Alternatively spliced p53 RNA in transformed and normal cells of different tissue types.

The alternatively spliced RNA species of tumor suppressor gene p53, containing an additional 96 bases derived from intron 10, is present at approximately 25 to 30% the level of regularly spliced p53 RNA in both normal epidermal and carcinoma cells. The presence of this alternatively spliced RNA in 10T1/2 fibroblast cells, mouse liver and testis suggests that this alternative splicing may be universal. The level of alternatively spliced p53 RNA was increased coordinately with that of regularly spliced p53 in 10T1/2 cells in response to epidermal growth factor. Immunoprecipitation analysis of epidermal cells using monoclonal antibodies which recognize different epitopes of p53 suggested that distinct p53 proteins may be translated from both RNA species. Considering previous observations on the potential importance of carboxyl terminal sequences in p53 function, knowledge of the ubiquitous presence of alternatively spliced p53 is important for future studies of p53 function in normal cells and in oncogenesis.     

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.     

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.     

Rescue of K562 cells from MDM2-modulated p53-dependent apoptosis by growth factor-induced differentiation

The wild-type human MDM2 protooncogene was tested for its ability to modulate apoptotic activity of the de novo expressed p53 tumor suppressor gene in K562 cells. We also studied the role of some cytokines in this phenomenon. K562, a human myeloid leukemia cell line, does not express p53 at the mRNA or protein level. In this study, we stably transfected K562 with eukaryotic vectors containing either normal p53 cDNA (pC53-SN3) or mutated p53 (143Val-->Ala) cDNA (pC53-SCX3). Transfectants expressing WT p53 or those expressing mutant p53 are called K562 SN and K562 SM respectively. Many leukemic cell lines undergo apoptosis when de novo WT p53 is expressed alone. In contrast, while the resulting clones (K562 SN and K562 SM) expressed p53, they did not undergo apoptosis. However, when treated with MDM2 mRNA antisense (MDM2 AS) oligodeoxynucleotides (ODNs), K562 SN demonstrated apoptotic features at both molecular and morphological levels. No change was observed when the other clones (K562 and K562 SM) were treated with MDM2 AS. Apoptosis induced in this manner was associated with a relatively small increase in intracellular calcium [Ca2+]i. Cells cultured in medium previously supplemented with recombinant human (rh) interleukin (IL)-3 and rh-erythropoietin (Epo) did not undergo apoptosis. Moreover, K562 SN cells were induced to differentiate. This differentiation was evaluated by measuring hemoglobin (Hb) level in cellular extracted proteins and by analyzing erythroid colony number and morphology. High Hb synthesis was obtained when K562 SN cells were cultured with cytokines (IL-3 + Epo) combined with MDM2 AS. Our results are consistent with the hypothesis that the function of the proto-oncogene MDM2 is to provide a 'feedback' mechanism for the p53-dependent pathway of apoptosis that could be shunted toward differentiation.     

Apoptosis of fetal testicular cells is regulated by both p53-dependent and independent mechanisms

A portion of fetal germ cells undergoes apoptosis in the physiological context, but the molecular mechanisms of their apoptosis are largely unknown. Because p53 tumor suppressor gene product promotes apoptosis in various types of cells, we have investigated the expression of p53 in fetal gonads and examined the influence of loss of p53 function in fetal gonad cells using mice deficient in the p53 gene. We found that the expression of p53 protein in fetal testis was induced after 15.5 dpc (days post coitum), while the expression was not detected in fetal ovary. The number of apoptotic cells found in the seminiferous tubules of fetal testes was not significantly different between p53-deficient and wild-type mice until 16.5 dpc. At 17.5 dpc, however, more apoptotic cells were observed in wild-type testes than in the p53-deficient mice. In contrast, a similar number of apoptotic cells was found in fetal ovaries throughout these developmental stages. These observations indicate that p53 promotes apoptosis of fetal testicular cells after 16.5 dpc.     

p53 and thymic 'death by neglect': thymic epithelial cell-induced apoptosis of CD4+8+ thymocytes is p53-independent

The involvement of the tumor suppressor protein, p53, in thymic epithelial cell-induced apoptosis of CD4+8+ (double positive) thymocytes, was studied in an in vitro model consisting of a thymic epithelial cell line (TEC) and thymocytes. p53 expression was not augmented in double positive (DP) thymocytes upon co-culturing with TEC, although extensive apoptosis was observed. In the same cells, p53 expression was upregulated in response to low ionizing irradiation, which was accompanied with massive apoptosis. Moreover, TEC induced apoptosis in two DP thymomas, derived from p53(-/-) mice, and in a double positive thymoma clone expressing mutant p53. The extent and kinetics of TEC-induced apoptosis was not affected by the status of p53 in the thymocytes tested. We conclude that thymic epithelial cell-induced apoptosis of immature DP thymocytes is p53-independent and apparently, involves a different apoptotic pathway than that triggered by DNA damage.     

A novel p53-inducible apoptogenic gene,PRG3, encodes a homologue of the apoptosis-inducing factor (AIF)

The p53 tumor suppressor protein induces cell cycle arrest or apoptosis in response to cellular stresses. We have identified PRG3 (p53-responsive gene 3), which is induced specifically under p53-dependent apoptotic conditions in human colon cancer cells, and encodes a novel polypeptide of 373 amino acids with a predicted molecular mass of 40.5 kDa. PRG3 has significant homology to bacterial oxidoreductases and the apoptosis-inducing factor, AIF, and the gene was assigned to chromosome 10q21.3-q22.1. Expression of PRG3 was induced by the activation of endogenous p53 and it contains a p53-responsive element. Unlike AIF, PRG3 localizes in the cytoplasm and its ectopic expression induces apoptosis. An amino-terminal deletion mutant of PRG3 that lacks a putative oxidoreductase activity retains its apoptotic activity, suggesting that the oxidoreductase activity is dispensable for the apoptotic function of PRG3. The PRG3 gene is thus a novel p53 target gene in a p53-dependent apoptosis pathway.     

p300 binding by E1A cosegregates with p53 induction but is dispensable for apoptosis.

E1A expression during adenovirus infection induces apoptosis. E1A expression causes accumulation of the p53 tumor suppressor protein, and E1A-induced apoptosis is p53 mediated in primary rodent cells, implying that p53 induction may be linked to apoptosis induction by E1A. Adenoviruses containing mutations in the E1A gene were tested for the ability to trigger both p53 accumulation and the appearance of enhanced cytopathy (cyt phenotype) and degradation of DNA (deg phenotype), indicative of apoptosis in infected HeLa cells. The adenoviruses had mutations which disrupted the pRb- and/or p300-binding activities of E1A so that the relationship between p53 induction and apoptosis and binding to these cellular proteins by E1A could be determined. An E1A mutation that specifically disrupted the p300-binding activity failed to induce p53 accumulation, whereas mutations in E1A which affected pRb binding induced p53 accumulation. Thus, p300 binding was required and pRb binding was dispensable for E1A-mediated accumulation of p53 in HeLa cells. All the E1A mutant viruses, regardless of the ability to induce p53 accumulation, induced the cyt and deg phenotypes, suggesting that p53 induction in infected HeLa cells was not essential for apoptosis, nor was binding of E1A to the pRb and/or p300 protein. The possibility that E1A induced a p53-independent apoptosis pathway was tested by analyzing the appearance of the cyt and deg phenotypes in Saos-2 cells, which were null for both alleles of p53, upon adenovirus infection. An adenovirus expressing wild-type 12S E1A induced both the cyt and deg phenotypes in Saos-2 cells, as did all the E1A mutant viruses. Thus, E1A expression during infection of human cells may trigger redundant p53-independent and -dependent apoptotic pathways.     

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.     

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.     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53-/- cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53- dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.