The candidate tumor suppressor gene, FHIT, encompasses the common human chromosomal fragile site at 3p14.2, the hereditary renal cancer translocation breakpoint, and cancer cell homozygous deletions. Fhit hydrolyzes dinucleotide 5,5ٟ-P 1 ,P 3 -triphosphate in vitro and mutation of a central histidine abolishes hydrolase activity. To study Fhit function, wild-type and mutant FHIT genes were transfected into cancer cell lines that lacked endogenous Fhit. No consistent effect of exogenous Fhit on growth in culture was observed, but Fhit and hydrolase ''dead'' Fhit mutant proteins suppressed tumorigenicity in nude mice, indicating that 5,5ٟ-P 1 ,P 3 -triphosphate hydrolysis is not required for tumor suppression.The structure and expression of the FHIT gene encompassing the FRA3B common fragile site frequently are altered in primary or cultured esophageal, head and neck, lung, gastric, breast, and cervical carcinomas (1-8). Structural alterations tend to be because of deletion within both FHIT alleles, resulting in loss of exons and concomitant absence of full-length FHIT transcript and protein (ref. 6; for review, ref. 9). It has been argued that the FHIT gene may be altered in cancer cells simply because it encompasses the fragile region and is likely to be very susceptible to breakage (7). We agree that the locus is highly susceptible to carcinogen damage, explaining why deletion is much more frequent than point mutation in the gene, but we argue that loss of Fhit function provides a selective advantage for the tumor cell; otherwise, frequent expansion of the deleted FHIT clones in tumors and tumor-derived cell lines would be difficult to explain.Fhit-related proteins have been found in mammals and yeasts (1, 10, 11) and constitute a branch of the histidine triad (HIT) superfamily (12). The Fhit branch includes the Schizosaccharomyces pombe diadenosine tetraphosphate hydrolase [dinucleoside 5Ј,5ٞ-P 1 ,P 4 -tetraphosphate (Ap 4 A) hydrolase] (10) to which Fhit is similar. Barnes et al. (13) have shown that Fhit behaves in vitro as a typical dinucleoside 5Ј,5ٞ-P 1 ,P 3 -triphosphate (Ap 3 A) hydrolase (EC 3.6.1.29); site-directed mutagenesis of FHIT demonstrated that the conserved histidines are required for full activity, and the central histidine of the triad is essential for Ap 3 A hydrolase activity.To investigate mechanisms for a selective growth advantage of Fhit negative tumors, we have prepared vectors for expression of Fhit in cancer-derived cells and have examined the phenotypes of the Fhit-expressing clones relative to the Fhit negative parental cells. To determine if the in vitro enzymatic activity was associated with a role in tumor suppression, the hydrolase ''dead'' mutant gene, FHITH96N, with the central histidine codon of the HIT changed to an asparagine codon, also was expressed in Fhit negative cancer cells. MATERIALS AND METHODSCells. The MKN74 cell line (kindly provided by Eiichi Tahara, University of Hiroshima, Japan), was derived from a gastric carcinoma (14) and forms tumors rapidly ...

Alteration of the FHIT (fragile histidine triad) gene occurs as an early and frequent event in lung carcinogenesis. FHIT gene transfer into lung cancer cell line H460 lacking Fhit protein expression resulted in reversion of tumorigenicity. To gain insight into the biological function of FHIT, we compared the H460 cell line with its Fhit transfectants (H460͞FHIT). A significant inhibition of cell growth was observed in H460͞FHIT cells. The analysis of apoptosis by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed a high rate of apoptosis-induced DNA strand breaks in stable clones. In situ results were confirmed by FACScan analysis that showed an apoptotic rate of 44-47% compared with a 15% level in the control H460 cells. Analysis of cell cycle-phase distribution indicated a significant G 0 ͞G 1 arrest and the presence of a sub-G 1 peak in the stable clones. No significant changes in Bcl2, BclX, and Bax protein expression level were observed in the transfected clones as compared with the control H460 cells whereas a 2-fold increase in Bak protein levels was noticed. An increased level of p21 waf protein paralleled by an up-regulation of p21 waf transcripts also was found in Fhit-expressing clones compared with the H460 cell line. No differences in p53 levels were observed in the same cells, suggesting a p53-independent effect. These data suggest that the observed growth-inhibitory effect in FHIT-reexpressing cells could be related to apoptosis and cell cycle arrest and link the tumor-suppressor activity of FHIT to its proapoptotic function.The FHIT (fragile histidine triad) gene (1), at 3p14.2, is a frequent target of deletions associated with abnormal RNA and protein expression in primary tumors and cell lines of lung, head and neck, kidney, cervix, and breast cancer (2-6). Stable FHIT-transduced clones expressing exogenous wild-type Fhit, isolated after transfection of various epithelial cell lines carrying inactivated endogenous Fhit, show reduced colonyformation efficiency in vitro and inhibition of tumor development in nude mice, indicating that FHIT acts as a tumorsuppressor gene (7). The Fhit protein is a diadenosine triphosphate (Ap 3 A) hydrolase belonging to the histidine triad superfamily (HIT) of nucleotide-binding proteins (8). Our observation that the His(96)Asn mutant, lacking hydrolytic activity, still inhibits tumor formation in vivo (7) suggests that the tumor-suppressing function of Fhit is not related to catalysis of nucleotide substrates. However, the biological mechanism of FHIT activity and the cellular pathways associated with its tumor-suppressor function are not known. Crystallographic studies suggested that Ap 3 A nucleotide binding is crucial for Fhit biological activity and that enzymesubstrate complexes may be a signaling form (9). Interestingly, it has been reported that apoptosis in human cultured cells is associated with a decrease of free Ap 3 A levels (10).To study a possible involvement of FHIT in cell growth control and apoptosis, we...

Literature on the cytogenetics of dermatofibrosarcoma protuberans (DFSP) is limited; only 10 cases with chromosome aberrations have been reported. They are karyotypically characterized by the presence of supernumerary ring(s), either as the sole cytogenetic abnormality or together with a few additional structural or numerical changes. We report the cytogenetic and fluorescence in situ hybridization (FISH) analysis of three new DFSP, one primary and two recurrent tumors. In two cases we found a supernumerary ring as the sole change, whereas the third had two copies of a marker chromosome and monosomy of chromosome 22. Sequences of chromosomes 17 and 22 were identified by FISH in the supernumerary rings and in the markers. The fluorescence pattern suggested that additional sequences were present in the two rings, but showed that the marker chromosomes were entirely painted by chromosome 17 and 22 probes. The findings indicate that juxtaposition and/or amplification of chromosome 17 and 22 sequences could be crucial in the pathogenesis of DFSP.