Inactivation of p53 gene expression by an insertion of Moloney murine leukemia virus-like DNA sequences.

Abstract

Analysis of Abelson murine leukemia virus-transformed L12 cells which lack the p53 cellular encoded tumor antigen revealed alterations in the p53-specific genomic DNA sequences. The active p53 gene, usually contained in a 16-kilobase EcoRI DNA fragment of p53 producer cells, went through major alterations leading to the appearance of a substantially larger 28.0-kilobase p53-specific EcoRI fragment. Detailed restriction enzyme analysis, with genomic probes spanning throughout the whole active p53 gene, indicated that the L12 p53 altered gene contains all the exons and principal introns of the normal p53 16.0-kilobase gene. However, its structure was interrupted by the integration of a novel DNA segment into the noncoding intervening sequences of the first p53 intron. Analysis of the inserted sequences revealed close homology to Moloney murine leukemia virus. This Moloney leukemia murine virus-like particle resides in a 5' to 3' transcriptional orientation, similar to the p53 gene, permitting the transcription of aberrant fused mRNA molecules detected in these cells.p53 is a cellular encoded protein that is overproduced in cancer cells. Accentuated concentrations of this protein were detected in a variety of cell lines and primary tumors of several species (6,7,9,16,17,30,33,39), suggesting that p53 may play a role in neoplastic transformation. In tumor cells this protein was found in its phosphorylated form (13,20,30,32), and in several instances it was found in a stable complex with viral tumor antigens (16,17,19,37). A limited occurrence of p53 was also observed in nontransformed cells, such as normal thymocytes (34), primary embryonic fibroblasts (24), and NIH-3T3 fibroblasts (27). It was suggested that p53 may display a function in the normal cell cycle (21,23,24).The function that p53 fulfills in transformed and nontransformed cells can be understood by comparing cells that express the protein and variant cells that do not express it. We have at our disposal a unique Abelson murine leukemia virus (Ab-MuLV)-transformed cell line, L12, which lacks detectable amounts of the p53 protein. Injection of these cells into syngeneic mice induced the development of tumors which were subsequently rejected, whereas other AbMuLV-transformed cells that were overproducing p53 developed into lethal tumors (31,45). This observation suggests a correlation between expression of p53 in tumor cells and their capacity to exhibit a fully transformed phenotype, evaluated as development of lethal tumors in syngeneic mice.In our previous studies employing specific cDNA probes (25, 26), we found that the inability of L12 cells to produce p53 is due to the absence of detectable mature p53-specific mRNA (46). Instead, these cells contain two major p53-specific mRNA species of a substantially larger size than the p53-specific mRNA in the p53 producing cells (46 RNA and DNA blot analysis. RNA was prepared according to the method of Auffray and Rougeon (1)

Alteration of p73 in acute myelogenous leukemia

“…Moreover, point mutations in p73 may not be the only way to abrogate or compromise p73 function. For example, p53 structure and functions can be altered by conformational changes in the p53 protein, slicing abnormalities, viral protein binding, and upregulation of intrinsic proteins such as MDM-2 [29][30][31][32][33][34][35]. Thus the absence of p73 coding mutations does not exclude p73 from playing a role in AML.…”

In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

“…We found that introducing a functional p53 gene into L12 (Abelson murine leukemia virus-transformed cells that lack p53 [32,34] changed their phenotype, and instead of regressing tumors, they caused lethal tumors in the hosts (33). Our conclusion was that L12 cells, which express the abl oncogene but lack p53 expression, are only partially transformed, and that the expression of a completely transformed phenotype requires the concomitant expression of abl and p53.…”

“…Heteroduplex analysis of the cloned human gene and the full-length human cDNA showed that it consists of about seven major exons. In the mouse there are two noncontiguous p53 genes (24,32,34,36), a principal active gene and a pseudogene, that map to different chromosomes (5,29). It is suggested therefore that in spite of the apparent resemblance between the mouse and human p53 proteins and the relatively conserved homology in their cDNA sequences (18), the structure of their p53 genes and their relative exon-intron localization seem to present unrelated patterns.…”

True

“…For example, p53 structure and function can be altered by conformational changes in the p53 protein, splicing abnormalities, viral protein binding, and upregulation of intrinsic proteins such as MDM-2. 6,8,17,[38][39][40][41][42][43][44][45][46][47][48][49][50] Thus, the absence of p73 coding mutations does not exclude p73 from playing a role in AML.…”