A 200-300 kb region of chromosome 3p14.2, including the fragile site locus FRA3B, is homozygously deleted in multiple tumor-derived cell lines. Exon amplification from cosmids covering this deleted region allowed identification of the human FHIT gene, a member of ther histidine triad gene family, which encodes a protein with 69% similarity to an S. pombe enzyme, diadenosine 5', 5''' P1, P4-tetraphosphate asymmetrical hydrolase. The FHIT locus is composed of ten exons distributed over at least 500 kb, with three 5' untranslated exons centromeric to the renal carcinoma-associated 3p14.2 breakpoint, the remaining exons telomeric to this translocation breakpoint, and exon 5 within the homozygously deleted fragile region. Aberrant transcripts of the FHIT locus were found in approximately 50% of esophageal, stomach, and colon carcinomas.

To determine the role of the FHIT gene, which encompasses the fragile site at 3p14.2, we analyzed 59 tumors of the small cell and non-small cell type by reverse transcription of FHIT mRNA, followed by PCR amplification and sequencing of products. Allelic losses affecting the gene were evaluated by microsatellite polymorphism analysis and genomic alterations by hybridization using cDNA and genomic probes. Small cell lung tumors (80%) and non-small cell lung cancers (40%) showed abnormalities in RNA transcripts of FHIT, and 76% of the tumors exhibited loss of FHIT alleles. Abnormal lung tumor transcripts lack two or more exons of the FHIT gene. Small cell lung cancer tumors and cell lines were analyzed by Southern blotting and showed rearranged BamHI fragments. These data suggest a critical role of the FHIT gene in lung carcinogenesis.

Human Fhit (fragile histidine triad) protein, encoded by the FHIT putative tumor suppressor gene, is a typical dinucleoside 5',5"'-P1,P3-triphosphate (Ap3A) hydrolase (EC 3.6.1.29) on the basis of its enzymatic properties we report here. Ap3A is the preferred substrate among ApnA (n = 3-6), and AMP is always one of the reaction products. Mn2+ and Mg2+ are equally stimulatory, while Zn2+ is inhibitory with Ap3A as the substrate. Values of the K(m) for Ap3A and Ap4A are 1.3 and 4.6 microM, respectively. Values of the specificity constant, kcat/K(m), for Ap3A and Ap4A are 2.0 x 10(6) and 6.7 x 10(3) s-1 M-1, respectively, for a glutathione S-transferase (GST)-Fhit fusion protein. Site-directed mutagenesis of FHIT demonstrated that all four conserved histidines are required for full activity, and the central histidine of the triad is absolutely essential for Ap3A hydrolase activity. This putative tumor suppressor is the first evidence for a connection between dinucleotide oligophosphate metabolism and tumorigenesis. Also, Fhit is the first HIT protein in which the histidine residues have been demonstrated by mutagenesis to be critical for function.

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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 ...