PolyADP-ribose-mediated regulation of p53 complexed with topoisomerase I following ionizing radiation.

This investigation demonstrates that the p53 and topoisomerase I (topo I) proteins which form a molecular complex in vivo are polyADP ribosylated following 1 Gy of gamma irradiation. Immunoprecipitations using a topo I monoclonal antibody were performed on protein extracts from gamma-irradiated TK6 human lymphoblastoid cells. Western blots on topo I immunoprecipitations probed with a polyADP-ribose polymer antibody demonstrated that several proteins, including p53, are co-immunoprecipitated with topo I. Furthermore, p53 and topo I are ADP ribosylated within 15 min following gamma irradiation. Unlike the other proteins within the complex, p53 is polyADP ribosylated at low levels in non-irradiated cells, and it is also the most heavily polyADP ribosylated following irradiation. Radiation induced polyADP ribosylation persists for at least 48 h following exposure. The DNA damage response does not involve the recruitment of free p53 to complex with topo I; the amount of p53 protein complexed with topo I was found to be independent of radiation exposure. It has recently been reported that p53 acts to catalytically stimulate the activity of topo I in the absence of DNA damage. We hypothesize that the rapid modification of the complex by polyADP ribosylation following radiation is a regulatory response to diminish topo I cleavage in the presence of DNA damage.     

Topotecan-triggered degradation of topoisomerase I is p53-dependent and impacts cell survival

The anticancer drug topotecan belongs to the group of topoisomerase I (topo I) inhibitors. In the presence of topotecan, topo I cleaves the DNA but is unable to religate the single-strand break. This leads to stabilization of topo I-DNA-bound complexes and the accumulation of DNA strand breaks that may interfere with DNA replication. The molecular mechanism of controlling the repair of topo I-DNA covalent complexes and its impact on sensitivity of cells to topotecan is largely unknown. Here, we used mouse embryonic fibroblasts expressing wild-type p53 and deficient in p53, in order to elucidate the role of p53 in topotecan-induced cell death. We show that p53-deficient mouse embryonic fibroblasts are significantly more sensitive to topotecan than wild-type cells, displaying a higher frequency of topotecan-induced apoptosis and DNA strand breaks. Treatment of p53 wild-type cells with pifithrin-alpha, an inhibitor of the trans-activating activity of p53, caused reversal of the phenotype, making wild-type cells more sensitive to topotecan. Upon topotecan treatment, topo I was degraded in wild-type but not in p53-deficient cells. Topo I degradation was attenuated by the proteosomal inhibitor MG132. Similar data were obtained with human glioblastoma cells. U138 cells (p53 mutated) were significantly more sensitive to topotecan than U87 cells (p53 wild-type). Furthermore, U87 cells showed significant degradation of topo I upon topotecan treatment, whereas in U138 cells, this response was abrogated. Topo I degradation was again attenuated by pifithrin-alpha. The data suggests that p53 causes resistance of cells to topo I inhibitors due to stimulation of topotecan-triggered topo I degradation which may impact topotecan-based cancer therapy.     

Induction of p53-dependent and p53-independent cellular responses by topoisomerase 1 inhibitors.

We have previously shown that loss of p53 function in A2780 human ovarian adenocarcinoma cells confers increased clonogenic resistance to several DNA-damaging agents, but not to taxol or camptothecin. We have now extended these studies, comparing wild-type p53-expressing A2780 cells with isogenic derivatives transfected with a dominant negative mutant (143; val to ala) p53. We show that, as well as retaining equivalent clonogenic sensitivity to camptothecin, mutant p53 transfectants of A2780 cells do not acquire significantly increased resistance to the camptothecin analogues topotecan and SN-38, the active metabolite of CPT-11. Compared with vector-alone transfectants they are, however, relatively (2.2-fold) resistant to GI 147211, a further camptothecin analogue undergoing clinical trial. Treatment of A2780 with camptothecin and each analogue produces an increase, maximal at 24-48 h after drug exposure, of cells in the G2/M phase of the cell cycle and a decrease in both G1 and S-phase cells. The G2 arrest is independent of p53 function for camptothecin and the three analogues. All four compounds can induce apoptosis in A2780, which is reduced in mutant p53 transfectants, as measured using the terminal DNA transferase-mediated b-d UTP nick end labelling (TUNEL) assay. Thus, although p53-dependent apoptosis is induced by camptothecin, topotecan and SN-38 in this human ovarian carcinoma cell line, these drugs induce p53-independent death, as measured by clonogenic assay.     

p53 disruption profoundly alters the response of human glioblastoma cells to DNA topoisomerase I inhibition

A critical challenge in cancer research is to identify genetic lesions that sensitize patients to chemotherapy. p53, which is mutated in nearly one-third to half of glioblastomas, may be such a lesion. In this paper, we demonstrate that p53 disruption dramatically sensitizes glioblastoma cells to DNA topoisomerase I inhibitor-mediated apoptosis. Using 19 glioblastoma cell lines, including 15 low-passage ex vivo cell lines derived from patients, as well as isogenic glioblastoma cells varying in p53 status, we show that clinically relevant levels of SN-38 potently induce cell cycle arrest and temporary senescence in glioblastoma cells with wild-type p53 while causing massive apoptosis in p53-deficient cells (P<0.0002). We demonstrate that glioblastoma cells with wild-type p53 proliferate when recultured in drug-free medium, whereas p53-deficient cells do not. We also show that p16 protein expression is neither necessary nor sufficient for initiation and/or maintenance of SN-38-induced arrest/senescence. These results indicate that p53 disruption has a dramatic effect on how glioblastoma cells process topoisomerase I inhibitor-mediated DNA damage.     

The tumor suppressor protein p53 stimulates the formation of the human topoisomerase I double cleavage complex in vitro

Previous studies have shown that human topoisomerase I interacts directly with the tumor-suppressor protein p53. In the past few years it has repeatedly been suggested that topoisomerase I and p53 may play a joint role in the response to genotoxic stress. This led to the suggestion that p53 and human topoisomerase I may cooperate in the process of DNA repair and/or apoptosis. Recently we have demonstrated that a human topoisomerase I cleavage complex can be recognized by an additional topoisomerase I molecule and thereby form a so-called double cleavage complex. The double cleavage complex creates an about 13 nucleotides long single-stranded gap that may provide an entry site for recombinational repair events. Here we demonstrate that p53 stimulates both the DNA relaxation activity as well as the formation of the human topoisomerase I double cleavage complex by at least a factor of six. Stimulation of topoisomerase I activity by p53 is mediated via the central part of topoisomerase I. We also show that human, bovine, and murine p53 stimulate human topoisomerase I relaxation activity equally well. From these results it is conceivable that p53's stimulatory activity on topoisomerase I may play a role in DNA recombination and repair as well as in apoptosis.     

Cellular stress triggers the human topoisomerase I damage response independently of DNA damage in a p53 controlled manner

The 'human topoisomerase I (htopoI) damage response' was reported to be triggered by various kinds of DNA lesions. Also, a high and persistent level of htopoI cleavage complexes correlated with apoptosis. In the present study, we demonstrate that DNA damage-independent induction of cell death using colcemid and tumor necrosis factor alpha is also accompanied by a strong htopoI response that correlates with the onset of apoptotic hallmarks. Consequently, these results suggest that htopoI cleavage complex formation may be caused by signaling pathways independent of the kind of cellular stress. Thus, protein interactions or signaling cascades induced by DNA damage or cellular stress might lead to the formation of stabilized cleavage complexes rather than the DNA lesion itself. Finally, we show that p53 not only plays a key role in the regulation of the htopoI response to UV-C irradiation but also to treatment with colcemid.     

Tissue-specific induction of p53 targets in vivo

The in vivo response to radiotherapy is not well understood but appears to involve the p53 tumor suppressor protein. We investigated the expression of apoptosis-inducing p53 target genes during gamma-irradiation-induced cell death in p53(+/+) or p53(-/-) mouse tissues using in situ hybridization. Our results reveal striking tissue specificity with distinct regulation of target p53-induced genes in different cells and tissue compartments, as well as variations in dependence on p53 for basal expression. p53-dependent induction of Puma occurred in the splenic white pulp, whereas Noxa and Bid were induced in the red pulp. These patterns correlated with activation of caspase-3 in both compartments. All apoptotic targets of p53 studied here (DR5, Bid, Puma, Noxa) were induced in the jejunum and ileum, which appeared to be the tissues most sensitive to irradiation. We also observed unexpected differences in p53 target gene activation between the transverse and descending colon. Finally, in the liver where irradiation did not lead to caspase-3 activation, we primarily observed p21(WAF1) induction as the major p53-dependent target gene response. Our findings indicate that the selectivity of p53 in transactivation following DNA damage in vivo results in unique tissue and cell type specificity, which may correlate with growth arrest or variable sensitivity to gamma-irradiation.     

Dissecting p53 tumor suppressor functions in vivo

Although the p53 tumor suppressor acts in a plethora of processes that influence cellular proliferation and survival, it remains unclear which p53 functions are essential for tumor suppression and, as a consequence, are selected against during tumor development. Using a mouse model harboring primary, genetically modified myc-driven lymphomas, we show that disruption of apoptosis downstream of p53 by Bcl2 or a dominant-negative caspase 9 confers-like p53 loss-a selective advantage, and completely alleviates pressure to inactivate p53 during lymphomagenesis. Despite their p53-null-like aggressive phenotype, apoptosis-defective lymphomas that retain intact p53 genes do not display the checkpoint defects and gross aneuploidy that are characteristic of p53 mutant tumors. Therefore, apoptosis is the only p53 function selected against during lymphoma development, whereas defective cell-cycle checkpoints and aneuploidy are mere byproducts of p53 loss.     

Multiple stress signals activate mutant p53 in vivo

p53 levels are tightly regulated in normal cells, and thus, the wild-type p53 protein is nearly undetectable until stimulated through a variety of stresses. In response to stress, p53 is released from its negative regulators, mainly murine double minute 2 (Mdm2), allowing p53 to be stabilized to activate cell-cycle arrest, senescence, and apoptosis programs. Many of the upstream signals that regulate wild-type p53 are known; however, limited information for the regulation of mutant p53 exists. Previously, we showed that wild-type and mutant p53R172H are regulated in a similar manner in the absence of Mdm2 or p16. In addition, this stabilization of mutant p53 is responsible for the gain-of-function metastatic phenotype observed in the mouse. In this report, we examined the role of oncogenes, DNA damage, and reactive oxygen species, signals that stabilize wild-type p53, on the stabilization of mutant p53 in vivo and the consequences of this expression on tumor formation and survival. These factors stabilized mutant p53 protein which oftentimes contributed to exacerbated tumor phenotypes. These findings, coupled with the fact that patients carry p53 mutations without stabilization of p53, suggest that personalized therapeutic schemes may be needed for individual patients depending on their p53 status.     

G2 arrest in response to topoisomerase II inhibitors: the role of p53

We have previously found that the overexpression of p53 causes G(2) arrest and represses the synthesis of cyclin-dependent kinase 1 and cyclin B1, two proteins required for cells to traverse from G(2) into M. G(2) arrest occurs in response to DNA damage caused by a variety of agents and treatments. Here, we investigate the role of p53 in the G(2) arrest that occurs in response to the topoisomerase inhibitors etoposide and merbarone. In HT1080 cells expressing a dominant-negative form of p53, treatment with etoposide still caused G(2) arrest, but the arrest could be overcome by additional treatment with caffeine, which inhibits the damage-responsive kinases ataxia telangiectasia mutated (ATM) and atm and rad3-related (ATR). However, caffeine could not overcome etoposide-induced G(2) arrest in HT1080 cells with functional p53. We conclude that etoposide activates two pathways, one of which depends on p53 and the other of which is sensitive to caffeine, and that either pathway is sufficient to activate G(2) arrest. Etoposide inhibits topoisomerase II by trapping the enzyme in a complex with cleaved DNA. Inhibition of topoisomerase II with merbarone, which does not stabilize a cleavage complex, causes G(2) arrest by a checkpoint that monitors the decatenation of chromatin. We find that caffeine can abrogate merbarone-induced G(2) arrest even in cells with functional p53, indicating that p53 does not contribute to the decatenation-sensitive response. Thus, p53 has a differential role in effecting G(2) arrest in response to topoisomerase II inhibitors, depending upon the mechanisms of action of the inhibitors tested.     

Antibody-mediated p53 protein therapy prevents liver metastasis in vivo

To evaluate the clinical efficacy of monoclonal antibody (mAb) 3E10 Fv antibody-mediated p53 protein therapy, an Fv-p53 fusion protein produced in Pichia pastoris was tested on CT26.CL25 colon cancer cells in vitro and in vivo in a mouse model of colon cancer metastasis to the liver. In vitro experiments showed killing of CT26.CL25 cells by Fv-p53 but not Fv or p53 alone, and immunohistochemical staining confirmed that Fv was required for transport of p53 into cells. Prevention of liver metastasis in vivo was tested by splenic injection of 100 nmol/L Fv-p53 10 min and 1 week after injection of CT26.CL25 cancer cells into the portal vein of BALB/c mice. Mice were sacrificed 1 week after the second injection of Fv-p53 and assigned a quantitative metastasis score. Control mice had an average metastasis score of 3.3 +/- 1.3, whereas mice treated with Fv-p53 had an average metastasis score of 0.8 +/- 0.4 (P = 0.004). These results indicate that Fv-p53 treatment had a profound effect on liver metastasis and represent the first demonstration of effective full-length p53 protein therapy in vivo. mAb 3E10 Fv has significant clinical potential as a mediator of intracellular and intranuclear delivery of p53 for prevention and treatment of cancer metastasis.     

Loss of atm sensitises p53-deficient cells to topoisomerase poisons and antimetabolites.

BACKGROUND: Ataxia-telangiectasia is a pleiotropic autosomal recessive disorder caused by mutations in the ATM gene. In addition to a profound cancer predisposition, another hallmark of ataxia-telangiectasia is radiosensitivity. Recently, p53-null mouse fibroblasts have been reported to be radiosensitised by the concurrent loss of ATM. MATERIALS AND METHODS: We compared the sensitivity of atm(+/+)/p53(-/-) and atm(-/-)/p53(-/-) mouse embryonic fibroblasts to different classes of chemotherapeutic agents using the MTT assay, Trypan Blue exclusion and fluorescence-activated cell sorting for cell cycle and apoptosis analyses. RESULTS: Loss of ATM function in p53-deficient cells resulted in a 2- to 4-fold increase in sensitivity to the topoisomerase I poisons camptothecin and topotecan, to the topoisomerase II poisons doxorubicin, epirubicin and etoposide, and to the antimetabolites 5-fluorouracil and gemcitabine, but not to the platinum compounds cisplatin, carboplatin and oxaliplatin, the taxanes docetaxel and paclitaxel, or to busulfan. Loss of ATM function did not result in increased apoptosis, but resulted in increased Trypan Blue staining in response to epirubicin, suggesting that processes other than apoptosis may mediate cytotoxicity. ATM deficiency did not alter the extent of G(1)/S or G(2)/M cell cycle phase accumulation produced by epirubicin, suggesting that enhanced sensitivity was not due to failure of checkpoint activation. CONCLUSIONS: We provide further evidence that ATM is involved in regulating cellular defences against some cytotoxic agents in the absence of p53. Tumour-targeted functional inhibition of ATM may be a valuable strategy for increasing the efficacy of anticancer agents in the treatment of p53-mutant cancers.     

mdmx is a negative regulator of p53 activity in vivo

Regulation of p53 protein activity is required for normal embryogenesis, tumor suppression, and cellular response to DNA damage. Here we report that loss of mdmx, a p53-binding protein, results in midgestational embryo lethality, a phenotype that is completely rescued by the absence of p53. Mice homozygous for both mdmx and p53 null mutations are viable and appear developmentally normal. Fibroblasts derived from embryos with reduced mdmx expression demonstrate a decreased growth rate and increased UV-induced apoptosis compared with wild-type cells and contain elevated levels of p53 and several p53 target proteins including the proapoptotic bax protein. These observations demonstrate that mdmx functions as a critical negative regulator of p53 in vivo.     

Dissecting the p53-Mdm2 feedback loop in vivo: uncoupling the role in p53 stability and activity

The p53-Mdm2 feedback loop is thought to be the main mechanism by which p53 autoregulates its levels and activity after DNA damage. We tested this paradigm in a genetically engineered mouse model in which the feedback loop was disrupted by point mutations in the p53 binding site of the Mdm2 promoter. We noted that while the p53-Mdm2 feedback loop is required to regulate p53 activity especially in the hematopoietic system in response to DNA damage, its role in development and in regulating the stability of p53 is dispensable. In the present study we have extended our characterization of this mouse model and show that the kinetics of p53 degradation is also unchanged in mouse embryonic fibroblasts (MEFs). Additionally, MG132 experiments indicate that other E3-ligases regulate p53 stability. Also, Mdm4 cooperates in inhibition of p53 activity and levels in these mice. Finally, we show in this system that enhanced acute p53 response does not promote aging or protect against late term tumorigenesis. We also discuss future perspectives for this study.     

p63 and p73 do not contribute to p53-mediated lymphoma suppressor activity in vivo

p53 is one of the most important tumor suppressor genes in human cancer, but the roles of its homologues p63 and p73 in tumor suppression, alone or in collaboration with p53, remains controversial. Both p63 and p73 can be deregulated after DNA damage, and induce cell cycle arrest and apoptosis, but mice carrying inactive alleles of these genes do not develop spontaneous tumors. Since heterozygous loss of p53 confers strong sensitization to radiation-induced lymphoma development, we investigated the possibility that radiation exposure may reveal previously undetected tumor suppressor properties in p63 or p73, alone or in combination with p53. Animals heterozygous for p63 or p73, as well as both double heterozygous p53/p63 or p53/p73 mice, showed no significant differences in tumor latency, spectrum or frequency after gamma-radiation, compared to their control counterparts. Deletions were found near the p63 locus on chromosome 16 in radiation-induced tumors, but these frequently included the knockout allele. No deletions or LOH involving the p73 gene were detected, and expression of both genes was maintained in the tumors. We conclude that p53 homologues do not contribute to p53 tumor suppressor activity in lymphoma development.