Multistage Friend erythroleukemia: independent origin of tumor clones with normal or rearranged p53 cellular oncogenes.

The erythroleukemia induced by Friend virus complex in adult mice is a multistage malignancy characterized by the emergence, late in the disease, of tumorigenic cell clones. We have previously shown that a significant proportion of these clones have unique rearrangements in their cellular p53 oncogene. The clonal relationships among Friend tumor cells isolated in the late stages of Friend erythroleukemia were analyzed by examining the unique integration site of Friend murine leukemia virus and the unique rearrangement in their cellular p53 oncogene. The majority of clones isolated from individual mice infected with Friend virus were clonally related as judged by the site of Friend murine leukemia virus integration. However, Southern gel analysis of DNA from individual Friend cell clones indicated that all of the clones with a normal p53 gene from the same mice were clonally related, but were unrelated to the Friend cell lines with a rearranged p53 gene. These results suggest that Friend tumor cells with rearrangements in their p53 gene arise as the result of a unique transformation event, rather than by progression from already existing tumor cells with a normal p53 gene. They also suggest that such rearrangements in the p53 gene confer a strong selective advantage to these cells in vivo.     

p16(Ink4a) interferes with Abelson virus transformation by enhancing apoptosis

Pre-B-cell transformation by Abelson virus (Ab-MLV) is a multistep process in which primary transformants are stimulated to proliferate but subsequently undergo crisis, a period of erratic growth marked by high levels of apoptosis. Inactivation of the p53 tumor suppressor pathway is an important step in this process and can be accomplished by mutation of p53 or down-modulation of p19(Arf), a p53 regulatory protein. Consistent with these data, pre-B cells from either p53 or Ink4a/Arf null mice bypass crisis. However, the Ink4a/Arf locus encodes both p19(Arf) and a second tumor suppressor, p16(Ink4a), that blocks cell cycle progression by inhibiting Cdk4/6. To determine if p16(Ink4a) plays a role in Ab-MLV transformation, primary transformants derived from Arf(-/-) and p16(Ink4a(-/-)) mice were compared. A fraction of those derived from Arf(-/-) animals underwent crisis, and even though all p16(Ink4a(-/-)) primary transformants experienced crisis, these cells became established more readily than cells derived from +/+ mice. Analyses of Ink4a/Arf(-/-) cells infected with a virus that expresses both v-Abl and p16(Ink4a) revealed that p16(Ink4a) expression does not alter cell cycle profiles but does increase the level of apoptosis in primary transformants. These results indicate that both products of the Ink4a/Arf locus influence Ab-MLV transformation and reveal that in addition to its well-recognized effects on the cell cycle, p16(Ink4a) can suppress transformation by inducing apoptosis.     

Friend leukemia virus infection enhances DNA damage-induced apoptosis of hematopoietic cells,causing lethal anemia in C3H hosts

Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53(-/-)) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53(-/-) --> C3H p53(+/+) bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.     

Attenuation markers of a candidate dengue type 2 vaccine virus, strain 16681 (PDK-53), are defined by mutations in the 5' noncoding region and nonstructural proteins 1 and 3

The genome of a candidate dengue type 2 (DEN-2) vaccine virus, strain PDK-53, differs from its DEN-2 16681 parent by nine nucleotides. Using infectious cDNA clones, we constructed 18 recombinant 16681/PDK-53 viruses to analyze four 16681-to-PDK-53 mutations, including 5' noncoding region (5'NC)-57 C-to-T, premembrane (prM)-29 Asp-to-Val (the only mutation that occurs in the structural proteins), nonstructural protein 1 (NS1)-53 Gly-to-Asp, and NS3-250 Glu-to-Val. The viruses were studied for plaque size, growth rate, and temperature sensitivity in LLC-MK(2) cells, growth rate in C6/36 cells, and neurovirulence in newborn mice. All of the viruses replicated to peak titers of 10(7.3) PFU/ml or greater in LLC-MK(2) cells. The crippled replication of PDK-53 virus in C6/36 cells and its attenuation for mice were determined primarily by the 5'NC-57-T and NS1-53-Asp mutations. The temperature sensitivity of PDK-53 virus was attributed to the NS1-53-Asp and NS3-250-Val mutations. The 5'NC-57, NS1-53, and NS3-250 loci all contributed to the small-plaque phenotype of PDK-53 virus. Reversions at two or three of these loci in PDK-53 virus were required to reconstitute the phenotypic characteristics of the parental 16681 virus. The prM-29 locus had little or no effect on viral phenotype. Sequence analyses showed that PDK-53 virus is genetically identical to PDK-45 virus. Restriction of the three major genetic determinants of attenuation markers to nonstructural genomic regions makes the PDK-53 virus genotype attractive for the development of chimeric DEN virus vaccine candidates.     

Moloney murine leukemia virus-induced lymphomas in p53-deficient mice: overlapping pathways in tumor development?

The effect of Moloney murine leukemia virus (MoMLV) infection was examined in mice lacking a functional p53 gene. Virus-infected p53-/- mice developed tumors significantly faster than uninfected p53-/- or virus-infected p53+/+ littermates. However, the degree of synergy between MoMLV and the p53 null genotype was weaker than the synergy between either of these and c-myc transgenes. A similar range of T-cell tumor phenotypes was represented in all p53 genotype groups, including p53-/- mice, which developed thymic lymphomas as the most common of several neoplastic diseases. Lack of p53 was associated with higher rates of metastasis and the ready establishment of tumors in tissue culture. Loss of the wild-type allele was a common feature of tumors in p53+/- mice and was complete in tumor cells in vitro, but this appeared to occur by a mechanism other than proviral insertion at the wild-type allele. A lower average MoMLV proviral copy number was observed in tumors of the p53 null and heterozygote groups, suggesting that the absence of a functional p53 gene reduced the number of steps required to complete the malignant phenotype. Mink cell focus-forming virus-like proviruses were detected in tumors of all infected mice but were relatively rare in p53 null mice. Analysis of c-myc, pim-1, and pal-1 showed that these loci were occupied by proviruses in some cases but at similar frequencies in p53 wild-type and null mice. In conclusion, while inactivation of p53 in the germ line predisposes mice to tumors similar in phenotype to those induced by MoMLV, it appears that virus-induced tumors generally occur without p53 loss. We speculate that a bcl-2-like function carried or induced by MoMLV may underlie this p53-independent pathway.     

The ability of large T antigen to complex with p53 is necessary for the increased life span and partial transformation of human cells by simian virus 40.

Simian virus 40 (SV40) T antigen binds to the tumor suppressor p53 protein, and this association may contribute to oncogenic transformation by the virus. We investigated the importance of this binding on transformation by examining three replication-competent mutants of SV40 (402DE, 402DN, and 402DH). These mutants express T antigens defective in binding to human and monkey p53s but retain some binding with mouse p53. All showed significant reduction in their ability to induce transformed cell foci of two normal human cell lines as well as a slight reduction with mouse embryo cells. Other comparable mutants which express T antigens retaining the ability to complex with p53 were able to induce foci at wild-type levels in both human and mouse cells. Further studies were performed with five T-antigen-positive clones isolated from the few human cell foci that appeared after transfection with 402 mutant DNAs. All five clones reached senescence at about the same point as did the parental untransformed cells. However, six other human cell clones obtained after transfection with DNA from nondefective mutants or wild-type virus were still growing well at more than 10 passages beyond their expected life span. These results suggest that the ability of T antigen to form stable complexes with p53 is necessary for SV40 to extend the life span and partially transform human cells in culture.     

High expression of MDM2 protein and low rate of p21(WAF1/CIP1) expression in SCID mice Epstein Barr virus-induced lymphoproliferation.

To study the prevalence of p53 inactivation and MDM2/p21(WAFI/CIP1) expression in severe combined immunodeficient (SCID) mice Epstein-Barr virus (EBV)-induced lymphoproliferation, 19 samples obtained after ip injection of peripheral blood mononuclear cells (PBMCs) from EBV-seropositive donors or lymphoblastoid cell lines (LCL) were analyzed. In all samples tested, overexpression of Ki-67 antigen was shown by immunohistochemistry, indicating a high proliferative index of SCID mice EBV-induced lymphoproliferation. P53 mutations were screened by functional assay in yeast in 14 samples. With this test, a p53-inactivating mutation was found in only one case; the remaining cases exhibited a wild-type p53 pattern. However, an accumulation of p53 protein was detected by immunohistochemistry in six of 19 samples. P21 expression was found in seven of 19 samples but was not correlated with the rate of p53 protein in tumors. In contrast, high levels of nuclear accumulation of MDM2 were found in all samples by immunohistochemistry. These results suggest that a high Ki-67 proliferative index in SCID mice EBV-induced lymphoproliferation is not due to the inactivation of p53 by mutation, but could be associated with an overexpression of MDM2, which would act by a p53-independent mechanism.(J Histochem Cytochem 47:1315-1321, 1999)     

Increased expression of Bcl-xL and c-Myc is associated with transformation by Abelson murine leukemia virus

Transformation mediated by the v-Abl oncoprotein, a tyrosine kinase encoded by the Abelson murine leukemia virus, is a multi-step process requiring genetic alterations in addition to expression of v-Abl. Loss of p53 or p19ARF was previously shown to be required for Abelson murine leukemia virus transformation of primary mouse embryonic fibroblasts (MEFs). By comparing gene expression patterns in primary p53-/- MEFs acutely infected with the v-Abl retrovirus, v-Abl-transformed MEF clones, and v-Abl-transformed MEF clones treated with Abl kinase inhibitor STI 571, we have identified additional genetic alterations associated with v-Abl transformation. Bcl-xL mRNA was elevated in three of five v-Abl-transformed MEF clones. In addition, elevated expression of c-Myc mRNA, caused either by c-myc gene amplification or by enhanced signaling via STAT3, was observed in five v-Abl-transformed MEF clones. The data suggest that increases in cell survival associated with Bcl-xL and increases in cell growth associated with c-Myc facilitate the transformation process dependent on constitutive mitogenic signaling by v-Abl.     

Loss of poly(ADP-ribose) polymerase-1 causes increased tumour latency in p53-deficient mice.

PARP-1-deficient mice display a severe defect in the base excision repair pathway leading to radiosensitivity and genomic instability. They are protected against necrosis induced by massive oxidative stress in various inflammatory processes. Mice lacking p53 are highly predisposed to malignancy resulting from defective cell cycle checkpoints, resistance to DNA damage-induced apoptosis as well as from upregulation of the iNOS gene resulting in chronic oxidative stress. Here, we report the generation of doubly null mutant mice. We found that tumour-free survival of parp-1(-/-)p53(-/-) mice increased by 50% compared with that of parp- 1(+/+)p53(-/-) mice. Tumour formation in nude mice injected with oncogenic parp-1(-/-)p53(-/-) fibroblasts was significantly delayed compared with parp-1(+/+)p53(-/-) cells. Upon gamma-irradiation, a partial restoration of S-phase radiosensitivity was found in parp-1(-/-)p53(-/-) primary fibroblasts compared with parp-1(+/+)p53(-/-) cells. In addition, iNOS expression and nitrite release were dramatically reduced in the parp-1(-/-)p53(-/-) mice compared with parp-1(+/+)p53(-/-) mice. The abrogation of the oxydated status of p53(-/-) cells, due to the absence of parp-1, may be the cause of the delay in the onset of tumorigenesis in parp-1(-/-)p53(-/-) mice.     

Hyperglycemia-induced apoptotic cell death in the mouse blastocyst is dependent on expression of p53

Murine preimplantation embryos exposed to hyperglycemia experience decreased glucose transport, and overexpression of the proapoptotic protein BAX, leading to increased apoptosis. These changes may account for the increased rates of miscarriages and malformations seen in women with diabetes mellitus. To test whether p53 expression is necessary for hyperglycemia-induced apoptosis, p53+/+, +/-, -/- embryos were obtained by superovulation. Two-cell embryos were cultured to a blastocyst stage in 52 mM D- or L-glucose. Apoptosis was detected using terminal dUTP nick end labeling (TUNEL) assays. In vivo studies were performed in the same manner using blastocysts recovered from streptozotocin-induced diabetic mothers. Both in vitro and in vivo studies showed that wildtype embryos had a significantly higher percentage of TUNEL-positive nuclei than p53+/- and -/- embryos. To test whether p53 is upstream of BAX, immunofluorescent confocal microscopy and immunoprecipitation/ immunoblotting were performed on blastocysts cultured in high vs. control glucose conditions. Blastocysts from p53+/+ mice exhibited increased BAX staining vs. p53+/- and -/- embryos. Next, to determine whether a decrease in glucose transport was upstream or downstream of p53, deoxyglucose transport was measured in individual blastocysts from p53+/+ and +/- diabetic vs. nondiabetic mice. Embryos from diabetic p53+/- mice exhibit a 44% decrease in glucose transport, similar to the 38% decrease seen in embryos from diabetic p53+/+ mice. Taken together, these results strongly indicate that p53 plays a role in hyperglycemia-induced apoptosis, upstream of BAX overexpression and downstream of the decrease in glucose transport experienced by the mouse preimplantation embryo.     

Elevated in vivo frequencies of mutant T cells with altered functional expression of the T-cell receptor or hypoxanthine phosphoribosyltransferase genes in p53-deficient mice

We have studied the effects of a defect in the p53 gene on spontaneous and radiation-induced somatic mutation frequencies in vivo by measuring T-cell receptor (TCR) and hypoxanthine phosphoribosyltransferase (HPRT) mutant frequencies (MFs) in p53 deficient mice both before and after exposure to X-irradiation. In the absence of irradiation, the TCR and HPRT mutant frequencies were roughly two-fold higher in p53 null (-/-) mice than in wild-type (+/+) mice. Unexpectedly, the TCR and HPRT MFs were slightly lower in heterozygote p53 (+/-) than in wild-type (+/+) mice, however. After 2 weeks 2Gy whole body irradiation the TCR and HPRT MFs were about two-fold higher in the p53 null (-/-) and p53 (+/-) mice than in the wild-type. Taken together, these findings suggest that a defect in the p53 gene may lead to TCR and HPRT mutants being recovered at higher frequencies in both irradiated and unirradiated mice, but it should be emphasized that the effects we have observed are not particularly strong, albeit that they are statistically significant. Interestingly, several of the highest TCR MF values that we observed in the course of our experiments were recorded in p53 (-/-) animals that had developed thymomas and hence appeared to be cancer prone.     

Astrocytes derived from p53-deficient mice provide a multistep in vitro model for development of malignant gliomas.

Loss or mutation of p53 is thought to be an early event in the malignant transformation of many human astrocytic tumors. To better understand the role of p53 in their growth and transformation, we developed a model employing cultured neonatal astrocytes derived from mice deficient in one (p53 +/-) or both (p53 -/-) p53 alleles, comparing them with wild-type (p53 +/+) cells. Studies of in vitro and in vivo growth and transformation were performed, and flow cytometry and karyotyping were used to correlate changes in growth with genomic instability. Early-passage (EP) p53 -/- astrocytes achieved higher saturation densities and had more rapid growth than EP p53 +/- and +/+ cells. The EP p53 -/- cells were not transformed, as they were unable to grow in serum-free medium or in nude mice. With continued passaging, p53 -/- cells exhibited a multistep progression to a transformed phenotype. Late-passage p53 -/- cells achieved saturation densities 50 times higher than those of p53 +/+ cells and formed large, well-vascularized tumors in nude mice. p53 +/- astrocytes exhibited early loss of the remaining wild-type p53 allele and then evolved in a manner phenotypically similar to p53 -/- astrocytes. In marked contrast, astrocytes retaining both wild-type p53 alleles never exhibited a transformed phenotype and usually senesced after 7 to 10 passages. Dramatic alterations in ploidy and karyotype occurred and were restricted to cells deficient in wild-type p53 following repeated passaging. The results of these studies suggest that loss of wild-type p53 function promotes genomic instability, accelerated growth, and malignant transformation in astrocytes.     

p53 deficiency increases transformation by v-Abl and rescues the ability of a C-terminally truncated v-Abl mutant to induce pre-B lymphoma in vivo

Abelson murine leukemia virus (A-MuLV) is an acute transforming retrovirus that preferentially transforms early B-lineage cells both in vivo and in vitro. Its transforming protein, v-Abl, is a tyrosine kinase related to v-Src but containing an extended C-terminal domain. Many mutations affecting the C-terminal portion of the molecule block the pre-B-transforming activity of v-Abl without affecting the fibroblast-transforming ability. In this study we have determined the abilities of both wild-type and C-terminally truncated (p90) forms of v-Abl to transform cells from p53(-/-) mice. Lack of p53 increases the susceptibility of bone marrow cells to transformation by v-Abl by a factor of more than 7 but does not alter v-Abl's preference for B220(+) IgM(-) pre-B cells. p53-deficient mice have earlier tumor onset, more rapid tumor progression, and decreased survival time following A-MuLV infection, but all of the tumors are pre-B lymphomas. Thus, p53-dependent pathways inhibit v-Abl transformation but play no role in conferring preferential transformation of pre-B cells. Surprisingly, the C-terminally truncated form of v-Abl (p90) transforms pre-B cells very efficiently in mice lacking p53, thus demonstrating that the C terminus of v-Abl does not determine preB tropism but is necessary to overcome p53-dependent inhibition of transformation.     

Chimeric dengue type 2 (vaccine strain PDK-53)/dengue type 1 virus as a potential candidate dengue type 1 virus vaccine

We constructed chimeric dengue type 2/type 1 (DEN-2/DEN-1) viruses containing the nonstructural genes of DEN-2 16681 virus or its vaccine derivative, strain PDK-53, and the structural genes (encoding capsid protein, premembrane protein, and envelope glycoprotein) of DEN-1 16007 virus or its vaccine derivative, strain PDK-13. We previously reported that attenuation markers of DEN-2 PDK-53 virus were encoded by genetic loci located outside the structural gene region of the PDK-53 virus genome. Chimeric viruses containing the nonstructural genes of DEN-2 PDK-53 virus and the structural genes of the parental DEN-1 16007 virus retained the attenuation markers of small plaque size and temperature sensitivity in LLC-MK(2) cells, less efficient replication in C6/36 cells, and attenuation for mice. These chimeric viruses elicited higher mouse neutralizing antibody titers against DEN-1 virus than did the candidate DEN-1 PDK-13 vaccine virus or chimeric DEN-2/DEN-1 viruses containing the structural genes of the PDK-13 virus. Mutations in the envelope protein of DEN-1 PDK-13 virus affected in vitro phenotype and immunogenicity in mice. The current PDK-13 vaccine is the least efficient of the four Mahidol candidate DEN virus vaccines in human trials. The chimeric DEN-2/DEN-1 virus might be a potential DEN-1 virus vaccine candidate. This study indicated that the infectious clones derived from the candidate DEN-2 PDK-53 vaccine are promising attenuated vectors for development of chimeric flavivirus vaccines.     

Chimeric dengue 2 PDK-53/West Nile NY99 viruses retain the phenotypic attenuation markers of the candidate PDK-53 vaccine virus and protect mice against lethal challenge with West Nile virus

Chimeric dengue serotype 2/West Nile (D2/WN) viruses expressing prM-E of WN NY99 virus in the genetic background of wild-type D2 16681 virus and two candidate D2 PDK-53 vaccine variants (PDK53-E and PDK53-V) were engineered. The viability of the D2/WN viruses required incorporation of the WN virus-specific signal sequence for prM. Introduction of two mutations at M-58 and E-191 in the chimeric cDNA clones further improved the viability of the chimeras constructed in all three D2 carriers. Two D2/WN chimeras (D2/WN-E2 and -V2) engineered in the backbone of the PDK53-E and -V viruses retained all of the PDK-53 vaccine characteristic phenotypic markers of attenuation and were immunogenic in mice and protected mice from a high-dose 10(7) PFU challenge with wild-type WN NY99 virus. This report further supports application of the genetic background of the D2 PDK-53 virus as a carrier for development of live-attenuated, chimeric flavivirus vaccines in general and the development of a chimeric D2/WN vaccine virus against WN disease in particular.     

C/EBPbeta represses p53 to promote cell survival downstream of DNA damage independent of oncogenic Ras and p19(Arf)

CCAAT/enhancer-binding protein-beta (C/EBPbeta) is a mediator of cell survival and tumorigenesis. When C/EBPbeta(-/-) mice are treated with carcinogens that produce oncogenic Ras mutations in keratinocytes, they respond with abnormally elevated keratinocyte apoptosis and a block in skin tumorigenesis. Although this aberrant carcinogen-induced apoptosis results from abnormal upregulation of p53, it is not known whether upregulated p53 results from oncogenic Ras and its ability to induce p19(Arf) and/or activate DNA-damage response pathways or from direct carcinogen-induced DNA damage. We report that p19(Arf) is dramatically elevated in C/EBPbeta(-/-) epidermis and that C/EBPbeta represses a p19(Arf) promoter reporter. To determine whether p19(Arf) is responsible for the proapoptotic phenotype in C/EBPbeta(-/-) mice, C/EBPbeta(-/-);p19(Arf-/-) mice were generated. C/EBPbeta(-/-);p19(Arf-/-) mice responded to carcinogen treatment with increased p53 and apoptosis, indicating p19(Arf) is not essential. To ascertain whether oncogenic Ras activation induces aberrant p53 and apoptosis in C/EBPbeta(-/-) epidermis, we generated K14-ER:Ras;C/EBPbeta(-/-) mice. Oncogenic Ras activation induced by 4-hydroxytamoxifen did not produce increased p53 or apoptosis. Finally, when C/EBPbeta(-/-) mice were treated with differing types of DNA-damaging agents, including alkylating chemotherapeutic agents, they displayed aberrant levels of p53 and apoptosis. These results indicate that C/EBPbeta represses p53 to promote cell survival downstream of DNA damage and suggest that inhibition of C/EBPbeta may be a target for cancer cotherapy to increase the efficacy of alkylating chemotherapeutic agents.     

Ser18 and 23 phosphorylation is required for p53-dependent apoptosis and tumor suppression

Mouse p53 is phosphorylated at Ser18 and Ser23 after DNA damage. To determine whether these two phosphorylation events have synergistic functions in activating p53 responses, we simultaneously introduced Ser18/23 to Ala mutations into the endogenous p53 locus in mice. While partial defects in apoptosis are observed in p53S18A and p53S23A thymocytes exposed to IR, p53-dependent apoptosis is essentially abolished in p53S18/23A thymocytes, indicating that these two events have critical and synergistic roles in activating p53-dependent apoptosis. In addition, p53S18/23A, but not p53S18A, could completely rescue embryonic lethality of Xrcc4(-/-) mice that is caused by massive p53-dependent neuronal apoptosis. However, certain p53-dependent functions, including G1/S checkpoint and cellular senescence, are partially retained in p53(S18/23A) cells. While p53(S18A) mice are not cancer prone, p53S18/23A mice developed a spectrum of malignancies distinct from p53S23A and p53(-/-) mice. Interestingly, Xrcc4(-/-)p53S18/23A mice fail to develop tumors like the pro-B cell lymphomas uniformly developed in Xrcc4(-/-) p53(-/-) animals, but exhibit developmental defects typical of accelerated ageing. Therefore, Ser18 and Ser23 phosphorylation is important for p53-dependent suppression of tumorigenesis in certain physiological context.