• Title, Summary, Keyword: cyclinD1-p16

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Cyclin D1, Retinoblastoma and p16 Protein Expression in Carcinoma of the Gallbladder

  • Srivastava, Vineeta;Patel, Brijesh;Kumar, Mohan;Shukla, Mridula;Pandey, Manoj
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.5
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    • pp.2711-2715
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    • 2013
  • Background: Cancer of the gallbladder is a relatively rare neoplasm with a poor prognosis. The exact mechanisms of its genesis are not known and very little information is available on molecular events leading to labeling this as an orphan cancer. Materials and Methods: In this prospective case control study we evaluated the expression of p16, pRb and cyclin D1 by immunohistochemistry to study the G1-S cell-cycle check point and its possible role in gallbladder carcinogenesis. A total of 25 patients with gallbladder carcinoma (group I), 25 with cholelithiasis (group II) and 10 normal controls. were enrolled Results: Cyclin D1 expression was seen in 10 (40%) patients each with carcinoma and cholelithiasis while only in 2 (20%) of the normal gallbladders but differences were not statistically significant (p value=0.488). p16 was expressed in 12% patients of carcinoma of the gallbladder and 28% of cholelithiasis, however this difference was not statistically significant (p value=0.095). Retinoblastoma protein was found to be expressed in 50% of normal gallbladders and 6 (24%) of carcinoma and 8 (32%) of gallstones. The present study failed to demonstrate any conclusive role of cyclin D1/RB/ p16 pathway in carcinoma of the gallbladder. Conclusions: The positive relation observed between tumor metastasis and cyclinD1 expression and p16 with nodal metastasis suggested that higher cyclin D1/p16 expression may act as a predictive biomarker for aggressive behavior of gallbladder malignancies.

Immunohistochemical Prognostic Factors in Soft Tissue Sarcoma (연부조직 육종에서 면역조직화학적 예후인자)

  • Choi, Kyung-Un;Kim, Jeung-Il;Moon, Nam-Hoon
    • The Journal of the Korean bone and joint tumor society
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    • v.14 no.2
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    • pp.106-118
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    • 2008
  • Purpose: Disturbed cell cycle regulatory proteins are key events underlying the development and/or progression of human malignancies. The aim of this study is to evaluate the protein expression status involved in G1/S cell cycle in human soft tissue sarcoma. Materials and Methods: We simultaneously evaluated the expression of Cyclin D1, Cyclin E, CDK4, CDK2, p16, p27, Rb, E2F1, p53 and Ki-67 by immunohistochemistry in 43 cases of soft tissue sarcoma Results: The Cyclin D1, Cyclin E, CDK4, CDK2, E2F1, and p53 were expressed in 25 (58.1%), 18 (41.9%), 13 (30.2%), 33 (76.7%), 20 (46.5%), and 18 cases (41.9%). The p16, p27, and Rb expressions were decreased in 26 (60.5%), 22 (51.2%) and 19 cases (44.2%). All of the cases showed alterations of more than one out of the above proteins. The increased Cyclin E expression and Ki-67 labeling index (LI) were significantly associated with histologic grade. The Cyclin E and E2F-1 expressions were increased in relapsed cases and the CDK4 expression was increased in cases of metastasis. Conclusion: Alterations of G1/S cell cycle regulatory proteins may play an important role in the tumoriogensis of soft tissue sarcomas. Our results suggest that increased expressions of Cyclin E, E2F1, and CDK4 were associated with tumor relapse or metastasis and could be considered as parameters of prognosis of soft tissue sarcoma.

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Rabbit Antibody Raised against Murine Cyclin D3 Protein Overexpressed in Bacterial System

  • Jun, Do-Youn;Kim, Mi-Kyung;Kim, Young-Ho
    • Journal of Microbiology and Biotechnology
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    • v.6 no.6
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    • pp.474-481
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    • 1996
  • Since the commercially available rabbit anti-cyclin D3, generated from c-terminal 16 amino acid residues which are common to human and murine cyclin D3, is highly cross-reactive with many other cellular proteins of mouse, a new rabbit polyclonal anti-cyclin D3 has been raised by using murine cyclin D3 protein expressed at a high level in Escherichia coli as the immunogen. To express murine cyclin D3 protein in E. coli, the cyclin D3 cDNA fragment encoding c-terminal 236 amino acid residues obtained by polymerase chain reaction (PCR) was inserted into the NcoI/BamHI site of protein expression vector, pET 3d. Molecular mass of the cyclin D3 overexpressed in the presence of IPTG (Isopropyl $\beta$-D-thiogalactopyranoside) was approximately 26 kDa as calculated from the reading frame on the DNA sequence, and the protein was insoluble and mainly localized in the inclusion bodies that could be easily purified from the other cellular soluble proteins. When renaturation was performed following denaturation of the insoluble cyclin D3 protein in the inclusion bodies using guanidine hydrochloride, 4.4 mg of soluble form of cyclin D3 protein was produced from the transformant cultured in 100ml of LB media under the optimum conditions. Four-hundred micrograms of the soluble form of cyclin D3 protein was used for each immunization of a rabbit. When the antiserum obtained 2 weeks after tertiary immunization was applied to Western blot analysis, it was able to detect 33 kDa cyclin D3 protein in both murine lymphoma cell line BW5147.G.1.4 and human Jurkat T cells at 3,000-fold dilution with higher specificity to murine cyclin D3, demonstrating that the new rabbit polyclonal anti-murine cyclin D3 generated against c-terminal 236 amino acid residues more specifically recognizes murine cyclin D3 protein than does the commercially available rabbit polyclonal antibody raised against c-terminal 16 amino acids residues.

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A Study of Influences of p53 Mutation, Cyclin D1 Over Expression, Ki67 Index, Mitotic Index on the Prognosis of Esophageal Squamous Cell Carcinoma (p53 변이, Cyclin D1의 과발현, Ki67 지수, 세포분열지수가 식도의 편평상피암의 예후에 미치는 영향)

  • Lee Hae Won;Cho Suk Ki;Sung Sook Whan;Lee Hyun Joo;Kim Young Tae;Kang Moon Chul;Kim Joo Hyun
    • The Korean Journal of Thoracic and Cardiovascular Surgery
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    • v.38 no.12
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    • pp.835-843
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    • 2005
  • Background: Cancer of the esophagus is one of the most malignant tumors with poor prognosis. The p53 gene alteration, over expression of Cyclin D1, and Ki67 index were thought to be the prognostic factors. However, their clinical significances in esophageal squamous cell carcinoma are controversial and p53 accumulation may not correlate with genetic mutation. The current study investigates their prognostic significance in squamous cell carcinoma of the esophagus. Material and Method: The Subjects studied were 124 esophageal squamous cell carcinoma patients who underwent esophagectomy. The mutation of p53, over expression of Cyclin D1, Ki67 labelling index, mitotic index were examined by using an immunohistochemical staining. We compared the results and investigated the correlation with the mutation of p53, overexpression of Cyclin D1, Ki67 labelling index, mitotic index and tumor size, and duration of survival. Result: There was no correlation between the results in immunohistochemical staining according to age, sex, tumor size, Iymph node status, and clinical stage of the disease. Mutant p53 protein was found in 69 cases (55.6$\%$). Median survival time was 21 months in cases with negative for mutant p53 protein and 22 months in positive cases. There was no significant difference in survival (p=0.46). Median survival time was 22 months in cases with negative for Cyclin D1 and 16 months in positive cases (p=0.18). Median and mean survival time was 22 months and 36 months when Ki67 labeling index was 40 or less (102 cases). Median and mean survival was 16 months and 23 months, when Ki67 labeling index was more than 40 (22 cases). There was significant difference in survival rate (p=0.011). Conciusion: Positivity of p53 and cyclin D1 was not useful in predicting the prognosis in our study. There was no significant correlation among mutant p53 protein accumulation, Cyclin D1 over expression, and Ki67 labeling index. However, in several studies, PCR single strand conformational polymorphism analysis of p53 showed a correlation to the prognosis. We thought that there was a significant discordance between p53 gene mutation and mutant p53 protein accumulation. When Ki67 labeling index was more than 40, prognosis was poorer, Ki67 seems to be a prognostic factor in our study. Therefore, we confirmed the possibility of using molecular markers as prognostic factors.

Anti-Proliferation Effects and Molecular Mechanisms of Action of Tetramethypyrazine on Human SGC-7901 Gastric Carcinoma Cells

  • Ji, Ai-Jun;Liu, Sheng-Lin;Ju, Wen-Zheng;Huang, Xin-En
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.8
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    • pp.3581-3586
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    • 2014
  • Aim: To investigate the effects of tetramethypyrazine (TMP) on proliferation and apoptosis of the human gastric carcinoma cell line 7901 and its possible mechanism of action. Methods: The viability of TMP-treated 7901 cells was measured with a 3-(4, 5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) and cell apoptosis was analyzed by flow cytometry. The distribution of cells in different phases of cell cycle after exposure of TMPs was analyzed with flow cytometry. To investigate the molecular mechanisms of TMP-mediated apoptosis, the expression of NF-${\kappa}Bp65$, cyclinD1 and p16 in SGC-7901 cells was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Results: TMP inhibited the proliferation of human gastric carcinoma cell line 7901 in dose and time dependent manners. Cell growth was suppressed by TMP at different concentrations (0.25, 0.5, 1.0, 2.0 mg/ml), the inhibition rate is 0.46%, 4.36%, 14.8%, 76.1% (48h) and 15.5%, 18.5%, 41.2%, 89.8% (72h) respectively. When the concentration of TMPs was 2.0mg/ml, G1-phase arrest in the SGC-7901 cells was significant based on the data for cell cycle distribution. RT-PCR demonstrated that NF-${\kappa}Bp65$ and cyclin D1 mRNA expression was significantly down-regulated in 7901 cells treated with 2.0 mg/ml TMP for 72h (p<0.05), while the p16 mRNA level was up-regulated (p<0.05). The protein expression of NF-${\kappa}Bp65$ and cyclin D1 decreased gradually with the increase in TMP concentration, compared with control cells (p<0.05), while expression of protein p16 was up-regulated (p<0.01). Conclusion: TMP exhibits significant anti-proliferative and pro-apoptotic effects on the human gastric carcinoma cell line SGC-7901. NF-${\kappa}Bp65$, cyclinD1 and p16 may also play important roles in the regulation mechanisms.

Retinoic Acid Increases the Cell Cycle Progression of Human Gingival Fibroblasts by Increasing Cyclin E and CDK 2 Expression and Decreasing $p21^{WAF1/CIP1}$ and $p16^{INK4A}$ Expression

  • You, Hyung-Keun;Seo, Se-Jeong;Kim, Kang-Ju;Choi, Na-Young;You, Yong-Ouk
    • International Journal of Oral Biology
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    • v.37 no.3
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    • pp.115-120
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    • 2012
  • Retinoic acid plays an important role in the regulation of cell growth and differentiation. In our present study, we evaluated the effects of all-trans retinoic acid (RA) on cell proliferation and on the cell cycle regulation of human gingival fibroblasts (HGFs). Cell proliferation was assessed using the MTT assay. Cell cycle analysis was performed by flow cytometry, and cell cycle regulatory proteins were determined by western blot. Cell proliferation was increased in the presence of a 0.1 nM to 1 ${\mu}M$ RA dose range, and maximal growth stimulation was observed in cells exposed to 1 nM of RA. Exposure of HGFs to 1 nM of RA resulted in an augmented cell cycle progression. To elucidate the molecular mechanisms underlying cell cycle regulation by RA, we measured the intracellular levels of major cell cycle regulatory proteins. The levels of cyclin E and cyclin-dependent kinase (CDK) 2 were found to be increased in HGFs following 1 nM of RA treatment. However, the levels of cyclin D, CDK 4, and CDK 6 were unchanged under these conditions. Also after exposure to 1 nM of RA, the protein levels of $p21^{WAF1/CIP1}$ and $p16^{INK4A}$ were decreased in HGFs compared with the control group, but the levels of p53 and pRb were similar between treated and untreated cells. These results suggest that RA increases cell proliferation and cell cycle progression in HGFs via increased cellular levels of cyclin E and CDK 2, and decreased cellular levels of $p21^{WAF1/CIP1}$ and $p16^{INK4A}$.

Ginsenoside Rh2 inhibits proliferation of human promyelocytic HL-60 leukemia cells via $G_0/G_1$ phase arrest and induction of differentiation

  • Cho, Seoung-Hee;Kim, Dong-Hyun;Lee, Kyung-Tae
    • Proceedings of the Ginseng society Conference
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    • pp.3-12
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    • 2006
  • 1 The present work was performed to investigate the effects of ginsenoside Rh2 on proliferation, cell cycle-regulation and differentiation of human leukemia HL-60 cells as well as the underlying mechanisms for these effects. 2 Ginsenoside Rh2 potently inhibited the proliferation of HL-60 cells in both a dose- and time-dependent manner with an $IC_{50}$, $20{\mu}M$. 3 DNA flow-cytometry indicated that ginsenoside Rh2 markedly induced a $G_1$ phase arrest of HL-60 cells. 4 Among the $G_1$ phase cell cycle-related proteins, the levels of cyclin-dependent kinase(CDK)4, 6 and cyclin D1, cyclin D2, cyclin D3 were reduced by ginsenoside Rh2, whereas the steadystate levels of CDK2 and cyclin E were unaffected. 5 The protein levels of a CDK inhibitor p16, $p21^{CIP1/WAF1}$ and $p27^{KIP1}$ were markedly increased by ginsenoside Rh2. 6 Ginsenoside Rh2 markedly enhanced the binding of $p21^{CIP1/WAF1}$ and $p27^{KIP1}$ with CDK2 and CDK6, resulting in the reduced activity of both kinases and the hypophosphorylation of Rb protein. 7 We furthermore suggest that ginsenoside Rh2 is a potent inducer of the differentiation of HL-60 cells, based on observations such as a reduction of the nitroblue tetrazolium level, an increase in the esterase activities and phagocytic activity, morphology changes, and the expression of CD11b, CD14, CD64 and CD66b surface antigens. 8 In conclusion, the onset of ginsenoside Rh2-induced the $G_0/G_1$ arrest of HL-60 cells prior to the differentiation is linked to a sharp up-regulation of the $p21^{CIP1/WAF1}$ level and a decrease in the CDK2, CDK4 and CDK6 activities. This is the first report demonstrating that ginsenoside Rh2 potently inhibits the proliferation of human promyelocytic HL-60 cells via the $G_1$ phase cell cycle arrest and differentiation induction.

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Tumor suppressor $p16^{INK4a}$ in Cancer

  • Lee, Mee-Hyun;Choi, Bu-Young;Surh, Young-Joon
    • Environmental Mutagens and Carcinogens
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    • v.25 no.3
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    • pp.87-96
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    • 2005
  • [ $p16^{INK4a}$ ] is a tumor suppressor that belongs to the INK4 family of the cyclin D-dependent kinases (cdk) inhibitors. It plays regulatory roles in cell proliferation and in tumorigenesis by interacting with Rb signaling. Abnormally elevated $p16^{INK4a}$ protein expression causes cell cycle arrest (G1/S transition) and loss of cyclin-cdk activity. In many cancers, $p16^{INK4a}$ is altered by mutation, deletion, and promoter methylation. This review summarizes the function of p16 as an important regulator of cancer pathobiology and a promising target fer developing cancer therapeutic and chemopreventive agents.

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Viscum Album Var Hot Water Extract Mediates Anti-cancer Effects through G1 Phase Cell Cycle Arrest in SK-Hep1 Human Hepatocarcinoma cells

  • Cruz, Joseph Flores dela;Kim, Yeon Soo;Lumbera, Wenchie Marie Lara;Hwang, Seong Gu
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.15
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    • pp.6417-6421
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    • 2015
  • Viscum album var (VAV) also known as mistletoe, has long been categorized as a traditional herbal medicine in Asia. In addition to its immunomodulating activities, mistletoe has also been used in the treatment of chronic hepatic disorders in China and Korea. There are numerous reports showing that VAV possesses anti-cancer effects, however influence on human hepatocarcinoma has never been elucidated. In the present study, hot water extracts of VAV was evaluated for its potential anti-cancer effect in vitro. SK-Hep1 cells were treated with VAV (50-400ug/ml) for both 24 and 48 hours then cell viability was measured by cell counting kit-8 (CCK-8). Flow cytometry analysis was used to measure the proportion of SK-Hep1 in the different stages of cell cycle. RT-PCR and Western blot analysis were conducted to measure expression of cell cycle arrest related genes and proteins respectively. VAV dose dependently inhibited the proliferation of SK-Hep1 cells without any cytotoxicity with normal Chang liver cell (CCL-13). Flow cytometry analysis showed that VAV extract inhibited the cell cycle of SK-Hep1 cells via G1 phase arrest. RT-PCR and Western blot analysis both revealed that cyclin dependent kinase 2 (Cdk2) and cyclin D1 gene expression were significantly down regulated while p21 was upregulated dose dependently by VAV treatment. Combined down regulation of Cdk2, Cyclin D1 and up regulation of p21 can result in cell death. These results indicate that VAV showed evidence of anti-cancer activity through G1 phase cell cycle arrest in SK-Hep1 cells.

Cha-ga Mushroom Water Extract induces G0/G1 Arrest in B16-F10 Melanoma cells (차가버섯추출물에 의한 흑색종의 세포주기 억제효과)

  • Youn, Myung-Ja;Song, Jeong-Hoon
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.21 no.1
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    • pp.204-208
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    • 2007
  • Chaga mushroom extract is well known as immune modulator and anti-cancer agent. However, the molecular mechanism by which Chaga exerts cell cycle arrest and apoptosis of cancer cells is poorly understood. In this study, we demonstrated anti-proliferative effects of Chaga extract on murine melanoma B16 cells. Chaga extract dose-dependently inhibited cell growth along with the arrest of G0/G1 phase and the induction of apoptotic cell death. Treatment with Chaga extract resulted in a decrease of cyclin E, cyclin D1, cdk 2, cdk 4 expression levels. Furthermore, in vivo inoculation study of B16 melanoma cells into Balb/c mice Chaga extract markedly suppressed the metastatic growth of tumor cells (6 folds, p<0.05,). These results indicate that Chaga mushroom extract induces apoptosis of B16 melanoma cells through arrest of G0/G1 phase in cell cycle.