title: Designed FHIT alleles establish that Fhit-induced apoptosis in cancer cells is limited by substrate binding.
abstract: 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 andor 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) and increases as large as 30-fold in K(M). Nonetheless, when mutant FHIT genes were expressed in two human cancer cell lines containing FHIT deletions, reductions in apoptotic activity correlated exclusively with K(M). Mutants with 2- and 7-fold increases 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.
authors.affiliation: Genetics and Molecular Biology Program, Kimmel Cancer Center, Philadelphia, PA 19107, USA.
authors.forename: Francesco
authors.initials: F
authors.lastname: Trapasso
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Krakowiak
authors.forename: Rossano
authors.initials: R
authors.lastname: Cesari
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Arkles
authors.forename: Sai
authors.initials: S
authors.lastname: Yendamuri
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ishii
authors.forename: Andrea
authors.initials: A
authors.lastname: Vecchione
authors.forename: Tamotsu
authors.initials: T
authors.lastname: Kuroki
authors.forename: Pawel
authors.initials: P
authors.lastname: Bieganowski
authors.forename: Helen C
authors.initials: HC
authors.lastname: Pace
authors.forename: Kay
authors.initials: K
authors.lastname: Huebner
authors.forename: Carlo M
authors.initials: CM
authors.lastname: Croce
authors.forename: Charles
authors.initials: C
authors.lastname: Brenner
mesh_terms: D017766:Acid Anhydride Hydrolases; D000483:Alleles; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D005434:Flow Cytometry; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D009369:Neoplasms / Q000235:genetics / Q000473:pathology*; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured; D000099170:Bis(5'-adenosyl)triphosphatase
chemical_list: D017766:Acid Anhydride Hydrolases; D009363:Neoplasm Proteins; D000099170:Bis(5'-adenosyl)triphosphatase