Lycopene inhibition of cell cycle progression in breast and endometrial cancer cells is associated with reduction in cyclin D levels and retention of p27(Kip1) in the cyclin E-cdk2 complexes.

Numerous studies have demonstrated the anticancer activity of the tomato carotenoid, lycopene. However, the molecular mechanism of this action remains unknown. Lycopene inhibition of human breast and endometrial cancer cell growth is associated with inhibition of cell cycle progression at the G(1) phase. In this study we determined the lycopene-mediated changes in the cell cycle machinery. Cells synchronized in the G(1) phase by serum deprivation were treated with lycopene or vehicle and restimulated with 5% serum. Lycopene treatment decreased serum-induced phosphorylation of the retinoblastoma protein and related pocket proteins. This effect was associated with reduced cyclin-dependent kinase (cdk4 and cdk2) activities with no alterations in CDK protein levels. Lycopene caused a decrease in cyclin D1 and D3 levels whereas cyclin E levels did not change. The CDK inhibitor p21(Cip1/Waf1) abundance was reduced while p27(Kip1) levels were unaltered in comparison to control cells. Serum stimulation of control cells resulted in reduction in the p27 content in the cyclin E--cdk2 complex and its accumulation in the cyclin D1--cdk4 complex. This change in distribution was largely prevented by lycopene treatment. These results suggest that lycopene inhibits cell cycle progression via reduction of the cyclin D level and retention of p27 in cyclin E--cdk2, thus leading to inhibition of G(1) CDK activities.     

BAD/BCL-[X(L)] heterodimerization leads to bypass of G0/G1 arrest

The pro-apoptotic molecule BAD binds BCL-[X(L)] or BCL2 and inactivates their survival function. In addition to their anti-apoptotic function, BCL2 and BCL-[X(L)] also delay cell cycle entry from quiescence. We found that the BH3-only molecule BAD also exerted a cell cycle effect. BAD expression resulted in failure to cell cycle block in growth arrest conditions. In low serum and in confluence, fibroblasts constitutively or inducibly expressing BAD persisted in S phase, continued to incorporate BrdU, and exhibited sustained cyclin E/cdk2 activity. Mutation analysis indicated that the cell cycle effect of BAD was not dependent on its phosphorylation status or subcellular localization, but strictly co-segregated with BCL-[X(L)] binding. bclx(-/-) MEFs expressing BAD and bad(-/-) MEFs both arrested in G0/G1 in low serum similar to wild-type controls, suggesting that the ability to overcome the G0/G1 checkpoint resulted from the presence of BAD/BCL-x(L) heterodimers, rather than the absence of BCL-[X(L)] or BAD. These data provide evidence that in addition to regulating apoptosis, the BAD/BCL-[X(L)] heterodimer has a novel cell cycle function.     

Pharmacologic inhibition of RAF-->MEK-->ERK signaling elicits pancreatic cancer cell cycle arrest through induced expression of p27Kip1

Expression of mutationally activated RAS is a feature common to the vast majority of human pancreatic adenocarcinomas. RAS elicits its effects through numerous signaling pathways including the RAF-->mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase [MEK]-->ERK MAP kinase pathway. To assess the role of this pathway in regulating cell proliferation, we tested the effects of pharmacologic inhibition of MEK on human pancreatic cancer cell lines. In eight cell lines tested, MEK inhibition led to a cessation of cell proliferation accompanied by G0-G1 cell cycle arrest. Concomitant with cell cycle arrest, we observed induced expression of p27Kip1, inhibition of cyclin/cyclin-dependent kinase 2 (cdk2) activity, accumulation of hypophosphorylated pRb, and inhibition of E2F activity. Using both antisense and RNA interference techniques, we assessed the role of p27Kip1 in the observed effects of MEK inhibition on pancreatic cancer cell proliferation. Inhibition of p27Kip1 expression in Mia PaCa-2 cells restored the activity of cyclin/cdk2, phosphorylation of pRb, and E2F activity and partially relieved the effects of U0126 on pancreatic cancer cell cycle arrest. Consistent with the effects of p27Kip1 on cyclin/cdk2 activity, inhibition of CDK2 expression by RNA interference also led to G0-G1 cell cycle arrest. These data suggest that the expression of p27Kip1 is downstream of the RAF-->MEK-->ERK pathway and that the regulated expression of this protein plays an important role in promoting the proliferation of pancreatic cancer cells. Moreover, these data suggest that pharmacologic inhibition of the RAF-->MEK-->ERK signaling pathway alone might tend to have a cytostatic, as opposed to a cytotoxic, effect on pancreatic cancer cells.     

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.     

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.     

Fludarabine-induced apoptosis of B chronic lymphocytic leukemia cells includes early cleavage of p27kip1 by caspases.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of growth arrested clonal B lymphocytes that undergo apoptosis when treated with fludarabine. To further explore the mechanism for the cell cycle arrest, we examined the expression and activity of cyclin-dependent kinases and inhibitors in primary B-CLL cells. We observed high levels of p27kip1, cyclin D2, cyclin E, cdk2, and cdk4 expression in freshly isolated B-CLL cells. Despite high levels of cyclins and cdks, little cdk2 or cdk4 activity was observed with p27kip1 in complex with cyclinD2/cdk4 and cyclin E/cdk2. Remarkably, when B-CLL cells were treated in vitro with fludarabine, p27kip1 underwent caspase-specific degradation accompanied by an increase in cdk4 activity. We conclude that the G0/G1 arrest of B-CLL cells may protect against apoptosis and that the decrease in p27kip1 expression by caspase cleavage may be a key step in chemotherapy-induced apoptosis in B-CLL.     

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.     

Expression of the cell cycle regulator p27(Kip1) in normal squamous epithelium, cervical intraepithelial neoplasia, and invasive squamous cell carcinoma of the uterine cervix. Immunohistochemistry and functional aspects of p27(Kip1).

BACKGROUND: Abnormality of cell cycle regulators and tumor suppressors, such as cyclin dependent kinase inhibitors (cdkIs), has been reported in malignant tumors. The current study was undertaken to examine the involvement of a cdkI, p27(Kip1) (p27), in the neoplastic process of the uterine cervical epithelium. METHODS: Immunohistochemical staining of p27 was performed in samples of normal cervical tissue (30 samples), cervical intraepithelial neoplasias (CINs; 17 samples), and invasive squamous cell carcinoma (SCC; 25 samples). The results were compared with the expression levels of Ki-67, cdk2, and cyclin E. The functional aspects of the p27 protein, such as its ability to bind to cdk2 and the phosphorylation activity of p27-bound cdk2, also were evaluated with an immunoprecipitation and histone H1 kinase assay. RESULTS: In normal cervical epithelia, the expression of p27 was strong in the intermediate and superficial cells but very weak in the parabasal cells. In CIN samples, the expression of p27 was negligible. The expression of p27 in these tissues showed an inverse topologic correlation to that of Ki-67, cdk2, and cyclin E. However, it is noteworthy that the number of p27 positive cells increased in SCC samples that also showed increased expression of Ki-67, cdk2, and cyclin E. The p27 protein in SCC samples was bound to cdk2 and cyclin E. However, cdk2 that was bound to p27 still possessed histone H1 kinase activity. CONCLUSIONS: The expression of p27 may be involved in the growth regulation of the normal squamous epithelium in the uterine cervix. However, aberrant function of p27 expression may occur in invasive SCC of the cervix.     

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.     

Relocalized p27Kip1 tumor suppressor functions as a cytoplasmic metastatic oncogene in melanoma

The p27 tumor suppressor negatively regulates G1 cell cycle progression. However, human malignancies rarely select for deletion/inactivation of p27, a hallmark of tumor suppressor genes. Instead, p27 is degraded or relocalized to the cytoplasm in aggressive malignancies, supporting the notion that p27 sequestration from its nuclear cyclin:cyclin-dependent kinase (cdk) targets is critical. However, emerging cell biology data suggest a novel cdk-independent cytoplasmic function of p27 in cell migration. Here, we find cytoplasmic p27 in 70% of invasive and metastatic melanomas. In contrast, no cytoplasmic p27 was detected in noninvasive, basement membrane-confined melanoma in situ, suggesting a late oncogenic role for cytoplasmic p27 in metastasis. Targeted cytoplasmic expression of wild-type or non-cdk-binding p27 at subphysiologic levels induced melanoma motility and resulted in numerous metastases to lymph node, lung, and peritoneum. These observations point to a prominent role of cytoplasmic p27 in metastatic disease that is independent of cyclin:cdk regulation or mere nuclear loss.     

Unusual deregulation of cell cycle components in early and frank estrogen-induced renal neoplasias in the Syrian hamster

There is strong evidence that estrogens are involved in theetiology, promotion and progression of a variety of cancers,including the cancers of the breast and endometrium. The Syrianhamster estrogen-induced, estrogen-dependent renal neoplasmis a well-established animal model used to elucidate the cellularand molecular mechanisms involved in solely estrogen-inducedcarcinogenic processes. G₁ cell cycle progression was studiedin estrogen-induced early renal tumor foci and in large kidneytumors of castrated male hamsters. Levels of cyclin D1, cyclinE and retinoblastoma (pRb) proteins were higher in these renalneoplasias than in adjacent uninvolved renal tissue and kidneysfrom untreated, age-matched animals. Of particular interestis the presence of a predominant 35 kDa cyclin E protein variantform in primary renal tumors. In addition, amounts of the phosphorylatedforms of cyclin-dependent kinases (cdk) 2 and 4 were decreased,and both RNA and protein levels of p27^kip1 (p27), a cyclin-dependentkinase inhibitor, were markedly higher in early and frank renaltumors than in adjacent uninvolved renal tissue and kidneysof untreated, age-matched animals. These changes in cell cyclecomponents coincided with a rise in renal tumor cell proliferation.Binding of the elevated p27 protein to cyclin E, cdk2 and cdk4,however, was not impaired, suggesting that this cell cycle suppressorprotein is functional. In addition, cyclin D1-, cdk2-, cdk4-and cyclin E-associated kinase activities were also lower inthese estrogen-induced renal neoplasms than in untreated, age-matchedkidneys. Interestingly, when compared with untreated kidneytissue, early and frank renal neoplasms had less of the 62 kDanative form of E2F1 and contained a 57 kDa variant form. Thuswe have characterized an unusual deregulation of the cell cycleduring estrogen-induced renal tumorigenesis in Syrian hamsterswhich still allows for estrogen-driven kidney tumor cell proliferationand may contribute to the early genomic instability found.     

A cyclin D1/cyclin-dependent kinase 4 binding site within the C domain of the retinoblastoma protein

Phosphorylation of the retinoblastoma protein (Rb) by the cyclin D1/cyclin-dependent kinase (cdk) 4 complex (cdk4/D1) is a key regulatory step for maintaining the orderly progression of the cell cycle. The B domain of Rb contains a site that recognizes and binds the LXCXE motif found in D-type cyclins. This interaction is important for phosphorylation of Rb by cdk4/D1, although in vitro the Rb C domain alone is efficiently phosphorylated by cdk4/D1. A mutation in the C domain of Rb, L901Q, has been identified that completely abolishes cdk4/D1 phosphorylation of the isolated C domain. By contrast, the L901Q mutation has no effect on phosphorylation by either cyclin E/cdk2 or cyclin B/cdk1, suggesting that the interaction between L901Q and cdk4/D1 is specific. Introduction of the L901Q mutation into Rb containing the A, B, and C domains results in phosphorylation becoming predominantly dependent on the LXCXE binding region. However, when the LXCXE binding region of Rb is mutated, phosphorylation becomes dependent on the L901 site within the C domain. The L901 binding site can supplant the LXCXE binding site for the cdk4/D1-dependent phosphorylation of S780 and S795 but not S807/S811. Despite the limited homology between C domains of Rb, p107, and p130, the L901 site is conserved and introduction of the L925Q mutation into the isolated C domain of p107 also inhibits phosphorylation by cdk4/D1. These data support a model for cdk4/D1 recognizing two independent binding sites in Rb and suggests a conservation of this C domain binding motif for cyclin D1/cdk4 kinase among the Rb family of proteins.     

Rhabdoid tumor growth is inhibited by flavopiridol.

PURPOSE: Rhabdoid tumors are aggressive and incurable pediatric malignancies. INI1/hSNF5, a tumor suppressor biallelically deleted/inactivated in rhabdoid tumors, directly represses cyclin D1. Rhabdoid tumors and cells are exquisitely dependent on cyclin D1 for genesis and survival, suggesting that targeting the cyclin/cyclin-dependent kinase (cdk) axis may be an effective therapeutic strategy for these tumors. Because cdk inhibitors have not been used for preclinical or clinical testing on rhabdoid tumors, we investigated the effect of flavopiridol, a pan-cdk inhibitor with promising clinical activity, on rhabdoid tumors. EXPERIMENTAL DESIGN: The effect of flavopiridol on rhabdoid cells was tested in vitro using survival, cell cycle, and apoptosis assays. Its effect was assessed in vivo using xenografted rhabdoid tumor models. Immunoblot and immunohistochemical analysis was used to assess the effect of flavopiridol on cyclin D1 and p21 expression in vitro and in vivo, respectively. RESULTS: Nanomolar concentrations of flavopiridol inhibited rhabdoid cell growth (IC(50) approximately 200 nmol/L), induced G(1) and G(2) arrest, and apoptosis in vitro in a concentration-dependent manner. These effects were correlated with the down-modulation of cyclin D1, up-regulation of p21, and induction of caspase 3/7 activities. Flavopiridol (at 7.5 mg/kg) significantly inhibited the growth of xenografted rhabdoid tumors, and its effect was correlated with the induction of p21 and down-modulation of cyclin D1. CONCLUSIONS: Flavopiridol is effective in inducing cell cycle arrest and cytotoxicity in rhabdoid tumors. Its effects are correlated with the down-regulation of cyclin D1 and the up-regulation of p21. Flavopiridol is potentially a novel chemotherapeutic agent for rhabdoid tumors.