Telomerase activity during spontaneous immortalization of Li-Fraumeni syndrome skin fibroblasts.

Li-Fraumeni Syndrome (LFS) is characterized by heterozygous germline mutations in the p53 gene. Accompanied by genomic instability and loss or mutation of the remaining wild type p53 allele, a low frequency of spontaneous immortalization in LFS fibroblasts occurs. It is believed that the loss of p53 wild type function contributes to immortalization of these LFS fibroblasts, but it is not clear if this is sufficient. Because stabilization of telomere length is also thought to be a necessary step in immortalization, telomerase activity, expression of the telomerase RNA component (hTR) and telomere length were anlaysed at various passages during the spontaneous immortalization of LFS skin fibroblasts. One LFS strain which immortalized, MDAH087 (087), had no detectable telomerase activity whereas another LFS strain which immortalized, MDAH041 (041), had detectable telomerase activity. In preimmortal cells from both strains, hTR was not detected by in situ hybridization. Immortal 087 cells remained negative for hTR, while immortal 041 cells demonstrated strong hTR in situ hybridization signals. 087 cells had long and heterogenous telomeres whereas telomeres of 041 cells had short, stable telomere lengths. Tumorigenicity studies in nude mice with ras-transformed 087 and 041 cells resulted in both cell lines giving rise to tumors and retaining telomerase status. Overall these results suggest that strain specificity may be important in telomerase re-activation and that both abrogation of p53 function and a mechanism to maintain telomeres are necessary for immortalization.

Analysis of genomic instability in Li-Fraumeni fibroblasts with germline p53 mutations.

Germline p53 mutations are frequently observed in the normal DNA of cancer-prone patients with Li-Fraumeni syndrome (LFS). Fibroblasts from LFS patients develop chromosomal aberrations, loss of cell cycle control, and spontaneous immortalization. We transfected four different mutant p53 genes into human skin fibroblasts from normal donors with two copies of wild-type p53 (p53(wt/wt)). Each mutant p53 expression-plasmid induced genomic instability equivalent to that seen in LFS cells. To test the role of wild-type and mutant p53 alleles in DNA replication and fidelity in LFS cells, we analysed the replication of the SV40-based shuttle vector pZ189 in four types of cells. We used p53(wt/mut) and p53(mut/-) LFS fibroblasts, and p53(-/-) non-LFS cells. Replication of pZ189 in vivo was significantly reduced by the presence of a p53(wt) allele. To show that this was not just due to inhibition of the function of T-antigen in SV40-based replication, we constructed a shuttle vector, pZ402, that contains a mutation in SV40 T-antigen which blocks its ability to interact with p53. Replication of pZ402 in LFS cells was also reduced by the presence of p53(wt), indicating that p53 can inhibit replication by interacting with proteins within the cellular replication machinery. Replicative errors in this shuttle vector are detected as mutations in a marker gene, supF. In addition to supF mutations, we observed deletion of a portion of the SV40 T-antigen gene in 100% of replicated plasmid pZ189 mutants (supF-) from the p53(wt/mut) fibroblasts and in 88% of the supF mutants from the p53(mut/-) (amino acid 175 arg to his) LFS cells. In one cell strain of immortal LFS cells, P53(mut/-) , containing a p53 frameshift mutation at amino acid 184, pZ189 replication yielded very few of these deleted shuttle vector plasmids (15%). These large deletions were not detected in plasmids replicated in p53(-/-) non-LFS cells, Saos-2 cells. Replicated plasmids with a normal supF gene were never found to have this large deletion regardless of the cell from which they were derived. Because the supF gene is not in the same region of the shuttle vector as the T-antigen gene it appears that second, independent gene deletions are frequent when replicative errors in supF occur in cells with a mutant p53. We conclude, therefore, that p53(wt/mut) LFS cells contain an activity that promotes mutations. Such an activity, which is likely to be due to the p53(mut), could result in the high rate of chromosomal instability and allelic loss of the wild-type p53 observed as these cells spontaneously immortalize.

Susceptibility to ras oncogene transformation is coregulated with signal transduction through growth factor receptors.

The human teratocarcinoma cell line PA-1 was derived from culturing ascites fluid cells from a patient with an ovarian germ line tumor. We previously described a non-neoplastic variant cloned from the PA-1 human teratocarcinoma cell line, clone 6, which at passage 40 was resistant to transformation by activated ras oncogenes. However, these cells could be transformed by a plasmid containing both myc and ras. Another PA-1 cell variant, clone 1, isolated at passage 63 and used 50 passages later becomes tumorigenic in nude mice after transfection with an activated ras oncogene (Tainsky et al., Anticancer Res., 8, 899-914, 1988). We report here that the progression from ras resistance to ras susceptibility occurs in both clone 1 and clone 6 cells during 25 passages in culture. In the presence of epidermal growth factor, transforming growth factor-alpha, and basic fibroblast growth factor, the ras-transformable cells exhibit anchorage independent growth, whereas the ras-resistant cells can not be growth stimulated by these growth factors. Similarly, ornithine decarboxylase (ODC) activity was inducible in ras susceptible and ras transformed cells by these growth factors, but not in the ras resistant cells. These differences are not due to the level and activity of epidermal growth factor receptor or to the level of expression of 25 proto-oncogenes.