It is important to understand the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death. Defects in apoptosis are implicated in both tumorigenesis and drug resistance, and these defects are cause of chemotherapy failures. These studies should explain the relationship between cancer genetics and treatment sensitivity, and should enable a more rational approach to anticancer drug design and therapy. Lung cancer is a major cause of cancer deaths throughout the world. Small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC) represent the two major categories of lung cancer that differ in their sensitivity to undergo apoptosis. The role of apoptosis regulation in lung cancer with major focus on the differential sensitivities of the major subtypes is reviewed.

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cells but not in normal cells. TRAIL is known to bind to 4 different receptors, 2 proapoptotic (DR4 andDR5Several members of the tumor necrosis factor receptor superfamily, which includes tumor necrosis factor receptor 1, Fas (Apo-1/CD95), and the decoy receptors for TRAIL (Apo-2L), regulate programmed cell death (apoptosis). Upon engagement by their respective ligands, tumor necrosis factor receptor 1 and Fas recruit adaptor molecules and activate a cascade of cysteine proteases (caspases), the proteolytic activity of which results in apoptosis. The TRAIL-activated DR4 (also TNFRSF10A, Apo-2, TRAIL-R1) 1,2 and DR5 (also TNFRSF10B, KILLER/DR5, TRICK2, TRAIL-R3) receptors 2-4 have also been shown to initiate a caspase-mediated apoptotic pathway. 4 Death inducing DR4 and DR5 and the decoy receptors DcR1 (also TNFRSF10C, TRID, TRAIL-R3) and DcR2 (also TNFRSF10D, TRUNDD, TRAIL-R4) are structurally related, especially in their extracellular domains and are all located at 8p 21-22. 5,6 Decoy receptor DcR1 completely lacks the intra-cellular death domain and DcR2 contains a truncated, nonfunctional death domain and appear unable to induce apoptosis. The extracellular domains of DcR1 or DcR2 compete with those of DR4 or DR5 for TRAIL binding. Thus, based on the current knowledge, DcR1 or DcR2 are believed to inhibit apoptosis induction mediated through DR4 and DR5. 5 Inactivation of genes may occur via point mutations, allelic loss (LOH), homozygous deletions or by aberrant methylation. 7-9 Aberrant methylation of 5Ј gene promoter regions associated with gene silencing is an epigenetic phenomenon involving many genes observed in almost all cancer types, including breast cancers, lung cancers and mesotheliomas. 9 -18 We have demonstrated loss of expression (at RNA and protein level) of DR4 and DR5 in lung cancer cell lines, although the mechanism was not via methylation. 19 Recently, 20 it was reported that DcR1 and DcR2 expression are frequently silenced by aberrant methylation of 5Ј regions of these genes in pediatric tumor cell lines and neuroblastomas. In our study, we examined the methylation and expression status of DcR1, DcR2, DR4 and DR5 in breast and lung cancers and malignant mesothelioma (MM). We also examined the methylation status of TRAIL receptor genes in many other types of malignancies. We tried to correlate methylation status with clinical features of patients including survival and histological type in lung breast MM and prostate cancer. MATERIAL AND METHODS Cell linesCell lines were initiated by AFG (breast, lung, some MMs) or HIP (most MMs). [21][22][23] Twenty-seven lung cancer cell lines and 23 breast cancer cell lines and were grown in RPMI-1640 medium (Life Technologies Inc., Rockville, MD) supplemented with 5% FBS and incubated in 5% CO 2 at 37°C. Four non-malignant mesothelial primary cell cultures (HCC3466, HCC3468, HCC3469,

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Purpose: Aberrant methylation of 5 gene promoter regions is an epigenetic phenomenon that is a major mechanism for silencing of tumor suppressor genes in many cancer types. There is limited information about the molecular changes involved in the pathogenesis of gallbladder carcinoma (GBC), including methylation status.Experimental Design: We investigated the aberrant promoter methylation profile of 24 known or suspected tumor suppressor genes in 50 GBCs and compared those results with the findings in 25 chronic cholecystitis (CC) specimens without cancer. The methylation-specific polymerase chain reaction and combined restriction analysis methods were used to detect methylation, and the results were confirmed by sequencing of cloned polymerase chain reaction products.Results: In GBC, gene methylation frequencies varied from 0% to 80%. Ten genes demonstrated relatively high frequencies of aberrant methylation: SHP1 (80%), 3-OST-2 (72%), CDH13 (44%), P15 INK4B (44%), CDH1 (38%), RUNX3 (32%), APC (30%), RIZ1 (26%), P16 INK4A (24%), and HPP1 (20%). Eight genes (P73, RAR␤2, SOCS-1, DAPK, DcR2, DcR1, HIN1, and CHFR) showed low frequencies (2-14%) of methylation, and no methylation of the remaining six genes (TIMP-3, P57, RASSF1A, CRBP1, SYK, and NORE1) was detected. In CC, methylation was detected for seven genes: SHP1 (88%), P15 INK4B (28%), 3-OST-2 (12%), CDH1 (12%), CDH13 (8%), DcR2 (4%), and P16 INK4A (4%). Significantly higher frequencies of methylation in GBC compared with CC were detected for eight genes (3-OST-2, CDH13, CDH1, RUNX3, APC, RIZ1, P16INK4A , and HPP1). Of those, four genes showed frequent methylation (>30%) in GBCs. The mean methylation index, an expression of the amount of methylated genes by case, was significantly higher in GBC (0.196 ؎ 0.013) compared with CC (0.065 ؎ 0.008; P < 0.001).Conclusions: Our study constitutes the most comprehensive methylation profile report available in GBC and demonstrates that this neoplasm has a distinct pattern of abnormal gene methylation. Whereas gallbladders from healthy individual were not available, our finding of methylation in CC cases without cancer suggests that this phenomenon represents an early event in the pathogenesis of GBC.

© 2 0 0 2 L a n d e s is an apoptosis inducing cysteine protease which is activated through the formation of a death-inducing signaling complex when death receptors are complexed to their specific ligands. Recent reports indicate that CASP8 expression is lost via a combination of promoter methylation and allelic loss in a subset of neuroblastomas. We investigated the state of the gene in lung tumors and cell lines. RT-PCR studies indicated that gene expression was lost in most (27 of 34, 79%) of small cell lung carcinoma (SCLC) cell lines, but expression was retained in all 22 non-SCLC (NSCLC) lines tested. Loss of gene expression at the RNA level was associated with absent protein expression by Western blotting and lack of CASP8 enzymatic activity. Methylation of the promoter region of the CASP8 gene was present in 16 of 27 (59%) of the SCLC lines lacking gene expression. All methylated cell lines lacked the presence of an unmethylated allele indicating biallelic methylation or loss of non-methylated allele. Promoter methylation was absent in all SCLC and NSCLC cell lines retaining gene expression, and all of these lines had the unmethylated form of the gene. One non-expressing SCLC cell line, NCI-H82, had a homozygous deletion at 2q33 encompassing the chromosomal location of the CASP8 gene. The mechanism of gene inactivation in the remaining 10 of 27 (37%) non-expressing SCLC cell lines is unknown. Using five polymorphic markers for 2q33 a high frequency of allelic loss was present in SCLC lines. Analyses of fresh tumors showed that 15 of 43 (35%) of the SCLC, seven of 40 (18%) of bronchial carcinoids and none of 44 NSCLC tumors had CASP8 promoter methylation. Because only approximately 60% of SCLC cell lines lacking CASP8 expression were methylated, extrapolating from the cell line data, we estimate that approximately 58% of SCLC and 30% of bronchial carcinoids lack CASP8 expression. Thus, CASP8 expression is absent in a subset of both high grade (SCLC) and low grade (carcinoid) neuroendocrine lung tumors but not in NSCLC, which usually lack neuroendocrine features. CASP8 may function as a tumor suppressor gene in neuroendocrine lung tumors. B i o s c i e n c e . N o t f o r d i s t r i b u t i o n .