposide or methotrexate. When caspase-3s was co-transfected, this cleavage was strongly reduced. Depletion of caspase-3 by RNA interference in HBL100 containing endogenous caspase-3s caused reduction in etoposide and methotrexate-induced apoptosis, whereas the depletion of caspase-3s sensitized cells to chemotherapy. In the presence of caspase-3s, a lack of interaction between caspase-3 and caspase-9 was observed. Immunoprecipitation assays showed that caspase-3s binds the pro-forms of caspase-3. This result suggested that the absence of interaction with caspase-9 when both variants of caspase-3 are present contribute to block the apoptosome assembly and inhibit apoptosis. These data support that caspases-3s negatively interferes with caspase-3 activation and apoptosis in breast cancer, and that it can play key roles in the modulation of response to chemotherapeutic treatments.

  18. The p53 Isoform Δ133p53β Promotes Cancer Stem Cell Potential

    Directory of Open Access Journals (Sweden)

    Nikola Arsic

    2015-04-01

    Full Text Available Cancer stem cells (CSC are responsible for cancer chemoresistance and metastasis formation. Here we report that Δ133p53β, a TP53 splice variant, enhanced cancer cell stemness in MCF-7 breast cancer cells, while its depletion reduced it. Δ133p53β stimulated the expression of the key pluripotency factors SOX2, OCT3/4, and NANOG. Similarly, in highly metastatic breast cancer cells, aggressiveness was coupled with enhanced CSC potential and Δ133p53β expression. Like in MCF-7 cells, SOX2, OCT3/4, and NANOG expression were positively regulated by Δ133p53β in these cells. Finally, treatment of MCF-7 cells with etoposide, a cytotoxic anti-cancer drug, increased CSC formation and SOX2, OCT3/4, and NANOG expression via Δ133p53, thus potentially increasing the risk of cancer recurrence. Our findings show that Δ133p53β supports CSC potential. Moreover, they indicate that the TP53 gene, which is considered a major tumor suppressor gene, also acts as an oncogene via the Δ133p53β isoform.

  19. 5' UTR control of native ERG and of Tmprss2:ERG variants activity in prostate cancer.

    Directory of Open Access Journals (Sweden)

    Francesca Zammarchi

    Full Text Available ERG, a member of the ETS transcription factor family, is frequently overexpressed in prostate cancer as a result of its fusion to the androgen-responsive Tmprss2 gene. Different genomic rearrangements and alternative splicing events around the junction region lead to multiple combination of Tmprss2:ERG fusion transcripts that correlate with different tumor aggressiveness, but their specific functions and biological activities are still unclear. The complexity of ERG expression pattern is compounded by the use of alternative promoters, splice sites, polyadenylation sites and translation initiation sites in both the native and fusion contexts. Our systematic characterization of native ERG and Tmprss2:ERG variants reveals that their different oncogenic potential is impacted by the status of the Ets domain and the configuration of the 5' UTR region. In particular, expression and activity of functional ERG and Tmprss2:ERG variants are influenced both by translation initiation signals within the different isoforms and by inhibitory upstream Open Reading Frames (uORF in their 5' UTRs. Stable expression of ERG and Tmprss2:ERG variants promoted cell migration/invasion, induced a block of proliferation and induced a senescence-like state, suggesting a role for these variants in the prostate tumorigenesis process. In addition to Tmprss2:ERG fusion products, a group of related native ERG isoforms is also highly over-expressed in fusion-carrying prostate cancers, and share the same translation initiation site (in ERG exon 4 with the commonly observed Tmprss2 exon1 joined to ERG exon 4 (T1:E4 fusion-derived variant. Usage of this ATG can be preferentially down-regulated by directed antisense-based compounds, possibly representing the basis of a targeted approach that distinguishes between tumor-associated and normal ERG.

  20. Characterization of four hemocyanin isoforms in Litopenaeus vannamei

    Institute of Scientific and Technical Information of China (English)

    XU Jingxiang; RUAN Lingwei; LI Zhen; YU Xiaoman; LI Sedong; SHI Hong; XU Xun

    2015-01-01

    In this study, the gene encoding hemocyanin subunit L, LvHcL, was cloned from Litopenaeus vannamei and the genomic organization was characterized. This gene was diverse with many SNPs and also had at least four isoforms, while one of them (LvHcL4) only had two exons and the exon2 was missed. Transcription analysis showed that these isoforms of LvHcL were up-regulated after WSSV challenge in WSSV-resistant shrimp, while the transcriptions were decreased constantly in WSSV-susceptible shrimp. It is suggested that the hemocyanin had rich polymorphism and was involved in the antiviral response. These results could extend our previous findings and provide insights into the immune feature of hemocyanin, which would be helpful for further studies aimed at antiviral mechanism in inver-tebrate.

  1. Differential regulation of renal phospholipase C isoforms by catecholamines.

    OpenAIRE

    Yu, P Y; Asico, L D; Eisner, G M; Jose, P A

    1995-01-01

    Dopamine and D1 agonists and NE all increase phosphatidyl inositol-specific phospholipase C (PLC) activity, but whereas dopamine produces a natriuresis, NE has an antinatriuretic effect. To determine if catecholamines differentially regulate the expression of PLC isoforms, we infused fenoldopam, a D1 agonist, or pramipexole, a D1/D2 agonist, intravenously or infused fenoldopam or NE into the renal artery of anesthetized rats. After 3-4 h of infusion, when the expected natriuresis (fenoldopam ...

  2. GABAB(1) receptor subunit isoforms differentially regulate stress resilience

    OpenAIRE

    O’Leary, Olivia F.; Felice, Daniela; Galimberti, Stefano; Savignac, Hélène M.; Bravo, Javier A.; Crowley, Tadhg; El Yacoubi, Malika; Vaugeois, Jean-Marie; Gassmann, Martin; Bettler, Bernhard; Dinan, Timothy G.; Cryan, John F.

    2014-01-01

    Stress can increase susceptibility to developing psychiatric disorders, including depression. Understanding the neurobiological mechanisms underlying stress resilience and susceptibility is key to identifying novel targets for the development of more effective treatments for stress-related psychiatric disorders. Here we show that specific isoforms of GABAB receptor subunits differentially regulate stress resilience. Specifically, GABAB(1a)−/− mice are more susceptible whereas GABAB(1b)−/− mic...

  3. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Directory of Open Access Journals (Sweden)

    Ralph D Hector

    Full Text Available Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5 cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR, which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  4. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse.

    Science.gov (United States)

    Hector, Ralph D; Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C; Bailey, Mark E S; Cobb, Stuart R

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3'-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders.

  5. Antagonistic functions of two stardust isoforms in Drosophila photoreceptor cells.

    Science.gov (United States)

    Bulgakova, Natalia A; Rentsch, Michaela; Knust, Elisabeth

    2010-11-15

    Membrane-associated guanylate kinases (MAGUKs) are scaffolding proteins that organize supramolecular protein complexes, thereby partitioning the plasma membrane into spatially and functionally distinct subdomains. Their modular organization is ideally suited to organize protein complexes with cell type- or stage-specific composition, or both. Often more than one MAGUK isoform is expressed by one gene in the same cell, yet very little is known about their individual in vivo functions. Here, we show that two isoforms of Drosophila stardust, Sdt-H (formerly called Sdt-B2) and Sdt-D, which differ in their N terminus, are expressed in adult photoreceptors. Both isoforms associate with Crumbs and PATJ, constituents of the conserved Crumbs-Stardust complex. However, they form distinct complexes, localized at the stalk, a restricted region of the apical plasma membrane. Strikingly, Sdt-H and Sdt-D have antagonistic functions. While Sdt-H overexpression increases stalk membrane length and prevents light-dependent retinal degeneration, Sdt-D overexpression reduces stalk length and enhances light-dependent retinal degeneration. These results suggest that a fine-tuned balance of different Crumbs complexes regulates photoreceptor homeostasis.

  6. Characterisation of CDKL5 Transcript Isoforms in Human and Mouse

    Science.gov (United States)

    Dando, Owen; Landsberger, Nicoletta; Kilstrup-Nielsen, Charlotte; Kind, Peter C.; Bailey, Mark E. S.; Cobb, Stuart R.

    2016-01-01

    Mutations in the X-linked Cyclin-Dependent Kinase-Like 5 gene (CDKL5) cause early onset infantile spasms and subsequent severe developmental delay in affected children. Deleterious mutations have been reported to occur throughout the CDKL5 coding region. Several studies point to a complex CDKL5 gene structure in terms of exon usage and transcript expression. Improvements in molecular diagnosis and more extensive research into the neurobiology of CDKL5 and pathophysiology of CDKL5 disorders necessitate an updated analysis of the gene. In this study, we have analysed human and mouse CDKL5 transcript patterns both bioinformatically and experimentally. We have characterised the predominant brain isoform of CDKL5, a 9.7 kb transcript comprised of 18 exons with a large 6.6 kb 3’-untranslated region (UTR), which we name hCDKL5_1. In addition we describe new exonic regions and a range of novel splice and UTR isoforms. This has enabled the description of an updated gene model in both species and a standardised nomenclature system for CDKL5 transcripts. Profiling revealed tissue- and brain development stage-specific differences in expression between transcript isoforms. These findings provide an essential backdrop for the diagnosis of CDKL5-related disorders, for investigations into the basic biology of this gene and its protein products, and for the rational design of gene-based and molecular therapies for these disorders. PMID:27315173

  7. Differences between disease-associated endoplasmic reticulum aminopeptidase 1 (ERAP1) isoforms in cellular expression, interactions with tumour necrosis factor receptor 1 (TNF-R1) and regulation by cytokines.

    Science.gov (United States)

    Yousaf, N; Low, W Y; Onipinla, A; Mein, C; Caulfield, M; Munroe, P B; Chernajovsky, Y

    2015-05-01

    Endoplasmic reticulum aminopeptidase 1 (ERAP1) processes peptides for major histocompatibility complex (MHC) class I presentation and promotes cytokine receptor ectodomain shedding. These known functions of ERAP1 may explain its genetic association with several autoimmune inflammatory diseases. In this study, we identified four novel alternatively spliced variants of ERAP1 mRNA, designated as ΔExon-11, ΔExon-13, ΔExon-14 and ΔExon-15. We also observed a rapid and differential modulation of ERAP1 mRNA levels and spliced variants in different cell types pretreated with lipopolysaccharide (LPS). We have studied three full-length allelic forms of ERAP1 (R127-K528, P127-K528, P127-R528) and one spliced variant (ΔExon-11) and assessed their interactions with tumour necrosis factor receptor 1 (TNF-R1) in transfected cells. We observed variation in cellular expression of different ERAP1 isoforms, with R127-K528 being expressed at a much lower level. Furthermore, the cellular expression of full-length P127-K528 and ΔExon-11 spliced variant was enhanced significantly when co-transfected with TNF-R1. Isoforms P127-K528, P127-R528 and ΔExon-11 spliced variant associated with TNF-R1, and this interaction occurred in a region within the first 10 exons of ERAP1. Supernatant-derived vesicles from transfected cells contained the full-length and ectodomain form of soluble TNF-R1, as well as carrying the full-length ERAP1 isoforms. We observed marginal differences between TNF-R1 ectodomain levels when co-expressed with individual ERAP1 isoforms, and treatment of transfected cells with tumour necrosis factor (TNF), interleukin (IL)-1β and IL-10 exerted variable effects on TNF-R1 ectodomain cleavage. Our data suggest that ERAP1 isoforms may exhibit differential biological properties and inflammatory mediators could play critical roles in modulating ERAP1 expression, leading to altered functional activities of this enzyme. PMID:25545008

  8. A Novel Splicing Variant of Mouse Interleukin (IL)-24 Antagonizes IL-24-induced Apoptosis*S⃞

    OpenAIRE

    Sahoo, Anupama; Jung, Yun Min; Kwon, Ho-Keun; Yi, Hwa-Jung; Lee, Suho; Chang, Sunghoe; Park, Zee-Yong; Hwang, Ki-Chul; Im, Sin-Hyeog

    2008-01-01

    Alternative splicing of mRNA enables functionally diverse protein isoforms to be expressed from a single gene, allowing transcriptome diversification. Interleukin (IL)-24/MDA-7 is a member of the IL-10 gene family, and FISP (IL-4-induced secreted protein), its murine homologue, is selectively expressed and secreted by T helper 2 lymphocytes. A novel splice variant of mouse IL-24/FISP, designated FISP-sp, lacks 29 nucleotides from the 5′-end of exon 4 of FISP. The level...

  9. Molecular mechanical differences between isoforms of contractile actin in the presence of isoforms of smooth muscle tropomyosin.

    OpenAIRE

    Lennart Hilbert; Genevieve Bates; Roman, Horia N.; Jenna L Blumenthal; Zitouni, Nedjma B.; Apolinary Sobieszek; Mackey, Michael C.; Anne-Marie Lauzon

    2013-01-01

    The proteins involved in smooth muscle's molecular contractile mechanism - the anti-parallel motion of actin and myosin filaments driven by myosin heads interacting with actin - are found as different isoforms. While their expression levels are altered in disease states, their relevance to the mechanical interaction of myosin with actin is not sufficiently understood. Here, we analyzed in vitro actin filament propulsion by smooth muscle myosin for [Formula: see text]-actin ([Formula: see text...

  10. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes.

    Science.gov (United States)

    Wu, I; Shin, S C; Cao, Y; Bender, I K; Jafari, N; Feng, G; Lin, S; Cidlowski, J A; Schleimer, R P; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expressing the GR-A, -B, or -C, but not the GR-D, isoform. cDNA microarray analyses of cells sensitive (GR-C3) and insensitive (GR-D3) to DEX revealed glucocorticoid-induced proapoptotic transcriptomes. Genes that were regulated by the proapoptotic GR-C3, but not by the GR-D3, isoform likely contributed to glucocorticoid-induced apoptosis. The identified genes include those that are directly involved in apoptosis and those that facilitate cell killing. Chromatin immunoprecipitation assays demonstrated that distinct chromatin modification abilities may underlie the distinct functions of GR isoforms. Interestingly, all GR isoforms, including the GR-D3 isoform, suppressed mitogen-stimulated cytokines. Furthermore, the GR-C isoforms were selectively upregulated in mitogen-activated primary T cells and DEX treatment induced GR-C target genes in activated T cells. Cell-specific expressions and functions of GR isoforms may help to explain the tissue- and individual-selective actions of glucocorticoids and may provide a basis for developing improved glucocorticoids. PMID:23303127

  11. Evolution of a TRIM5-CypA splice isoform in old world monkeys.

    Directory of Open Access Journals (Sweden)

    Ruchi M Newman

    2008-02-01

    Full Text Available The TRIM family proteins share a conserved arrangement of three adjacent domains, an N-terminal RING domain, followed by one or two B-boxes and a coiled-coil, which constitutes the tripartite-motif for which the family is named. However, the C-termini of TRIM proteins vary, and include at least nine evolutionarily distinct, unrelated protein domains. Antiviral restriction factor TRIM5alpha has a C-terminal B30.2/SPRY domain, which is the major determinant of viral target specificity. Here, we describe the evolution of a cyclophilin-A encoding exon downstream of the TRIM5 locus of Asian macaques. Alternative splicing gives rise to chimeric transcripts encoding the TRIM motif fused to a C-terminal CypA domain (TRIM5-CypA. We detected TRIM5-CypA chimeric transcripts in primary lymphocytes from two macaque species. These were derived in part from a CypA pseudogene in the TRIM5 locus, which is distinct from the previously described CypA insertion in TRIM5 of owl monkeys. The CypA insertion is linked to a mutation in the 3' splice site upstream of exon 7, which may prevent or reduce expression of the alpha-isoform. All pig-tailed macaques (M. nemestrina screened were homozygous for the CypA insertion. In contrast, the CypA-containing allele was present in 17% (17/101 of rhesus macaques (M. mulatta. The block to HIV-1 infection in lymphocytes from animals bearing the TRIM5-CypA allele was weaker than that in cells from wild type animals. HIV-1 infectivity remained significantly lower than SIV infectivity, but was not rescued by treatment with cyclosporine A. Thus, unlike owl monkey TRIMCyp, expression of the macaque TRIM5-CypA isoform does not result in increased restriction of HIV-1. Despite its distinct evolutionary origin, Macaca TRIM5-CypA has a similar domain arrangement and shares approximately 80% amino-acid identity with the TRIMCyp protein of owl monkeys. The independent appearance of TRIM5-CypA chimeras in two primate lineages constitutes a

  12. Yersinia pestis Caf1 Protein: Effect of Sequence Polymorphism on Intrinsic Disorder Propensity, Serological Cross-Reactivity and Cross-Protectivity of Isoforms.

    Science.gov (United States)

    Kopylov, Pavel Kh; Platonov, Mikhail E; Ablamunits, Vitaly G; Kombarova, Tat'yana I; Ivanov, Sergey A; Kadnikova, Lidiya A; Somov, Aleksey N; Dentovskaya, Svetlana V; Uversky, Vladimir N; Anisimov, Andrey P

    2016-01-01

    Yersinia pestis Caf1 is a multifunctional protein responsible for antiphagocytic activity and is a key protective antigen. It is generally conserved between globally distributed Y. pestis strains, but Y. pestis subsp. microtus biovar caucasica strains circulating within populations of common voles in Georgia and Armenia were reported to carry a single substitution of alanine to serine. We investigated polymorphism of the Caf1 sequences among other Y. pestis subsp. microtus strains, which have a limited virulence in guinea pigs and in humans. Sequencing of caf1 genes from 119 Y. pestis strains belonging to different biovars within subsp. microtus showed that the Caf1 proteins exist in three isoforms, the global type Caf1NT1 (Ala48 Phe117), type Caf1NT2 (Ser48 Phe117) found in Transcaucasian-highland and Pre-Araks natural plague foci #4-7, and a novel Caf1NT3 type (Ala48 Val117) endemic in Dagestan-highland natural plague focus #39. Both minor types are the progenies of the global isoform. In this report, Caf1 polymorphism was analyzed by comparing predicted intrinsic disorder propensities and potential protein-protein interactivities of the three Caf1 isoforms. The analysis revealed that these properties of Caf1 protein are minimally affected by its polymorphism. All protein isoforms could be equally detected by an immunochromatography test for plague at the lowest protein concentration tested (1.0 ng/mL), which is the detection limit. When compared to the classic Caf1NT1 isoform, the endemic Caf1NT2 or Caf1NT3 had lower immunoreactivity in ELISA and lower indices of self- and cross-protection. Despite a visible reduction in cross-protection between all Caf1 isoforms, our data suggest that polymorphism in the caf1 gene may not allow the carriers of Caf1NT2 or Caf1NT3 variants escaping from the Caf1NT1-mediated immunity to plague in the case of a low-dose flea-borne infection. PMID:27606595

  13. Reduced peripheral expression of the glucocorticoid receptor α isoform in individuals with posttraumatic stress disorder: a cumulative effect of trauma burden.

    Directory of Open Access Journals (Sweden)

    Hannah Gola

    Full Text Available BACKGROUND: Posttraumatic stress disorder (PTSD is a serious psychiatric condition that was found to be associated with altered functioning of the hypothalamic-pituitary-adrenal (HPA axis and changes in glucocorticoid (GC responsiveness. The physiological actions of GCs are primarily mediated through GC receptors (GR of which isoforms with different biological activities exist. This study aimed to investigate whether trauma-experience and/or PTSD are associated with altered expression of GR splice variants. METHODS: GRα and GRβ mRNA expression levels were determined by real-time quantitative PCR in whole blood samples of individuals with chronic and severe forms of PTSD (n = 42 as well as in ethnically matched reference subjects (non-PTSD, n = 35. RESULTS: Individuals suffering from PTSD exhibited significantly lower expression of the predominant and functionally active GRα isoform compared to non-PTSD subjects. This effect remained significant when accounting for gender, smoking, psychotropic medication or comorbid depression. Moreover, the GRα expression level was significantly negatively correlated with the number of traumatic event types experienced, both in the whole sample and within the PTSD patient group. Expression of the less abundant and non-ligand binding GRβ isoform was comparable between patient and reference groups. CONCLUSIONS: Reduced expression of the functionally active GRα isoform in peripheral blood cells of individuals with PTSD seems to be a cumulative effect of trauma burden rather than a specific feature of PTSD since non-PTSD subjects with high trauma load showed an intermediate phenotype between PTSD patients and individuals with no or few traumatic experiences.

  14. Analysis of stop-gain and frameshift variants in human innate immunity genes.

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    Antonio Rausell

    2014-07-01

    Full Text Available Loss-of-function variants in innate immunity genes are associated with Mendelian disorders in the form of primary immunodeficiencies. Recent resequencing projects report that stop-gains and frameshifts are collectively prevalent in humans and could be responsible for some of the inter-individual variability in innate immune response. Current computational approaches evaluating loss-of-function in genes carrying these variants rely on gene-level characteristics such as evolutionary conservation and functional redundancy across the genome. However, innate immunity genes represent a particular case because they are more likely to be under positive selection and duplicated. To create a ranking of severity that would be applicable to innate immunity genes we evaluated 17,764 stop-gain and 13,915 frameshift variants from the NHLBI Exome Sequencing Project and 1,000 Genomes Project. Sequence-based features such as loss of functional domains, isoform-specific truncation and nonsense-mediated decay were found to correlate with variant allele frequency and validated with gene expression data. We integrated these features in a Bayesian classification scheme and benchmarked its use in predicting pathogenic variants against Online Mendelian Inheritance in Man (OMIM disease stop-gains and frameshifts. The classification scheme was applied in the assessment of 335 stop-gains and 236 frameshifts affecting 227 interferon-stimulated genes. The sequence-based score ranks variants in innate immunity genes according to their potential to cause disease, and complements existing gene-based pathogenicity scores. Specifically, the sequence-based score improves measurement of functional gene impairment, discriminates across different variants in a given gene and appears particularly useful for analysis of less conserved genes.

  15. PKC isoforms interact with and phosphorylate DNMT1

    Directory of Open Access Journals (Sweden)

    Pradhan Sriharsa

    2011-05-01

    Full Text Available Abstract Background DNA methyltransferase 1 (DNMT1 has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization. Results Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity. Conclusions Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.

  16. Naturally occurring variants of human Α9 nicotinic receptor differentially affect bronchial cell proliferation and transformation.

    Directory of Open Access Journals (Sweden)

    Anna Chikova

    Full Text Available Isolation of polyadenilated mRNA from human immortalized bronchial epithelial cell line BEP2D revealed the presence of multiple isoforms of RNA coded by the CHRNA9 gene for α9 nicotinic acetylcholine receptor (nAChR. BEP2D cells were homozygous for the rs10009228 polymorphism encoding for N442S amino acid substitution, and also contained mRNA coding for several truncated isoforms of α9 protein. To elucidate the biologic significance of the naturally occurring variants of α9 nAChR, we compared the biologic effects of overexpression of full-length α9 N442 and S442 proteins, and the truncated α9 variant occurring due to a loss of the exon 4 sequence that causes frame shift and early termination of the translation. These as well as control vector were overexpressed in the BEP2D cells that were used in the assays of proliferation rate, spontaneous vs. tobacco nitrosamine 4-(methylnitrosamino-1-(3-pyridyl-1-butanone (NNK-induced cellular transformation, and tumorigenicity in cell culture and mice. Overexpression of the S442 variant significantly increased cellular proliferation, and spontaneous and NNK-induced transformation. The N442 variant significantly decreased cellular transformation, without affecting proliferation rate. Overexpression of the truncated α9 significantly decreased proliferation and suppressed cellular transformation. These results suggested that α9 nAChR plays important roles in regulation of bronchial cell growth by endogenous acetylcholine and exogenous nicotine, and susceptibility to NNK-induced carcinogenic transformation. The biologic activities of α9 nAChR may be regulated at the splicing level, and genetic polymorphisms in CHRNA9 affecting protein levels, amino acid sequence and RNA splicing may influence the risk for lung cancer.

  17. The FU gene and its possible protein isoforms

    Directory of Open Access Journals (Sweden)

    Nöthen Markus M

    2004-07-01

    Full Text Available Abstract Background FU is the human homologue of the Drosophila gene fused whose product fused is a positive regulator of the transcription factor Cubitus interruptus (Ci. Thus, FU may act as a regulator of the human counterparts of Ci, the GLI transcription factors. Since Ci and GLI are targets of Hedgehog signaling in development and morphogenesis, it is expected that FU plays an important role in Sonic, Desert and/or Indian Hedgehog induced cellular signaling. Results The FU gene was identified on chromosome 2q35 at 217.56 Mb and its exon-intron organization determined. The human developmental disorder Syndactyly type 1 (SD1 maps to this region on chromosome 2 and the FU coding region was sequenced using genomic DNA from an affected individual in a linked family. While no FU mutations were found, three single nucleotide polymorphisms were identified. The expression pattern of FU was thoroughly investigated and all examined tissues express FU. It is also clear that different tissues express transcripts of different sizes and some tissues express more than one transcript. By means of nested PCR of specific regions in RT/PCR generated cDNA, it was possible to verify two alternative splicing events. This also suggests the existence of at least two additional protein isoforms besides the FU protein that has previously been described. This long FU and a much shorter isoform were compared for the ability to regulate GLI1 and GLI2. None of the FU isoforms showed any effects on GLI1 induced transcription but the long form can enhance GLI2 activity. Apparently FU did not have any effect on SUFU induced inhibition of GLI. Conclusions The FU gene and its genomic structure was identified. FU is a candidate gene for SD1, but we have not identified a pathogenic mutation in the FU coding region in a family with SD1. The sequence information and expression analyses show that transcripts of different sizes are expressed and subjected to alternative splicing

  18. Functional differences between L- and T-plastin isoforms

    OpenAIRE

    1994-01-01

    Fimbrins/plastins are a family of highly conserved actin-bundling proteins. They are present in all eukaryotic cells including yeast, but each isoform displays a remarkable tissue specificity. T-plastin is normally found in epithelial and mesenchymal cells while L-plastin is present in hematopoietic cells. However, L-plastin has been also found in tumor cells of non-hematopoietic origin (Lin, C.-S., R. H. Aebersold, S. B. Kent, M. Varma, and J. Leavitt. 1988. Mol. Cell. Biol. 8:4659-4668; Lin...

  19. Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes: isolated isoforms and kinetics properties

    Directory of Open Access Journals (Sweden)

    Maria Regina de Aquino-Silva

    2008-01-01

    Full Text Available Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37 and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

  20. Enhanced expression of Rubisco activase splicing variants differentially affects Rubisco activity during low temperature treatment in Lolium perenne.

    Science.gov (United States)

    Jurczyk, Barbara; Pociecha, Ewa; Grzesiak, Maciej; Kalita, Katarzyna; Rapacz, Marcin

    2016-07-01

    Alternative splicing of the Rubisco activase gene was shown to be a point for optimization of photosynthetic carbon assimilation. It can be expected to be a stress-regulated event that depends on plant freezing tolerance. The aim of the study was to examine the relationships among Rubisco activity, the expression of two Rubisco activase splicing variants and photoacclimation to low temperature. The experiment was performed on two Lolium perenne genotypes with contrasting levels of freezing tolerance. The study investigated the effect of pre-hardening (15°C) and cold acclimation (4°C) on net photosynthesis, photosystem II photochemical activity, Rubisco activity and the expression of two splicing variants of the Rubisco activase gene. The results showed an induction of Rubisco activity at both 15°C and 4°C only in a highly freezing-tolerant genotype. The enhanced Rubisco activity after pre-hardening corresponded to increased expression of the splicing variant representing the large isoform, while the increase in Rubisco activity during cold acclimation was due to the activation of both transcript variants. These boosts in Rubisco activity also corresponded to an activation of non-photochemical mechanism of photoacclimation induced at low temperature exclusively in the highly freezing-tolerant genotype. In conclusion, enhanced expression of Rubisco activase splicing variants caused an increase in Rubisco activity during pre-hardening and cold acclimation in the more freezing-tolerant Lolium perenne genotype. The induction of the transcript variant representing the large isoform may be an important element of increasing the carbon assimilation rate supporting the photochemical mechanism of photosynthetic acclimation to cold. PMID:27152456

  1. Induction of Chemokine Expression by Adiponectin In Vitro is Isoform-Dependent

    OpenAIRE

    Song, Huijuan; Chan, James; Rovin, Brad H.

    2009-01-01

    Adiponectin is reported to have both pro- and anti-inflammatory effects. Because adiponectin circulates in isoforms of various sizes, and some responses to adiponectin are isoform-dependent, it was postulated that the pro-inflammatory effects of adiponectin may isoform-specific. To test this, peripheral blood mononuclear cells (PBMC), microvascular endothelial cells (MVEC), and human glomerular mesangial cells (HMC) were treated with high or low molecular weight (HMW, LMW) recombinant human a...

  2. MetaDiff: differential isoform expression analysis using random-effects meta-regression

    OpenAIRE

    Jia, Cheng; Guan, Weihua; Yang, Amy; Xiao, Rui; Tang, W. H. Wilson; Moravec, Christine S.; Margulies, Kenneth B.; Cappola, Thomas P.; Li, Mingyao; Li, Chun

    2015-01-01

    Background RNA sequencing (RNA-Seq) allows an unbiased survey of the entire transcriptome in a high-throughput manner. A major application of RNA-Seq is to detect differential isoform expression across experimental conditions, which is of great biological interest due to its direct relevance to protein function and disease pathogenesis. Detection of differential isoform expression is challenging because of uncertainty in isoform expression estimation owing to ambiguous reads and variability i...

  3. Selective glucocorticoid receptor translational isoforms reveal glucocorticoid-induced apoptotic transcriptomes

    OpenAIRE

    Wu, I; Shin, S. C.; Cao, Y; Bender, I K; N Jafari; Feng, G.; Lin, S.; Cidlowski, J. A.; Schleimer, R. P.; Lu, N Z

    2013-01-01

    Induction of T-cell apoptosis contributes to the anti-inflammatory and antineoplastic benefits of glucocorticoids. The glucocorticoid receptor (GR) translational isoforms have distinct proapoptotic activities in osteosarcoma cells. Here we determined whether GR isoforms selectively induce apoptosis in Jurkat T lymphoblastic leukemia cells. Jurkat cells stably expressing individual GR isoforms were generated and treated with vehicle or dexamethasone (DEX). DEX induced apoptosis in cells expres...

  4. Down-regulated expression of atypical PKC-binding domain deleted asip isoforms in human hepatocellular carcinomas

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Asip is a mammalian homologue of polarity protein Par-3 of Caenorhabditis elegans and Bazooka of Drosophila melanogaster. Asip/Par-3/Bazooka are PDZ-motif containing proteins that localize asymmetrically to the cell periphery and play a pivotal role in cell polarity and asymmetric cell division. In the present study, we have cloned human asip cDNA and its splicing variants by 5'-RACE and RT-PCR using candidate human EST clones which have a high homology to rat asip cDNA. The full-length cDNA of human asip encodes a 1,353 aa protein exhibiting 88% similarity to the rat one. Human asip is a single copy gene consisting of at least 26 exons and localizing in human chromosome 10, band p11.2, with some extraordinarily long introns. All exon/intron boundary nucleotides conform to the “gt-ag” rule. Three main transcripts were detected by Northern blot analysis, and at least five variants, from alternative splicing and polyadenylation, have been identified by RT-PCR and liver cDNA library screening. Exon 17b deleted asip mRNAs expressed ubiquitously in normal human tissues, including liver, on RT-PCR analysis. However, they were absent from most human liver cancer cell lines examined. More interestingly, the expression of exon 17b deleted variants was down regulated in 52.6% (10/19) clinic specimens of human hepatocellular carcinomas (HCCs), compared with the surrounding nontumorous liver tissues from the same patients. The presence of various splicing transcripts, the variation of their distribution among different tissues and cells, and their differential expressions in human HCCs suggest that human Asip isoforms may function in different context.

  5. IsoformEx: isoform level gene expression estimation using weighted non-negative least squares from mRNA-Seq data

    Directory of Open Access Journals (Sweden)

    Gupta Ravi

    2011-07-01

    Full Text Available Abstract Background mRNA-Seq technology has revolutionized the field of transcriptomics for identification and quantification of gene transcripts not only at gene level but also at isoform level. Estimating the expression levels of transcript isoforms from mRNA-Seq data is a challenging problem due to the presence of constitutive exons. Results We propose a novel algorithm (IsoformEx that employs weighted non-negative least squares estimation method to estimate the expression levels of transcript isoforms. Validations based on in silico simulation of mRNA-Seq and qRT-PCR experiments with real mRNA-Seq data showed that IsoformEx could accurately estimate transcript expression levels. In comparisons with published methods, the transcript expression levels estimated by IsoformEx showed higher correlation with known transcript expression levels from simulated mRNA-Seq data, and higher agreement with qRT-PCR measurements of specific transcripts for real mRNA-Seq data. Conclusions IsoformEx is a fast and accurate algorithm to estimate transcript expression levels and gene expression levels, which takes into account short exons and alternative exons with a weighting scheme. The software is available at http://bioinformatics.wistar.upenn.edu/isoformex.

  6. Data-variant kernel analysis

    CERN Document Server

    Motai, Yuichi

    2015-01-01

    Describes and discusses the variants of kernel analysis methods for data types that have been intensely studied in recent years This book covers kernel analysis topics ranging from the fundamental theory of kernel functions to its applications. The book surveys the current status, popular trends, and developments in kernel analysis studies. The author discusses multiple kernel learning algorithms and how to choose the appropriate kernels during the learning phase. Data-Variant Kernel Analysis is a new pattern analysis framework for different types of data configurations. The chapters include

  7. Prostaglandin D Synthase Isoforms from Cerebrospinal Fluid Vary with Brain Pathology

    Directory of Open Access Journals (Sweden)

    Michael G. Harrington

    2006-01-01

    Full Text Available Glutathione independent prostaglandin D synthase (Swissprot P41222, PTGDS has been identified in human cerebrospinal fluid and some changes in PTGDS in relation to disease have been reported. However, little is known of the extent that PTGDS isoforms fluctuate across a large range of congenital and acquired diseases. The purpose of this study was to examine changes in PTGDS isoforms in such a population. Spinal fluid from 22 healthy study participants (normal controls with no classifiable neurological or psychiatric diagnosis was obtained and PTGDS isoforms were identified by specific immunostaining and mass spectrometry after denaturing 2D gel electrophoresis. The PTGDS isoforms in controls consisted of five charge isoforms that were always present and a small number of occasional, low abundance isoforms. A qualitative survey of 98 different people with a wide range of congenital and acquired diseases revealed striking changes. Loss of the control isoforms occurred in congenital malformations of the nervous system. Gain of additional isoforms occurred in some degenerative, most demyelinating and vasculitic diseases, as well as in Creutzfeldt-Jakob disease. A retrospective analysis of published data that quantified relative amounts of PTGDS in multiple sclerosis, schizophrenia and Parkinson’s disease compared to controls revealed significant dysregulation. It is concluded that qualitative and quantitative fluctuations of cerebrospinal fluid PTGDS isoforms reflect both major and subtle brain pathophysiology.

  8. Proteomic Analysis of Cytokeratin Isoforms Uncovers Association with Survival in Lung Adenocarcinoma

    Directory of Open Access Journals (Sweden)

    Tarek G. Gharib

    2002-01-01

    Full Text Available Cytokeratins. (CK are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas. (64 stage I and 29 stage III and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, 19 occurred at significantly higher levels. (P<.05 in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms. (nos. 2165 and 2091, one of eight CK8 isoforms. (no. 439, one of three CK19 isoforms. (no. 1955 were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.

  9. Apical Scaffolding Protein NHERF2 Modulates the Localization of Alternatively Spliced Plasma Membrane Ca2+ Pump 2B Variants in Polarized Epithelial Cells*

    OpenAIRE

    Padányi, Rita; Xiong, Yuning; Antalffy, Géza; Lór, Krisztina; Pászty, Katalin; STREHLER, EMANUEL E.; Enyedi, Ágnes

    2010-01-01

    The membrane localization of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) in polarized cells is determined by alternative splicing; the PMCA2w/b splice variant shows apical localization, whereas the PMCA2z/b and PMCA2x/b variants are mostly basolateral. We previously reported that PMCA2b interacts with the PDZ protein Na+/H+ exchanger regulatory factor 2 (NHERF2), but the role of this interaction for the specific membrane localization of PMCA2 is not known. Here we show that co-expressio...

  10. A New View of Ras Isoforms in Cancers.

    Science.gov (United States)

    Nussinov, Ruth; Tsai, Chung-Jung; Chakrabarti, Mayukh; Jang, Hyunbum

    2016-01-01

    Does small GTPase K-Ras4A have a single state or two states, one resembling K-Ras4B and the other N-Ras? A recent study of K-Ras4A made the remarkable observation that even in the absence of the palmitoyl, K-Ras4A can be active at the plasma membrane. Importantly, this suggests that K-Ras4A may exist in two distinct signaling states. In state 1, K-Ras4A is only farnesylated, like K-Ras4B; in state 2, farnesylated and palmitoylated, like N-Ras. The K-Ras4A hypervariable region sequence is positively charged, in between K-Ras4B and N-Ras. Taken together, this raises the possibility that the farnesylated but nonpalmitoylated state 1, like K-Ras4B, binds calmodulin and is associated with colorectal and other adenocarcinomas like lung cancer and pancreatic ductal adenocarcinoma. On the other hand, state 2 may be associated with melanoma and other cancers where N-Ras is a major contributor, such as acute myeloid leukemia. Importantly, H-Ras has two, singly and doubly, palmitoylated states that may also serve distinct functional roles. The multiple signaling states of palmitoylated Ras isoforms question the completeness of small GTPase Ras isoform statistics in different cancer types and call for reevaluation of concepts and protocols. They may also call for reconsideration of oncogenic Ras therapeutics. PMID:26659836

  11. A Review of Metallothionein Isoforms and their Role in Pathophysiology

    Directory of Open Access Journals (Sweden)

    Senthil kumar M

    2011-05-01

    Full Text Available Abstract The Metallothionein (MT is a protein which has several interesting biological effects and has been demonstrated increase focus on the role of MT in various biological systems in the past three decades. The studies on the role of MT were limited with few areas like apoptosis and antioxidants in selected organs even fifty years after its discovery. Now acknowledge the exploration of various isoforms of MT such as MT-I, MT-II, MT-III and MT-IV and other isoforms in various biological systems. Strong evidence exists that MT modulates complex diseases and the immune system in the body but the primary function of MT still remains unknown. This review's main objective is to explore the capability to specifically manipulate MT levels in cells and in animals to provide answers regarding how MT could impact those complex disease scenarios. The experimental result mentioned in this review related among MT, zinc, cadmium, diabetic, heart disease, bone retardation, neuro toxicity, kidney dysfunction, cancer, and brain suggest novel method for exploration and contribute significantly to the growing scientist to research further in this field.

  12. Structures of alternatively spliced isoforms of human ketohexokinase.

    Science.gov (United States)

    Trinh, Chi H; Asipu, Aruna; Bonthron, David T; Phillips, Simon E V

    2009-03-01

    A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.

  13. Genome-Wide Functional Annotation of Human Protein-Coding Splice Variants Using Multiple Instance Learning.

    Science.gov (United States)

    Panwar, Bharat; Menon, Rajasree; Eksi, Ridvan; Li, Hong-Dong; Omenn, Gilbert S; Guan, Yuanfang

    2016-06-01

    The vast majority of human multiexon genes undergo alternative splicing and produce a variety of splice variant transcripts and proteins, which can perform different functions. These protein-coding splice variants (PCSVs) greatly increase the functional diversity of proteins. Most functional annotation algorithms have been developed at the gene level; the lack of isoform-level gold standards is an important intellectual limitation for currently available machine learning algorithms. The accumulation of a large amount of RNA-seq data in the public domain greatly increases our ability to examine the functional annotation of genes at isoform level. In the present study, we used a multiple instance learning (MIL)-based approach for predicting the function of PCSVs. We used transcript-level expression values and gene-level functional associations from the Gene Ontology database. A support vector machine (SVM)-based 5-fold cross-validation technique was applied. Comparatively, genes with multiple PCSVs performed better than single PCSV genes, and performance also improved when more examples were available to train the models. We demonstrated our p