The tumor-suppressor gene FHIT is involved in the regulation of apoptosis and in cell cycle control

Abstract

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...

Hint, Fhit, and GalT: Function, Structure, Evolution, and Mechanism of Three Branches of the Histidine Triad Superfamily of Nucleotide Hydrolases and Transferases

“…Though the kinetics of FHIT inactivation in the lung are inconsistent with this hypothesis (loss of Fhit protein occurs earlier and more frequently than TP53 mutation (56)), it was critically important to determine whether re-expression of the FHIT gene in fhit-cancer cell lines suppressed tumorigenicity. This has been demonstrated convincingly in several experimental systems (59)(60)(61)(62)(63)(64)(65)(66) and has been recently reviewed (67).…”

“…Because loss of FHIT in development of lung cancer occurs in preneoplastic lesions (56) and FHIT reexpression in fhit-/-cells suppresses tumor formation by induction of apoptosis (60,61,63,64,66), introduction of FHIT-expressing viruses has been explored as a preventative strategy in the NMBA-tumor induction model. Remarkably, administration of FHIT via viral vectors shortly after NMBA administration prevents tumor development (65), presumably by killing the cells that suffer their second hit at fhit.…”

“…Though FHIT re-expression in fhit-human or mouse cells induces death by apoptosis (60,61,63,64,66), relatively little can be said about the cellular pathway through which Fhit acts. There are three clues, however.…”

Frequent allelic deletion at the FHIT locus associated with p53 overexpression in squamous cell carcinoma subtype of Taiwanese non-small-cell lung cancers

“…The reported link of FHIT with apoptotic regulation (Ji et al, 1999;Sard et al, 1999) led us to examine its connection to the status of the p53 protein, a well-characterised antiproliferative factor, which induces growth arrest and apoptosis in response to various stimuli (Prives and Hall, 1999). It was previously indicated that the genetic changes at the FHIT locus may be correlated with p53 mutations (Burke et al, 1998;Marchetti et al, 1998;Chang et al, 2003), while other investigators found no statistically significant correlation between FHIT and p53 abnormalities in NSCLCs (Nelson et al, 1998;Geradts et al, 2000).…”

The role of human papillomavirus type 16 and the fragile histidine triad gene in the outcome of cervical neoplastic lesions

“…For cervical neoplasia, the chromosomal region of particular interest is 3p14.2 because it includes two known HPV16 integration sites (Thorland et al, 2000;Corbin et al, 2002) as well as the most commonly disrupted genomic fragile site, FRA3B (Glover and Stein, 1988). This site is sensitive to environmental carcinogens (Schantz et al, 2000) and is located between intron 3 and intron 7 of a putative tumour suppressor gene, fragile histidine triad (FHIT), which has proapoptotic activity (Ji et al, 1999;Sard et al, 1999;Dumon et al, 2001;Roz et al, 2002). One HPV insertion site is located in intron 4 and the other is centromeric to the FHIT gene.…”