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FHIT Gene Alterations in Head and Neck Squamous Cell Carcinomas (Fragile Site/Ap3a Hydrolase/Chromosome 3Pl4.2) LAURA VIRGILIO*, MICHELE Shustert, SUSANNE M

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FHIT Gene Alterations in Head and Neck Squamous Cell Carcinomas (Fragile Site/Ap3a Hydrolase/Chromosome 3Pl4.2) LAURA VIRGILIO*, MICHELE Shustert, SUSANNE M Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9770-9775, September 1996 Medical Sciences This contribution is part of the special series of Inaugural Articles by members of the National Academy of Sciences elected on April 30, 1996. FHIT gene alterations in head and neck squamous cell carcinomas (fragile site/Ap3A hydrolase/chromosome 3pl4.2) LAURA VIRGILIO*, MICHELE SHUSTERt, SUSANNE M. GOLLINt, MARIA LuISA VERONESE*, MASATAKA OHTA*, KAY HUEBNER*, AND CARLO M. CROCE* *Kimmel Cancer Institute and Kimmel Cancer Center, Jefferson Medical College, Philadelphia, PA 19107; and tDepartment of Human Genetics, University of Pittsburgh Graduate School of Public Health and the University of Pittsburgh Cancer Institute, Pittsburgh, PA 15261 Contributed by Carlo M. Croce, July 18, 1996 ABSTRACT To determine whether the FHIT gene at 3p25. All three regions present a loss of heterozygosity in 3pl4.2 is altered in head and neck squamous cell carcinomas 45-50% of the cases both at the precancerous and cancerous (HNSCC), we examined 26 HNSCC cell lines for deletions stages, with the exception of 3p2l.3, that shows a lower within the FHIT locus by Southern analysis, for allelic losses incidence (30%) in dysplastic lesions (18). Furthermore, pa- of specific exons FHIT by fluorescence in situ hybridization tients with dysplastic lesions with LOH either of 9p or 3p are (FISH) and for integrity ofFHIT transcripts. Three cell lines at higher risk of developing tumors. exhibited homozygous deletions within the FHIT gene, 55% Recently, we have cloned the FHIT gene, at 3pl4.2, which (15/25) showed the presence ofaberrant transcripts, and 65% encompasses the FRA3B fragile site, is disrupted by the t(3;8) (13/20) showed the presence of multiple cell populations with chromosomal translocation observed in a family with renal cell losses of different portions of FHIT alleles by FISH of FHIT carcinoma, and spans a region commonly deleted in cancer cell genomic clones to interphase nuclei. When the data obtained lines (20, 21). We have also shown that "80% of SCLC and by FISH and by reverse transcriptase-PCR analyses are 40% of NSCLC express aberrant FHIT transcripts (22), sug- combined, 22 of26 cell lines showed alterations of at least one gesting that the FHIT gene is a frequent target for alteration allele of the FHIT gene. Our data indicate that the FHIT gene in lung tumors. SCLC and HNSCC present the same type of is disrupted in HNSCCs and hence, loss ofFHIT function may histology, share major etiological factors such as tobacco (23, be important in the development and/or progression of head 24), and exhibit similar regions of LOH of the short arm of and neck cancers. chromosome 3. To determine whether the FHIT gene is also disrupted and aberrantly transcribed in HNSCCs, we studied Head and neck cancers represent 3% of all cancer in Western 26 early passage HNSCC-derived cell lines. 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). MATERIALS AND METHODS HNSCC has a high mortality rate with a 5 year survival of 40%. RNA Extraction and Reverse Transcription-PCR (RT- Tobacco and alcohol have been recognized as etiological PCR). Total RNA was extracted from cell lines using RNA- factors of these carcinomas, and the reported increase in the zol (Tel-Test, Friendswood, TX) and cDNA synthesized incidence of HNSCC by epidemiological studies is probably from 1 ,ug of total RNA. RT was performed in a 20 ,ul volume due to changes in consumption of these agents (2). of 1 x first strand buffer (GIBCO), 10 mM DTT, 500 mM of Several regions of loss of heterozygosity (LOH) have been each dNTP, 0.3 mg/ml random primers (GIBCO), and 300 identified in HNSCC recently, including regions of 3p, 9p, llq, units of SuperScript II (GIBCO) reverse transcriptase. The 13q, and 17p (3, 4). Among these loci, the 9p region presents samples were first incubated 5 min at 65°C and then at 37°C the highest rate of genetic alteration at 75%, followed by for 60 min. One microliter of cDNA was used for PCR alterations of 3p ranging between 45% and 55% (4, 5). LOH amplification with primers 5U2 and 3D2, from FHIT exons of 9p has been linked to the tumor suppressor gene, CDKN2 1 and 10, respectively (21), in a final reaction volume of 25 (6-8), whereas LOH on 3p has not yet been associated with ,ul containing 0.8 jig of each primer, 100 mM of each dNTP, specific genes. lx reaction buffer (Boehringer Mannheim), and 1.25 units Deletions of the short arm of chromosome 3 are common in Taq polymerase (Boehringer Mannheim). The PCR con- many human cancers, including sporadic and hereditary renal sisted of an initial denaturation step at 95°C for 3 min, carcinomas, small-cell lung carcinomas (SCLC) (9, 10) and followed by 25 cycles of 30 s at 94°C, 30 s at 62°C, and 40 s non-small-cell lung carcinomas (NSCLC) (11, 12) and carci- at 72°C. One microliter of the first PCR amplification nomas of the breast and cervix (13, 14). In this regard, it is product was used for a second PCR amplification reaction interesting to note that the most common fragile site in with primers 5U1 and 3D1, in FHIT exons 3 and 10 (21). The humans, FRA3B, is at 3pl4.2 (for reviews, see refs. 15 and 16), nested PCR amplification was for 30 cycles under the same and if, as has been suggested by Yunis and Soreng (17), fragile conditions as the first, and PCR products were resolved on sites are targeted areas of genetic lesions in human tumors, a 1.5% agarose gel. then 3pl4.2 may be the site of a gene involved in several types of cancers. LOH of alleles on the short arm of chromosome 3 in oral it has Abbreviations: HNSCC, head and neck squamous cell carcinomas; appears to be an early event carcinomas, since SCLC, small-cell lung carcinoma; NSCLC, non-small-cell lung carci- been reported in oral dysplastic lesions at the precancerous noma; LOH, loss of heterozygosity; FISH, fluorescence in situ hybrid- stage (18, 19). Three distinct regions of LOH on chromosome ization; RT-PCR, reverse transcription-PCR; UPCI, University of arm 3p have been identified in HNSCC, 3pl3-14, 3p2l.3, and Pittsburgh Head and Neck Tissue Culture Laboratory. 9770 Downloaded by guest on October 1, 2021 Medical Sciences: Virgilio et al. Proc. Natl. Acad. Sci. USA 93 (1996) 9771 DNA Sequencing. DNA bands corresponding to the normal 5 and flanking regions, and cosmid 05#7, spanning exons 9 and and abnormal sized FHIT transcripts were excised from the gel 10, were labeled by nick translation with biotin-16-dUTP and and the DNA purified from the agarose using the QIA quick digoxigenin-11-dUTP, respectively. The FISH probe nick gel extraction kit (Qiagen, Chatsworth, CA). Depending on the translation, chromosomal in situ suppression hybridization, amount recovered, the DNA fragment was either directly and detection reactions were carried out as described (26), sequenced, using the primer SU1, or was cloned in the vector, except that unincorporated nucleotides were removed from pCRII (Invitrogen), and individual clones sequenced. Se- the probe by ethanol precipitation. In addition to protease quencing was by dideoxynucleotide termination reaction pretreatment, the slides were aged in 2 x standard saline citrate chemistry for sequence analysis on the Applied Biosystems (SSC) for 30 min at 37°C, and treated with RNase A for 30 min models 373A and 377 DNA sequencers. at 37°C. Posthybridization washes were carried out three times Southern Blot Hybridization. Genomic DNA was purified for 3 min each in 50% formamide/2X SSC at 45°C, and 2x by standard phenol/chloroform extraction; 8 ,ug of DNA was SSC at 37°C. Detection was accomplished using 5 ,ug/ml of digested with BamHI, resolved on a 0.8% agarose gel, and fluorescein isothiocyanate-avidin and rhodamine-conjugated transferred to membrane by standard protocol (25). The anti-digoxigenin. Approximately 200 nuclei were counted for cosmids B4, c76, c63, and 05#1, covering portions of the FHIT each sample, along with a concurrent normal peripheral blood locus (21), were labeled by random primer extension (Strat- leukocyte control slide. Only subpopulations that represented agene), preannealed to sheared human genomic DNA, and at least 10% of the total number of nuclei were considered in hybridized to the filters, as described (22). the evaluation. Cell Lines. Cell lines were developed in the University of Pittsburgh Head and Neck Tissue Culture Laboratory (UPCI) (J. K. Reddy, K. M. Rossie, C. M. Lese, R. L. Wagner, J. T. RESULTS Johnson, and S.M.G., unpublished data). Briefly, primary Aberrant Transcripts in HNSCC Cell Lines. Twenty-six cell tumors biopsies were minced and plated in minimal essential lines established from HNSCC tumors were analyzed by medium supplemented with penicillin, streptomycin, neomy- RT-PCR for the presence of aberrant FHIT transcripts. Cell cin, L-glutamine, nonessential amino acids, and 10-15% fetal lines were all at relatively early passage in culture, mostly bovine serum. The individual passages of the cell lines are within the first 10-20 passages. Sites and stages of individual specified in the text. tumors are given in Table 1. RNA was isolated, reverse Fluorescence in Situ Hybridization (FISH) Analysis. Cells transcribed, and amplified by PCR using primers derived from were harvested for molecular cytogenetic studies by standard exons 1 and 10 followed by amplification with nested primers techniques. Briefly, cell cultures were incubated in the pres- in exons 3 and exon 10 of the FHIT gene (21, 22).
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