Two glucosylceramide synthase inhibitors attenuate doxorubicin-induced p21Cip1/Waf1 upregulation in HepG2 cells, irrespective of their differential chemosensitizing properties

We have previously reported that HepG2 human hepatocarcinoma cells are sensitized to doxorubicin-induced apoptosis by the glucosylceramide synthase inhibitor d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) but not by the more specific inhibitor d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol (PPPP). Herein we investigated whether the chemosensitizing action of PDMP impinged on any unspecific effect of this compound on doxorubicin-induced expression of p53 and/or p21(Cip1/Waf1), namely two proteins reported to modulate the apoptotic response to DNA-damaging agents, in a positive or negative fashion, respectively. We show that, in HepG2 cells, PDMP did not substantially affect doxorubicin-induced p53 upregulation, whereas drug-evoked upregulation of p21(Cip1/Waf1) was markedly attenuated. Although this outcome could be expected to account for the chemosensitizing effect of PDMP, impaired upregulation of p21(Cip1/Waf1), in the setting of unaltered p53 expression, was also observed in the case of PPPP. These results, while raising the possibility of a link between attenuation of drug-evoked p21(Cip1/Waf1) expression and redirection of (glyco)sphingolipid metabolism, show that, differently from other tumor systems, attenuation of doxorubicin-induced p21(Cip1/Waf1) expression is at least not sufficient to sensitize HepG2 cells to the apoptotic action of the drug.     

p53 dephosphorylation and p21Cip1/Waf1 translocation correlate with caspase-3 activation in TGF-β1-induced apoptosis of HuH-7 cells

The p53 tumor suppressor gene product plays an important role in the regulation of apoptosis. Transforming growth factor beta1 (TGF-beta1)-induced apoptosis in hepatic cells is associated with reduced expression of the retinoblastoma protein (pRb) and subsequent E2F-1-activated expression of apoptosis-related genes. In this study, we explored the potential role of p53 in TGF-beta1-induced apoptosis. HuH-7 human hepatoma cells were either synchronized in G1, S and G2/M phases, or treated with 1 nM TGF-beta1. The results indicated that greater than 90% of the TGF-beta1-treated cells were arrested in G1 phase of the cell cycle. This was associated with enhanced p53 dephosphorylation and p21(Cip1/Waf1) expression, which coincided with decreased Cdk2, Cdk4, and cyclin E expression, compared with synchronized G1 cells. In addition, p53 dephosphorylation coincided with caspase-3 activation, and translocation of p21(Cip1/Waf1) and p27(Kip1) into the cytoplasm, all of which were suppressed by caspase inhibition of TGF-beta1-induced apoptosis. Finally, phosphatase inhibition and pRb overexpression partially inhibited p53-mediated apoptosis. In conclusion, the results demonstrated that TGF-beta1-induced p53 dephosphorylation is associated with caspase-3 activation, and cytosolic translocation of p21(Cip1/Waf1) and p27(Kip1), resulting in decreased expression of Cdks and cyclins. Further, p53 appears to mediate TGF-beta1-induced apoptosis downstream of the pRb/E2F-1 pathway.     

Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression

The sphingoplipid ceramide is responsible for a diverse range of biochemical and cellular responses including a putative role in modulating cell cycle progression. Herein, we describe that an accumulation of ceramide, achieved through the exogenous application of C(6)-ceramide or exposure to sphingomyelinase, induces a G(2) arrest in Rhabdomyosarcoma (RMS) cell lines. Utilizing the RMS cell line RD, we show that this G(2) arrest required the rapid induction of p21(Cip1/Waf1) independent of DNA damage. This was followed at later time points (48 h) by the commitment to apoptosis. Apoptosis was prevented by Bcl-2 overexpression, but permitted the maintenance of elevated p21(Cip1/Waf1) protein expression and the stabilization of the G(2) arrest response. Inhibition of p21(Cip1/Waf1) protein synthesis with cyclohexamide (CHX) or silencing of p21(Cip1/Waf1) with siRNA, prevented ceramide-mediated G(2) arrest and the late induction of apoptosis. Further, adopting the recent discovery that murine double minute 2 (MDM2) controls p21(Cip1/Waf1) expression by presenting this CDK inhibitor to the proteasome for degradation, RD cells overexpressing MDM2 abrogated ceramide-mediated p21(Cip1/Waf1) induction, G(2) arrest and the late ensuing apoptosis. Collectively, these data further support the notion that ceramide accumulation can modulate cell cycle progression. Additionally, these observations highlight MDM2 expression and proteasomal activity as key determinants of the cellular response to ceramide accumulation.     

The molecular mechanisms of vitamin C on cell cycle regulation in B16F10 murine melanoma

Vitamin C has inconsistent effects on malignant tumor cells, which vary from growth stimulation to apoptosis induction. It is well known that melanoma cells are more susceptible to vitamin C than any other tumor cells, but the precise mechanism remains to be elucidated. In the present study, the proliferation of B16F10 melanoma cells was suppressed by vitamin C, which induced growth arrest in a dose-dependent manner without cytotoxic effects. Therefore, we investigated the changes in cell cycle distribution of B16F10 melanoma cells by staining DNAs with propidium iodide (PI). The growth inhibition of B16F10 melanoma by vitamin C was associated with an arrest of cell cycle distribution at G1 stage. In addition, the levels of p53-p21Waf1/Cip1 increased during G1 arrest, which were essential for vitamin C-induced cell cycle arrest. The increased p21Waf1/Cip1 inhibited CDK2. Moreover, the activity of p53-p21Waf1/Cip1 pathway was closely related with the activation of checkpoint kinase 2 (Chk2). Inhibitor of the PI3K-family, LY294002 and the ATM/ATR inhibitor, caffeine, blocked vitamin C-induced growth arrest in B16F10 melanoma cells. These results suggest that vitamin C might be a potent agent to inhibit proliferative activity of melanoma cells via the regulation of Chk2-p53-p21Waf1/Cip1 pathway.     

JNK supports survival in melanoma cells by controlling cell cycle arrest and apoptosis

JNK1/2 proteins belong to the family of stress-activated protein kinases. They play a complex role in growth regulation, inducing either cell death or growth support. In this report, we provide evidence that, in human melanoma cells, JNK inhibition with the small molecule inhibitor SP600125 induces either predominantly a G2/M arrest or apoptosis depending on the cell line. In 1205Lu cells, JNK inhibition induced cell cycle arrest through p53-dependent induction of p21 Cip1/Waf1 expression, while in WM983B cells, induction of apoptosis by JNK inhibition was accompanied by p53, Bad and Bax induction, not p21 Cip1/Waf1. JNK inhibition with the small molecule inhibitor SP600125 slowed growth of all cell lines, although the effect was markedly greater in cells exhibiting high phospho- (P-)JNK1 levels. Specific gene knockdown of JNK1 by means of siRNA oligonucleotides inhibited cell growth only in melanoma cell lines exhibiting high P-JNK1 levels. siRNAs directed against JNK2 did not reduce cell growth in any of the cell lines tested. Together, our findings demonstrate that JNK, and in particular the JNK1 isoform, support the growth of melanoma cells, by controlling either cell cycle progression or apoptosis depending on the cellular context.     

p53-independent induction of p21(waf1/cip1) contributes to the activation of caspases in GTP-depletion-induced apoptosis of insulin-secreting cells

We investigated the role of some key regulators of cell cycle in the activation of caspases during apoptosis of insulin-secreting cells after sustained depletion of GTP by a specific inosine 5'-monophosphate dehydrogenase inhibitor, mycophenolic acid (MPA). p21(Waf1/Cip1) was significantly increased following MPA treatment, an event closely correlated with the time course of caspase activation under the same conditions. MPA-induced p21(Waf1/Cip1) was not mediated by p53, since p53 mass was gradually reduced over time of MPA treatment. The increment of p21(Waf1/Cip1) by MPA was further enhanced in the presence of a pan-caspase inhibitor, indicating that the increased p21(Waf1/Cip1) may occur prior to caspase activation. This notion of association of p21(Waf1/Cip1) accumulation with caspase activation and apoptosis was substantiated by using mimosine, a selective p21(Waf1/Cip1) inducer independent of p53. Mimosine, like MPA, also increased p21(Waf1/Cip1), promoted apoptosis and simultaneously increased the activity of caspases. Furthermore, knocking down of p21(Waf1/Cip1) transfection of siRNA duplex inhibited caspase activation and apoptosis due to GTP depletion. In contrast to p21(Waf1/Cip1), a reduction in p27(Kip1) occurred in MPA-treated cells. These results indicate that p21(Waf1/Cip1) may act as an upstream signal to block mitogenesis and activate caspases which in turn contribute to induction of apoptosis.     

Senescence-like changes induced by expression of p21(waf1/Cip1) in NIH3T3 cell line

P21(Waf1/Cip1) is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21(Waf1/Cip1) involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidence for a link between p21(Waf1/Cip1) and cellular senescence. While in murine cells, the role of p21(Waf1/Cip1) is indefinite. We explored this issue using NIH3T3 cells with inducible p21(Waf1/Cip1) expression. Induction of p21(Waf1/Cip1) triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features, such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed that p21(Waf1/Cip1)-transduced NIH3T3 cells expressed beta-galactosidase activity at pH 6.0, which is known to be a marker of senescence. Our results suggest that p2l(Waf1/Cip1) can also induce senescence-like changes in murine cells.     

SarCNU-induced G2/M arrest in hepatoma cells is mediated by a p53-independent phosphorylation of cdc-2 at Tyr15

Hepatocellular carcinoma (HCC) is a major health problem in the Asia-Pacific region, with high incidence and mortality rate. There is currently no effective treatment for inoperable cases that represent the vast majority of patients. In the present study, we report that in vitro treatment of primary hepatoma, HepG2 (wild-type p53), PLC/PRF/5 (p53-mutant), and Hep3B (p53-deleted) cells with 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) resulted in upregulation of p53, p21(Cip1/Waf1), phosphorylated cdc-2 at Tyr15 in wild-type p53 cells and phosphorylation of cdc-2 at Tyr15 in p53-mutant or p53-deleted hepatoma cells. This was accompanied by the reduction in cdc-2 kinase activity and G(2)/M cell cycle arrest. These findings indicate that SarCNU-induced G(2)/M growth arrest in hepatoma cells by a p53-independent phosphorylation of cdc-2. Our data suggest the potential use of SarCNU in treatment of HCC.     

p21Waf1/Cip1 plays a critical role in modulating senescence through changes of DNA methylation

It has been reported that genomic DNA methylation decreases gradually during cell culture and an organism's aging. However, less is known about the methylation changes of age-related specific genes in aging. p21(Waf1/Cip1) and p16(INK4a) are cyclin-dependent kinase (Cdk) inhibitors that are critical for the replicative senescence of normal cells. In this study, we show that p21(Waf1/Cip1) and p16(INK4a) have different methylation patterns during the aging process of normal human 2BS and WI-38 fibroblasts. p21(Waf1/Cip1) promoter is gradually methylated up into middle-aged fibroblasts but not with senescent fibroblasts, whereas p16(INK4a) is always unmethylated in the aging process. Correspondently, the protein levels of DNA methyltransferase 1 (DNMT1) and DNMT3a increase from young to middle-aged fibroblasts but decrease in the senescent fibroblasts, while DNMT3b decreases stably from young to senescent fibroblasts. p21(Waf1/Cip1) promoter methylation directly represses its expression and blocks the radiation-induced DNA damage-signaling pathway by p53 in middle-aged fibroblasts. More importantly, demethylation by 5-aza-CdR or DNMT1 RNA interference (RNAi) resulted in an increased p21(Waf1/Cip1) level and premature senescence of middle-aged fibroblasts demonstrated by cell growth arrest and high beta-Galactosidase expression. Our results suggest that p21(Waf1/Cip1) but not p16(INK4a) is involved in the DNA methylation mediated aging process. p21(Waf1/Cip1) promoter methylation may be a critical biological barrier to postpone the aging process.     

The Cyclin-Dependent Kinase Inhibitor p21CIP1/WAF1 Blocks Paclitaxel-Induced G2M Arrest and Attenuates Mitochondrial Injury and Apoptosis in p53-Null Human Leukemia Cells

The functional significance of the cyclin-dependent kinase inhibitor (CDKI) p21Cip1/WAF1 in paclitaxel-mediated lethality was examined in p53-null human leukemia cells (U937 and Jurkat). In these cells, paclitaxel exposure failed to induce p21Cip1/Waf1 expression. Nevertheless, stable expression of U937 cells with a p21Cip1/WAF1 antisense construct blocked paclitaxel-induced G2M arrest and significantly, albeit modestly, increased mitochondrial injury, caspase activation, apoptosis, and loss of clonogenic potential. These protective effects were less than those observed in cells exposed to the antimetabolite ara-C. Consistent with these results, enforced expression of p21Cip1/WAF1 in Jurkat cells transfected with a construct driven by a doxycycline-responsive promoter increased the percentage of cells arrested in G2M, but attenuated paclitaxel-mediated mitochondrial injury and apoptosis. Unexpectedly, enforced expression of p21Cip1/WAF1 diminished paclitaxel-mediated inactivation of ERK, and reduced paclitaxel-induced activation of JNK as well as Bcl-2 phosphorylation. Together, these findings suggest that the CDKI p21Cip1/WAF1 modestly but significantly protects p53-null human leukemia cells from paclitaxel-mediated lethality, and raise the possibility that p21Cip1/WAF1-associated perturbations in signal transduction pathways as well as Bcl-2 phosphorylation status may play a role in this phenomenon.     

The p53/p21Cip1/ Waf1 pathway mediates the effects of SPARC on melanoma cell cycle progression

Secreted protein acidic and rich in cysteine (SPARC), or osteonectin, belongs to the family of matricellular proteins that modulate cell-matrix interactions and cellular functions. SPARC is highly expressed in melanoma, and we reported that SPARC promotes epithelial/mesenchymal-like changes and cell migration. Here, we used siRNA and conditional shRNA to investigate the contribution of tumor-derived SPARC to melanoma cell growth in vitro and in vivo. We found that depletion of SPARC induces G2/M cell cycle arrest and tumor growth inhibition with activation of p53 and induction of p21(Cip1/Waf1) acting as a checkpoint, preventing efficient mitotic progression. In addition, we demonstrate that reduced mesenchymal features and the invasive potential of SPARC-silenced cells are independent of p21(Cip1/Waf1) induction and cell cycle arrest. Importantly, overexpression of SPARC reduces p53 protein levels and leads to an increase in cell number during exponential growth. Our findings indicate that in addition to its well-known function as a mediator of melanoma cell migration and tumor-host interactions, SPARC regulates, in a cell-autonomous manner, cell cycle progression and proliferation through the p53/p21(Cip1/Waf1) pathway.     

Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A

Hepatitis C virus nonstructural protein, NS5A, is a phosphoprotein produced from the processing of the viral polyprotein precursor. NS5A associates with several cellular proteins in mammalian cells, and the biological consequences of this interaction are currently unknown. To this end, five stable NS5A-expressing murine and human cell lines were established. Tetracycline-regulated NIH3T3 cells and rat liver epithelial cells as well as the constitutive, NS5A-expressing, human Chang liver, HeLa, and NIH3T3 cells all exhibited cell growth retardation compared with the control cells. Cell cycle analysis by flow cytometry indicated that the NS5A-expressing human epitheloid tumor cells had a reduced S phase and an increase in the G(2)/M phase, which could be explained by a p53-dependent induction of p21(Waf1/Cip1) protein and mRNA levels. NS5A interacts with Cdk1 in vivo and in vitro, and a significant portion of the p21(Waf1/Cip1) was found to be in a complex with Cdk2 in the NS5A-expressing human hepatic cell line. Cdk1 and cyclin B1 proteins were also reduced in human Chang liver cells consistent with the increase in G(2)/M phase. Our results suggest that the NS5A protein causes growth inhibition and cell cycle perturbations by targeting the Cdk1/2-cyclin complexes.     

Susceptibility to killer T cells of gastric cancer cells enhanced by Mitomycin-C involves induction of ATBF1 and activation of p21 (Waf1/Cip1) promoter

Alpha-fetoprotein (AFP) expression is observed in embryonic tissues and, the expression of this protein is absent in normal adult tissues. The re-elevation of serum AFP strongly suggests generation of a malignant tumor in an adult. We demonstrated here that AFP-producing gastric cancer (AFP-gastric cancer) could be treated by a combination therapy with a low dose of Mitomycin-C (MMC) and lymphokineactivated killer T (LAK-T) cells. Treatment with MMC of AFP-gastric cancer cells enhanced their susceptibility to LAK-T cells and induced ATBF1 gene expression. We revealed here a novel signal pathway for regulation of the cell cycle of AFP-gastric cancer cells through ATBF1, which enhances the promoter activity of the p21 (Waf1/Cip1) gene. Immunoprecipitation revealed the direct interaction between ATBF1 and p53. Overexpressed ATBF1 stimulated p21 (Waf1/Cip1) promoter activity up to 4-fold compared with basal activity. The expression level of ATBF1 mRNA was doubled by MMC (0.05 microg/ml) treatment. The MMC treatment and ATBF1 overexpression synergistically activated the p21 (Waf1/Cip1) promoter activity in a dose-dependent manner up to 7-fold compared with basal activity.     

5-Fluorouracil (5-FU) induced apoptosis in gastric cancer cell lines: role of the p53 gene

We examined chemosensitivity to 5-fluorouracil (5-FU) in four human gastric cancer cell lines, by analyzing the expression of p53 and its related genes. Treatment with 1mM 5-FU induced variable degrees of apoptosis in the cultured cells. The apoptotic indices 72 h after treatment were approximately 14% in MKN-74 (wild-type p53 gene), 12% in MKN-45 (wild-type), 3% in MKN-28 (mutated) and 0.5% in KATO-III cells (deleted), respectively. On the other hand, 50 M 5-FU had little effect on the induction of apoptosis in MKN-74 cells, the value being approximately 2% after 72 h. Induction of P53 expression was noted 3 h after initiating the treatment, followed by the induction of P21/Waf1 after 6 h in both MKN-74 and MKN-45 cells. The same expression mode was noted in MKN-74 treated with 50 M 5-FU. Conversely, the level of P53 expression was constant in MKN-28 cells and absent in KATO-III cells, in which P21/Waf1 had never been induced. The Bax/Bcl-2 expression ratio was gradually elevated for up to 72 h in MKN-74 and MKN-45 cells treated with 1mM 5-FU; in contrast, it was unchanged in MKN-28 and KATO-III cells, and MKN-74 treated with 50 M 5-FU. These results might indicate that (1) 1mM 5-FU induces apoptosis in cultured gastric cancer cells carrying the wild-type p53 gene, but not those carrying the mutated type or a gene deletion, and (2) the elevated Bax/Bcl-2 expression ratio plays a more crucial role than the higher expression of P21/Waf1 in the induction of p53- gene dependent apoptosis.