Differential modulation of paclitaxel-mediated apoptosis by p21Waf1 and p27Kip1

The impact of the cyclin dependent kinase (CDK) inhibitors p21Waf1 and p27Kip1 on paclitaxel-mediated cytotoxicity was investigated in RKO human colon adenocarcinoma cells with the ecdysone-inducible expression of p21Waf1 or p27Kip1. Ectopic expression of p27Kip1 arrested cells at G1 phase, whereas p21Waf1 expression arrested cells at G1 and G2. Expression of p21Waf1 after paclitaxel treatment produced much greater resistance to paclitaxel than did expression of p27Kip1. We attributed this difference to the additional block at G2 induced by p21Waf1, which prevented cells from entering M phase and becoming paclitaxel susceptible. Expression of p21Waf1 inhibited p34cdc2 activity and markedly reduced paclitaxel-mediated mitotic arrest, from 87.5 to 23%. In contrast, p27Kip1 expression also inhibited p34cdc2 but reduced mitotic arrest only slightly, from 87. 4 to 74.5%. We concluded that the G2 block produced by p21Waf1, but not by p27Kip1, contributed to their unequal modulation of sensitivity to paclitaxel-mediated apoptosis in RKO cells, and there is no causal relationship between paclitaxel-mediated cytotoxicity and elevation of p34cdc2 activity.     

Up-regulation of cdc2 protein during paclitaxel-induced apoptosis

Microtubule damages induced by paclitaxel inhibit proteasome-dependent degradation of cyclin B, resulting in a sustained activation of cyclin B/cdc2 kinase and a cell cycle arrest in mitosis. It has been previously shown that this kinase activity is also required for paclitaxel-induced apoptosis. We found here that paclitaxel increased cdc2 mRNA and protein levels and led to an accumulation of cdc2 in the active dephosphorylated form in NIH-OVCAR-3 cells. The addition of cycloheximide inhibited the paclitaxel-induced increase in cdc2 protein level, further indicating that paclitaxel stimulates cdc2 synthesis. This increase in cdc2 synthesis is a consequence of paclitaxel-induced arrest in mitosis. Indeed, dual analysis of DNA and cdc2 protein contents indicated that cdc2 up-regulation occurred in cells arrested with a G2/M DNA content. Furthermore, no up-regulation of cdc2 protein was observed when paclitaxel-treated cells were prevented from entering mitosis by treatment with purvalanol A, a cyclin-dependent kinase (CDK) inhibitor, or stimulated to exit mitosis with 2-AP, a non-specific kinase inhibitor. In addition, when paclitaxel-induced apoptosis was inhibited by Bcl-2 over-expression, cdc2 up-regulation did not occur, leading to a lower level of activation of the cyclin B/cdc2 complex. Taken together, these results indicated that paclitaxel-induced cdc2 protein synthesis participates in a positive feedback loop designed to increase the activity of cyclin B/cdc2 kinase and thus may play a role in paclitaxel-induced apoptosis.     

Inhibition of p34cdc2 kinase activation, p34cdc2 tyrosine dephosphorylation, and mitotic progression in Chinese hamster ovary cells exposed to etoposide.

p34cdc2 kinase, an enzyme essential for mitosis in mammalian cells, may play a role in etoposide-induced G2 phase arrest of Chinese hamster ovary cells. In this study, etoposide is shown to cause inhibition of a specific p34cdc2 kinase activation pathway, that of tyrosine dephosphorylation. Exposure of asynchronously dividing cells to etoposide caused a simultaneous rapid decline of both mitotic index and p34cdc2 kinase activity, suggesting that the kinase was not activated and that the arrest point was in late G2 phase. Using synchronized cells, p34cdc2 kinase exhibited maximal activity at the G2/M transition. Activation of the kinase and the onset of mitosis were accompanied by increased electrophoretic mobility and tyrosine dephosphorylation of the p34cdc2 protein. A 1-h exposure to etoposide during early G2 phase inhibited p34cdc2 kinase activation, its shift in electrophoretic mobility, and its tyrosine dephosphorylation, all of which correlated with a delay in mitotic progression. The interaction between the p34cdc2 and cyclin B proteins appeared unaffected under etoposide exposure conditions which resulted in greater than 70% inhibition of p34cdc2 kinase activity and almost complete cessation of transition into mitosis. These data suggest that mammalian cells express a DNA damage-responsive mechanism which controls mitotic progression at the level of p34cdc2 tyrosine dephosphorylation.     

E2F-1 induces proliferation-specific genes and suppresses squamous differentiation-specific genes in human epidermal keratinocytes

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis. To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes. Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes. Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas. Oncogene (2000).     

Expression and prognostic role of cyclin-dependent kinase 1 (cdc2) in hepatocellular carcinoma

The expression of cyclin-dependent kinase 1 (cdc2), cyclin A and cyclin B1 was immunohistochemically studied in 101 hepatocellular carcinomas (HCC). cdc2 overexpression was directly related to advanced stage, portal invasion, intrahepatic metastasis, poor differentiation, high alpha-fetoprotein level, large size, high Ki-67 labeling index and poor prognosis. Cyclin A and B1 overexpression showed similar tendency to that of cdc2, but they were not recognized as independent prognostic factors by multivariate analysis. These findings suggest that cdc2 plays the most crucial role of the G2/M modulators in cell cycle progression and cell proliferation of HCC and significantly predicts the recurrence of this carcinoma.     

2-Methoxyestradiol and paclitaxel have similar effects on the cell cycle and induction of apoptosis in prostate cancer cells

2-Methoxyestradiol (2-ME) is an endogenous metabolite of estradiol with promise for cancer chemotherapy, including advanced prostate cancer. We have focused on events related to cell cycle arrest (G1 and G2/M) and induction of apoptosis in human prostate cancer cells. Treatment with 2-ME increased cyclin B1 protein and its associated kinase activity followed by later inhibition of cyclin A-dependent kinase activity and induction of apoptosis. Similar results were obtained with paclitaxel (taxol), a clinically relevant agent used to treat advanced prostate cancer. Cyclin-dependent kinase inhibitors prevented 2-ME and paclitaxel-mediated increase in cyclin B1-dependent kinase activity and blocked induction of apoptosis. Reduction of X-linked inhibitor of apoptosis (XIAP) protein by 2-ME and paclitaxel correlated with increased apoptosis. Lower doses of 2-ME and paclitaxel resulted in G1 (but not G2/M) cell cycle arrest in the p53 wild type LNCaP cell line, but with minimal induction of apoptosis. We suggest that 2-ME and paclitaxel-mediated induction of apoptosis in prostate cancer cells requires activation of cyclin B1-dependent kinase that arrests cells in G2/M and subsequently leads to the induction of apoptotic cell death.     

Constitutive activation of cyclin B1-associated cdc2 kinase overrides p53-mediated G2-M arrest

Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and that this function is required for the maintenance of genomic integrity. In this study, we investigated a regulatory role of p53 specifically in G2-M transition. Human bladder carcinoma cells lacking functional p53 were synchronized at G1-S, which is preceded by p53-mediated G1 arrest. p53 expression in the synchronized cells was induced by infection with a recombinant adenovirus that encodes p53. After release from the G1-S arrest, the cells progressed to S-phase and G2 but failed to enter mitosis. Biochemical analysis showed that p53 inhibits cell cycle-dependent expression of cdc2 and cyclin B1 and consequently inhibits cdc2 kinase. The role of cyclin B1-associated cdc2 kinase in p53-mediated G2-M arrest was further investigated by expression of both cyclin B1 and cdc2AF, in which inhibitory phosphorylation sites were substituted. The cells expressing both cdc2AF and cyclin B1 showed a constitutive activation of cdc2 kinase during cell cycle progression and passed through G2-M regardless of p53 expression. Therefore, inactivation of cdc2 kinase through cdc2 and cyclin B1 repression is an essential step in p53-mediated G2-M arrest.     

Flavopiridol, a cyclin-dependent kinase inhibitor, prevents spindle inhibitor-induced endoreduplication in human cancer cells.

Defects in cell cycle checkpoints can lead to chromosome abnormality, aneuploidy, and genomic instability, all of which can contribute to tumorigenesis. Recent studies and data presented in this study indicate that cells with compromised G1 checkpoint endoreduplicate and become polyploid in response to microtubule inhibitors. Previous studies have shown that polyploid cells are unstable and lose chromosomes randomly to give aneuploidy. In this study, we show that endoreduplication and polyploidation can be prevented by inhibiting the cyclin-dependent kinases (Cdks) by flavopiridol, a synthetic flavone presently undergoing phase II clinical trials. In our initial studies, we treated MCF-7 cells with paclitaxel, which results in the arrest of cells in G1 with 4n DNA content (pseudo G1). This was coincident with increased p53 and p21 protein expression and decreased cyclin E/Cdk2 kinase activity. In contrast, G1 checkpoint-compromised MDA-MB-468 (p53-/- and pRb-/-) and p21-/- HCT116 do not arrest in the pseudo G1 state after exposure to microtubule inhibitors and enter in the S phase with 4n DNA content. More than 60% of MDA-MB-468 cells accumulate with >4n DNA content after 72 h of nocodazole treatment. The MPM-2 labeling showed that 8n cells also undergo mitosis. These cells display deregulated and persistent activation of cyclin E/Cdk2 and cyclin B1/cdc2 kinase activity. Administration of flavopiridol after mitotic block results in the arrest of cells in the pseudo G1 state and the dramatic decrease in cells containing >4n DNA content in MDA-MB-468 cells. The cyclin E/Cdk2 and cyclin B1/cdc2 kinase activities remained low after exit from mitosis. Furthermore, pRb was hypophosphorylated after the addition of flavopiridol in p21-deficient HCT116 cells, indicating the arrest of cells at the pseudo G1 state. Based on these studies, we propose that flavopiridol preserves the genomic stability by preventing endoreduplication and polyploidy and thus has the potential to be used as a chemopreventive agent to prevent the occurrence of neoplasia.     

Bortezomib potentially inhibits cellular growth of vascular endothelial cells through suppression of G2/M transition

Bortezomib, a selective 26S proteasome inhibitor, has shown clinical benefits against refractory multiple myeloma. The indirect anti‐angiogenic activity of bortezomib has been widely recognized; however, the growth‐inhibitory mechanism of bortezomib on vascular endothelial cells remains unclear, especially on the cell cycle. Here, we showed that bortezomib (2 nM of the IC₅₀ value) potently inhibited the cellular growth of human umbilical vascular endothelial cells (HUVECs) via a vascular endothelial growth factor receptor (VEGFR)‐independent mechanism resulting in the induction of apoptosis. Bortezomib significantly increased the vascular permeability of HUVECs, whereas a VEGFR‐2 tyrosine kinase inhibitor decreased it. Interestingly, a cell cycle analysis using flow cytometry, the immunostaining of phospho‐histone H3, and Giemsa staining revealed that bortezomib suppressed the G2/M transition of HUVECs, whereas the mitotic inhibitor paclitaxel induced M‐phase accumulation. A further analysis of cell cycle‐related proteins revealed that bortezomib increased the expression levels of cyclin B1, the cdc2/cyclin B complex, and the phosphorylation of all T14, Y15, and T161 residues on cdc2. Bortezomib also increased the ubiquitination of cyclin B1 and wee1, but inhibited the kinase activity of the cdc2/cyclin B complex. These protein modifications support the concept that bortezomib suppresses the G2/M transition, rather than causing M‐phase arrest. In conclusion, we demonstrated that bortezomib potently inhibits cell growth by suppressing the G2/M transition, modifying G2/M‐phase‐related cycle regulators, and increasing the vascular permeability of vascular endothelial cells. Our findings reveal a cell cycle‐related mode of action and strongly suggest that bortezomib exerts an additional unique vascular disrupting effect as a vascular targeting drug. (Cancer Sci 2010)     

Extracellular signal-regulated kinase induces cyclin D1 and Cdk-2 expression and phosphorylation of retinoblastoma in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Southeast Asia. Hyperphosphorylation of retinoblastoma (pRB) by cyclin/CDKs in G1/S transition is required for its inactivation and cell cycle progression. In the present study, we report that phosphorylation of pRB at Ser780 and Ser795 was detected in 71% (33 of 46) and 63% (29 of 46) of HCCs examined respectively. pRB protein was undetectable in 13% (6 of 46) of HCCs examined. Phosphorylated pRB was localized in the nuclei of hepatocarcinoma cells. Benign hepatocytes exhibited very weakly or no nuclear staining for phosphorylated pRB. Over-expression of E2F-1, cyclin D1, Cdk-2, Cdk-4 and cyclin A was found in 64% (30 of 46), 43% (26 of 46), 28% (11 of 46), 71% (33 of 46) and 63% (29 of 46) of HCCs examined respectively and this was correlated with elevation of ERK. Treatment of HepG2 cells with MEK1/2 inhibitor U0126 resulted in cell cycle arrest, downregulation of cyclin D1 and Cdk-2 expression and inhibition of pRB phosphorylation at Ser780 and Ser795. Ectopic expression of activated MEK1 in HepG2 cells increased cyclin D1 and Cdk-2 expression, phosphorylation of pRB at Ser780 and Ser795, and percentage of cells in S phase. Our data indicate that activated ERK plays an important role in cyclin D1 and Cdk-2 expression and phosphorylation of pRB at Ser780 and Ser795 in liver cancer cells.