Theaflavins induce G2/M arrest by modulating expression of p21waf1/cip1, cdc25C and cyclin B in human prostate carcinoma PC-3 cells

Cancer of the prostate gland (PCA) is the most common invasive malignancy and is the second leading cause of cancer-related death in males. The polyphenolic constituents of black tea have gained considerable attention as chemopreventive agents. Many studies have shown that black tea reduces the risk of several cancer types. In the present study, we studied the effect of a black tea polyphenol, theaflavin (TF), on cellular proliferation and cell death in the human prostate cancer cell line, PC-3. We showed that TF inhibits cell proliferation in a dose- and time-dependent manner. Studies on cell cycle progression have shown that the anti-proliferative effect of TF is associated with an increase in the G2/M phase of PC-3 cells. Western blot results showed that TF-induced G2/M phase arrest was mediated through the inhibition of cyclin-regulated signaling pathways. TF induces cyclin kinase inhibitor p21(waf1/cip1) expression and inhibits cdc25C and cyclin B expression. Increased exposure time to TF caused apoptosis of PC-3 cells, which was associated with up-regulation of the pro-apoptotic proteins Bax, caspase-3 and caspase-9 and down-regulation of anti-apoptotic protein Bcl-2. The role of caspase-induced apoptosis was further confirmed by a reduction in mitochondria membrane potential and the appearance of a DNA laddering pattern. Thus, it can be concluded that TF acts as an effective anti-proliferative agent by modulating cell growth regulators in prostate cancer cells.     

MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation

The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion.     

The antiproliferative effect of beta-carotene requires p21waf1/cip1 in normal human fibroblasts.

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.     

Phosphorylation of the cyclin-dependent kinase inhibitor p21Cip1 on serine 130 is essential for viral cyclin-mediated bypass of a p21Cip1-imposed G1 arrest

K cyclin encoded by Kaposi's sarcoma-associated herpesvirus confers resistance to the cyclin-dependent kinase (cdk) inhibitors p16Ink4A, p21Cip1, and p27Kip1 on the associated cdk6. We have previously shown that K cyclin expression enforces S-phase entry on cells overexpressing p27Kip1 by promoting phosphorylation of p27Kip1 on threonine 187, triggering p27Kip1 down-regulation. Since p21Cip1 acts in a manner similar to that of p27Kip1, we have investigated the subversion of a p21Cip1-induced G1 arrest by K cyclin. Here, we show that p21Cip1 is associated with K cyclin both in overexpression models and in primary effusion lymphoma cells and is a substrate of the K cyclin/cdk6 complex, resulting in phosphorylation of p21Cip1 on serine 130. This phosphoform of p21Cip1 appeared unable to associate with cdk2 in vivo. We further demonstrate that phosphorylation on serine 130 is essential for K cyclin-mediated release of a p21Cip1-imposed G1 arrest. Moreover, we show that under physiological conditions of cell cycle arrest due to elevated levels of p21Cip1 resulting from oxidative stress, K cyclin expression enabled S-phase entry and was associated with p21Cip1 phosphorylation and partial restoration of cdk2 kinase activity. Thus, expression of the viral cyclin enables cells to subvert the cell cycle inhibitory function of p21Cip1 by promoting cdk6-dependent phosphorylation of this antiproliferative protein.     

Dendritic cell phenotype can be improved by certain chemotherapies and is associated with alterations to p21waf1/cip1

Introduction

Dendritic cells (DCs) possess the capacity to elicit immune responses against harmful antigens and have been used in DC-vaccines to stimulate the immune system to engage cancer cells. However, a lack of an appreciation of the quality of the DC that is used and/or the monocyte from which it is derived has limited their successful incorporation into treatment strategies.

Methods

In the current study, we explored the relationship between cytokine receptor expression on the monocytes and its subsequent development into DCs. The significance of p21 expression in DCs during differentiation was also studied, as was the effect that manipulating this with chemotherapy may have on DC quality.

Results

DCs separated into two groups based on their ability to respond to a maturation stimulus. This quality correlated with a particular receptor profile of granulocyte–macrophage colony-stimulating factor and interleukin 4 expressed on the monocytes from which they were derived. DC quality was also associated with p21 expression, and artificially increasing their levels in DCs by using some chemotherapy improved function.

Conclusions

Overall, these studies have highlighted a role for common chemotherapy in activating p21 in DCs, which is a prerequisite for good DC function.     

p21-dependent inhibition of colon cancer cell growth by mevastatin is independent of inhibition of G1 cyclin-dependent kinases

Mevastatin arrested HCT116 colon cancer cells at the G1/S transition and increased cellular levels of p21CIP1/WAF1. p21-deficient colon cancer cells continued to proliferate in the presence of mevastatin. Although p21 was necessary for the G1/S block, the G1 cyclin-dependent kinases (Cdks) cyclin E-Cdk2 and cyclin D-Cdk4 remained active. Despite the activity of the G1 Cdks the retinoblastoma protein was hypophosphorylated due to unknown mechanisms that were dependent on the p21 protein. The resulting decrease in cyclin A mRNA and protein led to a decrease in the activity of cyclin A-Cdk2. Therefore, although p21 was required for the G1/S arrest of HCT116 colon cancer cells by mevastatin, its mode of action was more complicated than the simple formation of a physical complex with cyclin-Cdk2. This mechanism of inhibition is different from that seen in prostate cancer cells (Ukomadu, C., and Dutta, A. (2003) J. Biol. Chem. 278, 4840-4846) where the activating phosphorylation of cyclin E-Cdk2 is suppressed and p21 is not required, suggesting the existence of cell line-specific differences in the mechanism by which statins arrest the cell cycle.     

The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression)

Butyrolactone I (BL) is a competitive inhibitor of ATP for binding and activation of cyclin-dependent kinases and is a potent inhibitor of cell cycle progression. Treatment of H460 human lung and SW480 human colon cancer cells with doses of BL that exceed the Ki for CDK inhibition but which are much lower than doses required to inhibit MAPK, PKA, PKC, or EGFR lead to a rapid significant reduction of endogenous p21 protein expression. BL-dependent inhibition of p21 expression appears to be p53-independent. BL-dependent p21 degradation was blocked by lactacystin, consistent with the hypothesis that there is accelerated p21 proteasomal degradation in the presence of BL. BL also inhibited the p53-dependent increase of p21 protein expression in cells exposed to the DNA damag-ing agent etoposide, and favored a greater G2/M arrest as compared to the non-BL exposed cells. BL accelerated the degradation of exogenously expressed p21 that was not observed with a C-terminal truncated form of p21. Degradation of exogenous p21 led to a shift to G2 accumulation in the cells exposed to BL. We conclude that BL has effects on the cell cycle beyond its role as a CDK inhibitor and can be used as a novel tool to study the mechanism of p21 degradation and the consequences towards p21- dependent checkpoints.     

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is an antiestrogen-regulated inhibitor of Cdk4 in human breast cancer cells.

The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Antiestrogen treatment of MCF-7 cells causes dramatic decreases in both Cdk4 and Cdk2 activities, which leads to a G(1) phase cell cycle arrest. In this report, we investigate the mechanism(s) by which Cdk4 activity is regulated in MCF-7 cells. Through time course analysis, we demonstrate that changes in Cdk4 activity in response to estrogen or antiestrogen treatment do not correlate directly with cyclin D1 protein levels or association. In contrast, Cdk4 activity does correlate with changes in the level of the Cdk inhibitor p21(WAF1/Cip1). Furthermore, we show that extracts of antiestrogen-treated cells contain a factor capable of inhibiting the Cdk4 activity present in extracts of estrogen-treated cells, and immunodepletion experiments identify this factor as p21(WAF1/Cip1). These results identify p21(WAF1/Cip1) as an important physiological regulator of Cdk4 complexes in human breast cancer cells.     

Lactoferrin inhibits G1 cyclin-dependent kinases during growth arrest of human breast carcinoma cells.

Lactoferrin inhibits cell proliferation and suppresses tumor growth in vivo. However, the molecular mechanisms underlying these effects remain unknown. In this in vitro study, we demonstrate that treatment of breast carcinoma cells MDA-MB-231 with human lactoferrin induces growth arrest at the G1 to S transition of the cell cycle. This G1 arrest is associated with a dramatic decrease in the protein levels of Cdk2 and cyclin E correlated with an inhibition of the Cdk2 kinase activity. Cdk4 activity is also significantly decreased in the treated cells and is accompanied by an increased expression of the Cdk inhibitor p21(CIP1). Furthermore, we show that lactoferrin maintains the cell cycle progression regulator retinoblastoma protein pRb in a hypophosphorylated form. Additional experiments with synchronized cells by serum depletion confirm the anti-proliferative activity of human lactoferrin. These effects of lactoferrin occur through a p53-independent mechanism both in MDA-MB-231 cells and other epithelial cell lines such as HBL-100, MCF-7, and HT-29. These findings demonstrate that lactoferrin induces growth arrest by modulating the expression and the activity of key G1 regulatory proteins.