Cyclin B and E2F-1 expression in prostate carcinoma cells treated with the novel retinoid CD437 are regulated by the ubiquitin-mediated pathway

E2F-1 and cyclin B are important regulators of the cell cycle, and their expressionand degradation are tightly regulated. Proteolysis of both molecules is mediated by the ubiquitin degradation pathway involving the activation of specific E3 ubiquitin ligases. Treatment of prostate carcinoma cells with the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (CD437/AHPN) results in the enhanced expression of E2F-1 and rapid degradation of cyclin B in the absence of the modulation of mRNA levels; this is accompanied by the S phase arrest of the cells and subsequent apoptosis. The elevated level of E2F-1 is because of the enhanced stability of the molecule, as indicated by pulse-labeling studies, demonstrating a prolonged half-life. The enhanced E2F-1 stability is associated with the concomitant acetylation of E2F-1, the disassociation of E2F-1 from the E2F-1 E3 ligase p45(SKP2), and decreased E2F-1 ubiquitination, suggesting CD437 inhibition of E-3 E2F-1 ligase activity. Exposure of the cells to CD437 also results in the enhanced association of the cyclin B E3 ligase APC with cyclin B and the rapid proteolysis of cyclin B. The CD437-enhanced proteolysis of cyclin B is blocked in the presence of the ubiquitin proteolysis inhibitor N-acetyl-leu-leu-norleu-al. Thus, CD437 modulates the expression of E2F-1 and cyclin B through the simultaneous stimulation and inhibition of the cyclin B and E2F-1 E3 ligases, respectively.     

Inhibition of cdk2 kinase activity by methylselenocysteine in synchronized mouse mammary epithelial tumor cells

Methylselenocysteine (MSC), an organic selenium compound has significant anticarcinogenic activity against mammary tumorigenesis. Previous experiments have demonstrated that MSC and inorganic selenite inhibit mammary cell (TM6 cell line) growth through different pathways. The present investigation demonstrated that MSC arrested cells in S phase during the TM6 cell cycle, which was followed by cells entering apoptosis at 48 h. Methylselenocysteine specifically affected the cdk2 kinase activity of the TM6 cells (54% reduction) at 16 h after release from growth arrest. The cdk4 kinase activity did not change during the cell cycle, confirming that cells had passed the G1 checkpoint and had entered S phase. The amount of cyclin E associated with cdk2 was increased by MSC by the 12 h time point, thereby facilitating entry of cells into S phase. Afterwards, cyclin E and cyclin A associated with cdk2 did not change for the remainder of the cell cycle. The data demonstrate that inhibition of mammary cell growth by MSC is mediated by alterations in progression of cells through S phase. The decrease in cdk2 kinase activity is coincident with prolonged arrest in S phase. One consequence of prolonged arrest may be apoptosis.      

Synthetic retinoid CD437 induces cell-dependent cycle arrest by differential regulation of cell cycle associated proteins

BACKGROUND: The synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene (CD437) exhibits a wide spectrum antitumor activity through induction of cellular apoptosis and cell cycle arrest. We investigated the effects and mechanisms of CD437 on cell cycle arrest of gastric cancer cells. MATERIALS AND METHODS: The activities of CD437 on cell growth were analyzed by measuring total cellular DNA. The effects of CD437 on cell cycle phase distribution were analyzed using flow cytometry. The levels of cell cycle associated proteins were analyzed by Western blot. The activities of cyclin-dependent kinases (cdks) were analyzed by phosphorylation of the histone H1 protein. RESULTS: CD437 at concentrations between 0.1 and 10 microM profoundly suppressed the growth of all six gastric cancer cell lines. Growth suppression associated with induction of G0/G1 or G2/M arrest was cell-line-dependent. CD437 decreased levels of cdk inhibitor p21 in G2/M-arrested SC-M1 cells. However, CD437 increased p21 levels in G0/G1-arrested AGS cells. Total and activated cyclin-dependent kinases were differentially regulated by CD437 in AGS and SC-M1 cells. CD437 (1 microM) induced activity of cdk2- and p34cdc2-associated H1 kinase by 14.6- and 1.8-fold, respectively, in SC-M1 cells. In contrast, CD437 slightly increased (1.6-fold) the cdk2-associated H1 kinase activity in AGS cells. CONCLUSION: CD437 profoundly suppressed the growth of gastric cancer cells, which was associated with cell-dependent induction of G0/G1 or G2/M arrest. The differential regulation of p21 that leads to alteration in the activity of cdks may play a critical role in cell-line-dependent regulation of cell cycle arrest following treatment with CD437.     

Mechanisms of suberoylanilide hydroxamic acid inhibition of mammary cell growth

 

The mechanism of suberoylanilide hydroxamic acid in cell growth inhibition involved induction of pRb-2/p130 interaction and nuclear translocation with E2F-4, followed by significant repression in E2F-1 and PCNA nuclear levels, which led to inhibition in DNA synthesis in mammary epithelial cell lines.

Synopsis

Background

Hybrid polar compounds (HPCs) have induced cell growth arrest, terminal differentiation and/or apoptosis in various transformed cell lines. We have previously reported that the prototype HPC (hexamethylene bisacetamide [HMBA]) was able to arrest the growth of transformed mammary (TM) 2H cells (p53 null), a highly tumorigenic mouse mammary epithelial cell line, by inhibiting G1 kinase activities, concomitant with an increase in the cyclin D2 protein level and hypophosphorylated isoforms of the three pRb pocket proteins, which led to the formation of stable cyclin D2/pRb complexes and G1 cell arrest. It has been reported that the second generation of HPCs (suberoylanilide hydroxamic acid [SAHA]), structurally related to but 2000-fold more potent than HMBA, was an inhibitor of histone deacetylase activity and caused accumulation of hyperacetylated histone H4 in murine erythroleukemia.

Objectives

To determine the mechanism of SAHA in cell growth inhibition in TM10 (p53 wt) and TM2H (p53 null) hyperplastic mouse mammary cell lines.

Methods

TM10 and TM2H cells were examined in the presence or absence of 2.5 μM SAHA for cell growth rate by [³H]-thymidine uptake, DNA synthesis by flow cytometry after cells were labeled with BrdU, G1/S cyclin-dependent kinase (cdk) activities, phosphorylation levels of pRb pocket proteins, protein levels of E2F-1, PCNA and p21, pRb-2/p130 interaction, and nuclear localization with E2F-4 by western blot, immunoprecipitation and immunostaining assays.

Results

SAHA was able to arrest cell growth at G1, and inhibited DNA synthesis in both TM10 and TM2H cell lines. Cell growth arrest was accompanied by increases in histone H3 and H4 protein and acetylation levels, a profound increase in the interaction and nuclear localization of pRb-2/p130–E2F-4 complexes, significant reductions in E2F-1 and PCNA protein levels, inhibition in G1/S cdk activities and increases in the levels of hypophosphorylated isoforms of three pRb pocket proteins.

Conclusion

A novel mechanism of SAHA mediated growth inhibition through significant increases in the formation and nuclear localization of pRb-2/p130–E2F-4 complexes, which resulted in cell growth arrest and significant repression in the levels of two key molecules, E2F-1 and PCNA, essential for DNA synthesis in two mouse mammary epithelial cell lines. These responses to SAHA were independent of the p53 status of the cell; however, reversibility of SAHA-mediated growth correlated with the wild type p53 status.     

Flavopiridol-induced apoptosis during S phase requires E2F-1 and inhibition of cyclin A-dependent kinase activity

Transformed cells are selectively sensitized to apoptosis induced by the cyclin-dependent kinase inhibitor flavopiridol after their recruitment to S phase. During S phase, cyclin A-dependent kinase activity neutralizes E2F-1 allowing orderly S phase progression. Inhibition of cyclin A-dependent kinase by flavopiridol could cause inappropriately persistent E2F-1 activity during S phase traversal and exit. Transformed cells, with high baseline levels of E2F-1 activity, may be particularly sensitive to cyclin A-dependent kinase inhibition, as the residual level of E2F-1 activity that persists may be sufficient to induce apoptosis. Here, we demonstrate that flavopiridol treatment during S phase traversal results in persistent expression of E2F-1. The phosphorylation of E2F-1 is markedly diminished, whereas that of the retinoblastoma protein is minimally affected, so that E2F-1/DP-1 heterodimers remain bound to DNA. In addition, manipulation of E2F-1 levels leads to predictable outcomes when cells are exposed to flavopiridol during S phase. Tumor cells expressing high levels of ectopic E2F-1 are more sensitive to flavopiridol-induced apoptosis during S phase compared with parental counterparts, and high levels of ectopic E2F-1 expression are sufficient to sensitize nontransformed cells to flavopiridol. Furthermore, E2F-1 activity is required for flavopiridol-induced apoptosis during S phase, which is severely compromised in cells homozygous for a nonfunctional E2F-1 allele. Finally, the response to flavopiridol during S phase is blunted in cells expressing a nonphosphorylatable E2F-1 mutant incapable of binding cyclin A, suggesting that the modulation of E2F-1 activity produced by flavopiridol-mediated cyclin-dependent kinase inhibition is critical for the apoptotic response of S phase cells.     

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.     

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.     

Cell cycle and transcriptional control of human myeloid leukemic cells by transforming growth factor beta

TGFbeta1 is a potent growth inhibitor of both primitive and more differentiated human myeloid leukemic cells. The extent of the growth inhibitory response to TGFbeta varies with cell type, and is not linked to stages of differentiation of cell lines. Downregulation of multiple cell cycle-regulatory molecules is a dominant event in TGFbeta1-mediated growth inhibition of human MV4-11 myeloid leukemia cells. Both G1-phase and G2-phase cyclins and cdks participate in the regulation of TGFbeta1-mediated growth inhibition of MV4-11 cells. By both depressing cdk2 synthesis and up-regulating cyclin E-associated p27, TGFbeta1 may magnify its inhibitory efficiency. TGFbeta1 also rapidly inhibits phosphorylation of pRb at several serine and threonine residues. The underphosphorylated pRb associates with E2F-4 in G1 phase, whereas the phosphorylated pRb mainly binds to E2F-1 and E2F-3 in proliferating MV4-11 cells. Since TGFbeta1 upregulates p130/E2F-4 complex formation and downregulates p107/E2F-4 complex formation, with E2F-4 levels remaining constant, our results suggest that E2F-4 is switched from p107 to pRb and p130 when cells exit from the cell cycle and arrest in G1 by TGFbeta1. In summary, TGFbeta1 inhibits growth of human myeloid leukemic cells through multiple pathways, whereas the "cdk inhibitor" p27 is both a positive and negative regulator.     

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.     

Adhesion-regulated G1 cell cycle arrest in epithelial cells requires the downregulation of c-Myc

Adhesion to the extracellular matrix is required for the expression and activation of the cyclin-cyclin-dependent kinase (CDK) complexes, and for G1 phase progression of non-transformed cells. However, in non-adherent cells no molecular mechanism has yet been proposed for the cell adhesion-dependent up-regulation of the p27 cyclin-dependent kinase inhibitor (CKI), and the associated inhibition of cyclin E-CDK2. We now show that in epithelial cells the expression of c-Myc is tightly regulated by cell-substrate adhesion. When deprived of adhesion, two independently derived mammary epithelial cell lines, 184A1N4 and MCF-10A, rapidly decrease their level of c-Myc mRNA and protein. This decrease in levels of c-Myc correlates with G1 phase arrest, as indicated by hypophosphorylation of pRb and inhibition of the activity of the cyclin E-CDK2 complex. In 184A1N4 cells, cell-substrate adhesion is required for the suppression of p27, and induction of cyclin E, E2F-1, but not cyclins D1 and D3. Enforced expression of c-Myc in non-adherent 184A1N4 and MCF-10A cells reverses the adhesion-dependent inhibition of cell cycle progression. Restoration of c-Myc in non-adherent cells induces the expression of E2F-1, and hyperphosphorylation of pRb in response to EGF treatment. In addition, expression of c-Myc results in the anchorage-independent activation of the CDK2 complex, the associated upregulation of cyclin E, and the destabilization and degradation of p27 by the ubiquitin-proteasome pathway. Our study thus suggests that c-Myc is the link between cell adhesion and the regulation of p27 and cyclin E-CDK2. Furthermore, we describe a role for c-Myc in adhesion-mediated regulation of E2F-1.     

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.     

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.     

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.     

Cell cyclins and cyclin-dependent kinase activities in mouse mammary tumor development

Breast cancer in humans, as in mice and rats, is thought tobe the result of sequential changes in the epithelial cellsof the mammalian glands. This study examines the altered expressionor activation of cell cycle related proteins in an in situ systemcomposed of hyperplasia, preneoplasia and neoplasia of mousemammary glands. The results showed a high level of cdc2/cdk2kinase activities in tumors compared to hyperplasias which wasindependent of cdc2/ cdk2 protein levels. Some of the cdk-associatedproteins which are thought to regulate cdk kinase activity wereexamined in these tissues. Cyclin A was overexpressed in allhyperplasias irrespective of their tumorigenic potentials. However,a number of alterations in cyclin E protein were associatedwith cdk2 and its associated kinase activity during mammarytumorigenesis. First, the level of normal cyclin E (p50) expressionwas positively correlated with the tumorigenic potentials ofdifferent hyperplasia lines. Second, several cyclin E isoforms(p48, p43, p35, p34, p32) were detected only in tumor tissues.Third, a 2.3- and 8.3-fold increase in cyclin E-associated cdk2kinase activity was present in highly tumorigenic hyperplasiasand neoplasias respectively compared to the low tumorigenichyperplasias. Polymorphic cell nuclear antigen (PCNA) proteinbound to cdk2 was a better indicator for cell proliferationand cdk2 kinase activity than the PCNA labeling index. Theseresults suggest a sequential pattern of multiple derangementsin factors regulating cdk2 protein function during mammary tumorigenesis.High levels of cdk2 kinase activity are observed only in tumorsand appear to be closely related to alterations in cyclin Eprotein expression.