Rat embryo fibroblasts immortalized with simian virus 40 large T antigen undergo senescence upon its inactivation.

Introduction of simian virus 40 T antigen into rodent fibroblasts gives rise to cells that can proliferate indefinitely but are dependent upon it for maintenance of their growth once the normal mitotic life span has elapsed. Inactivation of T antigen in these immortalized cells causes rapid and irreversible cessation of growth. To determine whether this growth arrest is associated with entry into senescence, we have undertaken a genetic and biological analysis of conditionally immortal (tsa) cell lines derived by immortalizing rat embryo fibroblasts with the thermolabile tsA58 T antigen. This analysis has identified the following parallels between the tsa cells after inactivation of T antigen and senescent rat embryo fibroblasts: (i) growth arrest is irreversible; (ii) it occurs in G1 as well as G2; (iii) the G1 block can be partially overcome by stimulation with 20% fetal calf serum, but the G2 block cannot be overcome; (iv) 20% fetal calf serum induces c-fos, but c-myc is unaltered; and (v) fibronectin and p21(Waf1/Cip1/Sdi1) are upregulated upon growth arrest. These results suggest that T-antigen-immortalized fibroblasts are committed to undergo senescence but are prevented from undergoing this process by T antigen. Inactivation of T antigen removes this block and results in senescence of the cells. Thus, these cell lines may represent a powerful system for study of the molecular basis of entry into senescence.

Nucleotide sequence polymorphism in a hotspot mutation region of the p53 gene.

By screening for mutations in the p53 coding sequence by means of single-strand conformation polymorphism (SSCP) in a series of breast tumors we detected a novel polymorphism. This change in the SSCP pattern was detected in 6.2% of the tumor DNAs analysed and implied an A to G substitution at the last base of codon 213, thus representing a neutral change. First suspecting a somatic mutation we confirmed its presence in matched sets of DNAs from normal tissues. Extending our study to a series of 60 ovarian carcinomas and 70 healthy blood donors we noticed that this polymorphism represented only 3% and 2.6% respectively. We wondered if the difference in frequency in the breast cancer population might not be related to familial breast cancer and analysed 26 DNAs from patients showing predisposition to the disease. Two patients presented this polymorphism and one corresponding kindred was analysed, revealing a mendelian mode of transmission but no correlation with the cancer phenotype.

p53 and RAS gene mutations in multiple myeloma.

We analysed genomic DNA from 30 patients with multiple myeloma (MM), searching for alterations in the p53 and RAS genes by a combination of polymerase chain reaction and single-strand conformation polymorphism techniques. Mutations in the p53 gene were observed in 20% (6 out of 30) of the patients, and were located in conserved sequence blocks within exons 5 and 7. These were single-nucleotide substitutions and consisted predominantly (4/6) of G:C to A:T transitions. Of the six patients with a mutated p53 gene, four were in the terminal phase of the disease. RAS gene mutations were found more frequently since they occurred in 47% (14 out of 30) of the patients. Mutations consisted of single-nucleotide substitutions, located in codons 12, 13 and 61 of either K- or N-RAS, to the exclusion of H-RAS. Moreover, one patient bore two simultaneous mutations, affecting simultaneously the K- and the N-RAS genes. RAS gene mutations were more frequently observed in patients with fulminating disease (10/15, 67%) than in patients with less aggressive forms of the disease (4/15, 26%). We also analysed genomic DNAs from 10 human myeloma cell lines, of which two bore mutations affecting codon 12 of the K-RAS gene, and one codon 12 of the N-RAS gene. The first two cell lines were obtained from freshly explanted tumor cells in which we observed identical mutations. Results presented here show that activating mutations in the RAS genes are, in MM, more frequent than those affecting the p53 gene and suggest that both events are related to terminal phases of the disease.

Mutations of the p53 gene in human myeloma cell lines.

Mutations affecting the p53 gene have been found associated with many human malignancies, but little is as yet known about multiple myeloma. We investigated p53 gene alterations in 10 human myeloma cell lines (HMCL), half of these being dependent upon exogenous interleukin 6 (IL-6) for in vitro growth, similar to freshly explanted myeloma cells. Using a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) approach, eight of the 10 HMCL were found to bear a mutated p53 gene. All the mutations were single base substitutions with a predominance of G:C to A:T transitions. There was no apparent relation between the presence of a mutation and IL-6 requirement of the cell line. Interestingly, in two cell lines (XG-2 and XG-4) the SSCP pattern showed the presence of both the wild-type and the mutated allele and, upon reverse PCR on RNA, both alleles were found to be concomitantly expressed at the RNA level. Moreover, three freshly explanted tumor samples had the same p53 gene status (mutated versus wild type) as the HMCL that were derived from them. These results show that p53 mutations are frequent in HMCL. Although no apparent relation could be evidenced with the loss of exogenous IL-6 requirement, it may prove interesting to investigate further potential relations between the presence of a mutated p53 allele and gradual autonomy for cell growth.

Expression of p24, a novel p21Waf1/Cip1/Sdi1-related protein, correlates with measurement of the finite proliferative potential of rodent embryo fibroblasts.

Normal mammalian fibroblasts undergo a limited number of divisions when cultured in vitro before entering a state of replicative senescence. The molecular basis for the determination of the finite mitotic potential is not known. Nevertheless, simian virus 40 T antigen, among other oncogenes, is able to prevent senescence in rodent embryo fibroblasts. T antigen immortalized cells are dependent upon this protein for maintaining growth once their normal mitotic life span has elapsed. Even though the mechanism that measures the finite mitotic potential of rodent fibroblasts is not known, it has been shown that it continues to function normally in the presence of this immortalizing gene. Accumulation of cyclin-dependent kinase inhibitors such as p21Waf1/Cip1/Sdi1 could potentially be a component of the mechanism that determines the finite life span. Here we show that accumulation of p21Waf1/Cip1/Sdi1 does not correlate with this biological counting mechanism, but we have identified p24, a p21Waf1/Cip1/Sdi1-related protein, whose accumulation does correlate with the measurement of the finite proliferative potential of rodent embryo fibroblasts and suggest that sequestration might be a mechanism by which its activity is regulated.