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 (H460/FHIT). A significant inhibition of cell growth was observed in H460/FHIT 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.

Figure 1

(A) In situ TUNEL staining of clone 2.I. Condensed and fragmented nuclei incorporated the fluorescein-labeled dUTP, thus indicating apoptosis-induced DNA strand breaks. (B) Double-immunofluorescent labeling of Fhit-reexpressing cells. Apoptotic nuclei are FITC-labeled. Fhit cytoplasmic expression is detected by rhodamine-conjugated polyclonal anti-Fhit antibody.

Figure 2

FACScan analysis of cell cycle and apoptosis. Cytofluorimetric histograms representative of different experiments are shown. Positive controls were 20,000-rad γ-irradiated cells (60–70%). MFI, mean fluorescence intensity.

Figure 3

Immunocytochemical analysis of Fhit expression in clone 2.3 cultured without (A) and with (B) FBS. Cytospins were fixed in 10% buffered formalin and stained with anti-Fhit polyclonal antibody at 1:500 dilution. Fhit immunoreactivity was present in the cytoplasm of transfected cells.

Figure 4

Western blot analysis of Bcl-2 (A), Bax (B), Bak (C), and p21^waf1 (D) in the H460 cell line and Fhit transfected clones 2.I and 2.3. Expression levels of Bak and p21^waf1 proteins were normalized on actin expression in the same lane. FBS-, cells cultured without FBS.

Figure 5

Expression of p21^waf1 mRNA in the control H460 cell line (lane 1), in FHIT transfected clones 2.I and 2.3 (lanes 2 and 3), and in the small-cell lung cancer cell line AFL, which lacks endogenous Fhit expression (lane 4). Total RNA (20 μg) was analyzed by Northern blotting, with successive hybridization to p21^waf1 and GAPDH cDNA probes.