Faculty Profiles - TOYOOKA Shinichi

Papers - TOYOOKA Shinichi

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P-594 非小細胞肺癌株におけるビスフォスフォネート製剤の直接増殖抑制効果の評価と検討(肺癌10)(一般示説60)

越宗龍一郎, 原文堅, 青江基, 豊岡伸一, 大谷裕, 平見有二, 花畑哲郎, 永広格, 佐野由文, 伊達洋至, 清水信義

日本呼吸器外科学会雑誌 18 ( 3 ) 485 - 485 2004

Establishment and validation of quantitative real-time pcr assay for aberrant methylation of 14-3-3û gene in breast and lung carcinoma

Ubaradka G. Sathyanarayana, Makoto Suzuki, Shinichi Toyooka, Asha Padar, Kiyomi O. Toyooka, Andrea L. Zern, Kuniharu Miyajima, Takashi Takahashi, Elizabeth Brambilla, Adi F. Gazdar

Cancer Genomics and Proteomics 1 ( 1 ) 1 - 8 2004

Language：English Publishing type：Research paper (scientific journal) Publisher：International Institute of Anticancer Research

Background: 14-3-3Û gene has been shown to be responsible for G2 cell cycle checkpoint control by p53 in response to DNA damage in human cells. In order to increase the potential utility of 14-3-3Û gene as a molecular marker in tumor analysis and prognosis, we established and validated a quantitative real-time MSP assay and correlated our findings with the standard MSP assay. Materials and Methods: We examined the expression of 14-3-3 Û gene by reverse transcription PCR (RT-PCR) in breast and lung cancer cell lines and control non-malignant tissue samples. To elucidate the mechanism of gene silencing, we studied the methylation patterns in cell lines, tumors and non-malignant control tissues of breast and lung using previously reported MSP assay. For fluorescence based quantitative Real-Time PCR assay, we designed primers and probe specific to 14-3-3Û gene, validated the assay in cell lines and non-malignant control tissues of breast and lung and extended the study to primary tumors and corresponding nonmalignant tissues. Results: The concordances between the standard MSP assay and the real-time assay were 95-100%. The overall concordances between standard MSP and real-time assay in 60 cell lines were 97%. By real-time assay, the differences in methylation frequencies between malignant and non-malignant breast and between malignant and nonmalignant lung tissues
between NSCLC and SCLC cell lines between MSP (-) and MSP (+) samples and between MSP (+) and MSP (++) samples were statistically significant. The mean real-time values for MSP (-), MSP (+) and MSP (++) samples were 2, 28 and 53 respectively. Conclusion: We conclude that promoter methylation is a valid pathway for silencing of 14-3- 3Û gene in primary breast and lung carcinomas. The real-time assay to distinguish the extent and degree of methylation of 14- 3-3Û gene among malignant and non-malignant tissues would potentially enhance the utility of this marker in breast and lung cancer analysis and prognosis.

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Alterations in the Mitochondrial Displacement Loop in Lung Cancers Reviewed

Makoto Suzuki, Shinichi Toyooka, Kuniharu Miyajima, Toshihiko Iizasa, Takehiko Fujisawa, Nebiyou B. Bekele, Adi F. Gazdar

Clinical Cancer Research 9 ( 15 ) 5636 - 5641 2003.11

Language：English Publishing type：Research paper (scientific journal)

Purpose: Alterations of the noncoding displacement (D) loop of mitochondrial DNA are present in many cancers. These alterations include numerical changes in a homopolymeric C tract (PCT) at positions 303-309 and single base substitutions (SBS). We determined the frequency of D-loop alterations in lung cancer cell lines and tumors, and related them to clinicopathologic features. Experimental Design: We sequenced the entire D-loop of 28 lung cancer cell lines [12 small cell lung cancer (SCLC) and 16 non-small cell lung cancer (NSCLC)] and matched B-lymphoblastoid cell lines. In 55 resected NSCLCs and corresponding nonmalignant lungs we determined the length of the PCT. Results: In nonmalignant cell lines and tissues the most frequent PCT repeat number was seven (36 of 83
43%) with a range of six to nine. Alterations, often multiple, were present in 17 of 28 (61%) of the cell lines, including 8 of 12 SCLC (67%) and 9 of 16 NSCLC (56%) lines. They consisted of SBS in 8 of 28 lines (29%), all of which were homoplasmic, and PCT changes in 14 of 28 (50%) lines, 8 of which were homoplasmic. Of interest, 95% (40 of 42) of the SBS were present within the two hypervariable regions in the D-loop. Because SBS were more frequent if PCT changes were present, only the PCT number was determined in resected samples. PCT changes were present in 11 of 55 (20%) of the NSCLC tumors. Changes were never noted in tumors when the PCT number in the nonmalignant tissue was seven, and only two tumor cell lines had changes when the PCT number in the matched lymphoblastoid cell line was seven. These changes were higher in squamous cell carcinomas (8 of 25
32%) than in adenocarcinomas (3 of 30
10%
P = 0.04) and in large tumors (T3 and T4
7 of 20
35%) compared with smaller tumors (T1 and T2
4 of 35
11%
P = 0.04). Smoking history, gender, age, and stage were not related to frequency of PCT change. Conclusions: Our findings indicate that D-loop alterations are frequent in lung cancers and their cell lines, and that these changes are weakly associated with certain clinical parameters. In tumors PCT changes were only present when the corresponding nonmalignant lung demonstrated a variation from the most common repeat number of seven.

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Aberrant methylation of multiple genes in the upper aerodigestive tract epithelium of heavy smokers Reviewed

S Zochbauer-Muller, S Lam, S Toyooka, AK Virmani, KO Toyooka, S Seidl, JD Minna, AF Gazdar

INTERNATIONAL JOURNAL OF CANCER 107 ( 4 ) 612 - 616 2003.11

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

An important method for silencing tumor suppressor genes in cancers is by aberrant methylation (referred to as methylation) of CpG islands in gene promoter regions. In lung cancer, methylation of the genes retinoic acid receptor beta-2 (RARbeta-2), CDH13 (H-cadherin), p16(INK4a) (p16), RASSFIA (RAS association domain family 1) is frequent. Thus, we investigated methylation of these genes in 4 different types of specimens (oropharyngeal brushes, sputum samples, bronchial brushes and bronchioloalveolar lavage [BAL] samples) of the upper aerodigestive tract epithelium from heavy smokers without evidence of cancer but with morphometric evidence of sputum atypia and compared the frequencies of methylation in the different types of specimens. In addition, we also analyzed sputum samples from 30 never smokers for methylation of these genes. Our major findings are: (i) At least one gene was methylated in one or more specimens from 48% of the smokers. However, methylation was statistically significant less frequently in never smokers compared to smokers. (ii) In general, methylation occurred more frequently in samples from the central airways (sputum, bronchial brushes) compared to the peripheral airways (BAL) and only occasionally in the oropharynx. (iii) RARbeta-2 was the most frequently methylated gene, whereas the frequency of methylation for the other genes was lower. (iv) Data from sputum samples and bronchial brushes were comparable. Our findings suggest that detection of methylation should be investigated as an intermediate marker for lung cancer risk assessment and response to chemopreventive regimens. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/ijc.11458

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The effect of epidermal growth factor receptor antisense morpholino oligomer on non-small cell lung cancer cell line Reviewed

K Washio, M Aoe, S Toyooka, H Mushiake, K Tsukuda, N Shimizu

ONCOLOGY REPORTS 10 ( 6 ) 1967 - 1971 2003.11

Language：English Publishing type：Research paper (scientific journal) Publisher：PROFESSOR D A SPANDIDOS

Overexpression of the epidermal growth factor receptor (EGFR) has been identified as a common component of various cancer types including lung cancer. Recently morpholino oligonucleotides appeared as a promising modification for antisense applications with few toxic effects and their stability. We investigated the effect of EGFR antisense morpholino oligomer on non-small cell lung cancer (NSCLC) cell line by evaluating EGFR mRNA, protein product and cell proliferation. The EGFR antisense morpholino oligomer was designed to target the translation start site in the EGFR mRNA. The four base-mismatch morphlino oligomer was designed as a control for EGFR antisense morpholino oligomer. These morpholino oligomers were introduced into NCI-H125 cell line which showed overexpression of EGFR. The EGFR mRNA and protein expression were quantified by real time RT-PCR and ELISA, respectively. The significant repression in both EGFR mRNA and protein expression was observed for three days after single treatment with EGFR antisense morpholino oligomer. Furthermore, the growth of NCI-H125 cell line was significantly inhibited with treatment by EGFR antisense morpholino oligomer. Our results indicate that EGFR antisense morpholino oligomer represses the EGFR expression at both mRNA and protein level and inhibits the proliferation of NSCLC cell line suggesting that it may be a promising strategy as one of antisense therapies for NSCLC.

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Detection of codon 61 point mutations of the K-ras gene in lung and colorectal cancers by enriched PCR. Reviewed

Toyooka S, Tsukuda K, Ouchida M, Tanino M, Inaki Y, Kobayashi K, Yano M, Soh J, Kobatake T, Shimizu N, Shimizu K

Oncology reports 10 ( 5 ) 1455 - 1459 2003.9

悪性中皮腫に対するin vitro及びin vivoのGefitinib(Iressa)の抗腫瘍効果(Antitumor effect by Gefitinib(Iressa)against malignant mesothelioma in vitro and in vivo)

原文堅, 青江基, 豊岡伸一, 平成人, 枝園忠彦, 高畠大典, 吉冨誠二, 高橋寛敏, 石部洋一, 小笠原豊, 土井原博義, 清水信義

日本癌治療学会誌 38 ( 2 ) 767 - 767 2003.9

Epigenetic down-regulation of death-associated protein kinase in lung cancers Reviewed

S Toyooka, KO Toyooka, K Miyajima, JL Reddy, M Toyota, UG Sathyanarayana, A Padar, MS Tockman, S Lam, N Shivapurkar, AF Gazdar

CLINICAL CANCER RESEARCH 9 ( 8 ) 3034 - 3041 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Purpose: Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine kinase involved in apoptosis. Aberrant methylation of DAPK was reported in lung cancers by methylation-specific PCR. However, we were unable to relate methylation with gene silencing with the same methodology. Our goals were to develop a methodology that related methylation with gene silencing and use it to study the state of the gene in lung cancers.
Experimental Design and Results: Using a semiquantitative real-time reverse transcription-PCR, DAPK expression was lower in lung cancers than in corresponding nonmalignant bronchial epithelial cells in five of six primary short-term cultures. In continuous cell lines, mRNA expression was down-regulated, as well as compared with nonmalignant bronchial epithelial cells, and its protein was not detected by Western blotting in 17 of 23 (74%) cell lines. We investigated methylation status of 5' flanking region of DAPK by combined bisulfite restriction analysis and bisulfited DNA sequencing. Aberrant methylation was detected in 21 of 48 (44%) cell lines, 2 of 6 primary cultured tumors, and 14 of 38,(37%) primary lung cancers, although varying degrees of methylation were noticed. Furthermore, bisufite sequence data suggested that aberrant methylation might occur selectively at some CpG dinucleotides in cell lines which had absent expression. Treatment with 5-aza-2'-deoxycytidine restored DAPK expression in heavily methylated cell lines tested, and histone deacetylase inhibitor trichostatin A alone restored DAPK expression in some methylated cell lines as well.
Conclusions: Our major. findings are: (a) DAPK expression is frequently down-regulated in lung cancers; (b) aberrant methylation of DAPK is frequent in lung cancers, although considerable heterogeneity of methylation is present, and some specific CpG dinucleotides are often methylated in expression negative lung cancers; and (c) besides methylation and histone deacetylation, there may be other mechanisms for down-regulation of DAPK expression.

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Aberrant methylation of TMS1 in small cell, non small cell lung cancer and breast cancer Reviewed

A Virmani, A Rathi, K Sugio, UG Sathyanarayana, S Toyooka, FC Kischel, Tonk, V, A Padar, T Takahashi, JA Roth, DM Euhus, JD Minna, AF Gazdar

INTERNATIONAL JOURNAL OF CANCER 106 ( 2 ) 198 - 204 2003.8

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

TMSI (target of methylation-induced silencing) is a CpG island-associated gene that functions in the regulation of apoptosis and encodes a caspase recruitment domain, a recently described motif found in apoptotic signaling molecules. Recent evidence suggests that silencing of genes in the apoptotic pathway contribute to human carcinogenesis. We examined the DNA methylation status of the TMSI promoter in lung and breast tumor tissues, tumor cell lines and nonmalignant tissues by methylation-specific polymerase chain reaction (MSP) and its mRNA expression by reverse transcription PCR. Aberrant methylation of TMSI was present in 70% (40 of 57) of small cell lung cancer (SCLC) cell lines and 41% (13 of 32) of SCLC tumor tissues, 48% (29 of 6 1) of non small cell lung cancer (NSCLC) cell lines and 40% (28 of 70) of NSCLC tumor tissues and 46% (12 of 26) of breast cancer cell lines and 32% (20 of 63) of breast tumor tissues. Methylation was absent in the peripheral blood lymphocytes and buccal epithelium from healthy volunteers, as well as in nonmalignant lung tissues and was rare in nonmalignant breast tissues 7% (2 of 30). DNA methylation was confirmed by sequence analysis and the methylation status correlated inversely with TMSI RNA expression in 18 cell lines tested. RNA expression was restored by treatment with the demethylating agent S-aza-2'-deoxycytidine, in 4 of 4 methylated cell lines that lacked the TMSI transcript. Our results suggest that methylation of TMSI may play a role in the pathogenesis of small cell and non small lung and breast cancers. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/ijc.11206

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Epigenetic inactivation of laminin-5-encoding genes in lung cancers Reviewed

Ubaradka G. Sathyanarayana, Shinichi Toyooka, Asha Padar, Takashi Takahashi, Elizabeth Brambilla, John D. Minna, Adi F. Gazdar

Clinical Cancer Research 9 ( 7 ) 2665 - 2672 2003.7

Language：English Publishing type：Research paper (scientific journal)

Purpose: We investigated the loss of expression of three laminin-5 (LN5)-encoding genes in lung cancer cell lines and elucidated the mechanism of inactivation of the genes in lung cancer cell lines and tumors. Experimental Design: We examined the expression of LN5-encoding genes by reverse transcription-PCR in 49 lung cancer cell lines. To elucidate the mechanism of gene silencing, we treated expression-negative cell lines (two for each gene) with a demethylating agent and examined the restoration of expression by reverse transcription-PCR. We dissected out the methylation patterns of CpG sites unique to the promoter regions of LN5-encoding genes by bisulfite genomic sequencing of expression-negative cell lines. We designed methylation-specific primers and validated the methylation status of the promoter regions in lung cancer cell lines using methylation-specific PCR. We further studied the methylation patterns of primary non-small cell lung cancer [NSCLC (n = 36)], small cell lung cancer [SCLC (n = 26)], and carcinoids (n = 24) tumors. Results: We observed frequent losses of expression in NSCLC (20-60%) and SCLC (65-86%) cell lines. Expression of one or more genes was lost in 90% of SCLC cell lines and 65% of NSCLC cell lines. Treatment of expression-negative cell lines with demethylating agent restored expression in all of the cases. Methylation of LN5-encoding genes was present more frequently in SCLC cell lines (60-80%) than in NSCLC cell lines (15-60%), and at least one gene was methylated in 95% of SCLC and 60% of NSCLC cell lines. The concordances between loss of expression and methylation in 40 lung cancer cell lines for the three genes (90-95%) were statistically significant. Methylation was more frequent in SCLC tumors (58-77%) than in NSCLC tumors (22-42%) and carcinoids (13-33%), and at least one gene was methylated in 92% of SCLC tumors, 47% of NSCLC tumors, and 33% of carcinoids. Conclusions: Our results demonstrate frequent epigenetic inactivation of LN5-encoding genes in lung cancers, and these findings are of biological interest and are potentially of clinical importance.

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Smoke exposure, histologic type and geography-related differences in the methylation profiles of non-small cell lung cancer Reviewed

S Toyooka, R Maruyama, KO Toyooka, D McLerran, ZD Feng, Y Fukuyama, AK Virmani, S Zochbauer-Muller, K Tsukuda, K Sugio, N Shimizu, K Shimizu, H Lee, CY Chen, KM Fong, M Gilcrease, JA Roth, JD Minna, AF Gazdar

INTERNATIONAL JOURNAL OF CANCER 103 ( 2 ) 153 - 160 2003.1

Language：English Publishing type：Research paper (scientific journal) Publisher：WILEY-LISS

Aberrant methylation of several known or putative tumor suppressor genes occurs frequently during the pathogenesis of lung cancers. There are major smoke exposure, histology, geography and gender-related changes in non-small cell lung cancer (NSCLC). We investigated smoking-related, histologic, geographic and gender differences in the methylation profiles of resected NSCLCs. We examined 514 cases of NSCLC and 84 corresponding nonmalignant lung tissues from 4 countries (USA, Australia, Japan and Taiwan) for the methylation status of 7 genes known to be frequently methylated in lung cancers [p16, RASSF1A (RAS association domain family 1), APC, RARbeta, CDH13, MGMT and GSTPI]. Multivariate analyses were used for data analysis. Adenocarcinoma was the major histologic type in women and never smokers; analyses that involved smoke exposure and gender were limited to this histology. Our major findings are a) methylation status of any single gene was largely independent of methylation status of other genes; b) the rates of methylation of p 16 and APC and the mean Methylation Index (MI), a reflection of the overall methylation status, were significantly higher in ever smokers than in never smokers; c) the mean MI of tumors arising in former smokers was significantly lower than the mean of current smokers; d) the methylation rates of APC, CDH13 and RARbeta were significantly higher in adenocarcinomas than in squamous cell carcinomas; e) methylation rates of MGMT and GSTP1 were significantly higher in the USA and Australian cases than in those from Japan and Taiwan; and (f) no significant gender-related differences in methylation patterns were noted. Our findings demonstrate important smoke exposure, histologic type and geography-related differences in the methylation profiles of NSCLC tumors. (C) 2002 Wiley-Liss, Inc.

DOI： 10.1002/ijc.10787

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日本人非小細胞癌におけるDNAメチル化と臨床病理学的因子の検討

豊岡伸一, 鈴木実, 丸山理一郎, 福山康朗, 飯笹俊彦, 藤沢武彦, 清水信義

日本呼吸器外科学会雑誌 17 ( 3 ) 281 - 281 2003

c-N0肺癌切除例の検討 : 縦隔鏡検査の適応について

三竿貴彦, 豊岡伸一, 中野秀治, 久保孝文, 林達朗

日本呼吸器外科学会雑誌 17 ( 3 ) 390 - 390 2003

A case of pancreatic adenocarcinoma with novel K-ras mutation and long term survival. Reviewed

Toyooka S, Tsukuda K, Mizuta M, Soh J, Shirakawa K, Shimizu K, Shimizu N

The American journal of gastroenterology 97 ( 7 ) 1852 - 1853 2002.7

Differential expression of FEZ1/LZTS1 gene in lung cancers and their cell cultures Reviewed

S Toyooka, Y Fukuyama, Wistuba, II, MS Tockman, JD Minna, AF Gazdar

CLINICAL CANCER RESEARCH 8 ( 7 ) 2292 - 2297 2002.7

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Purpose: The FEZ1/LZTS1 (FEZ1) gene, located on chromosome 8p22 (8p22), was identified recently as a candidate tumor suppressor gene. Because loss of heterozygosity at 8p21-22 is a frequent event in lung cancers, we studied FEZ1 alteration in short-term cultures of resected lung cancer tumors and cell lines.
Experimental Design: We examined FEZ1 expression in 17 non-small cell lung cancer (NSCLC), 19 small cell lung cancer (SCLC) cell lines, and 6 pairs of short-term cultures of resected NSCLCs and accompanying nonmalignant bronchial cells (NBECs) by reverse transcription-PCR and Western blotting. To investigate the mechanism for silencing, cells were cultured with 5-aza-2'-deoxycytidine or trichostatin A. We screened for genomic mutations by PCR-single-strand conformational polymorphism.
Results: Thirteen of 17 NSCLC (76%) and 3 of 19 SCLC (16%) of cell lines showed absent expression (P = 0.001). Of the paired NSCLC-NBEC cultures, 3 of 6 showed loss of expression in tumor cell cultures. In the cell lines retaining expression, the amplicon products in SCLCs were more intense than those of NSCLCs and NBECs. Expression of FEZ1 was not restored by 5aza-2'-deoxycytidine and trichostatin A. Although FEZ1 expression was moderately correlated with loss of heterozygosity of specific microsatellite makers at 8p21-22 in NSCLC cell lines, it was strongly correlated to D8S261 and LPL loci in SCLC cell lines. No mutation was found within cording region of FEZ1 by PCR-single-strand conformational polymorphism.
Conclusions: We found differential FEZ1 expression in NSCLC and SCLC cell lines, and the absent expression in 3 of 6 short-term cultures of NSCLC tumors. FEZ1 may be related to tumorigenesis of lung cancer.

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Aberrant methylation of the CDH13 (H-cadherin) promoter region in colorectal cancers and adenomas Reviewed

S Toyooka, KO Toyooka, K Harada, K Miyajima, P Makarla, UG Sathyanarayana, J Yin, F Sato, N Shivapurkar, SJ Meltzer, AF Gazdar

CANCER RESEARCH 62 ( 12 ) 3382 - 3386 2002.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Expression of the cadherin family member CDH13 (H-cadherin) is reduced in several human tumors, and it has been hypothesized that this gene functions as a tumor suppressor gene. Previously, we reported that the 5' region of CDH13 is frequently methylated in breast and lung cancers. Here we confirmed the promoter activity of 5' region of CDH13 by luciferase assay and examined its aberrant methylation in colorectal cancers, cell lines, and adenomas. Methylation status was investigated by methylation-specific PCR (MSP) and by bisulfite DNA sequencing of cloned DNA of PCR amplicons. In cell lines, we examined the correlation between methylation status and mRNA expression by reverse transcription-PCR. Aberrant methylation of CDH13 was present in 7 of 13 (54%) cell lines, and expression was absent in 6 of 13 (46%) cell lines. CDH13 expression was present in six cell lines that showed only the unmethylated form by MSP and in one cell line that showed both the methylated and unmethylated forms. Treatment with 5-aza-2'-deoxycytidine restored CDH13 expression in methylated cell lines. In surgically resected samples, 17 of 35 (49%) cases of primary colorectal cancer, 2 of 33 (6%) cases of corresponding nonmalignant colorectal mucosa, and 8 of 19 (42%) adenomas were methylated. Sequence data after bisulfite treatment indicated that primary cancers and two cell lines with loss of expression were highly methylated compared with nonmalignant colorectal epithelial cells, especially at the attachment sites of primers for NISP, although there was heterogeneity in methylation status. Our results suggest that CDH13 expression is frequently silenced by aberrant methylation in colorectal cancers and adenomas and that methylation of CDH13 commences at an early stage of multistep colorectal tumorigenesis.

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Differential inactivation of caspase-8 in lung cancers Reviewed

Narayan Shivapurkar, Shinichi Toyooka, Michael T. Eby, Chun Xian Huang, Ubaradka G. Sathyanarayana, H. Thomas Cunningham, Jyotsna L. Reddy, Elizabeth Brambilla, Takashi Takahashi, John D. Minna, Preet M. Chaudhary, Adi F. Gazdar

Cancer Biology and Therapy 1 ( 1 ) 65 - 69 2002

Language：English Publishing type：Research paper (scientific journal) Publisher：Landes Bioscience

Caspase-8 (CASP8) 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.

DOI： 10.4161/cbt.1.1.45

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DNA methylation profiles of lung tumors Reviewed

Shinichi Toyooka, Kiyomi O. Toyooka, Riichiroh Maruyama, Arvind K. Virmani, Luc Girard, Kuniharu Miyajima, Kenichi Harada, Yutaka Ariyoshi, Takashi Takahashi, Kenji Sugio, Elisabeth Brambilla, Michel Gilcrease, John D. Minna, Adi F. Gazdar

Molecular Cancer Therapeutics 1 ( 1 ) 61 - 67 2001.11

Language：English Publishing type：Research paper (scientific journal)

Aberrant methylation of CpG islands in promoter regions of tumor cells is one of the major mechanisms for silencing of tumor suppressor genes. We determined the frequency of aberrant promoter methylation of the p16, adenomatous polyposis coli (APC), H-cadherin (CDH13), glutathione S-transferase P1 (GSTP1), O6-methylguanine-DNA-methyltransferase (MGMT), retinoic acid receptor β-2 (RARβ), E-cadherin (CDH1), and RAS association domain family 1A (RASSF1A) genes in 198 tumors consisting of small cell lung cancers [SCLCs (n = 43)], non-small cell lung cancers [NSCLCs (n = 115)], and bronchial carcinoids (n = 40). The profile of methylated genes in the two neuroendocrine tumors (SCLC and carcinoids) were very different from that of NSCLC. However, whereas the overall pattern of aberrant methylation of carcinoids was similar to that of SCLC, carcinoids had lower frequencies of methylation for some of the genes tested. There were also significant differences in the methylation profiles between the two major types of NSCLC, adenocarcinoma and squamous cell carcinoma. We performed cluster analysis and found that SCLCs clustered with other SCLCs and carcinoids but not with NSCLCs, whereas the NSCLCs tended to cluster together. Within NSCLCs, adenocarcinomas and squamous cell carcinomas clustered with their respective histological types. Finally, we compared the methylation profiles of SCLC and NSCLC tumors and their respective cell lines (n = 44). In general, methylation frequencies were higher in tumor cell lines, but these differences were seldom significant. Thus, tumor cell lines appear to be suitable models to study aberrant DNA methylation. We conclude that SCLC, carcinoids, squamous cell carcinomas, and adenocarcinomas of the lung have unique profiles of aberrant methylation. Our findings should help us understand differences in the pathogenetic mechanisms of lung cancers. © 2001 American Association for Cancer Research.

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Loss of expression and aberrant methylation of the CDH13 (H-cadherin) gene in breast and lung carcinomas Reviewed

KO Toyooka, S Toyooka, AK Virmani, UG Sathyanarayana, DM Euhus, M Gilcrease, JD Minna, AF Gazdar

CANCER RESEARCH 61 ( 11 ) 4556 - 4560 2001.6

Language：English Publishing type：Research paper (scientific journal) Publisher：AMER ASSOC CANCER RESEARCH

Expression of some members of the cadherin family is reduced in several human tumors, and CDH13 (B-cadherin), located on chromosome 16q24.2-3, may function as a tumor suppressor gene. In human tumors, loss of expression of many tumor suppressor genes occurs by aberrant promoter region methylation. We examined the methylation status of the CDH13 promoter in breast and lung cancers and correlated it with mRNA expression using methylation-specific PCR and reverse transcription-PCR Methylation was frequent in primary breast tumors (18 of 55, 33%) and cell lines (7 of 20, 35%). In lung cancers, methylation was present more frequently in non-small cell lung cancer tumors (18 of 42, 43%) and cell lines (15 of 30, 50%) than in small cell Lung cancer cell Lines (6 of 30, 20%; P = 0.03). Only the methylated or unmethylated forms of the gene were present in most (73 of 80, 91%) tumor cell lines. CDH13 expression was present in 24 of 30 (80%) of nonmethylated tumor lines, All 18 methylated lines tested lacked expression irrespective of whether the unmethylated form was present, confirming biallelic inactivation in methylated lines, Gene expression was restored in all five methylated cell lines tested after treatment with the demethylating agent 5 ' -aza-2-deoxycytidine. Our results demonstrate frequent aberrant methylation of CDH13 in breast and lung cancers accompanied by loss of gene expression, although expression may occasionally be lost by other mechanisms.

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Three cases report of duplication of alimentary tract Reviewed

Shinichi Toyooka, Kazutoyo Shirakawa, Shinji Hato, Osanori Sogabe, Hiroya Maeda, Minoru Mizuta, Takashi Ohya, Ichizo Suemitsu

Japanese Journal of Gastroenterological Surgery 30 ( 1 ) 88 - 91 1997

Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Society of Gastroenterological Surgery

We have treated three cases of alimentary tract duplication in the last four years. Case 1 involved a 34-year-old man who had complained of abdominal pain since early childhood. On X-ray examination, an intra-abdominal calcification was detected. Although a definite diagnosis was not made, a laparotomy was performed because of the history of frequent severe abdominal pain. This led to diagnosis of duplication of the ileum. In case 2 a 13-year-old boy complained of abdominal pain and melena. Laparotomy was performed under a suspected diagnosis of Meckel's diverticulitis. A diagnosis of duplication of the ileum was then made. Case 3 involved a 67-year-old man who had undergone total gastrectomy because of gastric cancer. The operative findings included a tumor the size of a child's head was attached to the S-colon. We resected the tumor and made a diagnosis of duplication of the S-colon. The preoperative diagnosis of alimentary tract duplication is difficult. This potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause.

DOI： 10.5833/jjgs.30.88

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Papers - TOYOOKA Shinichi