Treatment of Chronic Myeloid Leukemia Cells with Imatinib (STI571) Impairs p53 Accumulation in Response to DNA Damage

Chronic myelogenous leukaemia (CML) is induced by the Bcr-Abl fusion protein. Inhibition of Bcr-Abl by STI571 is widely used to treat CML patients. Unlike in most cancer types, the frequency of p53 mutations in CML is low. Here, we investigated the effect of STI571 treatment of CML cells on p53 regulation. Exposure of CML cells, including established cell lines and freshly isolated cells from patients, to STI571 reduced p53 protein levels, and severely impaired its accumulation in response to DNA damage. This may be explained by the status of p53 serine 20 phosphorylation. In non-stressed CML cells, serine 20 of p53 is constitutively phosphorylated by Chk1, and is inhibited by STI571. In response to DNA damage, however, this phosphorylation is mediated by Chk1 and Chk2, and is only partially inhibited by STI571. CML cells expressing wild-type p53 are more resistant to treatment with STI571, but moderately more sensitive to DNA damage, than CML cells lacking p53. An enhanced induction of apoptosis by STI571 and DNA damage is observed in CML cells bearing wild-type p53, but not in cells lacking functional p53. This implies that the status of p53 may affect the response of CML cells to this combined treatment.     

Suppression of Polo like kinase 1 (PLK1) by p21(Waf1) mediates the p53-dependent prevention of caspase-independent mitotic death

Polo-like kinase 1 (Plk1) plays key roles in many aspects of mitosis. We have previously shown that induction of p21^Waf1 by p53 is responsible for protection of cells against adriamycin-induced polyploidy formation and mitotic catastrophe. Here we show that adriamycin treatment suppressed Plk1 expression in a p53- and p21^Waf1-dependent manner. Ablation of p21^Waf1 inhibited the adriamycin-induced p53 activation, and this inhibition was alleviated by knockdown of Plk1, suggesting that p21^Waf1-dependent suppression of Plk1 expression is responsible for maintaining p53 activation during stress response. Plk1 associated with p53 and disrupted its interaction with target gene promoters in cells treated with adriamycin. Overexpression of Plk1 inhibited the p53-mediated prevention of caspase-independent mitotic death, but not polyploidy formation, in adriamycin-treated cells. Together our results indicate that suppression of Plk1 by p21^Waf1 is responsible for p53-dependent protection against adriamycin-induced caspase-independent mitotic death.     

BCR/ABL Recruits p53 Tumor Suppressor Protein to Induce Drug Resistance

Tumors expressing the ABL oncoproteins (BCR/ABL, TEL/ABL, v-ABL) can avoidapoptosis triggered by DNA damaging agents. The tumor suppressor protein p53 is animportant activator of apoptosis in normal cells; conversely its functional loss may causedrug resistance. The ABL oncoprotein - p53 paradigm represents the relationship between anoncogenic tyrosine kinase and a tumor suppressor gene. Here we show that BCR/ABLoncoproteins employ p53 to induce resistance to DNA damage in myeloid leukemia cells.Cells transformed by the ABL oncoproteins displayed accumulation of p53 upon DNAdamage. In contrast, only a modest increase of p53 expression followed by activation ofcaspase-3 were detected in normal cells expressing endogenous c-ABL. Phosphatidylinositol-3 kinase-like protein kinases (ATR and also ATM) -dependent phosphorylation of p53-Ser15residue was associated with the accumulation of p53, and stimulation of p21Waf-1 andGADD45, resulting in G2/M delay in BCR/ABL cells after genotoxic treatment. Inhibition ofp53 by siRNA or by the temperature-sensitive mutation reduced G2/M accumulation anddrug resistance of BCR/ABL cells. In conclusion, accumulation of the p53 proteincontributed to prolonged G2/M checkpoint activation and drug resistance in myeloid cellsexpressing the BCR/ABL oncoproteins.     

Proteomic-based identification of Apg-2 as a therapeutic target for chronic myeloid leukemia

The oncogenic BCR/ABL tyrosine kinase induces constitutive enhanced “spontaneous” DNA damage and unfaithful repair in Philadelphia chromosome positive leukemia cells. Here, we investigated the changes of protein profile in H₂O₂-induced DNA damage/repair in BaF3-MIGR1 and BaF3-BCR/ABL cells through a proteomic strategy consisting of two-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry. In total, 41 spots were differentially expressed and 13 proteins were identified with further MS analysis. Two essential proteins, Proto-oncogene tyrosine–protein kinase ABL1 (c-ABL) and Heat shock 70 kDa protein 4 (Apg-2), were confirmed by Western blot and showed consistent changes with proteomic results. Moreover, functional analysis demonstrated that inhibition of Apg-2 not only decreased cell proliferation, but also induced cell apoptosis in BCR/ABL positive cells (BaF3-BCR/ABL, BaF3-BCR/ABL^T315I). We also proved that Apg-2 inhibition aggravated H₂O₂ induced damage in BCR/ABL positive cells, and enhanced the sensitivity of BaF3-BCR/ABL^T315I to STI571. Taken together, the findings in this work provide us with some clues to a better understanding of the molecular mechanisms underlying BCR/ABL in the DNA damage/repair processes and demonstrated that Apg-2 would be a valid target for anti-leukemia drug development.     

Characterization of the p53-Dependent Postmitotic Checkpoint following Spindle Disruption

The p53 tumor suppressor gene product is known to act as part of a cell cycle checkpoint in G₁ following DNA damage. In order to investigate a proposed novel role for p53 as a checkpoint at mitosis following disruption of the mitotic spindle, we have used time-lapse videomicroscopy to show that both p53^+/+ and p53^−/− murine fibroblasts treated with the spindle drug nocodazole undergo transient arrest at mitosis for the same length of time. Thus, p53 does not participate in checkpoint function at mitosis. However, p53 does play a critical role in nocodazole-treated cells which have exited mitotic arrest without undergoing cytokinesis and have thereby adapted. We have determined that in nocodazole-treated, adapted cells, p53 is required during a specific time window to prevent cells from reentering the cell cycle and initiating another round of DNA synthesis. Despite having 4N DNA content, adapted cells are similar to G₁ cells in that they have upregulated cyclin E expression and hypophosphorylated Rb protein. The mechanism of the p53-dependent arrest in nocodazole-treated adapted cells requires the cyclin-dependent kinase inhibitor p21, as p21^−/− fibroblasts fail to arrest in response to nocodazole treatment and become polyploid. Moreover, p21 is required to a similar extent to maintain cell cycle arrest after either nocodazole treatment or irradiation. Thus, the p53-dependent checkpoint following spindle disruption functionally overlaps with the p53-dependent checkpoint following DNA damage.     

Poly(ADP-ribose) polymerase inhibition enhances p53-dependent and -independent DNA damage responses induced by DNA damaging agent

Targeting DNA repair with poly(ADP-ribose) polymerase (PARP) inhibitors has shown a broad range of anti-tumor activity in patients with advanced malignancies with and without BRCA deficiency. It remains unclear what role p53 plays in response to PARP inhibition in BRCA-proficient cancer cells treated with DNA damaging agents. Using gene expression microarray analysis, we find that DNA damage response (DDR) pathways elicited by veliparib (ABT-888), a PARP inhibitor, plus topotecan comprise the G₁/S checkpoint, ATM and p53 signaling pathways in p53-wild-type cancer cell lines and BRCA1, BRCA2 and ATR pathway in p53-mutant lines. In contrast, topotecan alone induces the G₁/S checkpoint pathway in p53 wild-type lines and not in p53-mutant cells. These responses are coupled with G₂/G₁ checkpoint effectors p21^CDKN1A upregulation, and Chk1 and Chk2 activation. The drug combination enhances G₂ cell cycle arrest, apoptosis and a marked increase in cell death relative to topotecan alone in p53-wild-type and p53-mutant or -null cells. We also show that the checkpoint kinase inhibitor UCN-01 abolishes the G₂ arrest induced by the veliparib and topotecan combination and further increases cell death in both p53-wild-type and -mutant cells. Collectively, PARP inhibition by veliparib enhances DDR and cell death in BRCA-proficient cancer cells in a p53-dependent and -independent fashion. Abrogating the cell cycle arrest induced by PARP inhibition plus chemotherapeutics may be a strategy in the treatment of BRCA-proficient cancer.Key words: DNA damaging agent, G₂ arrest, microarray, PARP inhibition, p53, topotecan, veliparib (ABT-888)     

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.     

14-3-3Gamma inhibition of MDMX-mediated p21 turnover independent of p53

The stability of p21, a cyclin-dependent kinase inhibitor, is highly regulated by various protein molecules through the cell cycle and in response to extracellular signals. One of the p21 regulators is MDMX, which can directly bind to p21 and mediate its proteasomal degradation in an ubiquitination-independent fashion. The fact that 14-3-3γ binds to the MDMX domain adjacent to p21 binding suggests that this 14-3-3γ may affect MDMX-mediated p21 proteasomal turnover. Indeed, we found that overexpression of 14-3-3γ increased the level of both endogenous and exogenous p21 in p53-null cells by extending its half-life, leading to p21-dependent G1 arrest. Also, 14-3-3γ excluded p21 from binding to MDMX in a dose-dependent manner as determined by co-immunroprecipitation in vitro using purified proteins and in cells. In response to DNA damage, the level of the 14-3-3γ-MDMX complex increased whereas that of the MDMX-p21 complex declined as detected by co-immunoprecipitation assays, leading to the induction of p21 in p53-null cells. Knockdown of 14-3-3γ inversely alleviated the induction of p21 levels by DNA damage. Hence, our study as presented here unravels a new role for 14-3-3γ in protecting p21 from MDMX-mediated proteasomal turnover, which may partially account for DNA damage-induced elevation of p21 levels independent of p53.     

ATM-deficient human fibroblast cells are resistant to low levels of DNA double-strand break induced apoptosis and subsequently undergo drug-induced premature senescence

DNA DSBs are induced by IR or radiomimetic drugs such as doxorubicin. It has been indicated that cells from ataxia-telangiectasia patients are highly sensitive to radiation due to defects in DNA repair, but whether they have impairment in apoptosis has not been fully elucidated. A-T cells showed increased sensitivity to high levels of DNA damage, however, they were more resistant to low doses. Normal cells treated with combination of KU55933, a specific ATM kinase inhibitor, and doxorubicin showed increased resistance as they do in a similar manner to A-T cells. A-T cells have higher viability but more DNA breaks, in addition, the activations of p53 and apoptotic proteins (Bax and caspase-3) were deficient, but Akt expression was enhanced. A-T cells subsequently underwent premature senescence after treatment with a low dose of doxorubicin, which was confirmed by G2 accumulation, senescent morphology, and SA-β-gal positive until 15 days repair incubation. Finally, A-T cells are radio-resistant at low doses due to its defectiveness in detecting DNA damage and apoptosis, but the accumulation of DNA damage leads cells to premature senescence.     

Pharmacological activation of a novel p53-dependent S-phase checkpoint involving CHK-1

We have recently shown that induction of the p53 tumour suppressor protein by the small-molecule RITA (reactivation of p53 and induction of tumour cell apoptosis; 2,5-bis(5-hydroxymethyl-2-thienyl)furan) inhibits hypoxia-inducible factor-1α and vascular endothelial growth factor expression in vivo and induces p53-dependent tumour cell apoptosis in normoxia and hypoxia. Here, we demonstrate that RITA activates the canonical ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related DNA damage response pathway. Interestingly, phosphorylation of checkpoint kinase (CHK)-1 induced in response to RITA was influenced by p53 status. We found that induction of p53, phosphorylated CHK-1 and γH2AX proteins was significantly increased in S-phase. Furthermore, we found that RITA stalled replication fork elongation, prolonged S-phase progression and induced DNA damage in p53 positive cells. Although CHK-1 knockdown did not significantly affect p53-dependent DNA damage or apoptosis induced by RITA, it did block the ability for DNA integrity to be maintained during the immediate response to RITA. These data reveal the existence of a novel p53-dependent S-phase DNA maintenance checkpoint involving CHK-1.     

Effects of the environmental mammary carcinogen 6-nitrochrysene on p53 and p21(Cip1) protein expression and cell cycle regulation in MCF-7 and MCF-10A cells

The environmental pollutant 6-nitrochrysene (6-NC) is a potent mammary carcinogen in rats; it is more potent than numerous classical mammary carcinogens such as benzo[a]pyrene (BaP). The mechanisms that account for the remarkable carcinogenicity of 6-NC remain elusive. Similar to BaP, 6-NC is also known to induce DNA damage in rodents and in human breast tissues. As an initial investigation, we reasoned that DNA damage induced by 6-NC may alter the expression of p53 protein in a manner that differs from other DNA damaging carcinogens (e.g. BaP). Using human breast adenocarcinoma MCF-7 cells and immortalized human mammary epithelial MCF-10A cells, we determined the effects of 6-NC on the expression of p53 protein and its direct downstream target cyclin-dependent kinase inhibitor p21(Cip1) as well as on the cell cycle progression. Western blot analysis demonstrated that treatments of MCF-7 and MCF-10A cells with 6-NC for 12, 24 or 48h did not increase the level of total p53 protein; however, an increase of p21(Cip1) protein and a commitment increase of G(1) phase were observed in MCF-10A cells but not in MCF-7 cells. Further studies using 1,2-dihydroxy-1,2-dihydro-6-hydroxylaminochrysene (1,2-DHD-6-NHOH-C), the putative ultimate genotoxic metabolite of 6-NC, was conducted and showed a significant induction of p53 (p<0.05) in MCF-7 cells; however, this effect was not evident in MCF-10A cells, indicating the varied DNA damage responses between the two cell lines. By contrast to numerous DNA damaging agents such as BaP which is known to stimulate p53 expression, the lack of p53 response by 6-NC imply the lack of protective functions mediated by p53 (e.g. DNA repair machinery) after exposure to 6-NC and this may, in part, account for its remarkable carcinogenicity in the mammary tissue.