Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation

Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation. The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome). We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice. These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53. We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele. Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization. These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis.     

Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein

Cytoplasmic sequestration of the p53 tumor suppresser protein has been proposed as a mechanism involved in abolishing p53 function. However, the mechanisms regulating p53 subcellular localization remain unclear. In this report, we analyzed the possible existence of cis-acting sequences involved in intracellular trafficking of the p53 protein. To study p53 trafficking, the jellyfish green fluorescent protein (GFP) was fused to the wild-type or mutated p53 proteins for fast and sensitive analysis of protein localization in human MCF-7 breast cancer, RKO colon cancer, and SAOS-2 sarcoma cells. The wild-type p53/GFP fusion protein was localized in the cytoplasm, the nucleus, or both compartments in a subset of the cells. Mutagenesis analysis demonstrated that a single amino acid mutation of Lys-305 (mt p53) caused cytoplasmic sequestration of the p53 protein in the MCF-7 and RKO cells, whereas the fusion protein was distributed in both the cytoplasm and the nucleus of SAOS-2 cells. In SAOS-2 cells, the mutant p53 was a less efficient inducer of p21/CIP1/WAF1 expression. Cytoplasmic sequestration of the mt p53 was dependent upon the C-terminal region (residues 326-355) of the protein. These results indicated the involvement of cis-acting sequences in the regulation of p53 subcellular localization. Lys-305 is needed for nuclear import of p53 protein, and amino acid residues 326-355 can sequester mt p53 in the cytoplasm.     

Inactivation of tumor suppressor p53 by mot-2, a hsp70 family member

The mortalin genes, mot-1 and mot-2, are hsp70 family members that were originally cloned from normal and immortal murine cells, respectively. Their proteins differ by only two amino acid residues but exhibit different subcellular localizations, arise from two distinct genes, and have contrasting biological activities. We report here that the two proteins also differ in their interactions with the tumor suppressor protein p53. The pancytosolic mot-1 protein in normal cells did not show colocalization with p53; in contrast, nonpancytosolic mot-2 and p53 overlapped significantly in immortal cells. Transfection of mot-2 but not mot-1 resulted in the repression of p53-mediated transactivation in p53-responsive reporter assays. Inactivation of p53 by mot-2 was supported by the down-regulation of p53-responsive genes p21(WAF-1) and mdm-2 in mot-2-transfected cells only. Furthermore, NIH 3T3 cells transfected with expression plasmid encoding green fluorescent protein-tagged mot-2 but not mot-1 showed an abrogation of nuclear translocation of wild-type p53. These results demonstrate a novel mechanism of p53 inactivation by mot-2 protein.     

p53 activity is essential for normal development in Xenopus

BACKGROUND: The tumor suppressor p53 plays a key role in regulating the cell cycle and apoptosis in differentiated cells. Mutant mice lacking functional p53 develop normally but die from multiple neoplasms shortly after birth. There have been hints that p53 is involved in morphogenesis, but given the relatively normal development of p53 null mice, the significance of these data has been difficult to evaluate. To examine the role of p53 in vertebrate development, we have determined the results of blocking its activity in embryos of the frog Xenopus laevis. RESULTS: Two different methods have been used to block p53 protein activity in developing Xenopus embryos--ectopic expression of dominant-negative forms of human p53 and ectopic expression of the p53 negative regulator, Xenopus dm-2. In both instances, inhibition of p53 activity blocked the ability of Xenopus early blastomeres to undergo differentiation and resulted in the formation of large cellular masses reminiscent of tumors. The ability of mutant p53 to induce such developmental tumors was suppressed by co-injection with wild-type human or wild-type Xenopus p53. Cells expressing mutant p53 activated zygotic gene expression and underwent the mid-blastula transition normally. Such cells continued to divide at approximately normal rates but did not form normal embryonic tissues and never underwent terminal differentiation, remaining as large, yolk-filled cell masses that were often associated with the neural tube or epidermis. CONCLUSIONS: In Xenopus, the maternal stockpile of p53 mRNA and protein seems to be essential for normal development. Inhibiting p53 function results in an early block to differentiation. Although it is possible that mutant human p53 proteins have a dominant gain-of-function or neomorphic activity in Xenopus, and that this is responsible for the development of tumors, most of the evidence indicates that this is not the case. Whatever the basis of the block to differentiation, these results indicate that Xenopus embryos are a sensitive system in which to explore the role of p53 in normal development and in developmental tumors.     

Bcl-2 protects murine erythroleukemia cells from p53-dependent and -independent radiation-induced cell death

To better understand the molecular basis of radiation-induced cell death, we studied the role of the bcl-2 oncogene and the p53 tumor suppressor gene in this process. A temperature-sensitive mutant of murine p53 (p53Val-135) and/or bcl-2 was transfected into murine erythroleukemia cells (MEL, DP16-1, which are null in p53). We demonstrate that radiation-induced cell death occurs by both p53-dependent and -independent pathways and overexpression of bcl-2 modulates both pathways. When viability was measured 24 h post-radiation, cells that had been briefly exposed to wtp53 immediately after X-ray irradiation had decreased survival as compared to unirradiated cells expressing wtp53 or X-ray irradiated DP16-1 cells. However, at later times X-ray irradiated parental DP16-1 cells also had decreased survival compared to the unirradiated control. This decrease in survival began 48 h following radiation. Bcl-2 prevented radiation-induced cell death in DP16-1 cells expressing wtp53 and delayed radiation-induced cell death in DP16-1 cells without wtp53. X-ray irradiated cells expressing wtp53 displayed microscopic and biochemical characteristics consistent with cell death due to apoptosis. DP16-1 cells which were untransfected or co-transfected with wtp53 and bcl-2 displayed characteristics of cells undergoing necrosis. These results suggest that radiation-induced cell death occurs by both p53-dependent and p53-independent pathways. The p53-dependent pathway results in cell death via apoptosis and occurs approximately 24 h following radiation. The p53-independent pathway does not appear to involve apoptosis and occurs at a later time, starting 48 h after X-ray exposure. Thus, bcl-2 protects cells from p53-dependent radiation-induced apoptotic cell death and attenuates p53-independent radiation-induced cell death.     

Association among p53 gene mutation, nuclear accumulation of the p53 protein and aggressive phenotypes in breast cancer

In order to reveal whether differences in the type and site of p53 gene mutations influence the function of the gene and tumor phenotype, we examined nuclear accumulation of the p53 protein immunohistochemically, loss of the other p53 allele by restriction-fragment-length polymorphism analysis, and histological grade of atypia in 52 breast-cancer tissue specimens in which the position and pattern of the mutation were identified. When mis-sense point mutations or deletions of 3n bases (n = 1, 2, ...), which did not cause a frameshift downstream, occurred within codons 110-180 or 234-285, containing highly conserved regions, the p53 protein was almost always (92%) accumulated in nuclei in a majority of the cancer cells. When these mutations occurred outside these regions, only 46% of the cases showed nuclear accumulation of the protein in a majority of cancer cells. In tumors with non-sense point mutations or deletion of 3n + 1 or 3n + 2 bases (n = 0, 1, 2, ...), which caused a downstream frameshift, nuclear accumulation of the p53 protein was absent in 93% of cases. Irrespective of the mutation site or pattern, a majority of cases showing p53 mutation revealed loss of heterozygosity on 17p13 (83%), which suggested they do not carry wild-type p53 allele, and the highest histological grade of atypia (90%). Regardless of differences in their protein-expression pattern, a majority of the p53 gene mutations were suggested to function in a recessive mode and to be involved in the development of histologically aggressive breast cancer.     

DNA double-strand breaks, p53, and apoptosis during lymphomagenesis in scid/scid mice

The tumor-suppressing phenotype of p53 is thought to be due to its accumulation in response to DNA damage and resultant cell cycle arrest or apoptosis. scid/scid mice are defective in DNA double-strand break repair due to a mutation in DNA-dependent protein kinase (DNAPK). Treatment of scid/scid mice with gamma radiation or N-ethyl-N-nitrosourea resulted in approximately 86% incidence of T-cell lymphomas, compared with <6% in wild-type mice. The incidence of other tumor types was not increased in scid/scid mice, suggesting that the types of DNA double-strand break that are unrepaired in these mice are not strongly carcinogenic. To determine whether mutations in DNAPK and p53 interact, we examined mice deficient in both genes. Both scid/scid p53-/- and scid/scid p53+/- mice spontaneously developed lymphomas at shorter latency than did mice with either defect alone. Loss of the wild-type p53 allele was observed in 100% of tumors from scid/scid p53 +/- mice, indicating strong selection against p53. In contrast, p53 was not inactivated in lymphomas from scid/scid p53+/+ mice. Exposure of these tumor-bearing mice to gamma radiation resulted in p53 protein accumulation and high levels of apoptosis in all tumors that were not observed in tumors from scid/scid p53+/- mice. Thus, there was a bifurcation of molecular pathways to tumorigenesis. When p53 was heterozygous in the germ line, loss of the wild-type allele occurred, and the tumors became apoptosis resistant. When p53 was wild type in the germ line, p53 was not inactivated, and the tumors remained highly apoptosis sensitive.     

Loss of atm radiosensitizes multiple p53 null tissues

An unusual clinical finding in ataxia-telangiectasia, a human disorder caused by mutations in atm, is exquisite sensitivity to gamma irradiation. By contrast, homozygous deletion of p53 is marked by radiation resistance in certain tissue compartments. Previous studies (A. J. Levine, Cell, 88: 323-331, 1997) have shown that, in vitro, p53-deficient bone marrow cells are resistant to gamma irradiation. Furthermore, the gastrointestinal radiosensitization engendered by the loss of atm has recently been shown (C. H. Westphal et al., Nat. Genet., 16: 397-401, 1997) to be independent of p53. Expanding on previous work, we have looked at in vivo bone marrow resistance in p53-deficient mice. Our results indicate that inbred FVB strain p53 null mice survive lethal irradiation doses because of bone marrow resistance. Moreover, the deletion of atm radiosensitizes even p53 null bone marrow and mouse embryonic fibroblast cells. The results presented here argue that the loss of atm radiosensitizes multiple tissues in a p53-independent manner. Hence, functional inhibition of atm in p53 null and p53 wild-type human tumors may be a useful adjunct to gamma irradiation-based antitumor therapy.     

Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53

The p53 tumor suppressor gene has been shown to play an important role in determining cell fate. Overexpression of wild-type p53 in tumor cells has been shown to lead to growth arrest or apoptosis. Previous studies in fibroblasts have provided indirect evidence for a link between p53 and senescence. Here we show, using an inducible p53 expression system, that wild-type p53 overexpression in EJ bladder carcinoma cells, which have lost functional p53, triggers the rapid onset of G1 and G2/M growth arrest associated with p21 up-regulation and repression of mitotic cyclins (cyclin A and B) and cdc2. Growth arrest in response to p53 induction became irreversible within 48-72 h, with cells exhibiting morphological features as well as specific biochemical and ultrastructural markers of the senescent phenotype. These findings provide direct evidence that p53 overexpression can activate the rapid onset of senescence in tumor cells.     

Wild-type p53 and a p53 temperature-sensitive mutant suppress human soft tissue sarcoma by enhancing cell cycle control

Soft-tissue sarcomas are a heterogeneous group of tumors that are putatively mesenchymal in origin. Therapeutic advances in this disease have been limited over the past several decades. Approximately one-half of all patients will ultimately succumb, usually to uncontrollable pulmonary metastases. Although little is known about the underlying molecular determinants driving soft-tissue sarcoma inception, proliferation, and metastasis, mutation of the p53 gene is the most frequently detected molecular alteration in this disease. Accordingly, we were interested in determining whether transduction of wild-type (wt) p53 into soft-tissue sarcomas bearing mutated p53 genes might alter the malignant phenotype. SKLMS-1 is a human-derived leiomyosarcoma cell line with a codon 245 p53 point mutation. Cationic liposome was used to transfect wt p53 or 143Ala temperature-sensitive mutant p53 into this cell line. SKLMS-1 stable transfectants expressing wt p53 had decreased cell proliferation in vitro, decreased in vitro colony formation in soft agar, and decreased tumorigenicity in severe combined immunodeficient mice in vivo. Flow cytometric analysis of cell cycle components demonstrated markedly increased G1 cell cycle arrest and decreased entry into S phase, which corresponded to the induction of p21cip1 protein in the transfectants. Using SKLMS-1 stable transfectants expressing the 143Ala p53 temperature-sensitive mutant, we demonstrated the kinetics of and the causal relationship between wt p53 expression, the wt p53-dependent induction of cell cycle inhibitor p21cip1, and inhibition of cell cycle progression in p53-transfected SKLMS-1 cells. The ability to restore wt p53 growth-regulatory functions in soft-tissue sarcoma may ultimately be useful as a future therapy in patients with soft-tissue sarcomas.     

Wild-type p53 protein potentiates cytotoxicity of therapeutic agents in human colon cancer cells

Wild-type p53 is induced by DNA damage. In different cell types, this induction is suggested either to facilitate DNA repair by inducing a cell cycle pause or to potentiate cell death via apoptosis. Wild-type p53 in different cell types has similarly been associated with either enhancement of or increased resistance to the cytotoxicity of many cancer therapeutic agents. We have constructed a colorectal cancer cell line bearing, in addition to endogenous mutant p53 alleles, an exogenous wild-type p53 allele that is under the regulatable control of the lac repressor. Induction of wild-type p53 by isopropyl-beta-thiogalactopyranoside in these cells induces a reversible growth arrest but does not induce cell death. However, we find that the induction of wild-type p53 powerfully potentiates the cytotoxicity of both irradiation and 5-fluorouracil, two agents that are used clinically in the treatment of colorectal cancer. We also find that induction of wild-type p53 potentiates the cytotoxicity of topotecan, a member of the camptothecin family of drugs that also has clinical activity against colon cancer. These findings suggest that the common loss of wild-type p53 in many colorectal cancers may play a role in the clinical resistance of these tumors to anticancer agents. Although some cancer cells may not be directly killed by p53 gene therapy, our findings suggest that genetic alteration of some cancers to induce wild-type p53 may increase their sensitivity to cytotoxic gene therapy.     

Development and applications of enhanced green fluorescent protein mutants

The introduction of several mutations resulted in the generation of improved mutants of the green fluorescent protein (GFP). A strong green (GFPsg25) and blue (BFPsg50) fluorescent protein, gave 50-fold-100-fold brighter fluorescence compared to wild-type GFP and BFP (Tyr66His), respectively, upon expression in mammalian cells. GFPsg25 and BFPsg50 have different excitation and emission maxima. This allows their use as an efficient dual-color tagging system and their independent detection in living cells.     

Wild-type alternatively spliced p53: binding to DNA and interaction with the major p53 protein in vitro and in cells

A p53 variant protein (p53as) generated from alternatively spliced p53 RNA is expressed in normal and malignant mouse cells and tissues, and p53as antigen activity is preferentially associated with the G2 phase of the cell cycle, suggesting that p53as and p53 protein may have distinct properties. Using p53as and p53 proteins translated in vitro, we now provide evidence that p53as protein has efficient sequence-specific DNA-binding ability. DNA binding by p53 protein is inefficient in comparison and requires activation. Furthermore, p53as and p53 proteins formed hetero-oligomers when co-translated in vitro, resulting in inactivation of p53as DNA-binding activity. Gel filtration indicated that p53as translated in vitro, like p53, formed tetramers. In support of a functional role of p53as in cells, p53as/p53 hetero-oligomers were coimmunoprecipitated from mouse cells, and both protein forms were detectable in nuclear extracts by electrophoretic mobility shift assays. These results suggest that the biochemical functions of p53 are mediated by interaction between two endogenous protein products of the wild-type p53 gene.     

p53/E1b58kDa complex regulates adenovirus replication

We have explored a role for the adenovirus (Ad5) E1b58kDa/p53 protein complex in adenovirus replication. This was done by using virus mutants containing different defects in the E1b58kDa gene and cell lines that express either a wild-type p53 protein or a mutant p53 protein. We find that infection of wild-type p53-containing cells with wild-type Ad5 causes a shutoff of p53 and alpha-actin protein synthesis by distinct mechanisms, but neither occurs in mutant p53 cells. Our data also indicate that the shutoff is dependent on formation of the p53/E1b complex and may also involve another virus protein, E4ORF6. Following from these observations we asked whether failure to form the complex resulted in impaired adenovirus replication. Our experiments showed that neither wild-type Ad5 nor the E1b mutant dl338 could replicate in cells expressing a mutant p53 protein, but that wild-type adenovirus replicated well in wild-type p53-expressing cells. Collectively, our data suggest that the interaction between p53 and the E1b58kDa protein is necessary for efficient adenovirus replication. This is the first time such a direct link between the complex and virus replication has been demonstrated. These data raise serious questions about the usefulness of E1b-defective viruses in tumor therapy.