Showing papers by "Arnold J. Levine published in 1994"

p53 and E2F-1 cooperate to mediate apoptosis

Abstract: The tumor-suppressor protein p53 appears to function at the G1 phase of the cell cycle as a checkpoint in response to DNA damage. Mutations in the p53 gene lead to an increased rate of genomic instability and tumorigenesis. The E2F-1 transcription factor is a protein partner of the retinoblastoma-susceptibility gene product, RB. E2F-1 appears to function as a positive regulator or signal for entry into S phase. To explore possible interactions of p53 and E2F-1 in the cell cycle, a human E2F-1 expression plasmid was introduced into a murine cell line containing a temperature-sensitive p53 allele which produces a p53 protein in the wild-type conformation at 32 degrees C and the mutant form at 37.5 degrees C. Coexpression of the wild-type p53 protein and E2F-1 in these cells resulted in a rapid loss of cell viability through a process of apoptosis. Thus, the cell cycle utilizes an interacting or communicative pathway between RB-E2F-1 and p53.

Molecular abnormalities of mdm2 and p53 genes in adult soft tissue sarcomas

Abstract: Genetic alterations in the p53 and mdm2 genes have been reported to occur in soft tissue sarcomas. This study was designed to determine the prevalence and potential clinical value of detected molecular abnormalities and altered patterns of expression of mdm2 and p53 genes in adult soft tissue sarcomas. A cohort of 211 soft tissue sarcomas from adults that were both clinically and pathologically well characterized was analyzed. Monoclonal antibodies directed against mdm2 and p53 proteins were used to measure overexpression of these proteins in the nuclei of cells from sections of these tumors. Seventy-six of 207 tumors had abnormally high levels of mdm2 proteins and 56 of 211 tumors overexpressed p53 protein. Twenty-two cases had abnormally high levels of both mdm2 and p53 proteins based upon immunoreactivity with these antibodies. There was a striking statistically significant correlation between the overexpression of p53 and mdm2 proteins in the same tumor and poor survival (P < 0.05) of the patients. A group of 73 soft tissue sarcomas was chosen for analysis using Southern blots, single strand conformation polymorphisms, and direct DNA sequencing to confirm mdm2 gene amplifications and p53 mutations and correlate these with the results of the immunoreactivities. The overexpression of p53 and mdm2 proteins in the nuclei of tumor cells did not always correlate well with gene amplification at the mdm2 locus or mutation at the p53 gene. The possible reasons for these discrepancies are discussed.

The ribosomal L5 protein is associated with mdm-2 and mdm-2-p53 complexes.

Abstract: Throughout the purification of the mdm-2 or mdm-2-p53 protein complexes, a protein with a molecular weight of 34,000 was observed to copurify with these proteins. Several monoclonal antibodies directed against distinct epitopes in the mdm-2 or p53 protein coimmunoprecipitated this 34,000-molecular-weight protein, which did not react to p53 or mdm-2 polyclonal antisera in a Western immunoblot. The N-terminal amino acid sequence of this 34,000-molecular-weight protein demonstrated that the first 40 amino acids were identical to the ribosomal L5 protein, found in the large rRNA subunit and bound to 5S RNA. Partial peptide maps of the authentic L5 protein and the 34,000-molecular-weight protein were identical. mdm-2-L5 and mdm-2-L5-p53 complexes were shown to bind 5S RNA specifically, presumably through the known specificity of L5 protein for 5S RNA. In 5S RNA-L5-mdm-2-p53 ribonucleoprotein complexes, it was also possible to detect the 5.8S RNA which has been suggested to be covalently linked to a percentage of the p53 protein in a cell. These experiments have identified a unique ribonucleoprotein complex composed of 5S RNA, L5 protein, mdm-2 proteins, p53 protein, and possibly the 5.8S RNA. While the function of such a ribonucleoprotein complex is not yet clear, the identity of its component parts suggests a role for these proteins and RNA species in ribosomal biogenesis, ribosomal transport from the nucleus to the cytoplasm, or translational regulation in the cell.

The amino-terminal functions of the simian virus 40 large T antigen are required to overcome wild-type p53-mediated growth arrest of cells.

Abstract: High levels of the p53 tumor suppressor protein can block progression through the cell cycle. A model system for the study of the mechanism of action of wild-type p53 is a cell line (T64-7B) derived from rat embryo fibroblasts transformed by activated ras and a temperature-sensitive murine p53 gene. At 37 to 39 degrees C, the murine p53 protein is in a mutant conformation and the cells actively divide, whereas at 32 degrees C, the protein has a wild-type conformation and the cells arrest in the G1 phase of the cell cycle. Wild-type simian virus 40 large T antigen and a variety of T-antigen mutants were assayed for the ability to bypass the cell cycle block effected by the wild-type p53 protein to induce colony formation at 32 degrees C. The results indicate that two functions within the amino terminus of T antigen are essential to induce cell growth: (i) the ability to bind to the retinoblastoma protein, Rb, and (ii) the presence of a domain in the first exon that appears to interact with the cellular protein, p300. Thus, the cell cycle arrest triggered by wild-type p53 may be overcome by formation of a T-antigen complex with Rb, p300, or both that could then function to either remove p53-mediated negative growth regulatory signals or promote a positive cell growth signal. Surprisingly, T antigen-p53 complexes are not required to overcome the temperature-sensitive p53 block to the cell cycle in these cells. These data suggest that simian virus 40 T antigen associated with Rb, p300, or both proteins can communicate in a cell with the functions of the wild-type p53 protein.

The origins of the small DNA tumor viruses.

Abstract: Publisher Summary The chapter presents a description of the discovery of the origin of virus, namely smallpox virus and its vaccine. The chapter describes the cell culture and poliovirus. The viruses of monkey kidney cells are also discussed in the chapter. The use of monkey kidney cells on a scale of the size needed for vaccine trials rapidly led to considerable experience with these cells. The chapter also describes, in detail, the origins of the DNA tumor viruses, namely, the adenoviruses and simian virus 40 (SV40). The molecular biology of the DNA tumor viruses is also discussed in the chapter. The small DNA tumor viruses studied so intensively between 1960 and 1993 have contributed out of proportion to their size, to a fundamental body of knowledge about eukaryotes. From the perspective of the early days where searches for new viruses and vaccine development led to the discoveries of adenoviruses and SV40, no one could have predicted the central role these viruses would play in both basic biology and oncology. Such is the very nature and essence of science. The history of the discoveries of and studies with SV40, and the human adenoviruses teaches once again the powerful role of basic fundamental scientific research in showing the way to the application of medical needs. This history also shows how basic and applied research goals can go hand-in-hand to produce good science.

Methods for detecting mutant p53

Abstract: A panel of probes detects and distinguishes between sets of human p53 gene or protein mutations that frequently occur or are selected for in pre-cancer and cancer cells Each set of mutations gives rise to a phenotype that is different from that of wild-type p53 and of at least one other set of p53 mutations.

The Two Amino Terminal Transforming Functions of the SV40 Large T-Antigen are Required to Overcome P53 Mediated Growth Arrest

Abstract: About 60% of cancers from humans contain mutations at the p53 locus1,2. Most commonly, there is a missense mutation in one of the p53 alleles and a loss of the second allele resulting in a reduction to homozygosity of the mutant gene3. Thus p53 behaves like a tumor suppressor gene4 where a loss of function (recessive to wild-type) enhances the probability of cancerous growth. Returning the wild-type p53 gene into cells that are being transformed by an oncogene5 or are already transformed6 blocks the transformation process and, when p53 is expressed at high levels, inhibits cell division of transformed cells. Cells transformed with a temperature sensitive p53 mutant replicate at 37–39°C, where the p53 protein is in a mutant conformation, but fail to grow at 32°C, where the p53 protein is acting as a wild type tumor suppressor1–8. The wild-type p53 protein blocks progression through the cell cycle in G1 7,8. Thus, the wild-type protein can, under certain circumstances9, regulate progression of cells through the cell cycle while mutant p53 proteins fail to do this. The wild-type p53 protein, but not the mutant protein, can act as a transcription factor10–12 and so it is tempting to speculate that p53-mediated transcription of a set of genes can block progression through the cell cycle in the G1 phase of the cycle.