8-chloroadenosine induced HL-60 cell growth inhibition, differentiation, and G(0)/G(1) arrest involves attenuated cyclin D1 and telomerase and up-regulated p21(WAF1/CIP1)

8-Chloroadenosine, an active dephosphorylated metabolite of the antineoplastic agent 8-chloroadenosine 3',5'-monophosphate (8-Cl-cAMP), induces growth inhibition in multiple carcinomas. Here we report that 8-chloroadenosine inhibits growth in human promyelocytic leukemia HL-60 cells by a G(0)/G(1) phase arrest and terminates cell differentiation along the granulocytic lineage. The mechanism of 8-chloroadenosine-induced G(0)/G(1) arrest is independent of apoptosis. The expressions of cyclin D1 and c-myc in HL-60 are suppressed by 8-chloroadenosine, whereas the cyclin-dependent kinases inhibitor p21(WAF1/CIP1) is up-regulated. 8-Chloroadenosine has less effect on the expressions of cyclin-dependent kinase (cdk)2 and cdk4, G(1) phase cyclin-dependent kinases, and only moderately induces the expression of transforming growth factor beta1 (TGFbeta1) and the mitotic inhibitor p27(KIP1). Telomerase activity is reduced in extracts of 8-chloroadenosine treated HL-60 cells, but 8-chloroadenosine does not directly inhibit the catalytic activity of telomerase in vitro. Therefore, anti-proliferation of HL-60 cells by 8-chloroadenosine involves coordination of cyclin D1 suppression, reduction of telomerase activity, and up-regulation of p21(WAF1/CIP1) that arrest cell-cycle progression at G(0)/G(1) phase and terminate cell differentiation.     

Caspase 3-mediated cleavage of p21WAF1/CIP1 associated with the cyclin A-cyclin-dependent kinase 2 complex is a prerequisite for apoptosis in SK-HEP-1 cells

Apoptosis of SK-HEP-1 human hepatoma cells induced by treatment with ginsenoside Rh2 (G-Rh2) is associated with rapid and selective activation of cyclin A-associated cyclin-dependent kinase 2 (Cdk2). Here, we show that in apoptotic cells, the Cdk inhibitory protein p21(WAF1/CIP1), which is associated with the cyclin A-Cdk2 complex, undergoes selective proteolytic cleavage. In contrast, another Cdk inhibitory protein, p27(KIP1), which is associated with cyclin A-Cdk2 and cyclin E-Cdk2 complexes, remained unaltered during apoptosis. Ectopic overexpression of p21(WAF1/CIP1) suppressed apoptosis as well as cyclin A-Cdk2 activity induced by treatment of SK-HEP-1 cells with G-Rh2. The suppressive effects of p21(WAF1/CIP1) were much higher in the cells transfected with p21D112N, an expression vector that encodes a p21(WAF1/CIP1) mutant resistant to caspase 3 cleavage. Overexpression of cyclin A in SK-HEP-1 cells dramatically up-regulated cyclin A-Cdk2 activity and accordingly enhances apoptosis induced by treatment with G-Rh2. These up-regulating effects were blocked by coexpression of a dominant negative allele of cdk2. Furthermore, olomoucine, a specific inhibitor of Cdks, also blocked G-Rh2-induced apoptosis. These data suggest that the induction of apoptosis in human hepatoma cells treated with G-Rh2 occurs by a mechanism that involves the activation of cyclin A-Cdk2 by caspase 3-mediated cleavage of p21(WAF1/CIP1).     

Dissociation of bone morphogenetic protein-mediated growth arrest and apoptosis of mouse B cells by HPV-16 E6/E7

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-beta family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27KIP1, p16INK4a, or p15INK4b, but enhanced expression of p21(CIP1/WAF1). Accumulation of p21(CIP1/WAF1) resulted in increased binding of p21(CIP1/WAF1) to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21(CIP1/WAF1) and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21(CIP1/WAF1) is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.     

A pure estrogen antagonist inhibits cyclin E-Cdk2 activity in MCF-7 breast cancer cells and induces accumulation of p130-E2F4 complexes characteristic of quiescence

Estrogen antagonists inhibit cell cycle progression in estrogen-responsive cells, but the molecular mechanisms are not fully defined. Antiestrogen-mediated G(0)/G(1) arrest is associated with decreased cyclin D1 gene expression, inactivation of cyclin D1-cyclin dependent kinase (Cdk) 4 complexes, and decreased phosphorylation of the retinoblastoma protein (pRb). We now show that treatment of MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 results in inhibition of cyclin E-Cdk2 activity prior to a decrease in the G(1) to S phase transition. This decrease was dependent on p21(WAF1/Cip1) since treatment with antisense oligonucleotides to p21 attenuated the effect. Recruitment of p21 to cyclin E-Cdk2 complexes was in turn dependent on decreased cyclin D1 expression since it was apparent following treatment with antisense cyclin D1 oligonucleotides. To define where within the G(0) to S phase continuum antiestrogen-treated cells arrested, we assessed the relative abundance and phosphorylation state of pocket protein-E2F complexes. While both pRb and p107 levels were significantly decreased, p130 was increased 4-fold and was accompanied by the formation of p130.E2F4 complexes and the accumulation of hyperphophorylated E2F4, putative markers of cellular quiescence. Thus, ICI 182780 inhibits both cyclin D1-Cdk4 and cyclin E-Cdk2 activity, resulting in the arrest of MCF-7 cells in a state with characteristics of quiescence (G(0)), as opposed to G(1) arrest.     

Role for cyclin D1 in UVC-induced and p53-mediated apoptosis.

DNA damaging agents such as ultraviolet (UV) induce cell cycle arrest followed by apoptosis in cells where irreparable damage has occurred. Here we show that during early phase G1 arrest which occurs in UV-irradiated human U343 glioblastoma cells, there are (1) decreases in cyclin D1 and cdk4 levels which parallel a loss of S-phase promoting cyclin D1/cdk4 complexes, and (2) increases in p53 and p21 protein levels. We also show that the late phase UV-induced apoptosis of U343 cells occurs after cell cycle re-entry and parallels the reappearance of cyclin D1 and cdk4 and cyclin D1/cdk4 complexes. These findings suggest that cyclin D1 can abrogate UV-induced G1 arrest and that the p53-mediated apoptosis that occurs in these cells is dependent on cyclin D1 levels. We examined these possibilities using U343 cells that ectopically express cyclin D1 and found that indeed cyclin D1 can overcome the cell cycle arrest caused by UV. Moreover, the appearance of p53 protein and the induction of apoptosis in UV-irradiated cells was found to be dependent on the level of ectopically expressed cyclin D1. These findings, therefore, indicate that expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and p53-mediated apoptosis.     

Linkage of Curcumin-Induced Cell Cycle Arrest and Apoptosis by Cyclin-Dependent Kinase Inhibitor p21/WAF1/CIP1

We have recently shown that curcumin induces apoptosis in prostate cancer cells through Bax translocation to mitochondria and caspase activation, and enhances the therapeutic potential of TRAIL. However, the molecular mechanisms by which it causes growth arrest are not well-understood. We studied the molecular mechanism of curcumin-induced cell cycle arrest in prostate cancer androgen-sensitive LNCaP and androgen-insensitive PC-3 cells. Treatment of both cell lines with curcumin resulted in cell cycle arrest at G1/S phase and that this cell cycle arrest is followed by the induction of apoptosis. Curcumin induced the expression of cyclin-dependent kinase (CDK) inhibitors p16/INK4a, p21/WAF1/CIP1 and p27/KIP1, and inhibited the expression of cyclin E and cyclin D1, and hyperphosphorylation of retinoblastoma (Rb) protein. Lactacystin, an inhibitor of 26 proteasome, blocks curcumin-induced down-regulation of cyclin D1 and cyclin E proteins, suggesting their regulation at level of posttranslation. The suppression of cyclin D1 and cyclin E by curcumin may inhibit CDK-mediated phosphorylation of pRb protein. The inhibition of p21/WAF1/CIP1 by siRNA blocks curcumin-induced apoptosis, thus establishing a link between cell cycle and apoptosis. These effects of curcumin result in the proliferation arrest and disruption of cell cycle control leading to apoptosis. Our study suggests that curcumin can be developed as a chemopreventive agent for human prostate cancer.     

Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I

The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M(r)) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G(1), we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.     

Low levels of cyclin D and nonfunctional Rb protein affect cdk6 association with cyclin-dependent kinase inhibitor p27(Kip1)

p27(Kip1) associates with cyclin/cdk complexes and inhibiting cdk activity, and overexpression of p27(Kip1) induces G1 arrest. We found that p27(Kip1) overexpression inhibits cdk2 kinase activity, but not cdk6 kinase activity in HeLa cells. The amount of p27(Kip1) associated with cdk2 was significantly higher than that associated with cdk6. cdk6 complexes contained detectable amounts of p27(Kip1) in all human cell lines examined, except in HeLa cells where p27(Kip1) preferentially associated with cdk2. It appears that in HeLa cells overexpressed p27(Kip1) fails to inhibit cdk6 kinase activity because of low binding affinity of cdk6 to p27(Kip1). The low binding affinity is due to a low level of the cdk6/cyclin D complexes. Functional inactivation of pRb has an effect on p27(Kip1) association with cdk6/cyclin D complexes.     

Cell cycle dysregulation by green tea polyphenol epigallocatechin-3-gallate

Epidemiological, in vitro cell culture, and in vivo animal studies have shown that green tea or its constituent polyphenols, particularly its major polyphenol epigallocatechin-3-gallate (EGCG) may protect against many cancer types. In earlier studies, we showed that green tea polyphenol EGCG causes a G0/G1-phase cell cycle arrest and apoptosis of human epidermoid carcinoma (A431) cells. We also demonstrated that these effects of EGCG may be mediated through the inhibition of nuclear factor kappa B that has been associated with cell cycle regulation and cancer. In this study, employing A431 cells, we provide evidence for the involvement of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery during cell cycle deregulation by EGCG. As shown by immunoblot analysis, EGCG treatment of the cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, p16 and p18, (ii) downmodulation of the protein expression of cyclin D1, cdk4 and cdk6, but not of cyclin E and cdk2, (iii) inhibition of the kinase activities associated with cyclin E, cyclin D1, cdk2, cdk4 and cdk6. Taken together, our study suggests that EGCG causes an induction of G1-phase ckis, which inhibit the cyclin-cdk complexes operative in G0/G1 phase of the cell cycle thereby causing a G0/G1-phase arrest of the cell cycle, which is an irreversible process ultimately resulting in an apoptotic cell death. We suggest that the naturally occurring agents such as green tea polyphenols which may inhibit cell cycle progression could be developed as potent anticancer agents for the management of cancer.     

Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. 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 human prostate carcinoma cells by EGCG.     

Cyclin D-cdk6 complex is targeted by p21(WAF) in growth-arrested lymphoma cells

Normal human B lymphocytes are sensitive to the growth-inhibitory action of transforming growth factor beta1 (TGFbeta1) whereas malignant B lymphoma cells are mostly resistant to TGFbeta1 effects. We examined the phosphorylation status of retinoblastoma protein and the activity of G(1) cyclin-dependent kinases (cdk) in TGFbeta1-sensitive malignant follicular lymphoma cells during the TGFbeta1 treatment. The kinase activity of cdk2, cdk4, and cdk6 was significantly reduced and hypophosphorylation of pRb on serine 795 (S795) and threonine 373 (T373) was observed. We examined the composition of cdk complexes and the level of cdk inhibitors to explain the inhibitory action of TGFbeta1 toward cdk activity. Both cdk4 and cdk6 were notably dissociated from cyclin D cofactors, while cyclin E-cdk2 complexes remained coupled in TGFbeta1-treated cells. TGFbeta1-induced growth arrest was associated with notably increased binding of p21(WAF1) to cdk4 and cdk6. No induction of cdk-inhibitor molecules of INK family was observed in TGFbeta1-treated DoHH2 cells. As shown, TGFbeta1-induced growth arrest of malignant B cells was associated with the activation of CIP/KIP family members of cdk inhibitors.     

Tuning Cell Cycle Regulation with an Iron Key

Iron (Fe) is essential for cellular metabolism e.g., DNA synthesis and its depletion causes G1/S arrest and apoptosis. Considering this, Fe chelators have been shown to be effective anti-proliferative agents. In order to understand the anti-tumor activity of Fe chelators, the mechanisms responsible for G1/S arrest and apoptosis after Fe-depletion have been investigated. These studies reveal a multitude of cell cycle control molecules are regulated by Fe. These include p53, p27Kip1, cyclin D1 and cyclin-dependent kinase 2 (cdk2). Additionally, Fe-depletion up-regulates the mRNA levels of the cdk inhibitor, p21CIP1/WAF1, but paradoxically down-regulates its protein expression. This effect could contribute to the apoptosis observed after Fe-depletion. Iron-depletion also leads to proteasomal degradation of p21CIP1/WAF1 and cyclin D1 via an ubiquitin-independent pathway. This is in contrast to the mechanism in Fe-replete cells, where it occurs by ubiquitin-dependent proteasomal degradation. Up-regulation of p38 mitogen-activated protein kinase (MAPK) after Fe-depletion suggests another facet of cell cycle regulation responsible for inhibition of proliferation and apoptosis induction. Elucidation of the complex effects of Fe-depletion on the expression of cell cycle control molecules remains at its infancy. However, these processes are important to dissect for complete understanding of Fe-deficiency and the development of chelators for cancer treatment.     

Human papillomavirus type 16 E6 promotes retinoblastoma protein phosphorylation and cell cycle progression

We show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G(1)/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16(INK4a), CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16(INK4a). The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21(WAF1/CIP1) were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21(WAF1/CIP1) gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21(WAF1/CIP1) levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G(1) arrest imposed by oncogenic ras. Immunofluorescence staining of cells coexpressing ras and E6 from either HPV16 or HPV1 revealed that antiproliferative (p16(INK4a)) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.     

The lack of G1/S arrest of teratocarcinoma F9 cells is determinated by degradation of cyclin-dependent kinases inhibitor p21waf1/cip1

We have studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints following gamma-irradiation. Wild-type p53 protein is rapidly accumulated in F9 cells after gamma-irradiation, however this is not followed by G1/S arrest; there is just a reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells we investigated the levels of regulatory cell cycle proteins: G1-cyclins, cyclin dependent kinases and kinase inhibitor p21WAF1/CIP1. We have shown that in spite of p53-dependent activation of p21WAF1/CIP1 promoter, p21WAF1/CIP1 protein is not revealed by different polyclonal and monoclonal antibodies, either by immunoblotting or by immunofluorescent staining. However, when cells are treated with specific proteasome inhibitor lactacystin, p21WAF1/CIP1 protein is revealed. We therefore suggest that p21WAF1/CIP1 protein is subjected to proteasome degradation in F9 cells and probably the lack of G1/S arrest after gamma-irradiation is due to this degradation. Thus, it is the combination of functionally active p53 with low level expression of p21WAF1/CIP1 that causes a short delay of the cell cycle progression in G2/M, rather than the G1-arrest after gamma-irradiation of F9 cells.     

Post-proliferative cyclin E-associated kinase activity in differentiated osteoblasts: Inhibition by proliferating osteoblasts and osteosarcoma cells

Spontaneous differentiation of normal diploid osteoblasts in culture is accompanied by increased cyclin E associated kinase activity on (1) the retinoblastoma susceptibility protein pRB, (2) the p107 RB related protein, and (3) two endogenous cyclin E-associated substrates of 78 and 105 kD. Activity of the differentiation-related cyclin E complexes (diff.ECx) is not recovered in cdc2 or cdk2 immunoprecipitates. Phosphorylation of both the 105 kD endogenous substrate and the p107 exogenous substrate is sensitive to inhibitory activity (diff.ECx-i) present in proliferating osteoblasts. This inhibitory activity is readily recruited by the cyclin E complexes of differentiated osteoblasts but is not found in cyclin E immunoprecipitates of the proliferating cells themselves. Strong inhibitory activity on diff.ECx kinase activity is excerted by proliferating ROS 17/2.8 osteosarcoma cells. However, unlike the normal diploid cells, the diff.ECx-i activity of proliferating ROS 17/2.8 cells is recovered by cyclin E immunoprecipitation. The cyclin-dependent kinase inhibitor p21^CIP1/WAF1 inhibits diff.ECx kinase activity. Thus, our results suggest the existence of a unique regulatory system, possibly involving p21^CIP1/WAF1, in which inhibitory activity residing in proliferating cells is preferentially targeted towards differentiation-related cyclin E-associated kinase activity. J. Cell. Biochem. 66:141-152, 1997. © 1997 Wiley-Liss, Inc.     

Altered p27(Kip1) phosphorylation, localization, and function in human epithelial cells resistant to transforming growth factor beta-mediated G(1) arrest

p27(Kip1) is an important effector of G(1) arrest by transforming growth factor beta (TGF-beta). Investigations in a human mammary epithelial cell (HMEC) model, including cells that are sensitive (184(S)) and resistant (184A1L5(R)) to G(1) arrest by TGF-beta, revealed aberrant p27 regulation in the resistant cells. Cyclin E1-cyclin-dependent kinase 2 (cdk2) and cyclin A-cdk2 activities were increased, and p27-associated kinase activity was detected in 184A1L5(R) cells. p27 from 184A1L5(R) cells was localized to both nucleus and cytoplasm, showed an altered profile of phosphoisoforms, and had a reduced ability to bind and inhibit cyclin E1-cdk2 in vitro when compared to p27 from the sensitive 184(S) cells. In proliferating 184A1L5(R) cells, more p27 was associated with cyclin D1-cdk4 complexes than in 184(S). While TGF-beta inhibited the formation of cyclin D1-cdk4-p27 complexes in 184(S) cells, it did not inhibit the assembly of cyclin D1-cdk4-p27 complexes in the resistant 184A1L5(R) cells. p27 phosphorylation changed during cell cycle progression, with cyclin E1-bound p27 in G(0) showing a different phosphorylation pattern from that of cyclin D1-bound p27 in mid-G(1). These data suggest a model in which TGF-beta modulates p27 phosphorylation from its cyclin D1-bound assembly phosphoform to an alternate form that binds tightly to inhibit cyclin E1-cdk2. Altered phosphorylation of p27 in the resistant 184A1L5(R) cells may favor the binding of p27 to cyclin D1-cdk4 and prevent its accumulation in cyclin E1-cdk2 in response to TGF-beta.