IDENTIFICATION OF CHROMOSOMES IMPLICATED IN SUPPRESSION OF APOPTOSIS IN SOMATIC-CELL HYBRIDS

In vitro exposure of tumorigenic cell lines to the chemotherapeutic ag ent PALA (N-(phosphonoacetyl)-L-aspartate) usually results in cell dea th (shown here to be apoptosis), followed by clonal growth of rare sur vivors. On the other hand, normal diploid cells respond to PALA by arr esting in G(1) and G(2) of the cell cycle. It was previously suggested that growth control mechanisms might exist to prevent cells from ente ring S phase under toxic conditions and that genes involved in such me chanisms were mutated or deleted in tumor cells. Interestingly, the tu mor suppressor gene p53, a putative G(1) control gene, was shown to me diate PALA-induced growth arrest. However, growth arrest occurs in cel ls that lack wild-type p53, suggesting that other genes are involved a s well. To identify these genes, we have generated whole cell hybrids between mouse melanoma and normal human fibroblast cells. At early pas sage, a whole cell hybrid (BHF12) responds to PALA with growth arrest, while at later passage, the same hybrid undergoes apoptosis. To deter mine which human chromosomes are required for the PALE-induced growth arrest phenotype, we isolated subclones of the hybrid and tested them for their PALA response. FISH (fluorescence in situ hybridization) and PCR (polymerase chain reaction) amplification have been used to ident ify the human chromosome content of BHF12 and its subclones. Several h uman chromosomes, in addition to chromosome 17 (the location of p53), are consistently associated with the growth arrest phenotype. These fi ndings provide evidence that one or more genes in addition to p53 may be involved in the suppression of a drug-induced apoptosis pathway and may lie on one or more of these chromosomes