Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding

The FHIT gene is inactivated early in the development of many human tumors, andFhit-deficient mice have increased cancer incidence. Viral reexpression of Fhitkills Fhit-deficient cells by induction of apoptosis. Fhit, a member of branch 2 of the histidine-triad superfamily of nucleoside monophosphate hydrolases and transferases, is a diadenosine polyphosphate hydrolase, the active-site histidine of which is not required for tumor suppression. To provide a rigorous test of thehypothesis that Fhit function depends on forming a complex with substrates, we designed a series of alleles of Fhit intended to reduce substrate-binding and or hydrolytic rates, characterized these mutants biochemically, and then performed quantitative cell-death assays on cancer cells virally infected with each allele.The allele series covered defects as great as 100,000-fold in k(cat) andincreases as large as 30-fold in K(M). Nonetheless, when mutant FHIT genes wereexpressed in two human cancer cell lines containing FHIT deletions, reductions inapoptotic activity correlated exclusively with K(M). Mutants with 2- and 7-foldincreases in K(M) significantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in K(M) retained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.