Takefumi Kikuchi

Expression profiles of non-small cell lung cancers on cDNA microarrays: Identification of genes for prediction of lymph-node metastasis and sensitivity to anti-cancer drugs

To investigate genes involved in pulmonary carcinogenesis and those related to sensitivity of nonsmall cell lung cancers (NSCLCs) to therapeutic drugs, we performed cDNA microarray analysis of 37 NSCLCs after laser-capture microdissection of cancer cells from primary tumors. A clustering algorithm applied to the expression data easily distinguished two major histological types of non-small cell lung cancer, adenocarcinoma and squamous cell carcinoma. Subsequent analysis of the 18 adenocarcinomas identified 40 genes whose expression levels could separate cases with lymph-node metastasis from those without metastasis. In addition, we compared the expression data with measurements of the sensitivity of surgically dissected NSCLC specimens to six anti-cancer drugs (docetaxel, paclitaxel, irinotecan, cisplatin, gemcitabine, and vinorelbine), as measured by the CD-DST (collagen gel droplet embedded culture-drug sensitivity test) method. We found significant associations between expression levels of dozens of genes and chemosensitivity of NSCLCs. Our results provide valuable information for eventually identifying predictive markers and novel therapeutic target molecules for this type of cancer.

Frequent hypermethylation of CpG islands and loss of expression of the 14-3-3 σ gene in human hepatocellular carcinoma

The 14-3-3 σ gene has been implicated in G2/M cell cycle arrest by p53. Frequent inactivation of the 14-3-3 σ gene by hypermethylation of CpG islands has recently been reported in human breast carcinoma. The aim of this study was to examine the methylation status of CpG islands of the 14-3-3 σ gene in hepatocellular carcinoma (HCC). The methylation status of the 14-3-3 σ gene was evaluated in four normal liver tissues and 19 paired specimens of carcinoma and adjacent non-tumorous liver tissues using bisulfite-single strand conformation polymorphism (bisulfite-SSCP), a combination of sodium bisulfite modification and fluorescence-based polymerase chain reaction (PCR)-SSCP. The 14-3-3 σ protein expression was examined by immunohistochemical staining. Hypermethylation of CpG islands of the 14-3-3 σ gene was detected in 89% (17/19) of the HCC tissues but not in any of the four normal liver tissues. All of the 14 methylation-positive HCC samples analysed by immunohistochemistry showed loss of 14-3-3 σ expression, while both of the methylation-negative HCC samples retained the expression, and a significant correlation was found between methylation and loss of expression. Lower levels of methylation were detected in adjacent non-tumorous liver tissues (6/16 in cirrhotic tissues and 1/3 in chronic hepatitis tissues), but the 14-3-3 σ expression was retained in all of these tissues. In a methylation-positive HCC cell line, HLE, 5-aza-2′-deoxycytidine (5-aza-dC)-induced demethylation of CpG islands led to reactivation of gene expression, indicating that hypermethylation plays a causal role in inactivation of the 14-3-3 σ gene in HCC. Hypermethylation and the resulting loss of expression of the 14-3-3 σ gene corresponds to one of the most common abnormalities reported to date in HCC, suggesting their crucial role in the development and/or progression of HCC.

Epigenetic inactivation of CHFR in human tumors

Cell-cycle checkpoints controlling the orderly progression through mitosis are frequently disrupted in human cancers. One such checkpoint, entry into metaphase, is regulated by the CHFR gene encoding a protein possessing forkhead-associated and RING finger domains as well as ubiquitin–ligase activity. Although defects in this checkpoint have been described, the molecular basis and prevalence of CHFR inactivation in human tumors are still not fully understood. To address this question, we analyzed the pattern of CHFR expression in a number of human cancer cell lines and primary tumors. We found CpG methylation-dependent silencing of CHFR expression in 45% of cancer cell lines, 40% of primary colorectal cancers, 53% of colorectal adenomas, and 30% of primary head and neck cancers. Expression of CHFR was precisely correlated with both CpG methylation and deacetylation of histones H3 and H4 in the CpG-rich regulatory region. Moreover, CpG methylation and thus silencing of CHFR depended on the activities of two DNA methyltransferases, DNMT1 and DNMT3b, as their genetic inactivation restored CHFR expression. Finally, cells with CHFR methylation had an intrinsically high mitotic index when treated with microtubule inhibitor. This means that cells in which CHFR was epigenetically inactivated constitute loss-of-function alleles for mitotic checkpoint control. Taken together, these findings shed light on a pathway by which mitotic checkpoint is bypassed in cancer cells and suggest that inactivation of checkpoint genes is much more widespread than previously suspected.

DISTINCT METHYLATION PATTERN AND MICROSATELLITE INSTABILITY IN SPORADIC GASTRIC CANCER

Aberrant 5′ CpG island methylation is an alternative mechanism of gene inactivation during the development of cancer as demonstrated for several tumor‐suppressor genes. Also, marked relationship of microsatellite instability (MSI) and DNA methylation has been reported in sporadic colorectal cancer, which is a result of epigenetic inactivation of hMLH1 in association of promoter hypermethylation. In the present study, we investigated the 5′ CpG island hypermethylation of hMLH1, E‐cadherin and p16 in 61 primary gastric cancers (GCs) by using combined bisulfite restriction analysis (COBRA) and methylation‐specific PCR (MSP), and their MSI status. Of 61 GCs investigated, 5 (8.1%) tumors presented hMLH1 methylation, 16 (26.2%) and 25 (40.9%) showed E‐cadherin and p16 methylation respectively, and 8 (13.1%) presented high‐frequency MSI (MSI‐H). Of the 8 MSI‐H patients, 5 presented hMLH1 methylation, whereas no low‐frequency MSI (MSI‐L) and microsatellite stable (MSS) cases exhibited hMLH1 methylation (5/8 vs. 0/43, p < 0.00001). Furthermore, these patients also presented E‐cadherin and p16 hypermethylation. Our data showed a significant correlation between hMLH1 methylation and MSI in GC, and suggested that a common mechanism of aberrant de novo methylation can be postulated in these cancers. Int. J. Cancer 83:309–313, 1999.

Inactivation of p57KIP2 by regional promoter hypermethylation and histone deacetylation in human tumors

To clarify the role of DNA methylation in the silencing of the expression of cyclin-dependent kinase inhibitor p57KIP2 seen in certain tumors, we investigated the methylation status of its 5′ CpG island in various tumor cell lines and primary cancers. Dense methylation of the region around the transcription start site was detected in 1 out of 10 colorectal, 2 out of 8 gastric, and 6 out of 14 hematopoietic tumor cell lines and in 5 out of 35 (14%) gastric, 6 out of 20 (30%) hepatocellular, and 2 out of 18 (11%) pancreatic cancers; 7 out of 25 (28%) acute myeloid leukemia cases also showed methylation of the p57KIP2 gene, which strongly correlated with the CpG island methylator phenotype (P<0.001). Detailed mapping revealed that dense methylation of the region around the transcription start site (−300 to +400), but not of the edges of the CpG island, was closely associated with gene silencing. 5-aza-2′-deoxycytidine, a methyltransferase inhibitor, restored expression of p57KIP2, and chromatin immunoprecipitation using anti-histone H3 and H4 antibodies showed histone to be deacetylated in cell lines where p57KIP2 was methylated at the transcription start site. Regional methylation and histone deacetylation thus appear to be crucially involved in the silencing of p57KIP2 expression in human tumors.

DNA methylation and histone deacetylation associated with silencing DAP kinase gene expression in colorectal and gastric cancers

Death-associated protein kinase is a positive regulator of programmed cell death induced by interferon γ. To investigate the role of epigenetic inactivation of death-associated protein kinase in gastrointestinal cancer, we examined the methylation status of the 5′ CpG island of the death-associated protein kinase gene. Methylation of the 5′ CpG island was detected in 3 of 9 colorectal and 3 of 17 gastric cancer cell lines, while among primary tumours, it was detected in 4 of 28 (14%) colorectal and 4 of 27 (15%) gastric cancers. By contrast, methylation of the edge of the CpG island was detected in virtually every sample examined. Death-associated protein kinase expression was diminished in four cell lines that showed dense methylation of the 5′ CpG island, and treatment with 5-aza-2′-deoxycitidine, a methyltransferase inhibitor, restored gene expression. Acetylation of histones H3 and H4 in the 5′ region of the gene was assessed by chromatin immunoprecipitation and was found to correlate directly with gene expression and inversely with DNA methylation. Thus, aberrant DNA methylation and histone deacetylation of the 5′ CpG island, but not the edge of the CpG island, appears to play a key role in silencing death-associated protein kinase expression in gastrointestinal malignancies.

Aberrant methylation and histone deacetylation of cyclooxygenase 2 in gastric cancer

Cyclooxygenase 2 plays a critical role in the development of gastrointestinal cancers in both human and animal models. About 80% of the gastric cancer showed a high level of expression of cyclooxygenase 2, but a subset of cases do not express without unknown reason. Aberrant methylation of CpG island of COX‐2 was examined by using a series of gastric cancer cell lines and primary gastric cancers. Two out of 8 cell lines (25%) and 11 out of 93 (12%) primary cancers showed aberrant methylation of the 5′ region of COX‐2. Methylation of COX‐2 was closely associated with loss of expression and treatment of methylation inhibitor, 5‐deoxy‐2′‐azacytidine restored the expression of COX‐2. A combined treatment of 5‐deoxy‐2′‐azacytidine and a histone deacetylese inhibitor, trichostatin A, restored re‐expression of the gene synergistically and chromatin immunoprecipitation analysis revealed that histone of methylated COX‐2 promoter is deacetylated, indicating the role of cytosine methylation and histone deacetylation in the silencing of the gene. These results indicate that a subset of gastric cancer with COX‐2 methylation evolves through the pathway that is independent of COX‐2 expression and that COX‐2 inhibitor may not be useful to induce apoptosis in these cases.

Quantitative DNA methylation analysis by fluorescent polymerase chain reaction single-strand conformation polymorphism using an automated DNA sequencer.

A novel DNA methylation assay technique, termed bisulfite single‐strand conformation polymorphism (bisulfite‐SSCP), is a combination of sodium‐bisulfite modification and fluorescence‐based polymerase chain reaction (PCR)‐SSCP. After bisulfite treatment followed by PCR amplification, methylated and unmethylated alleles can be simultaneously separated in a nondenaturing gel using an automated DNA sequencer. Using bisulfite‐SSCP, methylation of hMLH1 was detected in a quantitative manner. This method is not only simple, quick, accurate, and quantitative, but detailed information about methylation is also available with less work. Methylation analysis of large numbers of samples for multiple loci will be facilitated by bisulfite‐SSCP.

DNA methylation changes in gastrointestinal disease

DNA methylation of the 5′ region of genes is often associated with gene silencing in X-chromosome inactivation and imprinting. Recent studies have indicated that altered DNA methylation plays a role in the inactivation of multiple tumor suppressor genes and DNA repair genes such as pl6INK4A and hMLHl. Colorectal adenomas have a relatively high frequency of methylation, and aberrant methylation is an early event during tumorigenesis. In aging patients, even colon epithelium which appears to be normal showed a significant amount of methylation in a subset of the genes. Colon mucosa from patients with inflammatory bowel disease also showed a high level of methylation. DNA methylation can be a specific diagnostic marker in gastrointestinal cancer and inflammatory bowel disease, for which there is no perfect marker for a noninvasive diagnosis.

Increased expression of T‐fimbrin gene after DNA damage in CHO cells and inactivation of T‐fimbrin by CpG methylation in human colorectal cancer cells

When DNA damage is induced by unprogrammed extrinsic events, activating‐cell‐cycle checkpoints delay cell‐cycle progression in the G1 or G2 phases and allow repair of a damaged template. In this study, we evaluated changes in gene expression upon radiation‐induced G2 cell‐cycle arrest using Chinese hamster ovary (CHO) cells. T‐fimbrin, an actin‐binding protein, was overexpressed in CHO cells in which G2 arrest had been induced by X‐radiation. Northern blot analysis revealed that T‐fimbrin gene expression was induced not only by X‐radiation but also by a topoisomerase II inhibitor, etoposide. Transfection of CHO cells with a vector encoding T‐fimbrin antisense RNA demonstrated that reduced T‐fimbrin expression induced alterations in cell‐cycle control; radiation‐induced G2 arrest was short and decreased in cells transfected with antisense T‐fimbrin. Additionally, T‐fimbrin gene expression was suppressed in a human colorectal cancer cell line, SW948, because of promoter‐specific DNA methylation. These results suggest that downregulation of T‐fimbrin may be involved in cancer development through G2/M cell‐cycle control in mammalian cells.