Structural and functional involvement of p53 in BER in vitro and in vivo.

p53 is involved in several DNA repair pathways. Some of these require the specific transactivation of p53-dependent genes and others involve direct interactions between the p53 protein and DNA repair associated proteins. Previously, we have shown that p53 acts directly in Base Excision Repair (BER) when assayed under in vitro conditions. Our present data indicate that this involvement is independent of the transcriptional activity of the p53 molecule. We found that under both in vitro and in vivo conditions, a p53 transactivation-deficient molecule, p53-22-23 was more efficient in BER activity than was wild type p53. However, mutations in the core domain or C-terminal alterations strongly reduced p53-mediated BER activity. These results are consistent with the hypothesis that the involvement of p53 in BER activity, a housekeeping DNA repair pathway, is a prompt and immediate one that does not involve the activation of p53 transactivation-dependent mechanisms, but rather concerns with the p53 protein itself. In an endogenous DNA damage status p53 is active in BER pathways as a protein and not as a transcription factor.

Change of the death pathway in senescent human fibroblasts in response to DNA damage is caused by an inability to stabilize p53.

The cellular function of p53 is complex. It is well known that p53 plays a key role in cellular response to DNA damage. Moreover, p53 was implicated in cellular senescence, and it was demonstrated that p53 undergoes modification in senescent cells. However, it is not known how these modifications affect the ability of senescent cells to respond to DNA damage. To address this question, we studied the responses of cultured young and old normal diploid human fibroblasts to a variety of genotoxic stresses. Young fibroblasts were able to undergo p53-dependent and p53-independent apoptosis. In contrast, senescent fibroblasts were unable to undergo p53-dependent apoptosis, whereas p53-independent apoptosis was only slightly reduced. Interestingly, instead of undergoing p53-dependent apoptosis, senescent fibroblasts underwent necrosis. Furthermore, we found that old cells were unable to stabilize p53 in response to DNA damage. Exogenous expression or stabilization of p53 with proteasome inhibitors in old fibroblasts restored their ability to undergo apoptosis. Our results suggest that stabilization of p53 in response to DNA damage is impaired in old fibroblasts, resulting in induction of necrosis. The role of this phenomenon in normal aging and anticancer therapy is discussed.

p53 modulates base excision repair activity in a cell cycle-specific manner after genotoxic stress.

To elucidate the nature of the cross-talk between the p53 protein and the DNA repair machinery, we have investigated the relationship between the two throughout the cell cycle. Base excision repair (BER) was analyzed in cell cycle phase-enriched populations of lymphoid cells expressing wild-type p53. Our study yielded the following novel findings: (a) BER exhibited two distinct peaks of activity, one associated with the G0-G1 checkpoint and the second with the G2-M checkpoint; (b) although the overall BER activity was reduced after exposure of cells to 400R, there was an augmentation of the G0-G1-associated BER activity and a reduction in the G2-M-associated BER activity; and (c) modulations in these patterns of BER after genotoxic stress were found to be p53 regulated. p53 protein levels induced after gamma-irradiation were distributed evenly in the various cell cycle populations (analyzed by the PAb-248 anti-p53 monoclonal antibody). However, both the dephosphorylation of serine 376 of p53 (contained in the PAb-421 epitope) and the specific DNA binding activity, as well as apoptosis, were enhanced toward the G2-M populations. Furthermore, inactivation of wild-type p53, mediated by mutant p53 expression, abolished the alterations in the BER pattern and showed no induction of a G2-M-associated apoptosis after gamma-irradiation. These results suggest that after genotoxic stress, stabilized p53 enhances the G0-G1-associated BER activity, whereas it predominantly reduces BER activity at the G2-M-enriched populations and instead induces apoptosis. After genotoxic stress, p53 functions as a modulator that determines the pattern of BER activity and apoptosis in a cell cycle-specific manner.

Activation of p53 protein by telomeric (TTAGGG)n repeats.

Genome instability is a primary factor leading to the activation of the p53 tumor suppressor protein. Telomeric repeat (TR) sequences are also responsible for genome integrity. By capping the termini of the chromosomes, TRs prevent them undergoing nucleolytic degradation, ligation or chromosome fusion. Interestingly, telomere shortening was suggested to activate p53, which in turn may cause primary cells to senesce. In order to elucidate the nature of a possible cross talk between the two, we introduced into cells TRs of defined length and investigated their effect on p53 activation and subsequent cellular response. We found that the introduction of a TR into cells leads to stabilization of the p53 protein. This stabilization was specific to TRs and was not observed in response to exposure of cells to plasmids containing non-TR sequences. p53 stabilization requires the presence of an intact p53 oligomerization domain. TR-activated p53 exhibited enhanced transcriptional activity. Eventually, TRs induced p53-dependent growth suppression, measured as a reduction in colony formation.

COOH-terminal domain of p53 modulates p53-mediated transcriptional transactivation, cell growth, and apoptosis.

The tumor suppressor protein p53 contributes to the control of cell cycle checkpoints and stress-induced apoptosis and is frequently mutated in many different types of human cancers. The COOH terminus of p53 modulates the transcriptional and apoptotic activities of the protein. Although COOH-terminal mutants of p53 are uncommon, we proposed that these p53 mutants nevertheless contributed to the selective clonal expansion of the cancer cells. Therefore, we analyzed the tumor-derived p53 COOH-terminal domain (CTD) mutants (352D/H, 356G/W, 342-stop, 360-del, and 387-del) functionally. The results have revealed that all mutants have impaired apoptotic activity when compared with wild-type p53. However, some of these mutants still transcriptionally transactivate p21Waf/Cip1 and inhibit cell growth. Interestingly, of the tumor-derived CTD mutants, oligomerization-defective mutant 342-stop was the only one that did not exhibit sequence-specific DNA binding or failed to transactivate p21Waf1/Cip1, Bax, and IGF-BP3 transcriptionally. The failure to inhibit cell growth by this tumor-derived CTD mutant supports the hypothesis that p53 sequence-specific transcriptional transactivity to p21Waf1/Cip1 is correlated with induction of cell cycle arrest and that the p53 transcriptional transactivity requires oligomerization of the p53 protein. These and other data indicate that the CTD of p53 is an important component of p53-mediated apoptosis and cell growth arrest and that inactivation of the apoptotic function, but not the inhibition of growth, is an important step during human tumorigenesis.