Abnormal structure, transcription and expression of the FHIT gene in lung cancer cell lines
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文摘
The most common genetic events in lung carcinogenesis are deletions on chromosome 3p which are felt to contribute to the inactivation of one or more tumour suppressor genes (TSG) at three distinct regions (3p14, p21 and p25). Allelic losses at 3p frequently occur in many tumours such as head and neck, breast and kidney suggesting the involvement of common TSG in these solid tumours. The FHIT gene at p14.2, spanning the t(3;8) renal cell carcinoma associated chromosomal translocation” the FRA3B common fragile site and homozygous deletions in cancer cell lines is a candidate tumour suppressor gene. It encodes the human diadenosine triphosphate hydrorase which produces ADP and AMP from the diadenosine substrate. We have reported that 80 % of SCLC and 40 % of NSCLC primary tumours express aberrant FHIT transcripts as well as LOH within the FHIT locus, suggesting that the FHIT gene is a frequent target for alterations in lung tumours. In order to correlate specific FHIT locus DNA lesions with their effects on transcription products and protein expression, we studied 11 lung cancer cell lines of different types by Southern blot analysis, reverse transcription-PCR, Western blot analysis and immunocytochemistry. Three cell lines (CALU1, CALU3, H460) showed abnormal restriction patterns after hybridization with cDNA and specific cosmid probes consisting in deletions or rearrangements involving exons 3, exon 4 and intron 5 as well as a more distal region surrounding exon 6 and 7, respectively. The RT-PCR analysis of RNAs revealed absence of normal FHIT transcript in three cell lines (CALU3, H460, AFL1). CALU3 showed lack of exon 4 only, whereas in H460 and AFL1 multiple abnormal transcripts were present and consisted in losses of exons 3 or 4 through exon 8 and 9. In six samples FHIT products of both normal and abnormal size were found and two cell lines showed the wild-type transcript only. Cloning and sequencing of the apparently normal sized transcripts in several cell lines revealed a mixture of different transcripts lacking crucial coding exons such as 5 or 8 often also exhibiting basepairs insertions of similar length. This multiplicity of genetic lesions may be explained by the fact that FHIT is located within the fragile 3B region which, by definition, is highly susceptible to breakage induced by carcinogens such as those contained in tobacco smoke. Western blot analysis, using a rabbit polyclonal antibody against the GST-FHIT fusion protein revealed that indeed 9 of the 11 cell lines were unable to produce the FHIT protein and immunocytochemistry performed on cytospins displayed that the only two cell lines positive in Western blot (CALU3, H146) also displayed a clear cytoplasmatic immunostaining. These data indicate that, due to the complexity of FHIT rearrangements, immunocytochemistry may be the best way to assess the level of involvement of FHIT in lung tumour and cell lines and that loss of function of FHIT is a crucial and frequent event in lung cancer.