Cellular senescence induced by p53-ras cooperation is independent of p21waf1 in murine embryo fibroblasts

Oncogenic activation in primary murine fibroblasts initiates a senescence-like cell cycle arrest that depends on the p53 tumor suppressor pathway. Conditional p53 activation efficiently induced a reversible cell cycle arrest but was unable to induce features of senescence. In contrast, coexpression of oncogenic ras with p53 produced an irreversible cell cycle arrest that displayed features of cellular senescence. Introduction of a conditional murine p53 allele (p53val135) into double p53/p21-null mouse embryonic fibroblasts showed that p21waf1 was not required for this effect, since p53-/-;p21-/- double-null cells undergo terminal growth arrest with features of senescence following coexpression of oncogenic Ras and p53. Our results indicate that oncogenic activation of the Ras pathway in murine fibroblasts converts p53 into a senescence inducer through a p21waf1-independent mechanism.     

Caffeine induces G2/M arrest and apoptosis via a novel p53-dependent pathway in NB4 promyelocytic leukemia cells

Methylxantine derivative, caffeine, is known to prevent the p53-dependent apoptosis pathway via inhibition of ATM (ataxia telangiectasia mutated) kinase, which activates p53 by phosphorylation of the Ser-15 residue. In contrast, it has been reported that caffeine induces p53-mediated apoptosis through Bax protein in non-small-cell lung cancer cells. Therefore, the effects of caffeine on cellular growth in malignant cells are controversial. We investigated the effects of caffeine on cell proliferation, cell cycle progression, and induction of apoptosis in NB4 promyelocytic leukemia cells containing wild-type p53. Caffeine suppressed the cellular growth of NB4 cells in a dose- and time-dependent manner. Caffeine induced G(2)/M phase cell cycle arrest in NB4 cells in association with the induction of phosphorylation at the Ser-15 residue of p53 and induction of tyrosine phosphorylation of cdc2. Expression of Bax protein was increased in NB4 cells after treatment with caffeine. Interestingly, the antisense oligonucleotides for p53 significantly reduced p53 expression and caffeine-induced G(2)/M phase cell cycle arrest in NB4 cells. These results suggest that caffeine induces cell cycle arrest and apoptosis in association with activation of p53 by a novel pathway to phosphorylate the Ser-15 residue and induction of phosphorylation of cdc 2 in leukemic cells with normal p53.     

Ganoderic acid Me induces G1 arrest in wild-type p53 human tumor cells while G1/S transition arrest in p53-null cells

The mechanism of cell cycle arrest of tumor cells induced by ganoderic acid Me (GA-Me) is not understood. In this work, GA-Me was found to possess remarkable cytotoxicity on highly metastatic lung carcinoma 95-D cell line in both dose- and time-dependent manners. The effect of GA-Me on cell cycle arrest was found in 95-D, p53-null lung cancer cells H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? human colon cancer cells. To obtain an insight into the role of p53 in cell cycle arrest by GA-Me, 95-D, H1299, HCT-116 p53^+/+ and HCT-116 p53^?/? cells were used for further investigation. GA-Me arrested cell cycle at G₁ phase in 95-D and HCT-116 p53^+/+ cells while S phase or G₁/S transition arrest in H1299 and HCT-116 p53^?/? cells. The results suggested that p53 may be a target of GA-Me, and it may be looked at as a new promising candidate for the treatment of carcinoma cells.     

The role of p21(CIP1/WAF1) in growth of epithelial cells exposed to hyperoxia

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21(CIP1/WAF1), which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2'-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.     

Synergy between the Mos/mitogen-activated protein kinase pathway and loss of p53 function in transformation and chromosome instability.

Constitutive activation of mitogen-activated protein kinase (MAPK) is a property common to many oncoproteins, including Mos, Ras, and Raf, and is essential for their transforming activities. We have shown that high levels of expression of the Mos/MAPK pathway in Swiss 3T3 fibroblast cause cells in S phase to undergo apoptosis, while cells in G1 irreversibly growth arrest. Interestingly, cells in G2 and M phases also arrest at a G1-like checkpoint after proceeding through mitosis. These cells fail to undergo cytokinesis and are binucleated. Thus, constitutive overexpression of Mos and MAPK cannot be tolerated, and fibroblasts transformed by Mos express only low levels of the mos oncogene product. Here, we show that p53 plays a key role in preventing oncogene-mediated activation of MAPK. In the absence of p53 (p53-/-), the growth arrest normally observed in wild-type p53 (p53+/+) mouse embryo fibroblasts (MEFs) is markedly reduced. The mos transformation efficiency in p53-/- MEFs is two to three orders of magnitude higher than that in p53+/+ cells, and p53-/- cells tolerate > 10-fold higher levels of both Mos and activated MAPK. Moreover, we show that, like Mos, both v-ras and v-raf oncogene products induce apoptosis in p53+/+ MEFs. These oncogenes also display a high transforming activity in p53-/- MEFs, as does a gain-of-function MAPK kinase mutant (MEK*). Thus, the p53-dependent checkpoint pathway is responsive to oncogene-mediated MAPK activation in inducing irreversible G1 growth arrest and apoptosis. Moreover, we show that the chromosome instability induced by the loss of p53 is greatly enhanced by the constitutive activation of the Mos/MAPK pathway.     

p53 independent G(1) arrest induced by DL-alpha-difluoromethylornithine

Ornithine decarboxylase (ODC), which catalyzes polyamine biosynthesis, plays an essential role in cell growth. DL-alpha-Difluoromethylornithine (DFMO), a synthetic inhibitor of ODC, inhibits cell growth. However, the exact mechanism by which polyamine depletion by DFMO results in growth inhibition remains to be elucidated. We clarified the mechanisms by which DFMO inhibits human gastric cancer cell (MKN45) growth. DFMO induced MKN45 cell G(1) phase arrest after 48 h, and the percentage of G(1) arrest cells continued to increase until 72 h. Expression of p21 and phosphorylation of Stat1 were significantly induced by DFMO at 24 h. Luciferase assay and gel shift assay showed specific binding of Stat1 to the p21 promoter, and promoter activity was activated at 24 h. In dominant negative p53 expressing cells, DFMO significantly induced p21 expression, arrested cells at G(1) phase, and suppressed cell growth effectively. These results suggest that DFMO induced MKN45 cell arrest at G(1) phase in a p53 independent manner, and Stat1 is, at least in part, involved in G(1) arrest.     

Cdc6 determines utilization of p21(WAF1/CIP1)-dependent damage checkpoint in S phase cells

When cells traversing G(1) are irradiated with UV light, two parallel damage checkpoint pathways are activated: Chk1-Cdc25A and p53-p21(WAF1/CIP1), both targeting Cdk2, but the latter inducing a long lasting arrest. In similarly treated S phase-progressing cells, however, only the Cdc25A-dependent checkpoint is active. We have recently found that the p21-dependent checkpoint can be activated and induce a prolonged arrest if S phase cells are damaged with a base-modifying agent, such as methyl methanesulfonate (MMS) and cisplatin. But the mechanistic basis for the differential activation of the p21-dependent checkpoint by different DNA damaging agents is not understood. Here we report that treatment of S phase cells with MMS but not a comparable dose of UV light elicits proteasome-mediated degradation of Cdc6, the assembler of pre-replicative complexes, which allows induced p21 to bind Cdk2, thereby extending inactivation of Cdk2 and S phase arrest. Consistently, enforced expression of Cdc6 largely eliminates the prolonged S phase arrest and Cdk2 inactivation induced with MMS, whereas RNA interference-mediated Cdc6 knockdown not only prolongs such arrest and inactivation but also effectively activates the p21-dependent checkpoint in the UV-irradiated S phase cells.     

SN2 DNA-alkylating agent-induced phosphorylation of p53 and activation of p21 gene expression

p53 is an important player in the cellular response to genotoxic stress whose functions are regulated by phosphorylation of a number of serine and threonine residues. Phosphorylation of p53 influences its DNA-binding and gene regulation activities. This study examines p53 phosphorylation in HCT-116 (MMR-deficient) and HCT-116+ch3 (MMR-proficient) human colon cancer cells treated with a S(N)2 DNA-alkylating agent, methylmethane sulfonate (MMS). MMS induces phosphorylation of p53 on Ser15 and Ser392 in a dose- and time-dependent manner. MMS-induced p53 phosphorylation is independent of DNA mismatch repair (MMR) activity. Nuclear extracts from MMS-treated HCT-116 cells had higher p21WAF1/Cip1 (p21) promoter DNA-binding activity in vitro opposed to untreated cells. After MMS treatment, the activation of the cloned p21 promoter in a transient transfection assay and endogenous p21 mRNA levels in HCT-116(p53+/+) versus HCT-116(p53-/-) cells increased, which correlates with an increased levels of phospho-p53(Ser15) and phospho-p53(Ser392). These results suggest that SN2 DNA-alkylating agent-induced phosphorylation of p53 on Ser15 and Ser392 increases its DNA-binding properties to cause an increased expression of p21 that may play a role in cell cycle arrest and/or apoptosis of HCT-116 cells.     

Characterization of adriamycin-induced G2 arrest and its abrogation by caffeine in FL-amnion cells with or without p53

We investigated the effect of Adriamycin on FL-amnion (FL) cells. After treatment with the drug, the cells arrested at G2, but we did not detect an increase in the p21 levels. We established a p53-deficient derivative of these cells, in which G2 arrest also occurred after treatment with Adriamycin, suggesting that the arrest we observed in these cells is independent of the p53 pathway. Low doses of Adriamycin (100-200 ng/ml) induced G2 arrest, while late S-phase arrest was observed at high doses (500-1000 ng/ml) in both FL and p53-deficient FL cells. Accumulation of cyclin B1 was detected only in cells arrested at G2, and not in those arrested at S phase, suggesting that the S-phase checkpoint functioned efficiently even in p53-deficient FL cells. In both cell lines, caffeine-induced activation of CDC2 kinase was detected only in cells arrested at G2 and CDC2 kinase-activated cells died exhibiting features of apoptosis. CDC2 kinase activation was inhibited by cycloheximide. Furthermore, cycloheximide inhibited activation of CDK2:cyclin A, which normally precedes CDC2 kinase activation in caffeine-treated cells. These results suggest that p53 and p21 do not have special roles in the S- and G2-phase checkpoints and that CDK2:cyclin A could be the target of the G2-phase DNA damage checkpoint.     

Concerted Regulation of Wild-Type p53 Nuclear Accumulation and Activation by S100B and Calcium-Dependent Protein Kinase C

The calcium ionophore ionomycin cooperates with the S100B protein to rescue a p53-dependent G(1) checkpoint control in S100B-expressing mouse embryo fibroblasts and rat embryo fibroblasts (REF cells) which express the temperature-sensitive p53Val135 mutant (C. Scotto, J. C. Deloulme, D. Rousseau, E. Chambaz, and J. Baudier, Mol. Cell. Biol. 18:4272-4281, 1998). We investigated in this study the contributions of S100B and calcium-dependent PKC (cPKC) signalling pathways to the activation of wild-type p53. We first confirmed that S100B expression in mouse embryo fibroblasts enhanced specific nuclear accumulation of wild-type p53. We next demonstrated that wild-type p53 nuclear translocation and accumulation is dependent on cPKC activity. Mutation of the five putative cPKC phosphorylation sites on murine p53 into alanine or aspartic residues had no significant effect on p53 nuclear localization, suggesting that the cPKC effect on p53 nuclear translocation is indirect. A concerted regulation by S100B and cPKC of wild-type p53 nuclear translocation and activation was confirmed with REF cells expressing S100B (S100B-REF cells) overexpressing the temperature-sensitive p53Val135 mutant. Stimulation of S100B-REF cells with the PKC activator phorbol ester phorbol myristate acetate (PMA) promoted specific nuclear translocation of the wild-type p53Val135 species in cells positioned in early G(1) phase of the cell cycle. PMA also substituted for ionomycin in the mediating of p53-dependent G(1) arrest at the nonpermissive temperature (37.5 degrees C). PMA-dependent growth arrest was linked to the cell apoptosis response to UV irradiation. In contrast, growth arrest mediated by a temperature shift to 32 degrees C protected S100B-REF cells from apoptosis. Our results suggest a model in which calcium signalling, linked with cPKC activation, cooperates with S100B to promote wild-type p53 nuclear translocation in early G(1) phase and activation of a p53-dependent G(1) checkpoint control.     

LYG-202 inhibits the proliferation of human colorectal carcinoma HCT-116 cells through induction of G1/S cell cycle arrest and apoptosis via p53 and p21(WAF1/Cip1) expression

We recently established that LYG-202, a new flavonoid with a piperazine substitution, exerts an anti-tumor effect in vivo and in vitro. In the present study, we demonstrate that LYG-202 induces G1/S phase arrest and apoptosis in human colorectal carcinoma HCT-116 cells. Data showed that the blockade of the cell cycle was associated with increased p21(WAF1/Cip1) and Rb levels and reduced expression of cyclin D1, cyclin E, and CDK4. Moreover, PARP cleavage, activation of caspase-3, caspase-8, and caspase-9, and an increased ratio of Bax/Bcl-2 were detected in LYG-202-induced apoptosis. Additionally, activation of p53 resulted in the up-regulation of its downstream targets PUMA and p21(WAF1/Cip1), as well as the down-regulation of its negative regulator MDM2, suggesting that the p53 pathway may play a crucial role in LYG-202-induced cell cycle arrest and apoptosis. Furthermore, siRNA knockdown of p53 attenuated the G1 cell cycle arrest and apoptosis induced by LYG-202, as the effects of LYG-202 on up-regulation of p21(WAF1/Cip1) and down-regulation of Bcl-2 and pro-caspase-3 were partly inhibited in p53 siRNA transfected cells compared with control siRNA transfected cells. Collectively, these data indicate that LYG-202 exerts its anti-tumor potency by activating the p53-p21 pathway for G1/S cell cycle arrest and apoptosis in colorectal cancer cells.     

ATR (ataxia telangiectasia mutated- and Rad3-related kinase) is activated by mild hypothermia in mammalian cells and subsequently activates p53

In vitro cultured mammalian cells respond to mild hypothermia (27-33?°C) by attenuating cellular processes and slowing and arresting the cell cycle. The slowing of the cell cycle at the upper range (31-33?°C) and its complete arrest at the lower range (27-28?°C) of mild hypothermia is effected by the activation of p53 and subsequent expression of p21. However, the mechanism by which cold is perceived in mammalian cells with the subsequent activation of p53 has remained undetermined. In the present paper, we report that the exposure of Chinese-hamster ovary-K1 cells to mildly hypothermic conditions activates the ATR (ataxia telangiectasia mutated- and Rad3-related kinase)-p53-p21 signalling pathway and is thus a key pathway involved in p53 activation upon mild hypothermia. In addition, we show that although p38MAPK (p38 mitogen-activated protein kinase) is also involved in activation of p53 upon mild hypothermia, this is probably the result of activation of p38MAPK by ATR. Furthermore, we show that cold-induced changes in cell membrane lipid composition are correlated with the activation of the ATR-p53-p21 pathway. Therefore we provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in mammalian cells leading to p53 and p21 activation, which is known to lead to cell cycle arrest.     

Diphenyleneiodonium induces ROS-independent p53 expression and apoptosis in human RPE cells

The diphenyleneiodonium (DPI) is widely used as an inhibitor of flavoenzymes, particularly NADPH oxidase. In this study, we investigated the effect of DPI on the apoptosis of human RPE cells. DPI treatment in ARPE-19 cells evoked a dose- and time-dependent growth inhibition, and also induced DNA fragmentation and protein content of the proapoptotic factor Bax. In addition, DPI significantly induced the expression and phosphorylation of p53, which induces proapoptotic genes in response to DNA damage or irreparable cell cycle arrest. ROS have been implicated as a key factor in the activation of p53 by many chemotherapeutic drugs. Recent data on the regulation of intracellular ROS by DPI are controversial. Therefore, we analyzed whether DPI could contribute to the generation of intracellular ROS. Although there was increase in ROS level from cells treated for 24h with DPI, it was not detectable at early time points, required to induce p53 expression. And DPI-induced p53 expression was not affected by the ROS scavenger NAC. We conclude that DPI induces the expression of p53 by ROS-independent mechanism in ARPE-19 cells, and renders cells sensitive to drug-induced apoptosis by induction of p53 expression.     

CtIP promotes G2/M arrest in etoposide-treated HCT116 cells in a p53-independent manner

Acquired resistance to cytotoxic antineoplastic agents is a major clinical challenge in tumor therapy; however, the mechanisms involved are still poorly understood. In this study, we show that knockdown of CtIP, a corepressor of CtBP, promotes cell proliferation and alleviates G2/M phase arrest in etoposide (Eto)-treated HCT116 cells. Although the expression of p21 and growth arrest and DNA damage inducible α (GADD45a), which are important targets of p53, was downregulated in CtIP-deficient HCT116 cells, p53 deletion did not affect G2/M arrest after Eto treatment. In addition, the phosphorylation levels of Ser317 and Ser345 in Chk1 and of Ser216 in CDC25C were lower in CtIP-deficient HCT116 cells than in control cells after Eto treatment. Our results indicate that CtIP may enhance cell sensitivity to Eto by promoting G2/M phase arrest, mainly through the ATR-Chk1-CDC25C pathway rather than the p53-p21/GADD45a pathway. The expression of CtIP may be a useful biomarker for predicting the drug sensitivity of colorectal cancer cells.