Use of a temperature-sensitive mutant to define the biological effects of the p210^BCR-ABL tyrosine kinase on proliferation of a factor-dependent murine myeloid cell line

The Philadelphia chromosome, detected in virtually all cases of chronic myelogenous leukemia, is formed by a reciprocal translocation between chromosomes 9 and 22 that fuses BCR encoded sequences upstream of exon 2 of c-ABL. This oncogene produces a fusion protein, p210^BCR-ABL, in which the ABL tyrosine kinase activity is elevated. This elevated kinase activity is essential for transformation, but the mechanisms involved are unknown. To investigate p210^BCR-ABL function we constructed a model system in which the tyrosine kinase activity of p210^BCR-ABL was inducible. Two amino acid substitutions, Arg to His at amino acid 457 and Tyr to His at amino acid 469 of c-abl, modeled on mutations known to render v-src temperature-sensitive for tyrosine kinase activity, were introduced into p210^BCR-ABL. This mutant was characterized in an IL-3 growth factor dependent murine myeloid cell line, 32Dc13. Cell lines expressing the temperature-sensitive mutant remained factor dependent at the non-permissive temperature, but at the permissive temperature displayed a marked reduction in cell death in the absence of growth factor and an exaggerated proliferative response to low levels of IL-3. Both the kinase activity of the mutant and the levels of tyrosine phosphorylated proteins are increased in the temperature-sensitive mutant at the permissive temperature. Further, tyrosine phosphorylation of potential substrates of the p210^BCR-ABL tyrosine kinase, p120 rasGAP and its associated proteins of p190 and p62, only occurs at the permissive temperature in cells expressing the temperature-sensitive mutant.