Properties of Li-Fraumeni Fibroblasts


We were interested in phenotypically characterizing normal cells from inherited cancer patients for the purposes of understanding mechanisms of carcinogenesis. As fibrosarcomas are frequently observed in Li-Fraumeni syndrome, fibroblasts from these patients offered an "at risk" target tissu e to observe phenotypic characteristics of this unusual syndrome. We have observed the selection of certain cells that spontaneously transform into infinite lifespan cell lines in culture under conditions in which cells from normal, unaffected controls we re clearly limited in their growth and number of cell divisions. Fibroblasts from affected individuals from Li-Fraumeni families develop changes in morphology, chromosomal abnormalities, and enter a growth crisis during which they begin to senesce (in a fashion similar to fibroblasts from normal donors) but then they recover. The cells then grow rapidly and maintain the morphology of a transformed cell. The chromosomal abnormalities are significant in that even prior to their emergence from senescence e ssentially all metaphases examined contained abnormal chromosome numbers, numerous damaged chromosomes and evidence for gene amplification in the form of double minute chromosomes and homogeneously staining regions. Thus, the spontaneous acquisition of a n infinite lifespan in culture of the skin fibroblasts with the associated aneuploidy, extremely rare events in cells derived from normal donors, occurs frequently in cells derived from Li-Fraumeni patients. Because these cells contain germline p53 mutations, we investigated the fate of the wild type and mutant alleles during the immortalization process. In particular, we tested whether both alleles were present after immortalization. Southern blot analysis of Rsa I digested genomic DNA hybridized to the polymorphic VNTR probe, pYNZ22, has shown loss of heterozygosity in a region close to p53 for patient fibroblasts, MDAH087; these cells contain a p53 germline mutation at amino acid 248 which changes the arg to trp, (CGG to TGG). In lymphoblast DNA and fibroblast DNA prior to and during cell growth crisis, i.e. pd 26, the p53 gene is heterozygous, as demonstrated by the Southern blot. The presence of both the mutant and wild type p53 genes in this DNA was confirmed by direct PCR-DNA sequence analysis. After 37 pd all fibrob last DNA samples analyzed were homozygous for the mutant p53 allele. Because, this is precisely the time at which this cell line begins to escape the slow growth crisis, it seems likely that the wild type p53 has a growth suppressive effect or an effect on cell death and its loss provides those cells with a significant growth advantage. These results support the dual involvement of germ line p53 mutations in the acquisition of aneuploidy and that loss of the wild type p53 allele may be necessary for immortalization of dermal fibroblasts derived from Li-Fraumeni cancer patients.