Differential Expression of FEZ1/LZTS1 Gene in Lung Cancers and Their Cell Cultures1
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2292 Vol. 8, 2292–2297, July 2002 Clinical Cancer Research Differential Expression of FEZ1/LZTS1 Gene in Lung Cancers and Their Cell Cultures1 Shinichi Toyooka, Yasuro Fukuyama, NSCLC cell lines, it was strongly correlated to D8S261 Ignacio I. Wistuba, Melvyn S. Tockman, and LPL loci in SCLC cell lines. No mutation was found John D. Minna, and Adi F. Gazdar2 within cording region of FEZ1 by PCR-single-strand con- formational polymorphism. Hamon Center for Therapeutic Oncology Research [S. T., Y. F., Conclusions: We found differential FEZ1 expression in J. D. M., A. F. G.], and Departments of Pathology [A. F. G.], Internal Medicine [J. D. M.], and Pharmacology [J. D. M.], University of NSCLC and SCLC cell lines, and the absent expression in 3 Texas Southwestern Medical Center, Dallas, Texas 75390-8593; of 6 short-term cultures of NSCLC tumors. FEZ1 may be Department of Pathology, Pontificia Universidad Catolica de Chile, related to tumorigenesis of lung cancer. Santiago, Chile [I. I. W.]; and Molecular Screening Laboratory, H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612-9497 [M .S. T.] INTRODUCTION Lung cancer is the most common cause of cancer deaths in the United States (1) and on clinicopathological grounds is ABSTRACT divided into two major types, NSCLCs and SCLCs. The mo- Purpose: The FEZ1/LZTS1 (FEZ1) gene, located on lecular genetic changes in these two types of lung cancer are chromosome 8p22 (8p22), was identified recently as a can- very different, including specific patterns of allelic loss (2–5). didate tumor suppressor gene. Because loss of heterozygos- Frequent allelic loss at the short arm of chromosome 8p21–23 ity at 8p21–22 is a frequent event in lung cancers, we studied (8p21–23) region has been reported in lung cancers (2, 3, 6–9). FEZ1 alteration in short-term cultures of resected lung In microdissected lung cancers, we found LOH3 in 86% of cancer tumors and cell lines. SCLCs, 100% of squamous cell carcinomas, and 81% of ade- Experimental Design: We examined FEZ1 expression in nocarcinomas (7). Furthermore, deletions commence early dur- 17 non-small cell lung cancer (NSCLC), 19 small cell lung ing the multistage development at the hyperplasia/metaplasia cancer (SCLC) cell lines, and 6 pairs of short-term cultures stage in NSCLC tumor patients and in smokers without cancer of resected NSCLCs and accompanying nonmalignant bron- (7). The deletions may persist for several decades after smoking chial cells (NBECs) by reverse transcription-PCR and West- cessation (7). ern blotting. To investigate the mechanism for silencing, Allelic losses on the 8p have also been reported as a ,cells were cultured with 5-aza-2-deoxycytidine or tricho- frequent event in several kinds of cancer including prostate statin A. We screened for genomic mutations by PCR-single- colon, breast, head and neck, urinary bladder, hepatocellular, strand conformational polymorphism. and cholangiocarcinomas (10–21). These findings suggest the Results: Thirteen of 17 NSCLC (76%) and 3 of 19 presence of TSGs in this region. Functional evidence for this hypothesis was provided by chromosome transfer and microcell ؍ SCLC (16%) of cell lines showed absent expression (P 0.001). Of the paired NSCLC-NBEC cultures, 3 of 6 fusion experiments (22–25). showed loss of expression in tumor cell cultures. In the At least two major candidate TSGs have been identified on cell lines retaining expression, the amplicon products in 8p (26, 27). One of these, the FEZ1/LZTS1 (FEZ1) gene located SCLCs were more intense than those of NSCLCs and on 8p22 encodes a Mr 67,000 leucine-zipper protein (27). NBECs. Expression of FEZ1 was not restored by 5- Genomic mutation and loss of expression of FEZ1 was reported aza-2-deoxycytidine and trichostatin A. Although FEZ1 in some solid tumors including esophagus, prostate, and gastric expression was moderately correlated with loss of hetero- cancers (27, 28). In this study, we examined the expression and zygosity of specific microsatellite makers at 8p21–22 in mutation status of FEZ1 gene in NSCLC and SCLC cell lines, and also primary cultures of NSCLC tumors and corresponding NBECs. Received 2/18/02; revised 4/15/02; accepted 4/15/02. MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the Lung Cancer Cell Lines. Thirty six lung cancer cell lines payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to (17 NSCLC and 19 SCLC cell lines) and corresponding B lym- indicate this fact. phoblastoid lines (n ϭ 30) were established by us (29). The 17 1 Supported by grants from the University of Texas Specialized Program of Research Excellence in Lung Cancer (P50CA70907) and the Early Detection Research Network (5U01CA8497102), National Cancer In- stitute, Bethesda, MD. 2 To whom requests for reprints should be addressed, at Hamon Center 3 The abbreviations used are: LOH, loss of heterozygosity; SCLC, small for Therapeutic Oncology Research, University of Texas Southwestern cell lung cancer; NSCLC, non-small cell lung cancer; TSG, tumor Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390- suppressor gene; RT-PCR, reverse transcription-PCR; NBEC, nonma- 8593.Phone:(214) 648-4921;Fax:(214) 648-4940;E-mail:Adi.gazdar@ lignant bronchial cell; 5-Aza-CdR, 5-aza-2Ј-deoxycytidine; TSA, tricho- UTsouthwestern.edu. statin A; SSCP, single-strand conformational polymorphism. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2002 American Association for Cancer Research. Clinical Cancer Research 2293 Table 1 Primer sequences for PCR-SSCP Exon Forward primer Reverse primer Size (bp)a 15Ј-TGCTATGACCTCAGTCCCCTC-3Ј 5Ј-CCATTTTGGAGCTGGACTTGCC-3Ј 280 15Ј-GACGGGCTGCTGAGGTTTGG-3Ј 5Ј-ACTTACCCTTGCCAGCGACC-3Ј 218 25Ј-GCATGAGTCACCGCGGTCCTT-3Ј 5Ј-GGGTGTGACCAGCGGGTCCA-3Ј 284 25Ј-ACACAGCACCAGCAGCAGCTA-3Ј 5Ј-CTGCGCTGCAGCTTCTGGAGG-3Ј 298 25Ј-AGAAGCTGTTGGAGAGGGAGG-3Ј 5Ј-CAAACCCATGAGCCCTGTGTG-3Ј 375 35Ј-ACTCACCTCTTGGCACTCTGTC-3Ј 5Ј-CTTCTTGCGCTGCAGCTCATTC-3Ј 266 35Ј-CCTGCGCACCAAGGGCCTG-3Ј 5Ј-ACATGGCCACGTAGCTCTGCT-3Ј 344 35Ј-GGTGATTCAGTACCAGAAACAGC-3Ј 5Ј-AGAGGGGTCTGAATTGCTGAGC-3Ј 269 a Size, PCR product size in bp. NSCLC cell lines consisted of 13 adenocarcinomas, 2 squamous 5-Aza-CdR and TSA Treatment. Six lung cancer cell cell carcinomas, and 2 large cell carcinomas. Most NSCLC cell lines (4 cases of NSCLC and 2 cases of SCLC) with loss of lines were derived from primary tumors, and most SCLC cell lines FEZ1 expression were incubated in culture medium with 5-Aza- were from metastases. Cells cultures were grown in RPMI 1640 CdR and TSA each (30). Drug treatment was accomplished by (Life Technologies, Inc., Rockville, MD) supplemented with 5% adding reagents to the culture medium to final concentrations as fetal bovine serum and incubated in 5% CO2 at 37°C. follows: (a) 5-Aza-CdR, 2 g/ml; and (b) TSA, 150 and 300 nM. Primary Culture of Resected Lung Cancer and NBECs. Cells were treated with 5-Aza-CdR for 5 days and TSA for Six NSCLC primary cells (2 cases each of adenocarcinomas, 2 24–48 h. Medium were changed every 48 h for 5-Aza-CdR and squamous cell carcinomas, and 2 large carcinomas) and its every 24 h for TSA. corresponding NBECs were cultured. Primary cultures of re- Western Blot Analysis. A sample of protein (30 g) sected nonmalignant lung tissue were selected for culture and from the cell lysates were separated by SDS-PAGE in 10% placed in trypsin in the cold room for 22–24 h. The next day, the polyacrylamide gels and transferred to nitrocellulose mem- specimen was removed from the cold room and 1 ml of medium branes. The membranes were incubated first with primary anti- was added. The medium was MCDB1532ϩ, consisting of body to Fez1 protein kindly given by Drs. Carlo Croce and MCDB 153 basal medium (Sigma Chemical Company, St. Hideshi Ishii (Kimmel Cancer Center, Jefferson Medical Col- Louis, MO) supplemented with growth factors in a collagen- lege of Thomas Jefferson University, Philadelphia, PA) and then coated dish. Every day, fresh medium was added to the dish. For with rabbit polyclonal antibody as secondary antibody coupled primary lung tumor cell culture, small tumor pieces were enzy- to horseradish peroxidase, after which the membranes were matically disassociated and cultured in MCDB153ϩ medium. developed by SuperSignal West Pico Chemiluminescent Sub- All of the culture pairs have been immunophenotyped with strate (PIERCE, Rockford, IL). vimentin, cytokeratin, and thyroid transcription factor. Early DNA Extraction and PCR-SSCP Assay. Genomic passages from all of the lines were preserved in liquid nitrogen DNA was isolated from all of the cultured cells by SDS/ until tested. proteinase K (Life Technologies, Inc.) digestion, phenol-chlo- RT-PCR Assay. The RT-PCR assay was used to exam- roform extraction, and ethanol precipitation (31). PCR-SSCP ine FEZ1 mRNA expression. Total RNA was extracted from the assay was performed for the cording region of FEZ1 gene. The samples (6 NSCLC primary cells and its corresponding NBECs, primer information used for PCR-SSCP is summarized in 17 NSCLC, and 19 SCLC cell lines) with Trizol (Life Technol- Table 1. PCR amplification was carried out 12 min for 95°C for ogies, Inc.) following the manufacturer’s instructions. Reverse initial denaturation, followed by 35 cycles of 94°C for 20 s, transcription reaction was performed on 2 g of total RNA with 60–64°C for 40 s, and 72°C for 40 s. The PCR products were the SuperScript II First-Strand Synthesis using oligodeoxythy- then denatured, loaded on 6% polyacrylamide gels with and midylic acid primer System (Life Technologies, Inc.), and ali- without 5% (vol/vol) glycerol, electrophoresed, and exposed to quots of the reaction mixture were used for the subsequent PCR X-ray film. amplification. The forward PCR amplification primer was Data Analysis.