p53 activates G1 checkpoint following DNA damage by doxorubicin during transient mitotic arrest

Recovery from DNA damage is critical for cell survival. The serious damage is not able to be repaired during checkpoint and finally induces cell death to prevent abnormal cell growth. In this study, we demonstrated that 8N-DNA contents are accumulated via re-replication during prolonged recovery period containing serious DNA damage in mitotic cells. During the incubation for recovery, a mitotic delay and initiation of an abnormal interphase without cytokinesis were detected. Whereas a failure of cytokinesis occurred in cells with no relation with p53/p21, re-replication is an anomalous phenomenon in the mitotic DNA damage response in p53/p21 negative cells. Cells with wild-type p53 are accumulated just prior to the initiation of DNA replication through a G₁ checkpoint after mitotic DNA damage, even though p53 does not interrupt pre-RC assembly. Finally, these cells undergo cell death by apoptosis. These data suggest that p53 activates G₁ checkpoint in response to mitotic DNA damage. Without p53, cells with mitotic DNA damage undergo re-replication leading to accumulation of damage     

CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.     

Radiation-induced phosphorylation of Chk1 at S345 is associated with p53-dependent cell cycle arrest pathways

Because DNA damage-inducible cell cycle checkpoints are thought to protect cells from the lethal effects of ionizing radiation, a better understanding of the mechanistic functions of cell cycle regulatory proteins may reveal new molecular targets for cancer therapy. The two major regulatory proteins of G2 arrest are Chk1 and p53. Yet, it is unclear how these two proteins interact and coordinate their functional roles during radiation-induced G2 arrest. To determine Chk1's role in p53-dependent G2 arrest, we used p53 proficient cells and examined expression of G2 arrest proteins under conditions in which G2 arrest was inhibited by the staurosporine analog, UCN-01. We found that UCN-01 inhibited both G1 and G2 arrest in irradiated p53 proficient cells. The arrest inhibition was associated with suppression of radiation-induced expression of both p21 and 14-3-3 sigma -- two known p53-dependent G2 arrest proteins. The suppression occurred despite normal induction of p53 and normal phosphorylation of p53 at S20 and Cdc25C at S216 -- the two known substrates of Chk1 kinase activity. In contrast, we showed that radiation-induced phosphorylation of Chk1 at S345 was associated with binding of Chk1 to p53, p21, and 14-3-3 sigma, and that UCN-01 inhibited S345 phosphorylation. We suggest that DNA damage-induced phosphorylation of Chk1 at S345, and subsequent p53 binding, links Chk1 with p53 downstream responses and may provide a coordinated interaction between DNA damage responses and cell cycle arrest functions.     

p53-dependent and p53-independent pathways for radiation-induced immature thymocyte differentiation

The pre-T-cell receptor (TCR) delivers essential survival/differentiation signals to the developing thymocytes. Severe combined immunodeficient (SCID) and recombination-activating gene (RAG)-deficient mice are unable to assemble antigen receptor genes, and therefore cannot express a pre-TCR. Consequently, T lymphocyte differentiation is arrested at an early stage in the thymus of these animals, and immature thymocytes are eliminated through apoptotic processes. This maturation arrest can be relieved and thymocyte differentiation rescued after the exposure of these mice to whole-body gamma-irradiation. Whereas the promotion of immature thymocyte survival/differentiation was shown to require p53 activity in irradiated SCID mice, it was suggested, on the other hand, that p53 activation prevents immature thymocytes survival/differentiation in irradiated RAG-deficient mice. However, SCID mice have impaired responses to ionizing radiation. In this paper, we analysed p53 requirement in radiation-induced thymocyte differentiation in CD3epsilon(Delta5/Delta5) mice, where pre-TCR deficiency also results in an early block of lymphocyte development. Our results show at the cellular and molecular levels that, in this DNA repair-proficient model, irradiation-induced thymocyte differentiation proceeds either by a p53-dependent or by a p53-independent pathway, which differ in their sensitivity to the radiation dose delivered.     

Jaceosidin induces p53-dependent G2/M phase arrest in U87 glioblastoma cells

Flavonoid compounds have been shown to trigger cell cycle arrest at G0/G1, S and G2/M checkpoints, allowing cells to repair DNA damage before entry into mitosis. Jaceosidin, a flavonoid compound, has been reported to induce apoptosis in various cancer cell lines. In our previous study, we established that jaceosidin induces apoptosis in U87 glioblastoma cells through G2/M phase arrest. However the molecular mechanisms oremained unclear. In the present study, mRNA and protein expression levels of major cell cycle regulatory genes were analyzed by semi-quantitative RT-PCR and Western blot studies respectively. The results demonstrated that jaceosidin-induced G2/M phase arrest in U87 cells is associated with DNA fragmentation, up-regulation of p53 and p21 and subsequent down-regulation of cyclin B1 and CDK1 expression at mRNA as well as at protein level. These findings provide insights into jaceosidin-induced G2/M phase arrest in U87 glioblastoma cells.     

The investigational new drug XK469 induces G(2)-M cell cycle arrest by p53-dependent and -independent pathways.

PURPOSE: XK469 (2-[4-(7-chloro-2-quinoxalinyloxy) phenoxy]propionic acid), a synthetic quinoxaline phenoxypropionic acid derivative, has broad activity against murine tumors and is entering Phase I clinical development as a topoisomerase IIbeta inhibitor. This study investigated the underlying molecular mechanism of XK469's effects on the cell cycle. EXPERIMENTAL DESIGN: Growth inhibition, cell cycle arrest, induction of p53 and p21 mRNA and protein, and cdc2 phosphorylation and kinase activity were studied in treated cells from the H460 lung cancer line and p21 and p53 knockout cells of the HCT 116 colon cancer line. RESULTS: XK469 arrested H460 cells at G(2)-M, which was associated with cdc2 phosphorylation and decreased cdc2 kinase activity. Moreover, XK469 stabilized p53 and subsequently increased p21(WAF1/CIP1). Furthermore, HCT116 p21(-/-) cells were less sensitive than wild-type cells to XK469-induced growth inhibition, but p53(+/+) and p53(-/-) cells were equally sensitive despite the absence of p21 induction in the p53(-/-) cells. CONCLUSIONS: When considered with published data, our study suggests a complex mechanism of XK469-mediated anticancer activity involving multiple pathways, including p53-dependent and -independent G(2)-M arrest via inactivation of cdc2-cyclin B1 kinase activity.     

Mechanism of G1-like arrest by low concentrations of paclitaxel: next cell cycle p53-dependent arrest with sub G1 DNA content mediated by prolonged mitosis

Paclitaxel (PTX) and other microtubule inhibitors cause mitotic arrest. However, low concentrations of PTX (low PTX) paradoxically cause G1 arrest (without mitotic arrest). Here, we demonstrated that unexpectedly, low PTX did not cause G1 arrest in the first cell cycle and did not prevent cells from passing through S phase and entering mitosis. Mitosis was prolonged but cells still divided, producing either two or three cells (tripolar mitosis), thus explaining a sub G1 peak caused by low PTX. Importantly, sub G1 cells were viable and non-apoptotic. Some cells fused back and then progressed to mitosis, frequently producing three cells again before becoming arrested in the next cell-cycle interphase. Thus, low PTX caused postmitotic arrest in second and even the third cell cycles. By increasing concentration of PTX, tripolar mitosis was transformed to mitotic slippage, thus eliminating a sub G1 peak. Time-lapse microscopy revealed that prolonged mitosis ensured a p53-dependent postmitotic arrest. We conclude that PTX directly affects cells only in mitosis and the duration of mitosis determines cell fate, including p53-dependent G1-like arrest.     

FGF1 inhibits p53-dependent apoptosis and cell cycle arrest via an intracrine pathway

We analysed the relationships between p53-induced apoptosis and the acidic fibroblast growth factor 1 (FGF1) survival pathway. We found that p53 activation in rat embryonic fibroblasts induced the downregulation of FGF1 expression. These data suggest that the fgf1 gene is a repressed target of p53. Unlike extracellular FGF1, which has no effect on p53-dependent pathways, intracellular FGF1 inhibits both p53-dependent apoptosis and cell growth arrest via an intracrine pathway. FGF1 increases MDM2 expression at both mRNA and protein levels. This increase is associated with an acceleration of p53 degradation, which may partly account for the ability of endogenous FGF1 to counteract p53 pathways. In the presence of FGF1, p53 was unable to transactivate bax, but no modification of p21 gene transactivation was observed. As Bax is an essential component of the p53-dependent apoptosis pathway, this suggests that intracellular FGF1 inhibits p53 pathways not only by decreasing the stability of p53, but also by modifying some of its transactivation properties. In conclusion, we showed that p53 and FGF1 pathways may interact in the cell to determine cell fate. Deregulation of one of these pathways modifies the balance between cell proliferation and cell death and may lead to tumor progression.     

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells.

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-kappa B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.     

ERK1/2 and p38 cooperate to induce a p21CIP1-dependent G1 cell cycle arrest

To study the mechanisms by which mitogen- and stress-activated protein kinases regulate cell cycle re-entry, we have used a panel of conditional kinases that stimulate defined MAPK or SAPK cascades. Activation of DeltaMEKK3:ER* during serum restimulation of quiescent cells causes a strong activation of JNK1 and p38alpha but only a modest potentiation of serum-stimulated ERK1/2 activity. In CCl39 cells this promoted a sustained G1 arrest that correlated with decreased expression of cyclin D1 and Cdc25A, increased expression of p21CIP1 and inhibition of CDK2 activity. In Rat-1 cells, in which p21(CIP1) expression is silenced by methylation, DeltaMEKK3:ER* activation caused only a transient delay in the S phase entry rather than a sustained G1 arrest. Furthermore, p21CIP1-/- 3T3 cells were defective for the DeltaMEKK3:ER*-induced G1 cell cycle arrest compared to their wild-type counterparts. These results suggest that activated DeltaMEKK3:ER* inhibits the G1 --> S progression by two kinetically distinct mechanisms, with expression of p21CIP1 being required to ensure a sustained G1 cell cycle arrest. The ERK1/2 and p38alphabeta pathways cooperated to induce p21CIP1 expression and inhibition of p38alphabeta caused a partial reversal of the cell cycle arrest. In contrast, selective activation of ERK1/2 by DeltaRaf-1:ER* did not inhibit serum stimulated cell cycle re-entry. Finally, selective activation of JNK by DeltaMEKK1:ER* failed to inhibit cell cycle re-entry, even in cells that retained wild-type p53, arguing against a major role for JNK alone in antagonizing the G1 --> S transition.     

p53-dependent G2 arrest associated with a decrease in cyclins A2 and B1 levels in a human carcinoma cell line

In vivo transfer of wild-type (wt) p53 gene via a recombinant adenovirus has been proposed to induce apoptosis and increase radiosensitivity in several human carcinoma models. In the context of combining p53 gene transfer and irradiation, we investigated the consequences of adenoviral-mediated wtp53 gene transfer on the cell cycle and radiosensitivity of a human head and neck squamous cell carcinoma line (SCC97) with a p53 mutated phenotype. We showed that ectopic expression of wtp53 in SCC97 cells resulted in a prolonged G1 arrest, associated with an increased expression of the cyclin-dependent kinase inhibitor WAF1/p21 target gene. A transient arrest in G2 but not in G1 was observed after irradiation. This G2 arrest was permanent when exponentially growing cells were transduced by Ad5CMV-p53 (RPR/INGN201) immediately after irradiation with 5 or 10 Gy. Moreover, levels of cyclins A2 and B1, which are known to regulate the G2/M transition, dramatically decreased as cells arrived in G2, whereas maximal levels of expression were observed in the absence of wtp53. In conclusion, adenoviral mediated transfer of wtp53 in irradiated SCC97 cells, which are mutated for p53, appeared to increase WAF1/p21 expression and decrease levels of the mitotic cyclins A2 and B1. These observations suggest that the G2 arrest resulted from a p53-dependent premature inactivation of the mitosis promoting factor.     

乙型肝炎病毒X基因经p53依赖性与非依赖性途径对p21WAF1表达的影响

背景与目的:研究表明,在不同情况下乙型肝炎病毒 X基因(HBx)对 p21WAF1的表达具有不同的影响,但作用机制仍不清楚.本研究的目的是探讨 HBx对 p53下游基因 p21WAF1的影响.方法:通过正、反义野生型 p53(wtp53)与 HBx单转染及共转染人肝癌细胞株 SMMC-7721细胞,以及 HBx转染不同内源性 p53状态的肝癌细胞株,检测 p21WAF1启动子荧光素酶基因表达,并用流式细胞仪观察细胞周期的变化 ,Western blot法比较 p53、p21WAF1表达水平的变化.结果:正、反义 wtp53与 HBx 单转染及共转染 SMMC-7721细胞 ,HBx与正义 wtp53共转染组(1.007± 0.098)与单转染正义 wtp53(0.490± 0.012)相比,p21WAF1启动子荧光素酶基因表达明显升高(P< 0.05),其余各组 HBx均可导致 p21WAF1启动子荧光素酶基因表达明显减少(P< 0.05).Western blot法表明 HBx可导致 p53的表达增加,但 p53表达较低的 SMMC-7721细胞转染 HBx后 p21WAF1蛋白的表达减弱 ,p21WAF1启动子荧光素酶基因表达(0.053± 0.010)也较对照组(0.094± 0.013)明显减少(P< 0.05),而 p53表达较强的 HepG2细胞转染 HBx后 p21WAF1蛋白的表达增强、p21WAF1启动子荧光素酶基因的表达(1.252± 0.052)较对照组明显升高(0.767± 0.031)(P< 0.05).细胞周期结果表明瞬时转染 HBx的 SMMC-7721和 Hep3B细胞停滞在 G0-G1期细胞数(42.31%、36.96%)较对照组(47.10%、42.90%)减少,而瞬时转染 HBx的 HepG2细胞(63.62%)停滞在 G0-G1期的细胞数较对照组(57.42%)增加,但稳定筛选后 ,pcDNA3HBxHepG2细胞(57.31%)停滞在 G0-G1期细胞数较对照组(61.49%)减少.结论:HBx一方面可能引起 p53蛋白在细胞内积聚导致 p21WAF1表达升高,另一方面可直接抑制 p21WAF1启动子的转录活性.     

Upregulated p53 expression activates apoptotic pathways in wild-type p53-bearing mesothelioma and enhances cytotoxicity of cisplatin and pemetrexed

The majority of malignant mesothelioma possesses the wild-type p53 gene with a homologous deletion of the INK4A/ARF locus containing the p14(ARF) and the p16(INK4A) genes. We examined whether forced expression of p53 inhibited growth of mesothelioma cells and produced anti-tumor effects by a combination of cisplatin (CDDP) or pemetrexed (PEM), the first-line drugs for mesothelioma treatments. Transduction of mesothelioma cells with adenoviruses bearing the p53 gene (Ad-p53) induced phosphorylation of p53, upregulated Mdm2 and p21 expression levels and decreased phosphorylation of pRb. The transduction generated cleavage of caspase-8 and -3, but not caspase-9. Cell cycle analysis showed increased G0/G1- or G2/M-phase populations and subsequently sub-G1 fractions, depending on cell types and Ad-p53 doses. Transduction with Ad-p53 suppressed viability of mesothelioma cells and augmented the growth inhibition by CDDP or PEM mostly in a synergistic manner. Intrapleural injection of Ad-p53 and systemic administration of CDDP produced anti-tumor effects in an orthotopic animal model. These data collectively suggest that Ad-p53 is a possible agent for mesothelioma in combination with the first-line chemotherapeutics.     

Interference of papilloma viruses with p53-dependent and -independent apoptotic pathways

Papillomavirus infection interferes with four distinct and dominant levels of intra- and intercellular control of oncogenesis, three of which are based on the induction of apoptosis. Papillomaviruses cause abrogation of the cellular senescence program and interfere with p53-dependent DNA repair or apoptosis triggered by DNA damage. As a result, spontaneous or induced mutations are not eradicated and the transformed state may be established through oncogene activation and tumor suppressor gene inactivation. The interference of papillomaviruses with a recently described p53-independent intercellular control step, in which nontransformed cells induce apoptosis specifically in their transformed neighbouring cells, allows survival of papillomavirus-expressing transformed cells. Finally, p53-dependent hypoxia-triggered apoptosis in microtumors may be overcome by papillomaviruses and thus lead to efficient and rapid tumor progression.     

Oxidative stress induces a prolonged but reversible arrest in p53-null cancer cells, involving a Chk1-dependent G2 checkpoint

Reactive oxygen species (ROS), the principal mediators of oxidative stress, induce responses such as apoptosis or permanent growth arrest/senescence in normal cells. Moreover, p53 activation itself contributes to ROS accumulation. Here we show that treatment of p53-null cancer cells with sublethal concentrations of ROS triggered an arrest with some morphological similarities to cellular senescence. Different from a classical senescent arrest in G(1), the ROS-induced arrest was predominantly in the G(2) phase of the cell cycle, and its establishment depended at least in part on an intact Chk1-dependent checkpoint. Chk1 remained phosphorylated only during the repair of double strand DNA breaks, after which Chk1 was inactivated, the G(2) arrest was suppressed, and some cells recovered their ability to proliferate. Inhibition of Chk1 by an RNAi approach resulted in an increase in cell death in p53-null cells, showing that the Chk1-dependent G(2) checkpoint protected cells that lacked a functional p53 pathway from oxidative stress. It has been proposed that the induction of a senescent-like phenotype by antineoplastic agents can contribute therapeutic efficacy. Our results indicate that oxidative stress-induced growth arrest of p53-null tumor cells cannot be equated with effective therapy owing to its reversibility and supports the concept that targeting Chk1 may enhance the effects of DNA-damaging agents on cancer progression in such tumors.     

Lack of p53-mediated G1 arrest in response to an environmental carcinogen.

The environmental carcinogen, 5-methylchrysene, is a component of cigarette smoke. Its reactive metabolite, anti-5-methylchrysene-1, 2-dihydrodiol-3,4-epoxide (5-MeCDE) mainly reacts with the N(2)-position of guanine residues in the DNA molecule. In this study, we demonstrate that the tumor suppressor protein p53 is stabilized in response to DNA damage by 5-MeCDE but fails to induce the cells' protective mechanism of G1 arrest in the human breast carcinoma cell line, MCF-7. In contrast, actinomycin D treatment of these cells did lead to G1 arrest. Western analyses revealed that, though both actinomycin D and 5-MeCDE treatment stabilized p53, only trace levels of p21(waf1/cip1) were seen in the latter case. This lack of p21(waf1/cip1) expression in 5-MeCDE-treated cells is attributed to a stealth characteristic of this environmental carcinogen that allows it to damage DNA and still escape the p53-mediated cellular defense mechanism of G1 arrest.     

2-Chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU) inhibits prostate carcinoma cell growth via p53-dependent and p53-independent pathways

BACKGROUND: Prostate carcinoma is the most commonly occurring malignancy in men. Although 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), an analog of the chloroethylnitrosoureas, has been used in the treatment of advanced solid tumors, the molecular mechanisms underlying the antineoplastic activity of this agent are not well understood. In the current study, the authors sought to investigate the effects of SarCNU on prostate carcinoma cell growth in vivo and in vitro. METHODS: Male SCID mice underwent subcutaneous implantation (on both flanks) of human CWR-22 and CWR-22R prostate carcinoma xenografts. Mice were treated with either vehicle or 60 or 80 mg SarCNU per kg body weight for 5 days, with tumor growth being assessed every 3 days. Animals were sacrificed 21 days after the final injection, and tumors subsequently were collected, weighed, and processed for analysis. Immunohistochemical analyses were performed to obtain data on the localization of p53 and p21Cip1/Waf1. Cell counting, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) assays, cell cycle analyses, Western blotting, and in vitro kinase assays were performed to determine the effects of SarCNU on growth, apoptosis, cell cycle arrest, cell cycle-regulated protein levels, and Cdc-2 activity, respectively. RESULTS: SarCNU reduced tumor incidence and inhibited the growth of CWR-22 and CWR-22R xenografts. In addition, treatment with this agent led to increases in p21Cip1/Waf1 levels and p53 phosphorylation at Ser15. In vitro administration of SarCNU to cells with wild-type p53 (LNCaP and primary CWR-22 cells) and cells with mutant p53 (PC-3 cells) resulted in G2/M arrest and the reduction of cellular Cdc-2 activity. Up-regulation of p53 levels, p53 phosphorylation at Ser15, and p21Cip1/Waf1 levels in primary CWR-22 and LNCaP cells, as well as up-regulation of Cdc-2 phosphorylation at Tyr15 in PC-3 cells, was detected. CONCLUSIONS: SarCNU induced G2/M arrest in prostate carcinoma cells via p53-dependent up-regulation of p21Cip1/Waf1 and p53-independent phosphorylation of Cdc-2 at Tyr15. These findings suggest a potential role for SarCNU in the treatment of prostate malignancies.