The aim of this proposal is to determine whether maintenance of the human cancer cell tumorigenic phenotype depends on the continued presence of activated ras oncogenes. Three different experimental protocols are proposed: 1) activated ras alleles will be inactivated by targeted homologous recombination, or by the expression of a doubly mutant ras dominant negative allele; 2) dominant negative alleles of proteins in the 2 well characterized ras signalling pathways will be used to selectively disrupt each pathway; and 3) ras antisense oligonucleotides will be used to modify the in vitro growth properties and tumorigenic potential of human tumor cells containing activated ras alleles. The human tumor cells used for these experiments are heterozygous for activating mutations in H-, K- or N-ras. Assays of genetically modified or oligonucleotide-treated cells will include biochemical examination of ras and ras-dependent signalling pathways, growth and morphology in vitro, and tumor formation in mice.
