D Wolf

Reconstitution of p53 expression in a nonproducer Ab-MuLV-transformed cell line by transfection of a functional p53 gene

L12 are Ab-MuLV-transformed cells that express the abl p120 oncogene product but lack the cellularly encoded p53. The functional p53 gene in these cells has been inactivated by the insertion of Moloney virus-like sequences into the first p53 intron. Transfection of L12 cells with a functional p53 gene, contained in a 16 kb Eco RI genomic cloned fragment gave rise to L12-derived cell lines with novel p53 sequences of various sizes and copy number. A high percentage of L12-derived clones efficiently transcribed p53 mRNA and synthesized the p53 protein. Whereas injection of L12 parental cells into syngeneic mice caused the development of local tumors that later regressed, L12-derived clones that expressed p53 caused lethal tumors in syngeneic mice, thus behaving similarly to other Ab-MuLV-transformed p53-producer cell lines. These results suggest that the expression of p53 is essential for tumor cells to exhibit a fully transformed phenotype, manifested in lethal tumors in syngeneic mice.

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.

Chromosomal assignment of the murine gene encoding the transformation-related protein p53.

p53 is a transformation-related protein that is encoded by the cellular genome and is synthesized at elevated levels in a wide range of different cell line types and in primary tumors of various species. By using several independently established anti-p53 monoclonal antibodies, it was possible to distinguish between p53 of mouse origin and p53 of Chinese hamster origin. By analysis of a series of mouse X Chinese hamster hybrid cell lines containing various mouse chromosomes, we mapped the p53 gene product to mouse chromosome 11.

Biological and molecular analysis of p53 cellular-encoded tumor antigen

Publisher Summary p53 was originally observed as a cellular product that formed a stable complex with the viral large T antigen expressed in Simian Virus 40 (SV40)-transformed cells. p53 is found in tumor cells in its phosphorylated form. The addition of radioactive phosphorus yields a phosphorylated p53 that immunoprecipitates with anti-p53 monoclonal antibodies. Under in vivo conditions, p53 phosphorylates onto a serine amino acid. Experiments showed that immunoprecipitation of p53 with a specific anti-p53 monoclonal antibody yields a p53 protein that also autokinases under in vitro conditions. In these experiments, p53 immunoprecipitated with several other anti-p53 monoclonal antibodies apparently did not bind the radioactive phosphate of γ-ATP under in vitro conditions. These conflicting results, concerning autokinase of p53 in vitro after immunoprecipitation with one reagent and not with another could be explained by the assumption that some monoclonal antibodies bind directly to the site of phosphorylation. The assumption that p53 is encoded by the normal cellular genome is based on the observation that it is expressed in several types of nontransformed cells. Several investigations suggest that the basis for the quantitative difference of p53 in transformed and nontransformed cells is due to posttranslational regulatory mechanisms.

Expression ofabl and other oncogenes is independent of metastatic potential in Abelson virus-transformed malignant murine large cell lymphoma

The role of oncogene expression in tumor metastasis was examined using the Abelson leukemia virus-transformed murine large cell lymphoma RAW117. Cell sublines of low and high metastatic potential expressed equallyabl oncogene-coded mRNA and its phosphoprotein product p160, and the capacity of p160 to become autophosphorylated withγ-[32P]ATP was the same among low and high metastatic cells. The expression of other oncogene-coded mRNAs (fos, myc, myb), if present, was also similar in low and high metastatic RAW117 cells. Although oncogene expression is thought to be important in initiating, and in some cases maintaining, the transformed phenotype, its expression in RAW117 lymphoma cells appears to be unrelated to metastatic phenotype.