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Jpn J Clin Oncol 1997;27:22–25 Decreased Expression of the p16/MTS1 Gene without Mutation is Frequent in Human Urinary Bladder Carcinomas , , , 1Chemotherapy Division, National Cancer Center Research Institute, Tokyo, 2Department of Urology, Nagoya City University Medical School, Nagoya and 3First Department of Pathology, Nagoya City University Medical School, Nagoya, Japan The p16 (CDKN2,MTS1) gene is located at 9p21 and its product, p16, inhibits the cyclin D/CDK4 complex. Loss of heterozygosity on chromosome 9p is very common in human bladder carcinomas and has been found in all stages of lesions, suggesting that it occurs early in bladder tumor progression. Several studies have revealed frequent homozygous deletion of the p16 gene in cell lines, and that such deletions are also common in some types of cancers. In addition, point mutations in the p16 gene have been identified in several types of neoplasia. In the present examination of urinary bladder tumors, no p16 gene mutations were detected, but nine cases out of 23 (39%) showed decreased mRNA expression, revealed by the reverse transcriptase polymerase chain reaction. There were no histological differences apparent between those cases with normal and those with decreased p16 expression. These results indicate that while p16 gene mutations may be rare, changes in the level of the p16 transcripts could play a role in human bladder carcinoma development. Key words: p16 – point mutation – gene expression – human urinary bladder carcinomas INTRODUCTION Loss of heterozygosity (LOH) of chromosome 9p21 has been found in several types of malignant tumors, including these arising in the urinary bladder (1–5). This indicates that the chromosomal region contains at least one tumor suppressor gene which may play an important role in development or progression of many types of tumors. In bladder carcinomas, LOH of chromosome 9 is the most frequent genetic change, deletion occurring in all grades and stages (6,7). Inactivation of a tumor suppressor gene on chromosome 9 is, therefore, a possible initiating event in bladder carcinogenesis. Recently, a gene encoding a 16 kDa protein was cloned from 9p21 to 2 (8,9). This p16 protein is an inhibitor of the cyclin dependent kinase which catalyzes the phosphorylation of retinoblastoma gene protein, releasing transcription factor E2F and resulting in progression from the G1 phase to the S phase of the cell cycle (10). Thus, the p16 protein can negatively regulate the Received July 4, 1996; accepted September 12, 1996 For reprints and all correspondence: Makoto Asamoto, Chemotherapy Division, National Cancer Center Research Institute, 1–1,Tsukiji 5-chome, Chuo-ku, Tokyo 104, Japan Abbreviations: LOH, Loss of heterozygosity; PCR, polymerase chain reaction; SSCP, single strand conformation polymorphism; RT, reverse transcriptase cell cycle and is a candidate 9p21 tumor suppressor gene. Homozygous deletions of the p16 gene have been observed frequently in cancer-cell lines established from many types of tissues, including the bladder (5,8), and germline mutations have been identified in patients in families with a genetic predisposition for melanoma development (11,12). Furthermore, somatic mutations have been found in primary esophageal (13), pancreatic (14) and lung carcinomas (15). To investigate the involvement of p16 gene alterations in primary bladder carcinomas, we identified somatic mutations in 23 cases using the polymerase chain reaction (PCR) and single strand conformation polymorphism (SSCP) method for all three exons of the p16 gene in addition to determining mRNA expression by the reverse transcriptase (RT) PCR technique. MATERIALS AND METHODS Twenty-three primary bladder carcinomas were obtained by trans-urethral resection in the Department of Urology, Nagoya City University Medical School. After removal of a biopsy, each specimen was immediately frozen in liquid nitrogen and stored at –80C. Total RNA and DNA samples were extracted simultaneously from frozen tissue using ISOGEN (Nippon Gene Co. Ltd. Toyama, Japan) according to the manufacturers instructions. Biopsies from each tumor were also processed for routine histological examination. All tumors were diagnosed as transitional cell carcinomas. ASPC-1 and T24 cell lines obtained from the 23 J Clin Oncol Nucleic AcidsJpn Research, 1994, 1997; Vol. 22,27(1) No. 1 ATCC and Japanese Cancer Research Resources Bank, respectively, were used as positive and negative controls for the p16 gene mutation analysis (14,16). To screen for p16 gene somatic mutations, PCR-SSCP was performed. First, exons 1 and 2 with flanking intronic sequences were amplified by PCR using the following primers: for exon 1, Ex1A (CGGAGAGGGGGAGAACAG) and Ex1B (TCCCCTTTTTCCGGAGAATCG) for exon 2, Ex2A (GCTCTACACAAGCTTCCTTTCC) and Ex2B (GGGCTGAACTTTCTGTGCTGG). The PCR conditions were one cycle at 95C (5 min); four cycles at 95C (30 s) with the annealing temperature (Tann) = 72C (30 s); four cycles with Tann = 68C (30 s); four cycles with Tann = 66C (30 s); four cycles with Tann = 64C (30 s); four cycles with Tann = 62C (30 s) ; and 30 cycles with Tann = 60C . Dimethyl sulfoxide was added to the reaction mixture at 5%. Secondary PCR was carried out using 1 µl of the diluted (1:100) first fragments with primers Ex1A and Ex1B for exon 1, or Ex2A and Ex2B for exon 2 as templates, 2 pmol of each primer, 25 µM of deoxynucleotide triphosphates, 2 µCi of α-32PdCTP (Amersham, 3000 µCi/mmol), 10 mM Tris (pH 8.3), 50 mM KCL, 1.5 mM MgCl2 , 12% DMSO (dimethyl sulphoxide) and 0.125 of a unit of Taq polymerase (Perkin-Elmer Cetus) in a final volume of 5 µl. Primer sequences and annealing temperatures were taken from published data (11). For exon 1, primers X1.31F (GGGAGCAGCATGGAGCCG) and X1.26R (AGTCGCCCGCCATCCCCT) were used. Exon 2 was divided into three parts and amplified with overlapping sequences to increase the sensitivity of detection of mutations by SSCP. For exon 2a, X2.62F (AGCTTCCTTCCGTCATGC) and 286R (GCAGCACCACCAGCGTG); for exon 2b, F200 (AGCCCAACTGCGCCGAC) and 346R (CCAGGTCCACGGGCAGA); and for exon 2c 305F (TGGACGTGCGCGATGC) and X2.42R (GGAAGCTCTCAGGGTACAAATTC) were used as primers. To analyze exon 3, the same conditions as for the secondary PCR for exons 1 and 2 were applied, except for application of genomic DNA as the PCR template instead of the PCR products, using primers X3.90F (CCGGTAGGGACGGCAAGAGA) and 530R (CTGTAGGACCCTCGGTGACTGATGA). Thirty cycles were performed of: 1 min at 94C, followed by 1 min at 63C for exon 1, at 55C for exon 2 and at 60C for exon 3, and finally 1.5 min at 72C. Forty-five µl of stop solution [95% formamide, 20 mM EDTA (ethylenediaminetetra-acetic acid), 0.05% bromophenol blue, 0.05% xylene cyanol] was then added to the reaction mixture and after heating at 80C for 2 min, 1 µl aliquots of samples were loaded onto 5% polyacrylamide gels (acrylamide:bis ratio, 49:1) with or without 5% glycerol. Gels 23 Figure 1. PCR-SSCP analysis of exons 1 and 2b (the middle part of exon 2) of the p16 gene in human bladder carcinomas, as well as T24 and ASPC-1 cells. Note the abnormal mobility shift in the bands for ASPC-1 indicating a point mutation in exon 2, but its lack in the bladder carcinoma and T24 cases. were run at 30 W with a water jacket at 25C for 3 h before drying at 80C and performance of autoradiography for 1–2 h. To investigate the mRNA expression, the RT-PCR method was applied. Total RNA (1 µg) treated with RNase-free DNase was incubated with oligo d (T)20 primers and AMV reverse transcriptase at 60C for 30 min. The 363 bp cDNA stretch corresponding to the first and second exons of p16 was amplified by primers p16U (GGGGTTCGGGTAGAGGAGGTG) and p16D (CATGGTTACTGCCTCTGGTG) with an RNA PCR kit (TaKaRa Co. Ltd., Otsu, Japan). The PCR conditions were the same as used for the exon 1 or 2 amplification. G3PDH expression was also examined as an internal control for RT-PCR with primers purchased from Clontech Laboratories, Inc. (Palo Alto, California, USA). Thirty cycles were performed at 94C, 60C, and 72C for 1, 1, and 1.5 min, respectively. Reaction products for p16 and the corresponding G3PDH were placed into the same wells and separated on 1% agarose gels before staining with ethidium bromide for visualization. RESULTS We investigated somatic mutations in all three exons of the p16 gene in 23 primary bladder carcinomas and the T24 bladder carcinoma cell line using the PCR-SSCP method. However, no mutations were detected in any of the tumors or the T24 cells (16). As a positive control, the pancreatic cell line ASPC-1 which has a mutation in exon 2 of the p16 gene was used. The presence of the mutation was confirmed (14). Representative photographs of the results of SSCP analysis for exons 1 and 2b (the middle part of exon 2) are shown in Fig. 1. Decreased expression of the p16 gene relative to G3PDH was noted in nine cases (39%) out of the 23 (Case nos 2,5,7,11,13,14,19,20 and 23) examined and also in the T24 cell line by the RT-PCR method (Fig. 2). 24 p16 in urinary bladder carcinoma by RT-PCR, the cycle number was minimized in the present study, with only one round of PCR performed, and the level of p16 in normal cells is known to be generally low (21–24). Therefore we believe that the signals observed were indeed from carcinoma as opposed to normal cells. The reasons for the decreased expression remain to be elucidated, but it could be due to methylation of the 5i CpG island of the p16 gene (25) or homozygous deletions of the gene (19,20). Whatever the reasons, the RT-PCR method described here is useful in detecting p16 gene abnormalities of any origin. Decreased expression of the p16 gene despite a lack of point mutations has also been reported for nasopharyngeal carcinomas (26) and high grade gliomas (27). Loss of p16 function is very likely to cause deregulation of G1/S phases in the cell cycle. This abnormality could be expected to endow a growth advantage or stimulate more proliferation. We have also conducted a preliminary study of cell proliferating activity in bladder carcinomas with or without p16 expression by PCNA staining (data not shown). However, we could not detect any clear correlation between the two parameters, indicating that a pathway independent of p16 may exist to regulate the cell cycle. Figure 2. mRNA expression of p16 and G3PDH in human bladder carcinomas, T24 and ASPC-1 cells revealed by RT-PCR. The upper bands correspond to G3PDH, and the lower bands to p16. In nine cases out of 23 (39%), decreased expression of p16 was observed relative to G3PDH (Case nos 2,5,7,11,13,14,19,20 and 23). M: DNA molecular weight markers, 123 bp DNA ladder (GibcoGRL, Life Technologies, Inc., Gaithersburg, MD, USA) There were no histological differences apparent between cases with normal and decreased p16 expression. DISCUSSION Although the mechanisms underlying development of bladder carcinogenesis remain unclear, several investigations have shown frequent gene alterations in the short arm of chromosome 9 in bladder carcinomas (1–4). The p16 gene in this location is in fact a candidate putative tumor suppressor gene in many types of tumors, including bladder carcinomas and their derived cell lines, many carrying homozygous deletions (8, 9). However, somatic mutations in the p16 gene may be rare except in hereditary melanomas, and in esophageal, pancreatic, and lung carcinomas (16,17). Our results are in agreement with the earlier reports of very low incidences of somatic mutations in primary bladder carcinomas (18,19). This is in clear contrast to the frequent occurrence of homozygous deletions (19,20). Primary epithelial tumors always contain contaminating normal cells e.g. stromal cells, endothelial cells and lymphocytes, and therefore it may be very difficult to find homozygous deletions using a PCR based technique. In this study, we applied a nested primed PCR method to detect mutations in order to increase efficiency of the technique, and found that the p16 gene could be amplified from all samples. This is not, however, conclusive proof of lack of a homozygous deletion, given the possible generation of a p16 gene product from contaminating normal cells. With regard to the apparent decrease in expression of the p16 gene in nine out of the 23 primary bladder carcinomas observed References 1. Knowles MA, Elder PA, Williamson M, Cairns JP, Shaw ME, Law MG. Allelotype of human bladder cancer. Cancer Res 1994;54:531–8. 2. Orlow I, Lianes P, Lacombe L, Dalbagni G, Reuter VE, Cordon CC. 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