Nava Almog

Mutant p53 protein expression interferes with p53-independent apoptotic pathways

Loss of normal p53 function was found frequently to interfere with response of cancer cells to conventional anticancer therapies. Since more than half of all human cancers possess p53 mutations, we decided to explore the involvement of mutant p53 in drug induced apoptosis. To further evaluate the relationship between the p53-dependent and p53-independent apoptotic pathways, and to elucidate the function of mutant p53 in modulating these processes, we investigated the role of a p53 temperature-sensitive (ts) mutant in a number of apoptotic pathways induced by chemotherapeutic drugs that are currently used in cancer therapy. To that end, we studied the M1/2, myeloid p53 non-producer cells, and M1/2-derived temperature-sensitive mutant p53 expressing clones. Apoptosis caused by DNA damage induced with γ-irradiation, doxorubicin or cisplatin, was enhanced in cells expressing wild type p53 as compared to that seen in parental p53 non-producer cells; mutant p53 expressing clones were found to be more resistant to apoptosis induced by these factors. Actinomycin D, a potent inhibitor of transcription, as well as a DNA damaging agent, abrogated the restraint apoptosis mediated by mutant p53. These observations suggest that while loss of wild type p53 function clearly reduces the rate of apoptosis, p53 mutations may result in a gain of function which significantly interferes with chemotherapy induced apoptosis. Therefore, to achieve a successful cancer therapy, it is critical to consider the specific relationship between a given mutation in p53 and the chemotherapy selected.

Role of wild type p53 in the G2 phase: regulation of the gamma-irradiation-induced delay and DNA repair.

Up-regulation of the p53 protein was found to induce cell cyle arrest at the G1/S border and in some cases at the G2/M border. Futhermore, it was suggested that p53 is associated with the induction of the various DNA repair pathways. Previously, we demonstrated that cells coexpressing endogenous wild type p53 protein, together with dominant negative mutant p53, exhibit deregulation of apoptosis, G1 arrest and delay in G2 following γ-irradiation. IN the present study, we investigated the role of p53 protein in the DNA damage response at the G2 phase. Using p53-null, wild type p53 and mutant p53-producer cell lines, we found that the two C-terminally spliced p53 forms could prevent γ-irradiation induced muatgenesis prior to mitosis, at the G2/M checkpoint. We found that at the G2 phase, p53 may facilitate repair of DNA breaks giving rise to micronuclei, and regulate the exit from the G2 checkpoint. At the G1 phase, only the regularly spliced form of p53 caused growth arrrest. In contrast, both the regularly and the alternatively spliced p53 forms directed postmitotic micronucleated cells towards apoptosis. These results provide a functional explanation for the cell cycle-independent expression of p53 in mnormal cycling cells, as well as in cells where p53 is up-regulated, following DNA damage.

Accentuated apoptosis in normally developing p53 knockout mouse embryos following genotoxic stress.

In order to identify the alternative pathways which may substitute for the p53 function during embryogenesis, we have focused our studies on p53−/− normally developing mouse embryos that survived a genotoxic stress. We assumed that under these conditions p53-independent pathways, which physiologically control genomic stability, are enhanced. We found that while p53+/+ mouse embryos elicited, as expected, a p53-dependent apoptosis, p53−/− normally developing mice exhibited an accentuated p53-independent apoptotic response. The p53-dependent apoptosis detected in p53+/+ embryos, was an immediate reaction mostly detected in the brain, whereas the p53-independent apoptosis was a delayed reaction with a prominent pattern observed in epithelial cells of most organs in the p53-deficient mice only. These results suggest that in the absence of p53-dependent apoptosis, which is a fast response to damaged DNA, p53-independent apoptotic pathways, with slower kinetics, are turned on to secure genome stability.

p53-dependent apoptosis is regulated by a C-terminally alternatively spliced form of murine p53.

It is now well accepted that the p53 C-terminus plays a central role in controlling the activity of the wild-type molecule. In our previous studies, we observed that a C-terminally altered p53 protein (p53AS), generated by an alternative spliced p53 mRNA, induces an attenuated p53-dependent apoptosis, compared to that induced by the regularly spliced form (p53RS). In the present study we analysed the interrelationships between these two physiological variants of wild-type p53, and found that in cells co-expressing both forms, in contrast to the expected additive effect on the induction of apoptosis, p53AS inhibits apoptosis induced by p53RS. This inhibitory effect is specific for p53-dependent apoptosis and was not evident in a p53-independent apoptotic pathway induced by growth factor deprivation. Furthermore, the expression of p53AS in transiently transfected cells caused both inhibition of apoptosis and inhibition of the p53RS-dependent transactivation of a number of p53 target genes. These results suggest that expression of an alternatively spliced p53 form may serve as an additional level in controlling the complexity of p53 function by the C-terminal domain.

The C-terminus of mutant p53 is necessary for its ability to interfere with growth arrest or apoptosis

The ability to suppress wild type p53-independent apoptosis may play an important role in the oncogenicity of p53 mutant proteins. However, structural elements necessary for this activity are unknown. Furthermore, it is unclear whether this mutant p53 mediated inhibition is specific to the apoptotic pathway or a more general suppression of the cellular response to stress. We observed that an unmodified C-terminus was required for the suppression of apoptosis by the p53 135(Ala to Val) oncogenic p53 mutant. It was also required for the novel activity of G2 arrest suppression, the predominant response at low levels of genotoxic stress. These observations are consistent with a model whereby mutant p53 suppressive activity is not specific to the apoptotic pathway, but rather increases the threshold of genotoxic stress needed for a DNA damage response to occur. Furthermore, these observations indicate that it may be possible to selectively kill mutant p53 expressing cells based on the lower sensitivity of their growth arrest response.