In vitro inhibition of the enzymatic activity of tumor suppressor FHIT gene product by carcinogenic transition metals

FHIT (Fragile Histidine Triad) is a human tumor suppressor gene. The Fhit protein is believed to inhibit tumor growth by inducing apoptosis through interaction with diadenosine triphosphate (APA). The latter is first sequestered and eventually hydrolyzed by Fhit to ADP and AMP. Thus, the balance between the cellular APA level and Fhit enzymatic activity may affect cell death or survival. Increasing the Ap(3)A level, e.g., by inhibition of the enzyme, should prevent apoptosis and thus sustain tumorigenesis. To test if certain carcinogenic transition metals could inhibit the enzymatic activity of Fhit, purified human Fhit protein [30 nM in 1.25 mM poly(vinylpyrrolidone)], expressed in and isolated from E. coli, was incubated at pH 6.8 (50 mM HEPES buffer in 150 mM NaCl) with 120 muM Ap3A in the presence of 5 mM Mg(II) (activating cation) and 0-100 muM Ni(II), Cu(II), Zn(II), Cd(II), Co(II), Cr(III), As(III), or As(V). The reaction mixtures were analyzed by HPLC. The results revealed a strong inhibitory potential of Cu(II) [0.4], followed by Ni(II) [3.5] : Zn(II) [7.0] much greater than Cr(III) [73] > Cd(II) [98] much greater than Co(II) [432] [the numbers in brackets are IC50 values, muM]. As(III) and As(V) had no effect. As revealed by spectrophotometry, mass spectrometry, and gel electrophoresis, the exceptionally strong inhibition by Cu (II) was associated with Fhit dimerization through formation of a disulfide bond. The other metals and also H2O2 and NO did not cause the dimerization. Thus, the effect of Cu(II) must be due to its reaction with Cys-39 bearing the only thiol group in Fhit monomer. Since Cys-39 is not readily accessible in the Fhit molecule, the reaction is most likely facilitated by conformational changes which follow the coordination of Cu(II) by the surface histidines 35, 94, and/or 96. The observed inhibition of Fhit may be mechanistically involved in metal- mediated toxicity and carcinogenesis.

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