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: Francesco Trapasso, Agnieszka Krakowiak, Rossano Cesari, Jeffrey Arkles, Sai Yendamuri, Hideshi Ishii, Andrea Vecchione, Tamotsu Kuroki, Pawel Bieganowski, Helen C Pace, Kay Huebner, Carlo M Croce, Charles Brenner
• Journal: Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci U S A) Vol. 100 Issue 4 Pg. 1592-7 (Feb 18 2003) ISSN: 0027-8424 [Print] United States
• PMID: 12574506 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Neoplasm Proteins
• fragile histidine triad protein
• Acid Anhydride Hydrolases

Topics

• Acid Anhydride Hydrolases
• Alleles
• Apoptosis (physiology)
• Cell Line
• Flow Cytometry
• Humans
• Kinetics
• Mutation
• Neoplasm Proteins (genetics, isolation & purification, metabolism, physiology)
• Neoplasms (genetics, pathology)
• Substrate Specificity
• Tumor Cells, Cultured