Cytokines inhibit p53-mediated apoptosis but not p53-mediated G1 arrest.

Murine erythroleukemia cells that lack endogenous p53 expression were transfected with a temperature-sensitive p53 allele. The temperature-sensitive p53 protein behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. Three independent clones expressing the temperature-sensitive p53 protein were characterized with respect to p53-mediated G1 cell cycle arrest, apoptosis, and differentiation. Clone ts5.203 responded to p53 activation at 32 degrees C by undergoing G1 arrest, apoptosis, and differentiation. Apoptosis was seen in cells representative of all phases of the cell cycle and was not restricted to cells arrested in G1. The addition of a cytokine (erythropoietin, c-kit ligand, or interleukin-3) to the culture medium of ts5.203 cells blocked p53-mediated apoptosis and differentiation but not p53-mediated G1 arrest. These observations indicate that apoptosis and G1 arrest can be effectively uncoupled through the action of cytokines acting as survival factors and are consistent with the idea that apoptosis and G1 arrest represent separate functions of p53. Clones ts15.15 and tsCB3.4 responded to p53 activation at 32 degrees C by undergoing G1 arrest but not apoptosis. We demonstrate that tsCB3.4 secretes a factor with erythropoietin-like activity and that ts15.15 secretes a factor with interleukin-3 activity and suggest that autocrine secretion of these cytokines blocks p53-mediated apoptosis. These data provide a framework in which to understand the variable responses of cells to p53 overexpression.     

Changes in Cyclins and Cyclin-Dependent Kinases Induced by DNA Damaging Agents in a Human Ovarian Cancer Cell Line Expressing Mutated or Wild-Type P53

The expression of cyclins, cyclin-dependent kinases (cdk), and cdk inhibitors was evaluated in clones from a human ovarian cancer cell line transfected with a temperature-sensitive mutant of p53, after treatment with the anticancer agents doxorubicin (DX) and AMSA. The two drugs were selected on the basis of their activity in these clones, since AMSA is equally active in cells expressing mutated or wild-type (wt) p53, while DX was much less cytotoxic in cells expressing wt p53. In untreated cells, the expression of wt p53 induced an accumulation of cells in the G2 and perhaps also the G1 phase of the cell cycle. Concomitantly cyclin B1 and cdc2 increased. Cyclin E and particularly D1 levels were also raised by wt p53 expression. Treatment of mutated p53-expressing cells (SK23a cells kept at 37°C) with DX or, more so, with AMSA, resulted in a strong accumulation of cyclin B1 and cdc2, in accordance with their ability to block cells in G2 phase of the cell cycle. Wt p53-expressing cells (SK23a cells kept at 32°C) treated with the drugs showed an increase in p21 expression and consequently decreased kinase activity after immunoprecipitation with p21 antibodies. Cdc2-associated kinase activity was also reduced in these conditions. We could also observe a decrease in the percentage of cells in G1 and G2 phases and an accumulation of cells in S phase after both DX and AMSA. Cdk2, retinoblastoma, and p27 levels did not change significantly. Treatment with DX or AMSA caused similar effects, suggesting that p53-induced changes in cyclin, cdk, and cdk inhibitors after DNA damage are not responsible for the marked reduction in the cytotoxicity of DX we observed in wt p53-expressing cells.     

p53‐mediated regulation of cell cycle progression: Pronounced impact of cellular microenvironment

Data on the biological effects of some overexpressed oncogenes and their cooperation with cellular factors are, at least partially, contradictory. There are reports on the strong pro‐apoptotic action of temperature‐sensitive (ts) p53^135val in transformed cells at permissive temperature. However, in our experience very high levels of p53^135val induce in transformed rat cells at permissive temperature cell cycle arrest but not apoptosis. Comparison of the experimental protocols reveals that cells used for transfection strongly differ. Therefore, we decided to explore the impact of primary cells used for generation of cell clones on the biological effects evoked by p53 and c‐Ha‐Ras. In the present study, we used primary rat cells (RECs) isolated from rat embryos of different age: at 13.5 gd (y) and 15.5 gd (o). We immortalized rat cells using ts p53^135val mutant and additionally generated transformed cells after co‐transfection with oncogenic Ras. The RECs were transfected with a constitutively activated Ha‐Ras protein, a mutation that is found in a wide variety of human tumors. The ts p53^135Val mutant, switching between wild‐type (wt) and mutant conformation, offers the possibility to study the escape from p53‐mediated cell cycle control in a model of malignant transformation in cells with the same genetic background. Surprisingly, the kinetics of cell proliferation at non‐permissive temperature and that of cell cycle arrest at 32°C strongly differed between cell clones established from yRECs and oRECs, thereby indicating that overexpression of genes such as ts p53^135Val mutant and oncogenic‐Ha‐Ras does not fully override the intrinsic cellular program. J. Cell. Physiol. 219: 459–469, 2009. © 2009 Wiley‐Liss, Inc.     

A role for Hsc70 in regulating nucleocytoplasmic transport of a temperature-sensitive p53 (p53Val-135)

Mouse temperature-sensitive p53(Val-135) accumulates in the nucleus and acts as a "wild-type" at 32 degrees C while it is sequestered in the cytoplasm at 37 degrees C. The cytoplasmic p53(Val-135) relocalized into the nucleus upon inhibition of the nuclear export at 37 degrees C, whereas a mutation in a major bipartite nuclear localization signal (NLS) caused constitutive cytoplasmic localization, indicating that it shuttled between the cytoplasm and the nucleus by its own nuclear export signal and NLS rather than tethered to cytoplasmic structures. Although the full-length p53(Val-135) did not bind the import receptor at 37 degrees C, a C-terminally truncated p53(Val-135) lacking residues 326-390 did bind it. Molecular chaperones such as Hsc70 were associated with p53(Val-135) at 37 degrees C but not at 32 degrees C. When the nuclear export was blocked by leptomycin B, only a fraction lacking Hsc70 was specifically accumulated in the nucleus. Immunodepletion of Hsc70 from the reticulocyte lysate caused p53(Val-135) to bind the import receptor. This binding was blocked by supplying the cell extract containing Hsc70 but not by the addition of recombinant Hsc70 alone. We suggest that the association with the Hsc70-containing complex prevents the NLS from the access of the import receptor through the C-terminal region of p53(Val-135) at 37 degrees C, whereas its dissociation at 32 degrees C allows rapid nuclear import.     

Temperature-dependent switching between "wild-type" and "mutant" forms of p53-Val135

The p53 gene is a suppressor of abnormal cell growth but is also subject to oncogenic activation by mutation. The mutant allele p53-Val135, has recently been discovered to be temperature-sensitive and functions as an oncogene at 37 degrees C and as a tumor suppressor at 32.5 degrees C. In order to investigate the molecular mechanism underlying the temperature sensitivity of p53-Val135 rabbit reticulocyte lysate was used to translate the p53 mRNAs in vitro at 37 degrees C and at 30 degrees C. The immunoreactivity and T antigen binding of wild-type protein p53-Ala135 were unaffected by temperature and were similar to wild-type p53 expressed in vivo. In contrast, the mutant p53-Val135 protein was markedly affected by temperature. At 37 degrees C p53-Val135 showed reduced T antigen binding and did not react with monoclonal antibodies PAb246 and PAb1620. At 30 degrees C, p53-Val135 behaved as the wild-type p53. Temperature also exerted a post-translational effect on p53-Val135 with complete conversion from wild-type to mutant phenotype within two minutes of temperature shift from 30 degrees C to 37 degrees C. There was incomplete conversion from mutant to wild-type phenotype when the temperature was shifted down from 37 degrees C to 30 degrees C. We propose that the temperature dependent forms of p53-Val135 represent conformational variants of the p53 protein with opposing functions in cell growth control.     

Growth suppression of Friend virus-transformed erythroleukemia cells by p53 protein is accompanied by hemoglobin production and is sensitive to erythropoietin.

The murine allele temperature-sensitive (ts) p53Val-135 encodes a ts p53 protein that behaves as a mutant polypeptide at 37 degrees C and as a wild-type polypeptide at 32 degrees C. This ts allele was introduced into the p53 nonproducer Friend erythroleukemia cell line DP16-1. The DP16-1 cell line was derived from the spleen cells of a mouse infected with the polycythemia strain of Friend virus, and like other erythroleukemia cell lines transformed by this virus, it grows independently of erythropoietin, likely because of expression of the viral gp55 protein which binds to and activates the erythropoietin receptor. When incubated at 32 degrees C, DP16-1 cells expressing ts p53Val-135 protein, arrested in the G0/G1 phase of the cell cycle, rapidly lost viability and expressed hemoglobin, a marker of erythroid differentiation. Erythropoietin had a striking effect on p53Val-135-expressing cells at 32 degrees C by prolonging their survival and diminishing the extent of hemoglobin production. This response to erythropoietin was not accompanied by down-regulation of viral gp55 protein.     

CDC37 is required for p60v-src activity in yeast.

Mutations in genes encoding the molecular chaperones Hsp90 and Ydj1p suppress the toxicity of the protein tyrosine kinase p60v-src in yeast by reducing its levels or its kinase activity. We describe isolation and characterization of novel p60v-src-resistant, temperature-sensitive cdc37 mutants, cdc37-34 and cdc37-17, which produce less p60v-src than the parental wild-type strain at 23 degrees C. However, p60v-src levels are not low enough to account for the resistance of these strains. Asynchronously growing cdc37-34 and cdc37-17 mutants arrest in G1 and G2/M when shifted from permissive temperatures (23 degrees C) to the restrictive temperature (37 degrees C), but hydroxyurea-synchronized cdc37-34 and cdc37-17 mutants arrest in G2/M when released from the hydroxyurea block and shifted from 23 to 37 degrees C. The previously described temperature-sensitive cdc37-1 mutant is p60v-src-sensitive and produces wild-type amounts of p60v-src at permissive temperatures but becomes p60v-src-resistant at its restrictive temperature, 38 degrees C. In all three cdc37 mutants, inactivation of Cdc37p by incubation at 38 degrees C reduces p60v-src-dependent tyrosine phosphorylation of yeast proteins to low or undetectable levels. Also, p60v-src levels are enriched in urea-solubilized extracts and depleted in detergent-solubilized extracts of all three cdc37 mutants prepared from cells incubated at the restrictive temperature. These results suggest that Cdc37p is required for maintenance of p60v-src in a soluble, biologically active form.     

Overexpressed poly(ADP-ribose) polymerase delays the release of rat cells from p53-mediated G(1) checkpoint

We have previously reported that in cells ectopically expressing temperature-sensitive p53(135val) mutant, p53 formed tight complexes with poly(ADP-ribose) polymerase (PARP). At elevated temperatures, p53(135val) protein, adopting the mutant phenotype, was localized in the cytoplasm and sequestered the endogenous PARP. To prove whether an excess of p53(135val) protein led to this unusual intracellular distribution of PARP, we have established cell lines overexpressing p53(135val) + c-Ha-ras alone or in combination with PARP. Interestingly, immunostaining revealed that PARP is sequestered in the cytoplasm by mutant p53 in cells overexpressing both proteins. Simultaneous overexpression of PARP had no effect on temperature-dependent cell proliferation and only negligibly affected the kinetics of p53-mediated G(1) arrest. However, if the cells were completely growth arrested at 32 degrees C and then shifted up to 37 degrees C, coexpressed PARP dramatically delayed the reentry of transformed cells into the cell cycle. Even after 72 h at 37 degrees C the proportion of S-phase cells was reduced to 20% compared to those expressing only p53(135val) + c-Ha-ras. The coexpressed PARP stabilized wt p53 protein and its enzymatic activity was necessary for stabilization.     

Enhanced binding of a 95 kDa protein to p53 in cells undergoing p53-mediated growth arrest.

To explore the biochemical functions of p53, we have initiated a search for cellular p53-binding proteins. Coprecipitation of three polypeptides was observed when cell lines overexpressing a temperature-sensitive (ts) p53 mutant were maintained at 32.5 degrees C (wild-type p53 activity, leading to growth arrest) but not at 37.5 degrees C (mutant p53 activity). One of these three proteins, designated p95 on the basis of its apparent molecular mass, was highly abundant in p53 immune complexes. We demonstrate herein that p95 is a p53-binding protein, which exhibits poor p53-binding in cells overproducing several distinct mutant p53 proteins. Yet, p95 associates equally well with both the wild-type (wt) and the mutant conformations of the ts p53 in transformed cells growth-arrested at 32.5 degrees C. On the basis of our findings we suggest that wt p53 activity increases p53-p95 complex formation and that such interaction may play a central role in p53 mediated tumour suppression.     

Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53

Mutant p53 can contribute to transformation, while wild-type (wt) p53 is not oncogenic and actually inhibits transformation. Furthermore, wt p53 may act as a suppressor gene in human carcinogenesis. We now describe the temperature-sensitive behavior of a particular mutant, p53val135. Like other p53 mutants, it can elicit transformation at 37.5 degrees C. However, at 32.5 degrees C it suppresses transformation, behaving like authentic wt p53. Moreover, the proliferation of transformed cells expressing p53val135 is dramatically inhibited at the permissive temperature. Significantly, the inhibition of both transformation and proliferation is reversible upon temperature upshift. These data demonstrate that the ability of wt p53 to suppress transformation is not due to a general lethal effect, but rather to a reversible growth arrest. p53val135 may prove instrumental for gaining insight into the cellular and molecular properties of wt p53.     

Identification of Pex5pM, and retarded maturation of 3-ketoacyl-CoA thiolase and acyl-CoA oxidase in CHO cells expressing mutant Pex5p isoforms

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem. Biophys. Res. Commun. 288, 321-327]. While analyzing the mutant cells, we found a novel Pex5p isoform (Pex5pM), which was shorter by seven amino acids than Pex5pL and longer by 30 amino acids than Pex5pS. Similar levels of mRNA syntheses for the PEX5 gene were observed in both the wild type and mutant cells, but the protein levels of Pex5p isoforms were markedly reduced in the mutant cells cultured at 37 degrees C and only slightly discernible at 30 degrees C, suggesting that they could be rapidly degraded. Furthermore, we characterized the peroxisomal localization of thiolase and acyl-CoA oxidase (Aox) in SK32 cells. The proteins in the organelle fraction were protected from proteinase K-digestion in the mutant cells, indicating that they were translocated inside peroxisomes. However, the conversion of Aox from component A to components B and C was completely prevented at both 30 and 37 degrees C, and the precursor form of thiolase was partially processed to the mature one in a temperature-sensitive manner. Transformed SK32 cells stably expressing one of the wild type Pex5p isoforms were isolated, and then the maturation steps for thiolase and Aox were examined. Pex5pM and S restored the processing of the two enzymes, but Pex5pL did not. In addition, Pex5pL prevented the maturation of thiolase observed at 30 degrees C. These results indicate that (i) the novel Pex5pM is functional and (ii) a seven amino acids-insertion, which is present in the L isoform but absent in the M isoform, plays some role in the process of maturation of thiolase and Aox.     

Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein.

We investigated the interaction between poly(ADP-ribose) polymerase-1 (PARP-1) and the product of the tumor suppressor gene p53 using two different approaches. In the first approach, we used primary and immortalized cells derived from wt and PARP-1 -/- mice. We examined whether PARP-1 deficiency would affect the expression of the wild-type (wt) p53 protein. The inactivation of the PARP-1 gene markedly affected the constitutive expression of the wt p53 protein. Interestingly, only the regularly spliced form of wt p53 was reduced to a barely detectable level in consequence to an approximately 8-fold shortening of its half-life, whereas the level of alternatively spliced p53 remained unchanged. Moreover, reconstitution of cells lacking the PARP-1 gene with the human counterpart restored the normal stability of the regularly spliced p53 protein. In the second approach, we performed experiments with c-Ha-ras transformed primary rat cells overexpressing the p53135val mutant alone or in combination with PARP-1. The advantage of this temperature sensitive p53135val mutant is its oncogenic character at 37 degrees C, connected with cytoplasmic localization of p53, and its tumor suppressor activity at 32 degrees C, accompanied by p53 translocation into the nucleus. No noticeable differences in proliferation and G1 accumulationwere observed between cells expressing p53135val with or without PARP-1. On the other hand, a comparison of the recovery of G1 arrested cells after a shift up to 37 degrees C for both cell lines showed dramatic differences in the kinetics. While cells expressing p53135val rapidly reached the characteristic S-phase level after a shift up to basal temperature, cells additionally expressing PARP-1 rested in G1 despite the temperature elevation. This coincided with exclusively cytoplasmic p53 protein in cells expressing p53135val and predominantly nuclear localization of p53 in p53135val +PARP-1 cells, as evidenced by immunostaining. Determination of the p53 level during the maintenance of cells at 32 degrees C revealed a marked decrease in the level of p53 in cells expressing p53135val alone, whereas in cells coexpressing PARP-1, the level of p53 remained largely unaffected. This indicates that the stability of wild-type p53 greatly differed between both cell lines. Furthermore, the inhibition of PARP-1 activity in G1 arrested cells by 3-aminobenzamide abolished its stabilizing effect on the wild-type p53 protein. Taken together, our results indicate that PARP-1 regulates the stability of the wt p53 protein and that its enzymatic activity is necessary for this stabilizing action.     

Modulation of caspase activation and p27(Kip1) degradation in the p53-induced apoptosis in IW32 erythroleukemia cells

To examine the p53-mediated biological activities and signalling pathways, we generated stable transfectants of the p53-null IW32 murine erythroleukemia cells expressing the temperature-sensitive p53 mutant DNA, tsp53(val135). Two clones with different levels of p53 protein expression were selected for further characterization. At permissive temperature, clone 1-5 cells differentiated along the erythroid pathway, and clone 3-2 cells that produced greater levels (3.5-fold) of p53 underwent apoptosis. Apoptosis of 3-2 cells was accompanied by mitochondrial cytochrome c release and caspase activation as well as by cleavage of caspase substrates. Bax protein was induced to a similar extent in these clones by wild-type p53; expression of p21(Cip1/Waf1) and p27(Kip1) proteins was also increased. However, significantly lesser extent of induction for both CDK inhibitors was detected in the apoptotic 3-2 clone. The general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD.fmk) blocked the p53-induced apoptosis in 3-2 cells, with a concomitant elevation of p27(Kip1), suggesting that p27(Kip1) protein underwent caspase-dependent proteolysis in the apoptotic 3-2 cells. Together these results linked a pathway involving cytochrome c release, caspase activation and p27(Kip1) degradation to the p53-induced apoptosis in IW32 erythroleukemia cells.     

Effects of exogenous wild-type p53 on a human lung carcinoma cell line with endogenous wild-type p53

Several studies have shown that expression of exogenous wild-type p53 is detrimental to the growth of cell lines with absent or mutant p53. In this study, wild-type p53 cDNA expression plasmids were transfected into A549 lung carcinoma cells which had previously been shown by sequencing to contain wild-type p53. When a constitutively expressed wild-type p53 plasmid containing the neomycin resistance gene was transfected into these cells, no G418-resistant colonies contained the exogenous p53 cDNA even though the neomycin resistance gene was integrated. When cells were transfected with a dexamethasone-inducible wild-type p53 cDNA expression plasmid, induction of p53 expression resulted in a decreased growth rate and a decreased proportion of S-phase cells. Continuous treatment with dexamethasone resulted in continued p53 expression for 16 days, but beyond that time expression ceased and could not be reinduced. These data indicated that although the A549 cell line could proliferate in the presence of endogenous wild-type p53 there was a strong selection pressure against continued expression of additional exogenous wild-type p53.     

Cell cycle analysis of p53-induced cell death in murine erythroleukemia cells.

A temperature-sensitive mutant of murine p53 (p53Val-135) was transfected by electroporation into murine erythroleukemia cells (DP16-1) lacking endogenous expression of p53. While the transfected cells grew normally in the presence of mutant p53 (37.5 degrees C), wild-type p53 (32.5 degrees C) was associated with a rapid loss of cell viability. Genomic DNA extracted at 32.5 degrees C was seen to be fragmented into a characteristic ladder consistent with cell death due to apoptosis. Following synchronization by density arrest, transfected cells released into G1 at 32.5 degrees C were found to lose viability more rapidly than did randomly growing cultures. Following release into G1, cells became irreversibly committed to cell death after 4 h at 32.5 degrees C. Commitment to cell death correlated with the first appearance of fragmented DNA. Synchronized cells allowed to pass out of G1 prior to being placed at 32.5 degrees C continued to cycle until subsequently arrested in G1; loss of viability occurred following G1 arrest. In contrast to cells in G1, cells cultured at 32.5 degrees C for prolonged periods during S phase and G2/M, and then returned to 37.5 degrees C, did not become committed to cell death. G1 arrest at 37.5 degrees C, utilizing either mimosine or isoleucine deprivation, does not lead to rapid cell death. Upon transfer to 32.5 degrees C, these G1 synchronized cell populations quickly lost viability. Cells that were kept density arrested at 32.5 degrees C (G0) lost viability at a much slower rate than did cells released into G1. Taken together, these results indicate that wild-type p53 induces cell death in murine erythroleukemia cells and that this effect occurs predominantly in the G1 phase of actively cycling cells.     

Kinetic instability of p53 core domain mutants: implications for rescue by small molecules

Oncogenic mutations in the tumor suppressor protein p53 are found mainly in its DNA-binding core domain. Many of these mutants are thermodynamically unstable at body temperature. Here we show that these mutants also denature within minutes at 37 degrees C. The half-life (t(1/2)) of the unfolding of wild-type p53 core domain was 9 min. Hot spot mutants denatured more rapidly with increasing thermodynamic instability. The highly destabilized mutant I195T had a t(1/2) of less than 1 min. The wild-type p53-(94-360) construct, containing the core and tetramerization domains, was more stable, with t(1/2) = 37 min at 37 degrees C, similar to full-length p53. After unfolding, the denatured proteins aggregated, the rate increasing with higher concentrations of protein. A derivative of the p53-stabilizing peptide CDB3 significantly slowed down the unfolding rate of the p53 core domain. Drugs such as CDB3, which rescue the conformation of unstable mutants of p53, have to act during or immediately after biosynthesis. They should maintain the mutant protein in a folded conformation and prevent its aggregation, allowing it enough time to reach the nucleus and bind its sequence-specific target DNA or the p53 binding proteins that will stabilize it.     

Hsp90 is essential for restoring cellular functions of temperature-sensitive p53 mutant protein but not for stabilization and activation of wild-type p53: implications for cancer therapy

Several signaling pathways that monitor the dynamic state of the cell converge on the tumor suppressor p53. The ability of p53 to process these signals and exert a dynamic downstream response in the form of cell cycle arrest and/or apoptosis is crucial for preventing tumor development. This p53 function is abrogated by p53 gene mutations leading to alteration of protein conformation. Hsp90 has been implicated in regulating both wild-type and mutant p53 conformations, and Hsp90 antagonists are effective for the therapy of some human tumors. Using cell lines that contain human tumor-derived temperature-sensitive p53 mutants we show that Hsp90 is required for both stabilization and reactivation of mutated p53 at the permissive temperature. A temperature decrease to 32 degrees C causes conversion to a protein conformation that is capable of inducing expression of MDM2, leading to reduction of reactivated p53 levels by negative feedback. Mutant reactivation is enhanced by simultaneous treatment with agents that stabilize the reactivated protein and is blocked by geldanamycin, a specific inhibitor of Hsp90 activity, indicating that Hsp90 antagonist therapy and therapies that act to reactivate mutant p53 will be incompatible. In contrast, Hsp90 is not required for maintaining wild-type p53 or for stabilizing wild-type p53 after treatment with chemotherapeutic agents, indicating that Hsp90 therapy might synergize with conventional therapies in patients with wild-type p53. Our data demonstrate the importance of the precise characterization of the interaction between p53 mutants and stress proteins, which may shed valuable information for fighting cancer via the p53 tumor suppressor pathway.