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, and Fhit-deficient mice have increased cancer incidence. Viral reexpression of Fhit kills 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 the hypothesis 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 kcatand increases as large as 30-fold in KM. Nonetheless, when mutant FHIT genes were expressed in two human cancer cell lines containing FHIT deletions, reductions in apoptotic activity correlated exclusively with KM. Mutants with 2- and 7-fold increases in KMsignificantly reduced apoptotic indices, whereas the mutant with a 30-fold increase in KMretained little cellular function. These data indicate that the proapoptotic function of Fhit is limited by substrate binding and is unrelated to substrate hydrolysis.