Stat1-dependent, p53-independent expression of p21(waf1) modulates oxysterol-induced apoptosis

7-Ketocholesterol (7kchol) is prominent in atherosclerotic lesions where apoptosis occurs. Using mouse fibroblasts lacking p53, p21(waf1), or Stat1, we found that optimal 7kchol-induced apoptosis requires p21(waf1) and Stat1 but not p53. Findings were analogous in a human cell system. Apoptosis was restored in Stat1-null human cells when wild-type Stat1 was restored. Phosphorylation of Stat1 on Ser(727) but not Tyr(701) was essential for optimum apoptosis. A neutralizing antibody against beta interferon (IFN-beta) blunted Ser(727) phosphorylation and apoptosis after 7kchol treatment; cells deficient in an IFN-beta receptor subunit exhibited blunted apoptosis. IFN-beta alone did not induce apoptosis; thus, 7kchol-induced release of IFN-beta was necessary but not sufficient for optimal apoptosis. In Stat1-null cells, expression of p21(waf1) was much less than in wild-type cells; introducing transient expression of p21(waf1) restored apoptosis. Stat1 and p21(waf1) were essential for downstream apoptotic events, including cytochrome c release from mitochondria and activation of caspases 9 and 3. Our data reveal key elements of the cellular pathway through which an important oxysterol induces apoptosis. Identification of the essential signaling events that may pertain in vivo could suggest targets for therapeutic intervention.     

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.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

Decreased translation of p21waf1 mRNA causes attenuated p53 signaling in some p53 wild-type tumors

DNA damage induces cell cycle arrest through both Chk1 and the p53 tumor suppressor protein, the latter arresting cells through induction of p21^waf1 protein. Arrest permits cells to repair the damage and recover. The frequent loss of p53 in tumor cells makes them more dependent on Chk1 for arrest and survival. However, some p53 wild type tumor cell lines, such as HCT116 and U2OS, are also sensitive to inhibition of Chk1 due to attenuated p21^waf1 induction upon DNA damage. The purpose of this study is to determine the cause of this attenuated p21^waf1 protein induction. We find that neither the induction of p21^waf1 mRNA nor protein half-life is sufficient to explain the low p21^waf1 protein levels in HCT116 and U2OS cells. The induced mRNA associates with polysomes but little protein is made suggesting these two cell lines have a reduced rate of p21^waf1 mRNA translation. This represents a novel mechanism for disruption of the p53-p21^waf1 pathway as currently known mechanisms involve either mutation of p53 or reduction of p53 protein levels. As a consequence, this attenuated p21^waf1 expression may render some p53 wild type tumors sensitive to a combination of DNA damage plus checkpoint inhibition.     

Caspase 2 is both required for p53-mediated apoptosis and downregulated by p53 in a p21-dependent manner

Upon treatment with some DNA damaging agents, human H1299 tumor-derived cells expressing inducible versions of wild-type or mutant p53 with inactive transactivation domain I (p53Q22/S23) undergo apoptosis. In cells expressing either version of p53, caspase 2 activation is required for release of cytochrome c and cell death. Furthermore, silencing of PIDD (a factor previously shown to be required for caspase 2 activation) by siRNA suppresses apoptosis by both wild-type p53 and p53Q22/S23. Despite the finding that caspase 2 is essential for DNA damage-facilitated, p53-mediated apoptosis, induction of wild-type p53 (with or without DNA damage) resulted in a reduction of caspase 2 mRNA and protein levels. In this study we sought to provide a mechanism for the negative regulation of caspase 2 by p53 as well as provide insight as to why p53 may repress a key mediator of p53-dependent apoptosis. Mechanistically, we show that DNA binding and/or transactivation domains of p53 are crucial for mediating transrepression. Further, expression of p21 (in p53-null cells inducibly expressing p21) is sufficient to mediate repression of caspase 2. Deletion of p21 or E2F-1 not only abrogated repression of caspase 2, but also stimulated the expression of caspase 2 above basal levels, implicating the requirement for an intact p21/Rb/E2F pathway in the down-regulation of caspase 2. As this p53/p21-dependent repression of caspase 2 can occur in the absence of DNA damage, caspase 2 repression does not simply seem to be a consequence of the apoptotic process. Down-regulation of caspase 2 levels by p53 may help to determine cell fate by preventing cell death when unnecessary.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

p53RDL1 regulates p53-dependent apoptosis

Although a number of targets for p53 have been reported, the mechanism of p53-dependent apoptosis still remains to be elucidated. Here we report a new p53 target-gene, designated p53RDL1 (p53-regulated receptor for death and life; also termed UNC5B). The p53RDL1 gene product contains a cytoplasmic carboxy-terminal death domain that is highly homologous to rat Unc5H2, a dependence receptor involved in the regulation of apoptosis, as well as in axon guidance and migration of neural cells. We found that p53RDL1 mediated p53-dependent apoptosis. Conversely, when p53RDL1 interacted with its ligand, Netrin-1, p53-dependent apoptosis was blocked. Therefore, p53RDL1 seems to be a previously un-recognized target of p53 that may define a new pathway for p53-dependent apoptosis. We suggest that p53 might regulate the survival of damaged cells by balancing the regulation of Netrin-p53RDL1 signalling, and cell death through cleavage of p53RDL1 for apoptosis.     

Thrombin inhibits tumor cell growth in association with up-regulation of p21(waf/cip1) and caspases via a p53-independent, STAT-1-dependent pathway

Thrombin, a multifunctional protein, has been found to be involved in cellular mitogenesis, tumor growth, and metastasis, in addition to its well known effects on the initiation of platelet aggregation and secretion and the conversion of fibrinogen to fibrin to form blood clots. These properties of thrombin rely on its action as a serine protease, which cleaves the N-terminal region of a 7-transmembrane G protein receptor (protease-activated receptor, PAR-1), thus exposing a tethered end hexapeptide sequence capable of activating its receptor. Little is known about its effect on genes that regulate the cell cycle. This study was undertaken to investigate the possible mechanisms by which thrombin regulates tumor cell growth in several tumor cell lines: human CHRF megakaryocyte, DU145 prostate, MDAMB231 and MCF7 breast, U3A fibrosarcoma, and 2 murine fibroblast cell lines, MEFp53(-/-) and CD STAT(-/-). We have found that thrombin under the conditions of culture employed inhibits cell growth by both up-regulation of p21(waf/cip1) and induction of caspases via its PAR-1 receptor. The increased expression of p21(waf/cip1) by thrombin was p53 independent, STAT1 dependent, and protein synthesis independent. This was associated with tyrosine phosphorylation of JAK2 and STAT1, and nuclear translocation of STAT1. Induction of apoptosis is also PAR-1-specific, STAT1-dependent, and associated with up-regulation of caspases 1, 2, and 3. Our study establishes, for the first time, a link between PAR-1 receptor activation with the STAT signal pathway, which leads to cell cycle control and apoptosis. This observation broadens our understanding of the mechanism of PAR-1 activation and its effect on cell growth, and could possibly lead to therapeutic approaches for the treatment of cancer.     

Vanadate-induced cell growth arrest is p53-dependent through activation of p21 in C141 cells

Vanadium is widely used in industry. It is a potent toxic agent and carcinogen. The mechanisms involved in its toxicity and carcinogenesis are still unclear. Improper cell growth is believed to be involved in cancer development. The present study investigated the regulation of p53 on vanadate-induced cell growth arrest using both p53 wild type C141 cells and p53 deficient embryo fibroblasts (p53 -/-). On vanadate stimulation, C141 cells exhibited a dose- and time-dependent S phase arrest as determined by DNA content analysis. In contrast, vanadate was unable to increase the percentage of S phase in p53 -/- cells. Luciferase assay showed that vanadate induced p53 activation in a dose- and time-dependent manner in p53 wild type C141 cells. Addition of pifithrin-alpha (PFT), a specific inhibitor of p53, reduced the activation of p53 with a concomitant decrease in growth arrest at S phase. Western blotting analysis demonstrated that vanadate caused a dose- and time-dependent increase of p21 level in C141 cells. Pretreatment of C141 cells with PFT decreased p21 expression induced by vanadate while the p21 expression did not vary in vanadate stimulated p53 -/- cells. The results obtained from the present study suggest that vanadate is able to induce S phase arrest through p53- and p21-dependent pathway.     

The antiproliferative activity of aloe-emodin is through p53-dependent and p21-dependent apoptotic pathway in human hepatoma cell lines

The aim of this study is to investigate the anticancer effect of aloe-emodin in two human liver cancer cell lines, Hep G2 and Hep 3B. We observed that aloe-emodin inhibited cell proliferation and induced apoptosis in both examined cell lines, but with different the antiproliferative mechanisms. In Hep G2 cells, aloe-emodin induced p53 expression and was accompanied by induction of p21 expression that was associated with a cell cycle arrest in G1 phase. In addition, aloe-emodin had a marked increase in Fas/APO1 receptor and Bax expression. In contrast, with p53-deficient Hep 3B cells, the inhibition of cell proliferation of aloe-emodin was mediated through a p21-dependent manner that did not cause cell cycle arrest or increase the level of Fas/APO1 receptor, but rather promoted aloe-emodin induced apoptosis by enhancing expression of Bax. These findings suggest that aloe-emodin may be useful in liver cancer prevention.     

Apoptotic effect of ethyl-4-isothiocyanatobutanoate is associated with DNA damage, proteasomal activity and induction of p53 and p21cip1/waf1

The effect of synthetic isothiocyanate ethyl-4-isothiocyanatobutanoate (E-4IB) on survival of mismatch repair-proficient TK6 and -deficient MT1 cell lines as well as the influence of proteasomal inhibitor MG132, caspase inhibitor Z-VAD-fmk, and ATM inhibitor caffeine on E-4IB modulation of cell cycle and apoptosis was evaluated. Flow cytometric analyses of DNA double strand breaks (γ-H2AX), mitotic fraction (phospho-histone H3), cell cycle modulation, apoptosis induction (sub-G₀ fraction and fluorescein diacetate staining), and dissipation of transmembrane mitochondrial potential (JC-1 staining) were performed. Western blotting was used for the evaluation of ERK activation, expression of p53, p21^cip1/waf1 and GADD45α proteins, as well as PARP fragmentation. Analysis of mitotic nuclei was performed for chromosomal aberrations assessment. MT1 cells were more resistant to E-4IB treatment then TK6 cells (IC₅₀ 8 μM vs. 4 μM). In both cell lines E-4IB treatment induced phosphorylation of H2AX, increase of p53 protein level, phospho-histone H3 staining, and G₂/M arrest. The sub-G₀ fragmentation was accompanied by PARP degradation, decreased mitochondrial transmembrane potential, and diminished p21^cip1/waf1 protein expression in TK6 cells. Caspase inhibitor Z-VAD-fmk decreased E-4IB induced sub-G₀ fragmentation and extent of apoptosis in TK6 cells, while proteasome inhibitor MG132 increased number of apoptotic cells in both cell lines tested. A number of aberrant metaphases and clastogenic effect of high E-4IB concentration was observed. The synthetic isothiocyanate E-4IB induced DNA strand breaks, increased mitotic fraction and apoptosis potentiated by MG132 inhibitor in both mismatch repair-proficient and -deficient cell lines. This work was supported in part by Slovak Governmental Research and Development sub-program Food-quality and safety No. 2003SP270280E010280E01, National Program “Use of Cancer Genomics to Improve the Human Population Health”, project 2003 SP 510280800/0280801, European Commission project (QLG1-CT-2000-01230), and VEGA projects 2/4069 and 2/3161/23.     

p53DINP1, a p53-inducible gene, regulates p53-dependent apoptosis

Using the differential display method combined with a cell line that carries a well-controlled expression system for wild-type p53, we isolated a p53-inducible gene, termed p53DINP1 (p53-dependent damage-inducible nuclear protein 1). Cell death induced by DNA double-strand breaks (DSBs), as well as Ser46 phosphorylation of p53 and induction of p53AIP1, were blocked when we inhibited expression of p53DINP1 by means of an antisense oligonucleotide. Overexpression of p53DINP1 and DNA damage by DSBs synergistically enhanced Ser46 phosphorylation of p53, induction of p53AIP1 expression, and apoptotic cell death. Furthermore, the protein complex interacting with p53DINP1 was shown to phosphorylate Ser46 of p53. Our results suggest that p53DINP1 may regulate p53-dependent apoptosis through phosphorylation of p53 at Ser46, serving as a cofactor for the putative p53-Ser46 kinase.