Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture

Epidemiological evidence suggests tea (Camellia sinensis L.) has chemopreventive effects against various tumors. Green tea contains many polyphenols, including epigallocatechin-3 gallate (EGCG), which possess anti-oxidant qualities. Reduction of chemically induced mammary gland carcinogenesis by green tea in a carcinogen-induced rat model has been suggested previously, but the results reported were not statistically significant. Here we have tested the effects of green tea on mammary tumorigenesis using the 7,12-dimethylbenz(a)anthracene (DMBA) Sprague-Dawley (S-D) rat model. We report that green tea significantly increased mean latency to first tumor, and reduced tumor burden and number of invasive tumors per tumor-bearing animal; although, it did not affect tumor number in the female rats. Furthermore, we show that proliferation and/or viability of cultured Hs578T and MDA-MB-231 estrogen receptor-negative breast cancer cell lines was reduced by EGCG treatment. Similar negative effects on proliferation were observed with the DMBA-transformed D3-1 cell line. Growth inhibition of Hs578T cells correlated with induction of p27(Kip1) cyclin-dependent kinase inhibitor (CKI) expression. Hs578T cells expressing elevated levels of p27(Kip1) protein due to stable ectopic expression displayed increased G1 arrest. Thus, green tea had significant chemopreventive effects on carcinogen-induced mammary tumorigenesis in female S-D rats. In culture, inhibition of human breast cancer cell proliferation by EGCG was mediated in part via induction of the p27(Kip1) CKI.     

Synthetic analogs of green tea polyphenols as proteasome inhibitors

BACKGROUND: Animal, epidemiological and clinical studies have demonstrated the anti-tumor activity of pharmacological proteasome inhibitors and the cancer-preventive effects of green tea consumption. Previously, one of our laboratories reported that natural ester bond-containing green tea polyphenols (GTPs), such as (-)-epigallocatechin-3-gallate [(-)-EGCG] and (-)-gallocatechin-3-gallate [(-)-GCG], are potent and specific proteasome inhibitors. Another of our groups, for the first time, was able to enantioselectively synthesize (-)-EGCG as well as other analogs of this natural GTP. Our interest in designing and developing novel synthetic GTPs as proteasome inhibitors and potential cancer-preventive agents prompted our current study. MATERIALS AND METHODS: GTP analogs, (+)-EGCG, (+)-GCG, and a fully benzyl-protected (+)-EGCG [Bn-(+)-EGCG], were prepared by enantioselective synthesis. Inhibition of the proteasome or calpain (as a control) activities under cell-free conditions were measured by fluorogenic substrate assay. Inhibition of intact tumor cell proteasome activity was measured by accumulation of some proteasome target proteins (p27, I kappa B-alpha and Bax) using Western blot analysis. Inhibition of tumor cell proliferation and induction of apoptosis by synthetic GTPs were determined by G(1) arrest and caspase activation, respectively. Finally, inhibition of the transforming activity of human prostate cancer cells by synthetic GTPs was measured by a colony formation assay. RESULTS: (+)-EGCG and (+)-GCG potently and specifically inhibit the chymotrypsin-like activity of purified 20S proteasome and the 26S proteasome in tumor cell lysates, while Bn-(+)-EGCG does not. Treatment of leukemic Jurkat T or prostate cancer LNCaP cells with either (+)-EGCG or (+)-GCG accumulated p27 and IkappaB-alpha proteins, associated with an increased G(1) population. (+)-EGCG treatment also accumulated the pro-apoptotic Bax protein and induced apoptosis in LNCaP cells expressing high basal levels of Bax, but not prostate cancer DU-145 cells with low Bax expression. Finally, synthetic GTPs significantly inhibited colony formation by LNCaP cancer cells. CONCLUSIONS: Enantiomeric analogs of natural GTPs, (+)-EGCG and (+)-GCG, are able to potently and specifically inhibit the proteasome both, in vitro and in vivo, while protection of the hydroxyl groups on (+)-EGCG renders the compound completely inactive.     

Docking studies and model development of tea polyphenol proteasome inhibitors: applications to rational drug design

Previously, we demonstrated that natural and synthetic ester bond-containing green tea polyphenols were potent and specific non-peptide proteasome inhibitors. However, the molecular mechanism of inhibition is currently unknown. Here, we report that inhibition of the chymotrypsin activity of the 20S proteasome by (-)-epigallocatechin-3-gallate (EGCG) is time-dependent and irreversible, implicating acylation of the beta5-subunit's catalytic N-terminal threonine (Thr 1). This knowledge is used, along with in silico docking experiments, to aid in the understanding of binding and inhibition. On the basis of these docking experiments, we propose that (-)-EGCG binds the chymotrypsin site in an orientation and conformation that is suitable for a nucleophilic attack by Thr 1. Consistently, the distance from the electrophilic carbonyl carbon of (-)-EGCG to the hydroxyl group of Thr 1 was measured as 3.18 A. Furthermore, the A ring of (-)-EGCG acts as a tyrosine mimic, binding to the hydrophobic S1 pocket of the beta5-subunit. In the process, the (-)-EGCG scissile bond may become strained, which could lower the activation energy for attack by the hydroxyl group of Thr 1. This model is validated by comparison of predicted and actual activities of several EGCG analogs, either naturally occurring, previously synthesized, or rationally synthesized.     

The role of cyclin-dependent kinase inhibitor p27Kip1 in anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition

Cyclin-dependent kinase (CDK) inhibitor p27Kip1 binds to the cyclin E.CDK2 complex and plays a major role in controlling cell cycle and cell growth. Our group and others have reported that anti-HER2 monoclonal antibodies exert inhibitory effects on HER2-overexpressing breast cancers through G1 cell cycle arrest associated with induction of p27Kip1 and reduction of CDK2. The role of p27Kip1 in anti-HER2 antibody-induced cell cycle arrest and growth inhibition is, however, still uncertain. Here we have provided several lines of evidence supporting a critical role for p27Kip1 in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition. Induction of p27Kip1 and G1 growth arrest by anti-HER2 antibody, murine 4D5, or humanized trastuzumab (Herceptin) are concentration-dependent, time-dependent, irreversible, and long-lasting. The magnitude of G1 cell cycle arrest induced by trastuzumab or 4D5 is well correlated with the level of p27Kip1 protein induced. Up-regulation of p27Kip1 and G1 growth arrest could no longer be removed with as little as 14 h of treatment with trastuzumab. Anti-HER2 antibody-induced p27Kip1 protein, G1 arrest, and growth inhibition persist at least 5 days after a single treatment. The magnitude of growth inhibition of breast cancer cells induced by anti-HER2 antibody closely parallels the level of p27Kip1 induced. Induced expression of exogenous p27Kip1 results in a p27Kip1 level-dependent G1 cell cycle arrest and growth inhibition similar to that obtained with anti-HER2 antibodies. Reducing p27Kip1 expression using p27Kip1 small interfering RNA blocks anti-HER2 antibody-induced p27Kip1 up-regulation and G1 arrest. Treatment with anti-HER2 antibody significantly increases the half-life of p27Kip1 protein. Inhibition of ubiquitin-proteasome pathway, but not inhibition of calpain and caspase activities, up-regulates p27Kip1 protein to a degree comparable with that obtained with anti-HER2 antibodies. We have further demonstrated that anti-HER2 antibody significantly decreases threonine phosphorylation of p27Kip1 protein at position 187 (Thr-187) and increases serine phosphorylation of p27Kip1 protein at position 10 (Ser-10). Expression of S10A and T187A mutant p27Kip1 protein increases the fraction of cells in G1 and reduces a further antibody-induced G1 arrest. Consequently, p27Kip1 plays an important role in the anti-HER2 antibody-induced G1 cell cycle arrest and tumor growth inhibition through post-translational regulation. Regulation of the phosphorylation of p27Kip1 protein is one of the post-translational mechanisms by which anti-HER2 antibody upregulates the protein.     

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.     

Inducible p27(Kip1) expression inhibits proliferation of K562 cells and protects against apoptosis induction by proteasome inhibitors

Overexpression of the cyclin-dependent kinase inhibitor p27(Kip1) has been demonstrated to induce cell cycle arrest and apoptosis in various cancer cell lines, but has also been associated with the opposite effect of enhanced survival of tumor cells and increased resistance towards chemotherapeutic treatment. To address the question of how p27(Kip1) expression is related to apoptosis induction, we studied doxycycline-regulated p27(Kip1) expression in K562 erythroleukemia cells. p27(Kip1) expression effectively retards proliferation, but it is not sufficient to induce apoptosis in K562 cells. p27(Kip1)-expressing K562 cells, however, become resistant to apoptosis induction by the proteasome inhibitors PSI, MG132 and epoxomicin, in contrast to wild-type K562 cells that are efficiently killed. Cell cycle arrest in the S phase by aphidicolin, which is not associated with an accumulation of p27(Kip1) protein, did not protect K562 cells against the cytotoxic effect of the proteasome inhibitor PSI. The expression levels of p27(Kip1) thus constitute an important parameter, which decides on the overall sensitivity of cells against the cytotoxic effect of proteasome inhibitors.     

Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.     

ErbB2 potentiates breast tumor proliferation through modulation of p27(Kip1)-Cdk2 complex formation: receptor overexpression does not determine growth dependency

Overexpression of the ErbB2 receptor, a major component of the ErbB receptor signaling network, contributes to the development of a number of human cancers. ErbB2 presents itself, therefore, as a target for antibody-mediated therapies. In this respect, anti-ErbB2 monoclonal antibody 4D5 specifically inhibits the growth of tumor cells overexpressing ErbB2. We have analyzed the effect of 4D5-mediated ErbB2 inhibition on the cell cycle of the breast tumor cell line BT474. 4D5 treatment of BT474 cells resulted in a G(1) arrest, preceded by rapid dephosphorylation of ErbB2, inhibition of cytoplasmic signal transduction pathways, accumulation of the cyclin-dependent kinase inhibitor p27(Kip1), and inactivation of cyclin-Cdk2 complexes. Time courses demonstrated that 4D5 treatment redirects p27(Kip1) onto Cdk2 complexes, an event preceding increased p27(Kip1) expression; this correlates with the downregulation of c-Myc and D-type cyclins (proteins involved in p27(Kip1) sequestration) and the loss of p27(Kip1) from Cdk4 complexes. Similar events were observed in ErbB2-overexpressing SKBR3 cells, which exhibited reduced proliferation in response to 4D5 treatment. Here, p27(Kip1) redistribution resulted in partial Cdk2 inactivation, consistent with a G1 accumulation. Moreover, p27(Kip1) protein levels remained constant. Antisense-mediated inhibition of p27(Kip1) expression in 4D5-treated BT474 cells further demonstrated that in the absence of p27(Kip1) accumulation, p27(Kip1) redirection onto Cdk2 complexes is sufficient to inactivate Cdk2 and establish the G(1) block. These data suggest that ErbB2 overexpression leads to potentiation of cyclin E-Cdk2 activity through regulation of p27(Kip1) sequestration proteins, thus deregulating the G(1)/S transition. Moreover, through comparison with an ErbB2-overexpressing cell line insensitive to 4D5 treatment, we demonstrate the specificity of these cell cycle events and show that ErbB2 overexpression alone is insufficient to determine the cellular response to receptor inhibition.     

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.     

Induction of G1 arrest and apoptosis in human jurkat T cells by pentagalloylglucose through inhibiting proteasome activity and elevating p27Kip1, p21Cip1/WAF1, and Bax proteins

Pentagalloylglucose, which is found in many medicinal plants, can arrest the cell cycle at G(1) phase through down-regulation of cyclin-dependent kinases 2 and 4 and up-regulation of the cyclin-dependent kinase inhibitors p27(Kip1) and p21(Cip1/WAF1) in human breast cancer cells. Pentagalloylglucose also induces apoptosis in human leukemic cells. However, the mechanisms by which pentagalloylglucose induces these effects is unclear. We now show that pentagalloylglucose inhibits the activities of purified 20 and 26 S proteasomes in vitro, the 26 S proteasome in Jurkat T cell lysates, and chymotrypsin-like activity of the 26 S proteasome in intact Jurkat T cells. The turnover of p27(Kip1) and p21(Cip1/WAF1), which is necessary for cell cycle progression mediated by proteasome degradation, was disrupted by treatment of human Jurkat T cells with pentagalloylglucose. This was shown by cycloheximide treatment and in vivo pulse-chase labeling experiments, and this effect correlated with the arrest of proliferation of Jurkat T cells at G(1). Inhibition of the proteasome by pentagalloylglucose and by the proteasome inhibitor MG132 caused accumulation of ubiquitin-tagged proteins in Jurkat T cells. The addition of pentagalloylglucose to Jurkat T cells enhanced the stability of the proteasome substrate Bax and increased cytochrome c release and apoptosis. Our findings suggest a mechanism for the effect of pentagalloylglucose on the cell cycle in human leukemic cells: that pentagalloylglucose down-regulates proteasome-mediated pathways because it is a proteasome inhibitor.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Blockade of the extracellular signal-regulated kinase pathway induces marked G1 cell cycle arrest and apoptosis in tumor cells in which the pathway is constitutively activated: up-regulation of p27(Kip1)

Constitutive activation of the ERK pathway is associated with the neoplastic phenotype of a relatively large number of human tumor cells. Blockade of the ERK pathway by treatment with PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase/ERK kinase (MEK), completely suppressed the growth of tumor cells in which the pathway is constitutively activated (RPMI-SE and HT1080 cells). Consistent with its prominent antiproliferative effect, PD98059 induced a remarkable G(1) cell cycle arrest, followed by a modest apoptotic response, in these tumor cells. Selective up-regulation of p27(Kip1) was observed after PD98059 treatment of RPMI-SE and HT1080 cells. Overexpression in RPMI-SE cells of either a kinase-negative form of MEK1 or wild-type MAP kinase phosphatase-3 also induced up-regulation of p27(Kip1). The up-regulation of p27(Kip1) correlated with increased association of p27(Kip1) with cyclin E-cyclin-dependent kinase (CDK) 2 complexes, a concomitant inhibition of cyclin E-CDK2 kinase activity, and a consequent decrease in the phosphorylation state of retinoblastoma protein, which would culminate in the marked G(1) cell cycle arrest observed in these tumor cells. These results suggest that the complete growth suppression that follows specific blockade of the ERK pathway in tumor cells in which the pathway is constitutively activated is mediated by up-regulation of p27(Kip1).     

Methylation suppresses the proteasome-inhibitory function of green tea polyphenols

Under physiological conditions, biotransformation reactions, such as methylation, can modify green tea polyphenols (GTPs) and therefore limit their in vivo cancer-preventive activity. Although a recent study suggested that methylated polyphenols are less cancer-protective, the molecular basis is unknown. We previously reported that ester bond-containing GTPs, for example (-)-epigallocatechin-3-gallate [(-)-EGCG] or (-)-epicatechin-3-gallate [(-)-ECG], potently and specifically inhibit the proteasomal chymotrypsin-like activity. In this study, we hypothesize that methylated GTPs have decreased proteasome-inhibitory abilities. To test this hypothesis, methylated (-)-EGCG and (-)-ECG analogs that can be found in vivo were synthesized and studied for their structure-activity relationships (SARs) using a purified 20S proteasome. The addition of a single methyl group on (-)-EGCG or (-)-ECG led to decreased proteasome inhibition and, as the number of methyl groups increased, the inhibitory potencies further decreased. These SARs were supported by our findings from in silico docking analysis published recently. Previously, we synthesized a peracetate-protected (-)-EGCG molecule, Pro-EGCG (1), to enhance its cellular permeability and stability, and current HPLC analysis confirms conversion of Pro-EGCG (1) to (-)-EGCG in cultured human leukemic Jurkat T cells. Furthermore, in this study, peracetate-protected forms of methylated GTPs were added in intact Jurkat T cells to observe the intracellular effects of methylation. Peracetate-protected, monomethylated (-)-EGCG induced greater cellular proteasome inhibition and apoptosis than did peracetate-protected, trimethylated (-)-EGCG, consistent with the potencies of the parent methylated analogs against a purified 20S proteasome. Therefore, methylation on GTPs, under physiological conditions, could decrease their proteasome-inhibitory activity, contributing to decreased cancer-preventive effects of tea consumption.     

The p42/p44 mitogen-activated protein kinase activation triggers p27Kip1 degradation independently of CDK2/cyclin E in NIH 3T3 cells.

The p42/p44 mitogen-activated protein (MAP) kinase is stimulated by various mitogenic stimuli, and its sustained activation is necessary for cell cycle G(1) progression and G(1)/S transition. G(1) progression and G(1)/S transition also depend on sequential cyclin-dependent kinase (CDK) activation. Here, we demonstrate that MAP kinase inhibition leads to accumulation of the CDK inhibitor p27(Kip1) in NIH 3T3 cells. Blocking the proteasome-dependent degradation of p27(Kip1) impaired this accumulation, suggesting that MAP kinase does not act on p27(Kip1) protein synthesis. In the absence of extracellular signals (growth factors or cell adhesion), genetic activation of MAP kinase decreased the expression of p27(Kip1) as assessed by cotransfection experiments and by immunofluorescence detection. Importantly, MAP kinase activation also decreased the expression of a p27(Kip1) mutant, which cannot be phosphorylated by CDK2, suggesting that MAP kinase-dependent p27(Kip1) regulation is CDK2-independent. Accordingly, expression of dominant-negative CDK2 did not impair the down-regulation of p27(Kip1) induced by MAP kinase activation. These data demonstrate that the MAP kinase pathway regulates p27(Kip1) expression in fibroblasts essentially through a degradation mechanism, independently of p27(Kip1) phosphorylation by CDK2. This strengthens the role of this CDK inhibitor as a key effector of G(1) growth arrest, whose expression can be controlled by extracellular stimuli-dependent signaling pathways.     

Non-malignant and tumor-derived cells differ in their requirement for p27Kip1 in transforming growth factor-beta-mediated G1 arrest

Transforming growth factor beta (TGF-beta) induces G(1) arrest in susceptible cells by multiple mechanisms that inhibit the G(1) cyclin-dependent kinases (Cdks), including Cdk2, Cdk4, and Cdk6. TGF-beta treatment of early passage finite lifespan human mammary epithelial cells (HMECs) led to an accumulation of p27(Kip1) in cyclin E1-Cdk2 complexes and kinase inhibition. The requirement for p27 in the G(1) arrest by TGF-beta was assessed by transfection of antisense p27 (ASp27) oligonucleotides into TGF-beta-treated HMECs. Despite a reduction in total and cyclin E-Cdk2 bound p27 after ASp27 transfection, HMECs remained arrested in the G(1) phase. Maintenance of the G(1) arrest was accompanied by increased association of the Cdk inhibitor p21(WAF-1/Cip-1) and the retinoblastoma family member p130(Rb2) in cyclin E1-Cdk2 complexes along with kinase inhibition. In contrast to the findings in HMECs, p27 was essential for G(1) arrest by TGF-beta in two tumor-derived lines. ASp27 transfection into two TGF-beta-responsive, cancer-derived lines was not associated with increased compensatory binding of p21 and p130 to cyclin E1-Cdk2, and these cell lines failed to maintain G(1) arrest despite the continued presence of TGF-beta. Progressive cell cycle deregulation leading to impaired checkpoint controls during malignant tumor progression may alter the role of p27 from a redundant to an essential inhibitor of G(1)-to-S phase progression.     

sst2 somatostatin receptor mediates cell cycle arrest and induction of p27(Kip1). Evidence for the role of SHP-1.

Activation of the somatostatin receptor sst2 inhibits cell proliferation by a mechanism involving the stimulation of the protein-tyrosine phosphatase SHP-1. The cell cycle regulatory events leading to sst2-mediated growth arrest are not known. Here, we report that treatment of Chinese hamster ovary cells expressing sst2 with the somatostatin analogue, RC-160, led to G1 cell cycle arrest and inhibition of insulin-induced S-phase entry through induction of the cyclin-dependent kinase inhibitor p27(Kip1). Consequently, a decrease of p27(Kip1)-cdk2 association, an inhibition of insulin-induced cyclin E-cdk2 kinase activity, and an accumulation of hypophosphorylated retinoblastoma gene product (Rb) were observed. However, RC-160 had no effect on the p21(Waf1/Cip1). When sst2 was coexpressed with a catalytically inactive mutant SHP-1 in Chinese hamster ovary cells, mutant SHP-1 induced entry into cell cycle and down-regulation of p27(Kip1) and prevented modulation by insulin and RC-160 of p27(Kip1) expression, p27(Kip1)-cdk2 association, cyclin E-cdk2 kinase activity, and the phosphorylation state of Rb. In mouse pancreatic acini, RC-160 reverted down-regulation of p27(Kip1) induced by a mitogen, and this effect did not occur in acini from viable motheaten (mev/mev) mice expressing a mutant SHP-1 with markedly deficient enzymes. These findings provide the first evidence that sst2 induces cell cycle arrest through the up-regulation of p27(Kip1) and demonstrate that SHP-1 is required for maintaining high inhibitory levels of p27(Kip1) and is a critical target of the insulin, and somatostatin signaling cascade, leading to the modulation of p27(Kip1).     

p27 Kip1 nuclear localization and cyclin-dependent kinase inhibitory activity are regulated by glycogen synthase kinase-3 in human colon cancer cells

The cellular mechanisms regulating intestinal differentiation are poorly understood. Sodium butyrate (NaBT), a short-chain fatty acid, increases p27 Kip1 expression and induces cell cycle arrest associated with intestinal cell differentiation. Here, we show that treatment of intestinal-derived cells with NaBT induced G0/G1 arrest and intestinal alkaline phosphatase, a marker of differentiation, activity and mRNA expression; this induction was attenuated by inhibition of glycogen synthase kinase-3 (GSK-3). Moreover, treatment with NaBT increased the nuclear, but not the cytosolic, expression and activity of GSK-3beta. NaBT decreased cyclin-dependent kinase CDK2 activity and induced p27 Kip1 expression; inhibition of GSK-3 rescued NaBT-inhibited CDK2 activity and blocked NaBT-induced p27 Kip1 expression in the nucleus but not in the cytoplasm. In addition, we demonstrate that NaBT decreased the expression of S-phase kinase-associated protein 2 (Skp2), and this decrease was attenuated by GSK-3 inhibition. Furthermore, NaBT increased p27 Kip1 binding to CDK2, which was completely abolished by GSK-3 inhibition. Overexpression of an active form of GSK-3beta reduced Skp2 expression, increased p27 Kip1 in the nucleus and increased p27 Kip1 binding to CDK2. Our results suggest that GSK-3 not only regulates nuclear p27 Kip1 expression through the downregulation of nuclear Skp2 expression but also functions to regulate p27 Kip1 assembly with CDK2, thereby playing a critical role in the G0/G1 arrest associated with intestinal cell differentiation.     

Accumulation of a form of p27(Kip1) not associated with Cdk-cyclin complexes in transforming growth factor-beta-arrested Mv1Lu cells

The p27(Kip1) cyclin-dependent kinase inhibitor translocates in response to transforming growth factor-beta to a Cdk2-cyclin E complex inhibiting its catalytic activity, but the p27(Kip1) protein levels are unaffected [1]. We show here that transforming growth factor-beta induces the accumulation of a form of p27(Kip1) representing a subpopulation of total p27(Kip1) in growth-arrested Mv1Lu epithelial cells. The inducible p27(Kip1) is detectable only by a specific p27(Kip1) monoclonal antibody recognizing a native form of p27(Kip1). The increase in this subset of p27(Kip1) correlates with G(1) arrest and withdrawal of the cells from the cycle induced by transforming growth factor-beta, serum starvation, or contact inhibition. In contrast to the majority of p27(Kip1) in the cells, the transforming growth factor-beta-inducible p27(Kip1) is devoid of cyclin-dependent kinase/cyclin interactions. The results indicate that growth arresting treatments induce the accumulation of non-cyclin-dependent kinase-bound p27(Kip1), which may function as a reservoir for inhibition of Cdk2-cyclin E activities.     

Regulation of p27Kip1 accumulation in murine B-lymphoma cells: role of c-Myc and calcium.

IgM cross-linking induces G1 arrest and apoptosis in murine B-lymphoma cells. It prevents pRb phosphorylation by decreasing cyclin-dependent kinase 2 activity via the up-regulation of cyclin kinase inhibitor p27Kip1. Anti-IgM also causes an increase in cytosolic free calcium and a loss of c-myc mRNA and protein. This down-regulation of c-Myc is prevented by CD40L, which rescues cells from anti-IgM-induced apoptosis. In this study, we addressed the mechanism(s) of anti-IgM-induced p27Kip1 accumulation. We examined effects of early events in B-cell receptor-mediated signaling, c-Myc down-regulation, and an increase in free calcium on p27Kip1. Down-regulation of c-myc alone had no effect on p27Kip1; neither did an increase in free calcium alone. Together, these two events led to p27Kip1 induction, growth arrest, and apoptosis. CD40L, the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, and cyclosporin A all prevented anti-IgM-induced p27Kip1 accumulation, suggesting that both the decrease in c-Myc expression and an increase in free calcium are necessary for p27Kip1 up-regulation.