[CANCER RESEARCH57. 504-512, February 1. 1997] ABSTRACT The FHIT gene, encoded by 10 exons in a 1.1-kb transcript, encom passes approximately 1 Mb of genomic DNA, which includes the heredi tary RCC t(3;8) translocation break at 3p14.2, the FRA3B common fragile region, and homozygous deletions in various cancer-derived cell lines. Because some of these genetic landmarks (e.g., the t(3;8) break between untranslated FHIT exons 3 and 4, a major fragile region that Includes a viral integration site between exons 4 and 5, and cancer cell homozygous deletions in intron 5) do not necessarily affect coding exons and yet apparently affect expression of the gene product, we examined the FHIT locus and its expression in detail in more than 10 tumor-derived cell lines to clarify mechanisms underlying aberrant expression. We observed some cell lines with apparently continuous large homozygous deletions, which includedoneormorecodingexons celllineswithdiscontinuousdeletions, some of which included or excluded coding exons and cell lines that exhibited heterozygous and/or homozygous deletions, by Southern blot analysis for the presence of specific exons. Most of the cell lines that exhibited genomic alterations showed alteration of FHIT transcripts and absence or diminution of Fhlt protein. INTRODUCTION The FHJT gene, spanning the t(3;8) (3p14.2;q24) RCC5-associated chromosome translocation, the FRA3B common fragile region, and homozygous deletions in cancer cells, is a potential tumor suppressor gene (1) that encodes the human diadenosine triphosphate hydrolase (2). The in vivo function of this enzyme, which produces ADP and AMP in vitro from the diadenosine substrate, is not known. The 1.1-kb FHIT cDNA is encoded by 10 small exons distributed over a genomic locus of about 1 Mb; the t(3;8) break falls between untrans lated 5' exons 3 and 4. The gene overlaps homozygous deletions observed in various cancer cell lines, which often include exon 5, the first protein-coding exon, and usually include portions of the >200-kb intron 5 (1, 3). The FHIT gene is apparently expressed at low levels in most adult tissues but is absent or undetectable by Northern blot in some tumor cell lines with deletions (1). We have previously investigated the integrity of the FHJT transcript in small tissue samples from primary tumors by RT from total RNA or mRNA followed by PCR amplifi cation, using primers in exons 1 and 10; this amplification product Received 1017/96;accepted 12/19/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by United States Public Health Service Grants CA51083, CAll 124,andCA39860.KimmelCancerCenterCoreGrantCA56336, and a gift from R. R. M. Carpenter Ill and Mary K. Carpenter. 2 Present address: Department of Genetics and Pathology, Medical Academy, Al. Powstancow Wlkp. 72, 70-111 Szczecin, Poland. 3 Present address: Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-33 Japan. Phone:011-81-298-77-2000; Fax:011-81-298-77-2024. 4 To whom requests for reprints should be addressed, at Kimmel Cancer Institute, BLSB, Room 1008, 233 South 10th Street, Philadelphia, PA 19107. Phone: (215) 503-4656; Fax: (215) 923-4498. 5 The abbreviations used are: RCC, renal cell carcinoma; SSCP, sIngle-strand confor mation polymorphism; RT, reverse transcription; STS, sequence-tagged site; SCC, aqua mous cell carcinoma; HNSCC, head and neck 5CC; GDB, Genome Data Base; LINE, long Interspersedelement YAC, yeastartificialchromosome;GST, glutathioneS-transferase. was then reamplified using primers nested inside the original primers in exons 2 and 9 (1, 4—6).These studies, in parallel with preliminary investigations of the integrity of the FHJT genomic locus, suggested that lesions in the FHJT genomic locus often resulted in shorter aberrant RT-PCR products and further suggested that aberrant FHJT RT-PCR products might be diagnostic of a DNA lesion in cases in which the FHIT genomic locus was not or could not be studied in detail. Such aberrant FHIT RT-PCR products have been observed in a variety of types of tumors and tumor cell lines, as have FHJT genomic lesions, and have been useful in suggesting which cancer cell lines are likely to exhibit alterations within the FHJT locus, although Thiagalingam et a!. (7) have found a lower frequency of involvement of FHIT in colon cancer xenografts and have cautioned that nested RT-PCR amplification can produce artifactual aberrant products. In ongoing detailed analyses of the FHJT locus, we have undertaken a study to correlate specific FHJT locus DNA lesions with their effects on RT-PCR products and Fhit protein expression. The intron-exon structure of the FHJT gene was determined, and intron-exon boundaries sequenced in order to amplify and sequence individual exons that show SSCPs. Ends of cosmids in the FHJT contig were sequence tagged (STSs) so that numerous probes through out the FHJT locus could be tested for homozygous deletion. Finally, rabbit antibodies specific for the Fhit protein were generated and used to study Fhit expression in cancer cell lines. MATERIALS AND METHODS Cells. Cancer-derived cell lines were obtained from American Type Cul tare Collection or were kindly donated by Drs. Edward Lattime (RCC cells), Dolly Huang (HK1, CNE1, and CNE2 nasopharyngeal carcinoma cells), Linda Cannizzaro (9944 and 9542 lymphoblastoidcell lines carrying the t(3;8) translocation), or other colleagues and were maintained as described (8). Cos/Fhit-Flag transient tmansfected cells were previously described (2). The DT36 cell line was established from a laryngeal SCC from a patient who had been treated for multiple head and neck tumors. DT36 cells used in this study had been subcultured between 10 and 20 times. RNA Extraction, RT, and RT-PCR Amplification. mRNA was isolated from cell lines and tissues after treatment with 4 M guanidinium isothiocyanate, followed by phenol-chloroform extraction and isopropanol precipitation, or total RNAs were prepared using the RNAzol kit (Tel-Test, Inc., Friendswood, Texas), according to the manufacturer' s instructions. RTwas performedin 30 @.d finalvolumeof 50 mMTris-HC1, pH 8.3, 75 nmi KC1,3 mMMgCl2,10nmiDli', 0.125 maieachdNTP,500 ng ofoligo-dT, 600 units of Moloney mm-incleukemia virus-reverse transcriptase (Life Technol ogies, Inc.), 40 units of RNasin (Promega), and 2 @sg of RNA. This reaction was incubated at 37°C for 90 mm and boiled for 5 mm. RT-PCR primers are listed inTable1. PCRAmplification.Theoligonucleotides forgenerating PCRproducts for agarose gel analysis or SSCP analysis were designed using the computer program Oligo 4.0 (National Biosciences) or were taken from the 0DB. PCR amplifications were carried out in 12.5 il final volume with 100 ng of genomic DNA template, 20 ng of primers, 10 mr@i Tris-HC1, pH 8.3, 50 m@i KC1,0.1 mg/mIgelatin, 1.5 mistMgCl2,0.2 mMeach dNTP, and0.5 units of Taq polymerase (ABI). The amplifications were performed in a Perkin-Elmer Cetus thermal cycler for 30 cycles of 94°C for 30 s (for denaturation), 57°C 504 Structure and Expression of the Human FHIT Gene in Normal and Tumor Cells1 Teresa Druck, Piotr Hadaczek,2 Tie.Bo Fu, Masataka Ohta,3 Zurab Siprashvili, Raffaele Baffa, Massimo Negrini, Kumar Kastury, Maria Luisa Veronese, David Rosen, Jay Rothstein, Peter McCue, Maria Grazia Cotticelli, Hiroshi Inoue, Carlo M. Croce, and Kay Huebner4 DepamnentsofMicrobiologyandlmmunology(TD., PH., T-B.F., MO., ZS., RB., MN., KK., M.LV., M.G.C., H.!., C.M.C., K.H.J, Otolaryngology(D.R., JR.], and Surgical Pathology (P. MI, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107 Research. on November 29, 2021. © 1997 American Association for Cancer cancerres.aacrjournals.org Downloaded from