Involvement of p27Kip1 in G1 arrest by high dose 5 alpha-dihydrotestosterone in LNCaP human prostate cancer cells

The cell cycle is governed by cyclin dependent kinases (cdks), which are activated by binding of cyclins, inhibited by cdk inhibitors and regulated by phosphorylation and dephosphorylation. Exposure to high dose dihydrotestosterone (DHT) inhibits population growth of the human prostate carcinoma cell line, LNCaP. To determine the mechanism of growth arrest by high dose DHT, we assayed the changes in cell cycle profile and the cell cycle regulators that mediate these effects. Treatment of asynchronously growing LNCaP cells with 100 nM DHT caused a G1 arrest. The proportion of cells in S phase fell from 22 to 2%, while the G1 fraction rose from 74 to 92% by 24 h. Loss of phosphorylation of the retinoblastoma protein was noted and cdk4 and cyclin E/ cdk2 activities fell. Inhibition of these G1 cyclin dependent kinases was not due to loss of either cyclin or cdk proteins nor to increases in the cdk inhibitors p16INK4A and p21CiP1. p21Cip1 protein levels remained constant, and cyclin E-associated p21CiP1 fell, suggesting that p21CiP1 is not relevant to this form of cyclin E/cdk2 inhibition. Of note, total p27KiP1 levels and cyclin E-associated p27Kip1 increased as cells arrested and the amount of the CAK activated cdk2 bound to cyclin E decreased. p27KiP1 immunodepletion experiments demonstrated that the DHT-mediated increase in p27Kip1 was sufficient to fully saturate and inhibit target cyclin E/ cdk2. The inhibition of cyclin E/cdk2 by p27Kip1 contributes to G1 arrest of LNCaP following high dose DHT. p27KiP1 may be a key effector of androgen dependent growth modulation in prostate cancer cells.     

UVB induced cell cycle checkpoints in an early stage human melanoma line, WM35

The activation of cell cycle checkpoints in response to genotoxic stressors is essential for the maintenance of genomic integrity. Although most prior studies of cell cycle effects of UV irradiation have used UVC, this UV range does not penetrate the earth's atmosphere. Thus, we have investigated the mechanisms of ultraviolet B (UVB) irradiation-induced cell cycle arrest in a biologically relevant target cell type, the early stage human melanoma cell line, WM35. Irradiation of WM35 cells with UVB resulted in arrests throughout the cell cycle: at the G1/S transition, in S phase and in G2. G1 arrest was accompanied by increased association of p21 with cyclin E/cdk2 and cyclin A/cdk2, increased binding of p27 to cyclin E/cdk2 and inhibition of these kinases. A loss of Cdc25A expression was associated with an increased inhibitory phosphotyrosine content of cyclin E- and cyclin A-associated cdk2 and may also contribute to G1 arrest following UVB irradiation. The association of Cdc25A with 14-3-3 was increased by UVB. Reduced cyclin D1 protein and increased binding of p21 and p27 to cyclin D1/cdk4 complexes were also observed. The loss of cyclin D1 could not be attributed to inhibition of either MAPK or PI3K/PKB pathways, since both were activated by UVB. Cdc25B levels fell and the remaining protein showed an increased association with 14-3-3 in response to UVB. Losses in cyclin B1 expression and an increased binding of p21 to cyclin B1/cdk1 complexes also contributed to inhibition of this kinase activity, and G2/M arrest. Oncogene (2000) 19, 4480 - 4490.     

Resveratrol causes WAF-1/p21-mediated G(1)-phase arrest of cell cycle and induction of apoptosis in human epidermoid carcinoma A431 cells.

Resveratrol (trans-3,4',5,-trihydroxystilbene), a phytoalexin found in grapes, nuts, fruits, and red wine, is a potent antioxidant with cancer-preventive properties. The mechanism by which resveratrol imparts cancer chemopreventive effects is not clearly defined. Here, we demonstrate that resveratrol, via modulations in cyclin-dependent kinase (cdk) inhibitor-cyclin-cdk machinery, results in a G(1)-phase arrest of the cell cycle followed by apoptosis of human epidermoid carcinoma (A431) cells. Resveratrol treatment (1-50 microM for 24 h) of A431 cells resulted in a dose-dependent (a) inhibition of cell growth as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, (b) G(1)-phase arrest of the cell cycle as shown by DNA cell cycle analysis, and (c) induction of apoptosis as assessed by ELISA. The immunoblot analysis revealed that resveratrol treatment causes a dose- and time-dependent (a) induction of WAF1/p21; (b) decrease in the protein expressions of cyclin D1, cyclin D2, and cyclin E; and (c) decrease in the protein expressions of cdk2, cdk4, and cdk6. Resveratrol treatment was also found to result in a dose- and time-dependent decrease in kinase activities associated with all of the cdks examined. Taken together, our study suggests that resveratrol treatment of the cells causes an induction of WAF1/p21 that inhibits cyclin D1/D2-cdk6, cyclin D1/D2-cdk4, and cyclin E-cdk2 complexes, thereby imposing an artificial checkpoint at the G(1)-->S transition of the cell cycle. This series of events results in a G(1)-phase arrest of the cell cycle, which is an irreversible process that ultimately results in the apoptotic death of cancer cells. To our knowledge, this is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of cancer cells by resveratrol.     

Alterations of p53, pRb, cyclin D(1) and cdk4 in human oral and pharyngeal squamous cell carcinomas

Alteration of expression of tumour suppressor genes and cell cycle regulators may be responsible for oral and pharyngeal cancer development. We have studied the expression of p53, pRb, cyclin D(1) and cdk4 in 53 cases of oral and pharyngeal squamous cell carcinomas using immunohistochemistry. Tumour expression of all nuclear proteins was scored according to the percentage of positive cancer nuclei. Positive p53 expression was detected in 27/53 (50.94%) cases. Lack of pRb immunostaining was observed in 39/53 (73.58%) cases. Overexpression of cyclin D(1) was shown in 21 (39.62%) tumours. The overexpression of cdk4 was detected in 43/53 (81.13%) cases. There was no significant association among these cell cycle regulatory proteins. This implies that the aberration of an important cell cycle regulator may be sufficient to disrupt regulatory mechanism in a manner favouring tumourigenesis. In summary, our results suggest that inappropriate expression of p53, pRb, cyclin D(1) or cdk4 is likely to contribute to the development of oral and pharyngeal cancers. The lack of pRb expression and/or overexpression of cdk4 play a crucial role in the development of this malignancy.     

Expression of G1 phase regulators in MG-63 osteosarcoma cell line.

Cyclins and cyclin-dependent kinases (cdks) form complexes that govern transitions during cell cycle phases. In this study we characterized a human osteosarcoma cell line, MG-63, for the expression level of cyclin D1, cyclin E, cdk4, cdk2, and cell cycle inhibitors pRb and p21. To investigate the role of these proteins we treated MG-63 cells with tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6). Cell proliferation analysis demonstrated an increased proliferation of MG-63 cells with IL-6, while TNF-alpha acted as an anti-proliferative agent. Immunoblotting revealed an increased expression of p21 with TNF-alpha and its complex with cdk2. TNF-alpha reduced the expression of the cyclin E-cdk2 complex. TNF-alpha did not affect the amount of cyclin D1, cyclin E, cdk4, cdk2, and of cyclin D1-cdk4 complex. IL-6 decreased p21 expression and its complex with cdk2, while it increased the cyclin E-cdk2 complex. Cyclin D1 and cdk4 expression and their complex did not change after IL-6 treatment, nor did cyclin E and cdk2 protein expression. Hyperphosphorylated/dephosphorylated Rb protein ratio was reduced with TNF-alpha whereas it increased with IL-6. These results may suggest an important role of p21 and of cyclin E-cdk2 complex in the G1 phase regulation through pRb phosphorylation in MG-63 cells.     

cyclin E和cdk2在肺癌中表达及其临床意义

目的 :探讨细胞周期调控因子在肺癌发病中的作用。方法 :应用免疫组化方法检测细胞周期蛋白E(cyclinE)和细胞周期蛋白依赖性激酶 2 (cdk2 )在 38例肺癌和 6例正常肺组织中的表达状况。结果 :cyclinE和cdk2在肺癌中阳性率分别为 6 0 .5%、6 5.8% ,显著高于正常肺组织 (P <0 .0 5) ;cdk2表达与肺癌分期密切相关 (P <0 .0 5) ,而cyclinE表达与肺癌分期、淋巴结转移和病理分型均无相关性 (P>0 .0 5) ;肺癌中两者共表达率为 31.8%。结论 :cyclinE和cdk2参与肺癌的发生发展过程 ,但可能不是反映肺癌生物学特征有价值的标记物。     

PKCeta associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/cdk2/p21 complex. Among the 10 isoforms of PKC, PKCeta is predominantly expressed in squamous cell epithelia and induces terminal differentiation of keratinocytes. PKCeta that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCeta and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCeta with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCeta with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCeta associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated. Oncogene (2000) 19, 6334 - 6341.     

Monoterpenes inhibit proliferation of human colon cancer cells by modulating cell cycle-related protein expression

The monoterpene perillyl alcohol (POH) is a naturally occurring anti-cancer compound which is effective against a variety of rodent organ-specific tumor models. To establish the molecular mechanisms of POH and its major metabolite perillic acid (PA) as anti-proliferative agents, their effects on cell proliferation, cell cycle and cell cycle regulatory proteins were studied in HCT 116 human colon cancer cells. POH, and to a lesser extent, PA, exerted a dose-dependent inhibitory effect on cell growth correlated with a G1 arrest. Analysis of G1 cell cycle regulators expression revealed that monoterpenes increased expression of cdk inhibitor p21(Waf1/Cip1) and cyclin E, and decreased expression of cyclin D1, cyclin-dependent kinase (cdk) 4 and cdk2. Our results suggest that monoterpenes induce growth arrest of colon cancer cells through the up-regulation of p21(Waf1/Cip1) and the down-expression of cyclin D1 and its partner cdk4.     

Cell cycle genes as targets of retinoid induced ovarian tumor cell growth suppression.

We have examined the effect of all-trans-retinoic acid (RA) on cell cycle gene expression in RA sensitive CA-OV3 and RA resistant SK-OV3 ovarian carcinoma cell lines. Gene expression was analysed by multiprobe RNAse protection, Western blotting and in vitro kinase assays. No differences were observed between RA sensitive and RA resistant ovarian carcinoma cells in the levels of expression of many cell cycle genes including cyclin A, B and E, cdk 2,4 and 6, E2F-1, E2F-2, E2F-3, E2F-4, E2F-5, DP-1 and DP-2. However, RA sensitive CA-OV3 cells expressed higher levels of p53, p27, p21, and p16 compared to RA resistant SK-OV3 cells. In addition, RA treatment of CA-OV3 cells resulted in a significant decrease in hyperphosphorylated RB and RB-2/p130 and corresponding significant increases in the levels of hypophosphorylated and/or partially phosphorylated RB-2/p130 protein and hypophosphorylated RB. Also, RA treatment increased expression of the cdk inhibitor p27 and decreased activity of cdk 2, cdk 4 and cdk 6. Finally, amounts of p27-cyclin E and RB-2/p130-E2F4 complexes were found to increase in CA-OV3 cells growth arrested by RA. These results suggest that the pocket protein pathways are critical targets for retinoid suppression of ovarian carcinoma cell growth.     

Lower cyclin H and cyclin-dependent kinase-activating kinase activity in cell cycle arrest induced by lack of adhesion to substratum

Knowledge about adhesion checkpoints is important to counteract dissemination of cells from solid tumors. Lack of anchorage in adherent cells is associated with growth arrest and inhibition of cyclin-dependent kinases (cdks) required to drive cell cycle progression. Because cyclin-cdk complex activation requires CDK-activating kinase comprising cdk7 and cyclin H, we now investigated their relationship to decreased proliferation by lack of cell spreading. This report shows that either UV irradiation on an adhesive substrate or culture on a nonadhesive substrate produced K1735 melanoma growth arrest. Inhibition of proliferation by UV primarily induced the cdk inhibitor p21WAF1 without a significant effect on cyclin H and cdk7. In contrast, lack of adhesion to substratum decreased cyclin H but not cdk7 with accumulation of a slower migrating, presumably unphosphorylated cdk4 isoform. These results were paralleled by decreased cdk7-mediated phosphorylation of GST-cdk2 and lower activation of a baculovirus-derived cdc2-cyclin B kinase complex. This is the first report showing that cyclin H-mediated down-regulation of cdk-activating kinase activity is involved in growth arrest induced by lack of anchorage.     

Analysis of cyclin E and CDK2 in ovarian cancer: Gene amplification and RNA overexpression

Cyclins and their associated kinases (cdks) play a key role in controlling the cell cycle, a process whose disregulation can potentially lead to uncontrolled cell growth and hence to cancer. We have studied the role of both cyclin E and its associated kinase cdk2 in ovarian cancer. Primary, metastatic, recurrent and benign ovarian tumors were screened for cyclin E and cdk2 gene amplification. Cyclin E was shown to be amplified in 21% and cdk2 in 6.4% of the cases analyzed. Cyclin E and cdk2 RNA expression levels were determined by semi-quantitative RT-PCR analysis in a partially overlapping series of samples and compared to the expression levels of normal ovarian surface epithelial cells. Cyclin E RNA was overexpressed in 29.5% and cdk2 in 6.5% of ovarian tumors tested. We determined that in most cases gene amplification leads to higher RNA levels for cyclin E and that the overall levels of cyclin E and cdk2 RNA were correlated. We hypothesize that cyclin E and cdk2 are, in part co-regulated and that they may concur to ovarian tumor development. Int. J. Cancer 75:34–39, 1998.© 1998 Wiley-Liss, Inc.     

Cell cycle regulatory protein expression in fresh acute myeloid leukemia cells and after drug exposure.

Characteristics of treatment-induced cell cycle arrest are important for in vitro and in vivo sensitivity of acute myeloid leukemia (AML) cells to cytotoxic drugs. We analyzed the expression of the major G1 cell cycle regulators (p21Cip1, p27Kip1, cyclins D, cyclin E and pRb) in 41 fresh AML cell samples. The level of p27 expression was the only factor correlated with the response to chemotherapy, a high level of p27 expression being predictive of complete remission. There was a close relation between expression of pRb, cyclin D2 and FAB subtype, illustrated by the absence of both proteins in most samples having a monocytic component (M4, M5). We also assessed the expressions of pRb, cyclin E, p21 and p27 and the activity of cdk2, the major regulator of S-phase entry, after exposure to cytosine-arabinoside (AraC) and daunorubicin (DNR), and found these proteins could characterize time- and dose-dependent cellular response to each drug. We observed hyperphosphorylated pRb, increased levels of cyclin E and a high cdk2 activity, but no p21 induction, in AML cells exposed to 10(-6) M AraC. After exposure to 10(-5) M AraC, corresponding to the serum concentration reached in high-dose AraC regimens (HDAraC), a strong p21 induction was observed, associated with similarly overexpressed cyclin E and even higher cdk2 activity than after 10(-6) M AraC, while apoptosis was significantly increased. These data suggest that cdk2 activity is likely to play a role in AraC-induced apoptosis in AML cells. This mechanism may account for high efficacy of HDAraC in cells showing little sensitivity to conventional AraC doses.     

p16INK4A mediates cyclin dependent kinase 4 and 6 inhibition in senescent prostatic epithelial cells

The senescence checkpoint constrains the proliferative potential of normal cells in culture to a finite number of cell doublings. In this study, we investigated the mechanism of cyclin dependent kinase (cdk) inhibition in senescent human prostatic epithelial cells (HPECs). Progression of HPECs from early passage to senescence was accompanied by a gradual loss of cells in S phase and an accumulation of cells containing 2N DNA. Furthermore, G1-S phase-associated kinase activities progressively diminished with increasing cell passage. In senescent HPECs, cdk4 and cyclin E1- and A-associated kinases were catalytically inactive. In contrast to observations in senescent fibroblasts, levels of the kinase inhibitor protein (KIP) inhibitor p21CIP1 diminished over the proliferative life span of HPECs. p27KIP1 levels fell as cells approached senescence, and the association of both p21CIP1 and p27KIP1 with cdk4/6 complexes was decreased. However, the level of cyclin E1-associated KIP molecules was unaltered as cells progressed into senescence. Progression to senescence was accompanied by a progressive increase in both the level of p16(INK4A) and in its association with cdk4 and cdk6. As HPECs approached senescence, cdk4- and cdk6-bound p16(INK4A) showed a shift to a slower mobility due to a change in its phosphorylation profile. As p16(INK4A) increased in cdk4 and cdk6 complexes, there was a loss of cyclin D1 binding. The altered phosphorylation of p16(INK4A) in senescent prostatic epithelial cells may facilitate its association with cdk4 and cdk6 and play a role in the inactivation of these kinases.     

cdk2 和cyclin E 在肾细胞癌中表达的意义

 目的 探讨细胞周期蛋白依赖性激酶2(cdk2)和细胞周期蛋白E(cyclin E)在肾细胞癌发病机制中的作用及其临床意义。方法 应用免疫组化方法检测cdk2、cyclin E和增殖细胞核抗原(PCNA)在48例肾细胞癌和12例正常骨组织中的表达。结果 肾细胞癌中cdk2和cyclin E表达均显著高于正常肾组织(P<0．05)。且与PCNA指数密切显著相关。但与肿瘤分级、分期、淋巴结转移及病理类型之间无显著性相关。肾细胞癌中cdk2和cyclin E共同高表达率达47．1％，且与肿瘤分级、分期和淋巴结转移之间呈正相关。结论 cdk2／cyclin E复合高表达可能在肾细胞癌发病过程中起着重要作用，联合检测两者表达水平对判断肾细胞癌生物学行为有较重要的意义。