[CANCER RESEARCH 63, 3352–3355, June 15, 2003] Allelic Loss on Chromosome 3p21.3 and Promoter Hypermethylation of Semaphorin 3B in Non-Small Cell Lung Cancer 1 Tamotsu Kuroki, Francesco Trapasso, Sai Yendamuri, Ayumi Matsuyama, Hansjuerg Alder, Noel N. Williams, Larry R. Kaiser, and Carlo M. Croce 2 Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 [T. K., F. T., S. Y., A. M., H. A., C. M. C.], and Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104 [N. N. W., L. R. K.] ABSTRACT The aim of this study was to evaluate the promoter methylation status and loss of heterozygosity (LOH) of the SEMA3B in non-small cell lung cancers (NSCLCs). We analyzed the methylation status of semaphorin 3B (SEMA3B) promoter and LOH at 3p21.3 in eight NSCLC cell lines and 27 primary tumors. Hypermethylation of SEMA3B was found in 50% of the cell lines and 41% of the primary tumors studied. The presence of hypermethylation was statistically associated with loss of SEMA3B expres- sion in both cell lines (P  0.02) and primary tumors (P < 0.01). There was no correlation between SEMA3B and tumor stage. On the other hand, the correlation between SEMA3B methylation status and LOH at 3p21.3 was significant (P  0.02). Notably, 7 of 8 tumors with both hypermethy- lation and LOH of SEMA3B showed the absence of the expression. Treat- ment with 5-AZAC restored SEMA3B expression in NSCLC cell line. These results indicate that SEMA3B gene alterations may play a impor- tant role in the malignant transformation of NSCLC via a two-hit mech- anism, including epigenetic changes and allelic loss, for tumor suppressor gene inactivation. INTRODUCTION Allelic loss on chromosome 3p is a frequent event in the multistep carcinogenesis of many types of cancer, including NSCLCs 3 (1). Deletion of the region 3p21.3 is one of the most frequent and early event in NSCLC development, indicating that inactivation of tumor suppressor genes on 3p21.3 may play an important role in early steps of NSCLC carcinogenesis (2). Recent studies have suggested that SEMA3B, located at 3p21.3, is a candidate tumor suppressor gene that is frequently inactivated in lung cancer cell lines by promoter hyper- methylation (3). Furthermore, introduction and expression of SEMA3B in a human ovarian cancer cell line resulted in suppression of tumor formation (4). SEMA3B is a member of the semaphorin family (5). The semaphorin family of proteins plays a critical role in axonal guidance and contains a highly conserved 500 amino acid semaphorin domain at NH 2 -terminal (6, 7). Semaphorins have been classified into eight subclasses and five subclasses, classes 3–7 are found in vertebrates (8). Two class 3 semaphorin genes, SEMA3B and SEMA3F, reside at 3p21.3 and have been shown to play a potential tumor suppressive role in tumorigenesis (3, 5, 9). In this study, we analyzed the promoter methylation status of SEMA3B in NSCLCs by using MSP. We also examined these samples for LOH at the SEMA3B locus by analysis of microsatellite markers flanking 3p21.3. The frequency and potential clinical implications of hypermethylation of SEMA3B and the correlation between methylation status and LOH at 3p21.3 in NSCLCs were investigated in this study. MATERIALS AND METHODS Cell Lines and Tissues. Human lung cancer cell lines Calu-3, A549, NCI-H23, NCI-H460, NCI-H522, NCI-H1299, NCI-H1573, and NCI-H1650 were obtained from the American Type Culture Collection. All cell lines were maintained in RPMI 1640 with 10% fetal bovine serum at 37°C and 5% CO 2. The tumor samples and their corresponding noncancerous tissues were ob- tained from 27 patients who underwent surgery for NSCLC at the Hospital of the University of Pennsylvania (18 male and 9 female; median age, 67.6 years; range, 51– 80 years). Of these 27 samples, 11 were squamous cell carcinomas, 10 were adenocarcinomas, 4 were poorly differentiated carcinomas, and 2 samples had other histologies. Seventeen of them were stage I, 7 were stage II, and 3 were stage III, according to the TNM classification. DNA and RNA Extraction. The tissue samples were excised and imme- diately stored at -80°C. DNA and RNA were extracted from each of the eight cell lines and from the 27 paired lung tissues according to methods described previously (10). LOH Analysis. Allelic losses were analyzed by a PCR approach with primers amplifying polymorphic microsatellites flanking the SEMA3B gene at loci D3S1568, D3S1621, and D3S4597. The primer sequences were obtained from the Genome Database, and primers were labeled using 5'-fluorescein phosphoramidite or 5'-tetrachlorofluorescein phosphoramidite for microsatel- lite loci, as described by Ishii et al. (11). PCR was performed on the genomic DNA samples using the following conditions: 50 ng of genomic DNA tem- plate, 10 pmol of each primer, 2.5 mM MgCl 2 , 1.5 mM deoxynucleoside triphosphate mix, 1 PCR buffer, and 0.5 unit of AmpliTaq Gold (Perkin- Elmer, Branchburg, NJ) in a 20-l final volume. PCR cycles included one cycle of 95°C for 12 min followed by 35 cycles consisting of 10 cycles at 94°C for 15 s, 55°C for 15 s, and 72°C for 30 s and 25 cycles at 89°C for 15 s, 55°C for 15 s, and 72°C for 30 s, followed by 72°C for 10 min in a Perkin-Elmer Gene Amp PCR system 9600. PCR products were denatured in formamide for 5 min at 95°C and then loaded on a 6% denaturing gel on the Applied Biosystems 373 DNA sequencer. LOH was analyzed by using the Applied Biosystems Prism Genescan and the Applied Biosystems Prism Genetyper Analysis software (Perkin-Elmer/Applied Biosystems). Cases were defined as LOH when an allele peak signal from tumor DNA was reduced by 50% compared with the normal counterpart. Methylation Analysis. The methylation status in the promoter region of the SEMA3B was determined by MSP as described previously (12). DNA methylation patterns in the promoter region of the SEMA3B gene were deter- mined by sequencing bisulfite-treated DNA (3). Primer sequences for methy- lation analysis of SEMA3B promoter region were for the unmethylated reaction (product size, 135 bp): 5'-GTGGTTAGGTGGGGTATTTTT-3' (sense) and 5'-ATCAACAATAAAAACAAAAACA-3' (antisense) and for the methylated reaction (product size, 133 bp): 5'-TGGTTAGGCGGGGTATTTTC-3' (sense) and 5'-TCAACAATAAAAACGAAAACG-3' (antisense). Briefly, 1 g of the genomic DNA was denatured by 2 M NaOH and then incubated in 3 M sodium bisulfite and 10 mM hydroquinone for 16 h at 55°C. Bisulfite-treated DNA was extracted using a genomic DNA clean-up kit (Promega, Madison, WI). Mod- ified DNA was amplified by two different set of primers specific for unmethy- lated and methylated SEMA3B promoter region sequences. Human genomic DNA (Clontech, Palo Alto, CA) treated in vitro with SssI methylase (New England Biolabs, Inc., Beverly, MA) was used as a positive control. PCR was performed in 50-l reaction volumes containing 1 PCR buffer II (Perkin- Elmer, Branchburg, NJ), 2 mM MgCl 2 , 0.2 mM of each deoxynucleoside Received 12/19/02; accepted 4/15/03. 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. 1 This work was in part supported by United States Public Health Service Grant CA56036 from the National Cancer Institute. 2 To whom requests for reprints should be addressed, at Kimmel Cancer Institute, Jefferson Medical College, Thomas Jefferson University, BLSB Room 1050, 233 South 10th Street, Philadelphia, PA 19107-5799. Phone: (215) 503-4645; Fax: (215) 923-3528; E-mail: croce@calvin.jci.tju.edu. 3 The abbreviations used are: NSCLC, non-small cell lung cancer; SEMA3B, sema- phorin 3B; MSP, methylation-specific PCR; LOH, loss of heterozygosity; TNM, Tumor- Node-Metastasis; RT-PCR, reverse transcription-PCR; 5-AZAC, 5-aza-2'-deoxycytidine. 3352 Research. on October 1, 2021. © 2003 American Association for Cancer cancerres.aacrjournals.org Downloaded from