Upregulation of survivin during immortalization of nontransformed human fibroblasts transduced with telomerase reverse transcriptase.

These investigations demonstrate that expression of the inhibitor of apoptosis family member, survivin, is dramatically increased during immortalization of nontransformed human fibroblasts that were transduced with telomerase reverse transcriptase (hTERT). Expression of survivin in immortalized fibroblasts peaked during G(2)/M phase of the cell cycle. However, the upregulation of survivin was dissociated from the rate of proliferation and proportion of G(2)/M cells. Depletion of survivin from immortal fibroblasts increased sensitivity to stress-induced apoptosis and resulted in an accumulation of cells with 4N DNA content. Conversely, overexpression of survivin in mortal fibroblasts conferred resistance to apoptosis. In contrast, very low levels of survivin in proliferating parental fibroblasts had no bearing on sensitivity to apoptosis. The upregulation of survivin did not appear to be a direct consequence of hTERT transduction. However, repression of hTERT resulted in the rapid downregulation of survivin in telomerase-immortalized fibroblasts and tumor cell lines, but not in cells immortalized via an Alternative Lengthening of Telomeres mechanism. These results have important therapeutic implications, as telomerase and survivin are both broadly expressed in human cancers. Selection during the immortalization process for cells expressing high levels of survivin may account for the abundance of survivin in diverse tumor types.

Amyloid-beta precursor-like protein APLP1 is a novel p53 transcriptional target gene that augments neuroblastoma cell death upon genotoxic stress.

The tumor suppressor p53 is a key modulator of the cellular stress response, inducing cell-cycle arrest, apoptosis, senescence and cell differentiation. To evaluate further the molecular mechanism underlying p53 function, the transcriptional profiles of proliferating and senescent WI-38 cells, both wild-type p53 expressers and counterparts with an inactivated p53, were compared by DNA microarray analysis. In particular, the amyloid-beta precursor-like protein 1 (APLP1) is induced in senescent cells in a p53-dependent manner. APLP1 was confirmed to be a novel transcriptional target of p53 by in vivo and in vitro characterization of a p53 responsive element found in the first intron of the APLP1 gene locus. APLP1 knockdown experiments demonstrate that APLP1 is required for the proliferation of fibroblastic and epithelial cells. Moreover, depletion of APLP1 expression diminishes stress-induced apoptosis of neural cells, whereas ectopic APLP1 expression augments apoptosis. Based on these data, a mechanism is proposed whereby p53-dependent induction of APLP1 is involved in neural cell death, and which may exacerbate neuronal cell loss in some acute or chronic neurodegenerative disorders.

p53 mediates density-dependent growth arrest.

While the stress-response-associated importance of the p53 tumor suppressor is well established, recent studies have also linked p53 with several basic parameters in the normal behavior of cells. Here, we present evidence that basal p53 expression in WI38 human embryonic lung fibroblasts restricts growth rate and mediates density-dependent inhibition of growth and the associated G1 phase arrest of the cell cycle by affecting the density-dependent regulation of p16/INK4a. Additionally, we show that prolonged culturing of hTert-immortalized WI38 cells leads to a loss of density-dependent growth inhibition that correlates with p27/KIP deregulation as well as the previously shown INK4a locus silencing, and to an onset of contact-induced, p53-dependent cell death.

p53 is a regulator of macrophage differentiation.

While it is well accepted that p53 plays a role in apoptosis, less is known as to its involvement in cell differentiation. Here we show that wild-type p53 facilitates IL-6-dependent macrophage differentiation. Treatment of M1/2 cells expressing the temperature-sensitive p53 143 (Val to Ala) mutant, at the wild-type conformation, facilitated the appearance of mature macrophages that exhibited phagocytic activity. Enhancement of differentiation by the p53 143 (Val to Ala) in the wild-type conformation was coupled with the inhibition of apoptosis induction by this protein. In agreement with previous studies, we found that p53 levels were reduced during p53-dependent macrophage differentiation. This occurred when p53 levels before IL-6 stimuli were high. Interestingly, the p53 143 (Val to Ala) protein, at the mutant conformation, enhanced macrophage differentiation, as did the wild-type conformation, whereas the p53 273 (Arg to His) core mutant exerted an inhibitory effect on this pathway. The transcription-deficient p53 molecules, p53 (22-23) and p53 22,23,143, could not induce p53-dependent differentiation. Moreover, the p53 (22-23) protein inhibited the p53-independent differentiation pathway. Interestingly, the p53 (22-23) protein not only blocked IL-6-mediated differentiation, but also induced significant apoptotic cell death, upon IL-6 stimulation. Taken together, our data show that wild-type p53 enhances macrophage differentiation, while various p53 mutant types exert different effects on this differentiation pathway.

Integrity of the N-terminal transcription domain of p53 is required for mutant p53 interference with drug-induced apoptosis.

The present study examined whether the ability of mutant p53 to block apoptosis depended on its transcriptional activity. A core domain mutant p53 (143 Val to Ala), in which two N-terminal residues (22 and 23) essential for transactivation were also mutated (Leu to Glu and Trp to Ser, respectively), was examined. While p53 containing only the core mutation efficiently interfered with drug-induced apoptosis, further modification at the N-terminus abolished this blocking activity. Furthermore, expression of c-myc, a suggested target for core mutant p53 transactivation, was elevated in the core mutant p53-expressing cells, but was abolished in the presence of the transcription-deficient p53 core mutant. In addition, wild-type p53, mutated in the N-terminus (residues 22 and 23), was unable to induce apoptosis by itself. Nevertheless, it synergized with drugs in the induction of apoptosis. This suggests that the integrity of the N-terminus is essential for both the activity of wild-type p53 in apoptosis and for mutant p53-mediated block of drug-induced apoptosis. This supports the notion that core p53 mutants act via a gain of function mechanism.

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.

The role of the C' terminus of murine p53 in the p53/mdm-2 regulatory loop.

Mdm-2 plays a central role in the regulation of p53 protein level and activity. Although the interaction of mdm-2 and p53 occurs through the N-terminus of the p53 protein, our present data suggest that the C' terminus plays an important role in the regulation of the p53/mdm-2 loop. Comparative analysis of the murine regularly spliced form of p53 (RSp53) and a physiological C-terminally modified p53 protein, which results from alternative splicing of the p53 mRNA (ASp53), indicated that the two isoforms behave differently in the p53/mdm-2 loop. We found that ASp53 can preferentially induce higher levels of the mdm-2 protein, compared with RSp53. Although the transactivation capacity of both forms is inhibited by mdm-2, only RSp53 is directed to proteolytic degradation by mdm-2, while ASp53 is relatively resistant. We present evidence that suggests that ASp53 protein levels determine the biological activities mediated by RSp53, such as the induction of apoptosis, through the mdm-2/p53 regulatory loop. We suggest, therefore, a new mechanism for the regulation of p53, and show that alteration of the p53 extreme C' terminus can significantly change the transcription activity and the resistance to degradation properties of the p53 protein.

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.