FHIT gene alterations in head and neck squamous cell carcinomas.

Abstract

To determine whether the FHIT gene at 3pl4.2 is altered in head and neck squamous cell carcinomas (HNSCC), we examined 26 HNSCC cell lines for deletions within the FHIT locus by Southern analysis, for allelic losses of specific exons FHIT by fluorescence in situ hybridization (FISH) and for integrity of FHIT transcripts. Three cell lines exhibited homozygous deletions within the FHIT gene, 55% (15/25) showed the presence of aberrant transcripts, and 65% (13/20) Head and neck cancers represent 3% of all cancer in Western countries (1), and in some geographical regions such as India, the incidence is as high as 45% (2); 90-95% of these tumors are head and neck squamous cell carcinomas (HNSCC). HNSCC has a high mortality rate with a 5 year survival of 40%. Tobacco and alcohol have been recognized as etiological factors of these carcinomas, and the reported increase in the incidence of HNSCC by epidemiological studies is probably due to changes in consumption of these agents (2).Several regions of loss of heterozygosity (LOH) have been identified in HNSCC recently, including regions of 3p, 9p, llq, 13q, and 17p (3, 4). Among these loci, the 9p region presents the highest rate of genetic alteration at 75%, followed by alterations of 3p ranging between 45% and 55% (4, 5). LOH of 9p has been linked to the tumor suppressor gene, CDKN2 (6-8), whereas LOH on 3p has not yet been associated with specific genes.Deletions of the short arm of chromosome 3 are common in many human cancers, including sporadic and hereditary renal carcinomas, small-cell lung carcinomas (SCLC) (9, 10) and non-small-cell lung carcinomas (NSCLC) (11, 12) and carcinomas of the breast and cervix (13,14). In this regard, it is interesting to note that the most common fragile site in humans, FRA3B, is at 3pl4.2 (for reviews, see refs. 15 Recently, we have cloned the FHIT gene, at 3pl4.2, which encompasses the FRA3B fragile site, is disrupted by the t(3;8) chromosomal translocation observed in a family with renal cell carcinoma, and spans a region commonly deleted in cancer cell lines (20,21). We have also shown that "80% of SCLC and 40% of NSCLC express aberrant FHIT transcripts (22), suggesting that the FHIT gene is a frequent target for alteration in lung tumors. SCLC and HNSCC present the same type of histology, share major etiological factors such as tobacco (23,24), and exhibit similar regions of LOH of the short arm of chromosome 3. To determine whether the FHIT gene is also disrupted and aberrantly transcribed in HNSCCs, we studied 26 early passage HNSCC-derived cell lines. MATERIALS AND METHODS RNA Extraction and Reverse Transcription-PCR (RT-PCR). Total RNA was extracted from cell lines using RNAzol (Tel-Test, Friendswood, TX) and cDNA synthesized from 1 ,ug of total RNA. RT was performed in a 20 ,ul volume of 1 x first strand buffer (GIBCO), 10 mM DTT, 500 mM of each dNTP, 0.3 mg/ml random primers (GIBCO), and 300 units of SuperScript II (GIBCO) reverse transcriptase. The samples were first incubated 5 min at 65°C and then at 37°C ...

Aberrant splicing of the TSG101 and FHIT genes occurs frequently in multiple malignancies and in normal tissues and mimics alterations previously described in tumours

“…It remains to be seen whether truncated TSG101 and FHIT transcripts are important in tumour progression in a wide variety of malignancies or whether these truncations are merely the consequence of altered mRNA splicing ®delity that occurs more commonly in tumour cells. In the case of FHIT, FISH and Southern blot analysis have shown that in some tumours and cell lines the same exons that are missing from the truncated transcripts appear to be deleted in genomic DNA Virgilio et al, 1996). Despite this, it has not been possible to clone any of the putative genomic breakpoints and so the evidence that FHIT is the real target of the genomic deletions seen at the fragile site locus that encompasses this gene is lacking.…”

Frequent breakpoints in the region surrounding FRA3B in sporadic renal cell carcinomas

“…The noncoding exon 4 of the FHIT gene is distal to the marker D3S1481 (Figure 1). Aberrant transcripts of this gene were detected in several di erent digestive tract cancers (50%) , lung tumors both SCLC (80%) and NSCLC (40%) (Sozzi et al, 1996a), in 57% of Merkel cell carcinoma (Sozzi et al, 1996b), in 20% of breast cancers , and more recently in 55% of squamous cell carcinoma of the head and neck (Virgilio et al, 1996). Sequence analysis of the RT-PCR rescued transcripts of the FHIT gene revealed that in all cases complete exons were lost.…”

FHIT gene alterations in esophageal cancer and ulcerative colitis (UC)

“…Representational di erence analysis demonstrated a frequent site of homozygous deletion at the FHIT locus in esophageal, gastric and colorectal carcinomas (Lisitsyn et al, 1994). Several recent papers have reported alterations of FHIT in several tumor types, frequently characterized by abnormal-sized transcripts (Virgilio et al, 1996;Sozzi et al, 1996;Yanagisawa et al, 1996;Mao et al, 1996;Ohta et al, 1996;Druck et al, 1997). However, some investigators have found a completely normal FHIT transcript and coding DNA sequence in colorectal carcinoma cell lines and xenografts, suggesting either a di erent inactivation mechanism or a minor role for FHIT in the genesis of colorectal cancer (Thiagalingam et al, 1996).…”