p53 is dispensable for UV-induced cell cycle arrest at late G(1) in mammalian cells

Genotoxic agents, including gamma-rays and UV light, induce transient arrest at different phases of the cell cycle. These arrests are required for efficient repair of DNA lesions, and employ several factors, including the product of the tumor suppressor gene p53 that plays a central role in the cellular response to DNA damage. p53 protein has a major function in the gamma-ray-induced cell cycle delay in G(1) phase. However, it remains uncertain as to whether p53 is also involved in the UV-mediated G(1) delay. This report provides evidence that p53 is not involved in UV-induced cellular growth arrest in late G(1) phase. This has been demonstrated in HeLa cells synchronized at the G(1)/S border by aphidicolin, followed by UV exposure. Interestingly, the length of this p53-independent G(1) arrest has been shown to be UV dose-dependent. Similar results were also obtained with other p53-deficient cell lines, including human promyelocytic leukemia HL-60 and mouse p53 knock-out cells. As expected, all of these cell lines were defective in gamma-ray-induced cell growth arrest at late G(1). Moreover, it is shown that in addition to cell cycle arrest, HL-60 cells undergo apoptosis in G(1) phase in response to UV light but not to gamma-rays. Together, these findings indicate that p53- compromised cells have a differential response following exposure to ionizing radiation or UV light.     

Mdm2 antagonists induce apoptosis and synergize with cisplatin overcoming chemoresistance in TP53 wild‐type ovarian cancer cells

Ovarian cancer (OVCa) is the leading cause of death from gynecological malignancies. Although treatment for advanced OVCa has improved with the introduction of taxane–platinum chemotherapy, the majority of patients will develop resistance to the treatment, leading to poor prognosis. One of the causes of chemoresistance is the reduced ability to undergo apoptosis. Cisplatin is a genotoxic drug that leads cells to apoptosis through the activation of the p53 pathway. Defective signaling in this pathway compromises p53 function, and thus cisplatin does not induce apoptosis. A new group of nongenotoxic small molecules called Nutlins have been developed to inhibit p53‐Mdm2 binding, inducing apoptosis in chemoresistant tumors through the activation of the p53 pathway. The wild‐type p53 cisplatin‐resistant ovarian cancer cell‐line A2780cis was used to test the effect of Nutlin‐3a (Nut3a) on apoptosis response. The results showed that Nut3a synergized with cisplatin, inducing cell‐cycle arrest in G2/M and potentiating apoptotic cell death. Increased apoptosis was also induced in wild‐type TP53 primary OVCa cultures by double cisplatin–Nut3a treatment. In conclusion, Nut3a appears to sensitize chemoresistant OVCa cells to cisplatin, inducing apoptosis. As increased response was generalized in primary tumors, this cisplatin–Nut3a combination could be useful for the treatment of patients harboring wild‐type TP53 who do not respond to standard chemotherapy.     

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.     

A role for mismatch repair in control of DNA ploidy following DNA damage

Many reports have shown a link between mismatch repair (MMR) deficiency and loss of normal cell cycle control, particularly loss of G2 arrest. However almost all of these studies utilized transformed cell lines, and thus the involvement of other genes in this phenotype cannot be excluded. We have examined the effects of cisplatin treatment on primary embryo fibroblasts (MEFs) derived from mice in which the MMR gene Msh2 had been inactivated (Msh2(-/-)). This analysis determined that both primary Msh2(-/-) and wild type (WT) fibroblasts exhibited an essentially identical G2 arrest following cisplatin treatment. Similarly, we observed a cisplatin-induced G2 arrest in immortalized MMR deficient (Mlh1(-/-) and Pms2(-/-)) and WT MEFs. p53 deficient primary MEFs (p53(-/-)) exhibited both a clear G2 arrest and an increase in cells with a DNA content of 8N in response to cisplatin. When the Msh2 and p53 defects were combined (p53(-/-)/Msh2(-/-)) the G2 arrest was essentially identical to the p53(-/-) fibroblasts. However, the p53(-/-)/Msh2(-/-) fibroblasts demonstrated a further increase in cells with an 8N DNA content, above that seen in the p53(-/-) fibroblasts. These results suggest that loss of MMR on its own is not enough to overcome G2 arrest following exposure to cisplatin but does play a role in preventing polyploidization, or aberrant DNA reduplication, in the absence of functional p53.     

Down-regulation of survivin by ultraviolet C radiation is dependent on p53 and results in G(2)-M arrest in A549 cells

Deregulation of survivin expression is implicated in tumorigenesis. To examine the regulation of survivin expression in response to DNA damage, we exposed A549 human lung cancer cells to ultraviolet C (UVC) radiation, which induces DNA single-strand breakage. UVC irradiation induced G(2)-M arrest that was accompanied by accumulation of p53 and subsequent down-regulation of survivin. Depletion of p53 by RNA interference prevented the UVC-induced down-regulation of survivin. Furthermore, depletion of survivin resulted in G(2)-M arrest, suggesting that down-regulation of survivin by p53 contributes to the p53-dependent G(2)-M checkpoint triggered by DNA damage.     

Sequential activation and inactivation of G2 checkpoints for selective killing of p53-deficient cells by microtubule-active drugs

By inducing p53-dependent G2 arrest, the pretreatment with low concentrations of DNA damaging drugs (e.g., doxorubicin, DOX) can prevent cell death caused by microtubule-active drugs (e.g., paclitaxel, PTX), thus potentially permitting selective killing of p53-deficient cancer cells. However, DOX still protects a subset of tumor cell lines lacking wt p53 (HL60 and Jurkat leukemia cells), thus limiting the utility of protection of cells with wt p53 (e.g., normal cells). The present work overcomes this obstacle by adding an abrogator of p53-independent checkpoint (e.g., UCN-01) to the DOX-PTX sequence. By inhibiting a p53-independent pathway, UCN-01 overrode DOX-induced G2 arrest and instead induced G1 arrest in HL60 and Jurkat, thus propelling these p53-deficient cells from G2 to G1. Once they entered mitosis, cells were killed by PTX. Induction of G2 arrest with sequential abrogation of a p53-independent checkpoint allows pharmacological manipulation of Raf-1/Bcl-2 hyperphosphorylation, PARP and Rb cleavage and cell death caused by PTX in p53-deficient cells. Unlike previous approaches, this strategy is intended to increase selectivity, not the cytotoxicity of PTX. This rational sequence of agents that induces p53-dependent and abrogates p53-independent arrest represents a cancer-selective strategy for treatment of p53-deficient tumors.     

Schedule-dependent synergy between the heat shock protein 90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin and doxorubicin restores apoptosis to p53-mutant lymphoma cell lines.

PURPOSE: Loss of p53 function impairs apoptosis induced by DNA-damaging agents used for cancer therapy. Here, we examined the effect of the heat shock protein 90 (HSP90) inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) on doxorubicin-induced apoptosis in lymphoma. We aimed to establish the optimal schedule for administration of both drugs in combination and the molecular basis for their interaction. EXPERIMENTAL DESIGN: Isogenic lymphoblastoid and nonisogenic lymphoma cell lines differing in p53 status were exposed to each drug or combination. Drug effects were examined using Annexin V, active caspase-3, cell cycle, and cytotoxicity assays. Synergy was evaluated by median effect/combination index. Protein expression and kinase inhibition provided insight into the molecular mechanisms of drug interaction. RESULTS: Presence of mutant p53 conferred increased survival to single agents. Nevertheless, DMAG showed synergistic toxicity with doxorubicin independently of p53 status. Synergy required exposure to doxorubicin before DMAG. DMAG-mediated down-regulation of CHK1, a known HSP90 client, forced doxorubicin-treated cells into premature mitosis followed by apoptosis. A CHK1 inhibitor, SB-218078, reproduced the effect of DMAG. Administration of DMAG before doxorubicin resulted in G1-S arrest and protection from apoptosis, leading to additive or antagonistic interactions that were exacerbated by p53 mutation. CONCLUSIONS: Administration of DMAG to doxorubicin-primed cells induced premature mitosis and had a synergistic effect on apoptosis regardless of p53 status. These observations provide a rationale for prospective clinical trials and stress the need to consider schedule of exposure as a critical determinant of the overall response when DMAG is combined with chemotherapeutic agents for the treatment of patients with relapsed/refractory disease.     

Genetic status of p53 and induction of apoptosis by radiation or isoflavones in human gastric carcinoma cell lines

We have shown previously that various human cancer cell lines undergo morphological changes and internucleosomal DNA fragmentation characteristic of apoptosis after exposure to ionizing radiation or isoflavones. Here, we assessed the role of p53 gene in cell cycle and apoptosis following treatment of 11 gastric carcinoma cell lines with gamma-rays, genistein, biochanin A, or daidzein. Cell survival was measured by trypan blue staining, and apoptosis was assessed by fluorochrome staining. The rate of cell survival and apoptosis of the cells by gamma-irradiation or isoflavones did not correlate with p53 gene abnormalities. Flow cytometric measurement of DNA content demonstrated that while gamma-irradiation and genistein induced G(2) arrest, biochanin A and daidzein blocked the cell cycle of all carcinoma cells at G(1) phase. At multiple time points following irradiation, G(2) arrest was observed at 12-16 h in the wild-type and mutant p53 cell lines. Induction of p53 and p21 proteins was not observed in wild-type p53 lines after exposure to gamma-irradiation or isoflavones by Western blotting. Moreover, transfection of the wild-type p53 gene into MKN-1 cells failed to induce G(1) arrest by gamma-irradiation and genistein. Based on these results, we hypothesize that gastric cancer cells may possess a signal pathway which is different from the usual mechanisms of the p53-mediated DNA damage response in normal or hematopoietic tumor cells.     

Hsp90-inhibitor geldanamycin abrogates G2 arrest in p53-negative leukemia cell lines through the depletion of Chk1

Checkpoint protein Chk1 has been identified as an Hsp90 client. Treatment with 100 nM geldanamycin (GM) for 24 h markedly reduced the Chk1 amount in Jurkat and ML-1 leukemia cell lines. Because Chk1 plays a central role in G2 checkpoint, we added GM to G2-arrested Jurkat and HL-60 cells pretreated with 50 nM doxorubicin for 24 h. GM slowly released both cell lines from doxorubicin-induced G2 arrest into G1 phase. GM also abrogated ICRF-193-induced decatenation G2 checkpoint in Jurkat and HL-60 cells. Western blot analysis showed that addition of GM attenuates doxorubicin- and ICRF-193-induced Chk1 phosphorylation at Ser345. GM, however, failed to abrogate G2 arrest in p53-positive ML-1 cells maybe due to the p21 induction. GM released HeLa cells from doxorubicin-induced G2 arrest but trapped them at M phase. Flow cytometric analysis showed that addition of GM converted doxorubicin-induced necrosis into apoptosis in Jurkat cells. Colony assay indicated that although GM has a weak cytotoxic effect as a single agent, it dramatically intensifies the cytotoxicity of doxorubicin and ICRF-193 in Jurkat and HL-60 cells. These results suggest that abrogation of G2 checkpoint by GM may play a central role in sensitizing p53-negative tumor cells to DNA-damaging and decatenation-inhibiting agents.     

Stress-specific signatures: expression profiling of p53 wild-type and -null human cells

Gene expression responses of human cell lines exposed to a diverse set of stress agents were compared by cDNA microarray hybridization. The B-lymphoblastoid cell line TK6 (p53 wild-type) and its p53-null derivative, NH32, were treated in parallel to facilitate investigation of p53-dependent responses. RNA was extracted 4 h after the beginning of treatment when no notable decrease in cell viability was evident in the cultures. Gene expression signatures were defined that discriminated between four broad general mechanisms of stress agents: Non-DNA-damaging stresses (heat shock, osmotic shock, and 12-O-tetradecanoylphorbol 13-acetate), agents causing mainly oxidative stress (arsenite and hydrogen peroxide), ionizing radiations (neutron and gamma-ray exposures), and other DNA-damaging agents (ultraviolet radiation, methyl methanesulfonate, adriamycin, camptothecin, and cis-Platinum(II)diammine dichloride (cisplatin)). Within this data set, non-DNA-damaging stresses could be discriminated from all DNA-damaging stresses, and profiles for individual agents were also defined. While DNA-damaging stresses showed a strong p53-dependent element in their responses, no discernible p53-dependent responses were triggered by the non-DNA-damaging stresses. A set of 16 genes did exhibit a robust p53-dependent pattern of induction in response to all nine DNA-damaging agents, however.     

Mitogen requirement for cell cycle progression in the absence of pocket protein activity

Primary mouse embryonic fibroblasts lacking expression of all three retinoblastoma protein family members (TKO MEFs) have lost the G1 restriction point. However, in the absence of mitogens these cells become highly sensitive to apoptosis. Here, we show that TKO MEFs that survive serum depletion pass G1 but completely arrest in G2. p21CIP1 and p27KIP1 inhibit Cyclin A-Cdk2 activity and sequester Cyclin B1-Cdk1 in inactive complexes in the nucleus. This response is alleviated by mitogen restimulation or inactivation of p53. Thus, our results disclose a cell cycle arrest mechanism in G2 that restricts the proliferative capacity of mitogen-deprived cells that have lost the G1 restriction point. The involvement of p53 provides a rationale for the synergism between loss of Rb and p53 in tumorigenesis.     

Induction of apoptosis by cisplatin and its effect on cell cycle-related proteins and cell cycle changes in hepatoma cells.

We investigated cisplatin-induced apoptosis and the effects on cell cycle-related proteins and cell cycle changes. Two human hepatoma cell lines, HepG2 (with wild-type p53) and Hep3B (with deleted p53), were treated with different concentrations of cisplatin. Cisplatin induced apoptosis in both cell lines as assessed by cell morphology, DNA fragmentation analysis,TdT-mediated dUTP nick end labeling assay and flow cytometry. HepG2 cells were more sensitive to cisplatin than Hep3B. Low-dose cisplatin induced a transient G(1) arrest, S phase block and upregulation of p53 and p21(WAF1/CIP1) expression in HepG2, but not in Hep3B cells. With cisplatin at a high dose, both cell lines underwent apoptosis that was accompanied by downregulation of p27(KIP1) and Bcl-x(L). In HepG2, upregulation of p53 and p21(WAF1/CIP1) was observed before apoptosis occurred, suggesting that cisplatin-induced apoptosis in HepG2 might be p53-dependent. Expression of Fas was also increased following cisplatin treatment in HepG2. However, there was no induction of p53, p21(WAF1/CIP1) and Fas observed in Hep3B cells. In conclusion, cisplatin induced apoptosis in hepatoma cells via both p53-dependent and -independent pathways.     

p53/p21(CIP1) cooperate in enforcing rapamycin-induced G(1) arrest and determine the cellular response to rapamycin

The relationship between G(1) checkpoint function and rapamycininduced apoptosis was examined using two human rhabdomyosarcoma cell lines, Rh1 and Rh30, that express mutated p53 alleles. Serum-starved tumor cells became apoptotic when exposed to rapamycin, but were completely protected by expression of a rapamycin-resistant mutant mTOR. Exposure to rapamycin (100 ng/ml) for 24 h significantly increased the proportion of Rh1 and Rh30 cells in G(1) phase, although there were no significant changes in expression of cyclins D1, E, or A in drug-treated cells. To determine whether apoptosis was associated with continued slow progression through G(1) to S phase, cells were exposed to rapamycin for 24 h, then labeled with bromodeoxyuridine (BrdUrd). Histochemical analysis showed that >90% of cells with morphological signs of apoptosis had incorporated BRDURD: To determine whether restoration of G(1) arrest could protect cells from rapamycin-induced apoptosis, cells were infected with replication-defective adenovirus expressing either p53 or p21(CIP1). Infection of Rh30 cells with either Ad-p53 or Ad-p21, but not control virus (Ad-beta-gal), induced G(1) accumulation, up-regulation of p21(CIP1), and complete protection of cells from rapamycin-induced apoptosis. Within 24 h of infection of Rh1 cells with Ad-p21, expression of cyclin A was reduced by >90%. Similar results were obtained after Ad-p53 infection of Rh30 cells. Consistent with these data, incorporation of [(3)H]thymidine or BrdUrd into DNA was significantly inhibited, as was cyclin-dependent kinase 2 activity. These data indicate that rapamycin-induced apoptosis in tumor cells is a consequence of continued G(1) progression during mTOR inhibition and that arresting cells in G(1) phase, by overexpression of p53 or p21(CIP1), protects against apoptosis. The response to rapamycin was next examined in wild-type or murine embryo fibroblasts nullizygous for p53or p21(CIP1). Under serum-free conditions, rapamycin-treated wild-type MEFs showed no increase in apoptosis compared to controls. In contrast, rapamycin significantly induced apoptosis in cells deficient in p53 ( approximately 2.4-fold) or p21(CIP1) ( approximately 5.5-fold). Infection of p53(-/-) MEFs with Ad-p53 or Ad-p21 completely protected against rapamycin-induced apoptosis. Under serum-containing conditions, rapamycin inhibited incorporation of BrdUrd significantly more in wild-type murine embryo fibroblasts (MEFs) than in those lacking p53 or p21(CIP1). When BrdUrd was added 24 h after rapamycin, almost 90% and 70% of cells lacking p53 or p21(CIP1), respectively, incorporated nucleoside. In contrast, only 19% of wild-type cells incorporated BrdUrd in the presence of rapamycin. Western blot analysis of cyclin levels showed that rapamycin had little effect on levels of cyclins D1 or E in any MEF strain. However, cyclin A was reduced to very low levels by rapamycin in wild-type cells, but remained high in cells lacking p53 or p21(CIP1). Taken together, the data suggest that p53 cooperates in enforcing G(1) cell cycle arrest, leading to a cytostatic response to rapamycin. In contrast, in tumor cells, or MEFs, having deficient p53 function the response to this agent may be cell cycle progression and apoptosis.