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 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.
Trapasso
Francesco
F
Genetics and Molecular Biology Program, Kimmel Cancer Center, Philadelphia, PA 19107, USA.
Krakowiak
Agnieszka
A
Cesari
Rossano
R
Arkles
Jeffrey
J
Yendamuri
Sai
S
Ishii
Hideshi
H
Vecchione
Andrea
A
Kuroki
Tamotsu
T
Bieganowski
Pawel
P
Pace
Helen C
HC
Huebner
Kay
K
Croce
Carlo M
CM
Brenner
Charles
C
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
D017766:Acid Anhydride Hydrolases; D009363:Neoplasm Proteins; D000099170:Bis(5'-adenosyl)triphosphatase