Mutational analysis of death receptor genes Fas, TRAILR1 and TRAILR2 in prostate carcinomas

Apoptosis is a cellular event essential for normal tissue homeostasis and development (1). As one of the main apoptosis pathways, the death receptor (extrinsic) pathway induces apoptosis by interaction of death ligands Fas ligand and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) with death receptors Fas and TRAIL receptors, respectively (1). Of the death receptors, Fas, TRAIL receptor 1 (TRAILR1) and TRAIL receptor 2 (TRAILR2) are considered the main apoptosis inducers (1). Evasion of apoptosis is considered a hallmark of cancer (1). Failure of apoptosis could allow the survival of transformed cells that are prone to undergo further genetic damage and play an important role in the pathogenesis of cancers. Death receptormediated apoptosis in cancers is known to be blocked by several mechanisms, including somatic mutations of Fas, TRAILR1 and TRAILR2 genes (1–5). Somatic mutation of Fas is widespread in human cancers, including those of the lung and urinary bladder, leukemia and multiple myeloma (1–3). Somatic mutation of TRAILR2 has been detected in lung cancer, breast cancer and lymphoma (4–6), while somatic mutation ofTRAILR1 has been detected in breast cancer and lymphoma (4, 5). In an earlier study, Fas gene mutation was reported in prostate intraepithelial neoplasm (PIN), but not in prostate carcinoma (7). Mutational status of TRAILR1 andTRAILR2 genes in prostate carcinomas has not been reported. The aim of this study was to explore whether death receptor genes Fas, TRAILR1 and TRAILR2 are somatically mutated in prostate carcinomas. For this, formalin-fixed tissues of 45 prostate carcinomas were randomly selected for the mutational analysis by polymerase chain reaction (PCR) and subsequent single-strand conformation polymorphism (SSCP). This method was previously proven to be adequate for the detection of Fas, TRAILR1 and TRAILR2 mutations in cancer tissues (2–6). All of the prostate tissues were obtained by radical prostatectomy. Ages of the patients ranged from 44 to 68 years with an average of 62.5 years. Sizes of the cancers ranged from 1.0 to 4.1 cm in diameter with an average of 2.3 cm. All of the prostate carcinomas were adenocarcinomas, and consisted of 15 Gleason score 7, 11 Gleason score 8 and 19 Gleason score 9 cancers. Of these, 37 (82.2%) specimens had PIN. Tumour, lymph node and metastasis (TNM) stages of the prostate carcinomas were 31 stage II and 14 stage III. Prostate-specific antigen of the patients ranged from 0.75 to 130.0 ng ⁄mL with an average of 22.85 ng ⁄mL. Malignant cells and normal cells from the same patients were selectively procured from hematoxylin and eosinstained slides using a 30G1 ⁄2 hypodermic needle (2, 3). We have analyzed entire coding regions of the three death receptor genesFas (12 primer pairs covering exons 1–9), TRAILR1 (11 primer pairs covering exons 1–8) and TRAILR2 (12 primer pairs covering exons 1–8). For Fas, we also analyzed a polymorphism in the promoter region at position -670 by SSCP.GenomicDNA fromboth tumor cells and corresponding normal cells was amplified using the primer pairs by PCR. Radioisotope ([P]dCTP) was incorporated into the PCR products for detection by SSCP autoradiogram.After SSCP,migrationof thePCRproducts on the SSCP was analyzed by visual inspection. Direct DNA sequencing reactions were performed in cancers with the mobility shifts in the SSCP. Other procedures of the PCR and SSCP weredescribed inour previous studies (2, 3). On the SSCP autoradiograms, all of the PCR products from the coding regions were clearly seen. However, we did not observe any aberrantly migrating bands in the tumors compared with the bands in the corresponding normal tissues in Fas or TRAILR1 or TRAILR2 gene (Fig. 1). The PIN lesions did not show any abnormality in the SSCP, either. Of the 45 prostate cancers, 21 (46.7%) were informative (two heterozygous alleles in the patients’ normal DNA) for the polymorphism at position -670 in the promoter. Among the informative cases, only one (4.8%) showed allelic loss. Detailed data will willingly be provided on request. Failure of apoptosis allows survival of cells and plays an important role in the development