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Lysyl oxidase (LOX) and HRAS-like suppressor (HRASLS) are silenced in human gastric cancers and are reported to have growth-suppressive activities in ras-transformed mouse/rat fibroblasts. Here, we analyzed whether or not LOX and HRASLS are tumor suppressor genes in human gastric cancers. Loss of heterozygosity and promoter methylation of LOX were detected in 33% (9 of 27) and 27% (26 of 96) of gastric cancers, respectively. Biallelic methylation and loss of heterozygosity with promoter methylation were also demonstrated in gastric cancers. Silencing of LOX was also observed in colon, lung, and ovarian cancer cell lines. As for mutations, only one possible somatic mutation was found by analysis of 96 gastric cancer samples and 58 gastric and other cancer cell lines. When LOX was introduced into a gastric cancer cell line, MKN28, in which LOX and HRASLS were silenced, it reduced the number of anchorage-dependent colonies to 57 to 61%, and the number of anchorage-independent colonies to 11 to 23%. Sizes of tumors formed in nude mice were reduced to 19 to 26%. Growth suppression in soft agar assay was also observed in another gastric cancer cell line, KATOIII. On the other hand, neither loss of heterozygosity nor a somatic mutation was detected in HRASLS, and its introduction into MKN28 did not suppress the growth in vitro or in vivo. These data showed that LOX is a tumor suppressor gene inactivated by methylation and loss of heterozygosity in gastric cancers, and possibly also in other cancers.

Aberrant CpG methylations play important roles in cancer development and progression. In this study, aberrant methylations in human breast cancer were searched for using methylation-sensitive representational difference analysis (MS-RDA). A CpG island (CGI) in the 5 0 region of the heparan sulfate d-glucosaminyl 3-Osulfotransferase-2 (3-OST-2) gene was found to be hypermethylated, while its exon 2 was hypomethylated. In seven breast cancer cell lines, hypermethylation of the 5 0 region and loss of 3-OST-2 expression were observed. Treatment with a demethylating agent, 5-aza-2 0 -deoxycytidine, removed the methylation of the CGI in the 5 0 region and restored its expression, demonstrating silencing of the 3-OST-2 gene. Methylation-specific PCR (MSP) analysis in 85 primary breast cancers showed that the hypermethylation of the CGI in the 5 0 region was present in 75 (88%) of them. Quantitative reverse transcriptase-PCR (RT-PCR) analysis in 37 primary breast cancers showed that the average expression level was decreased in them. Further, MSP analysis in primary colon, lung and pancreatic cancers showed that hypermethylation of the CGI in the 5 0 region was present in the colon (8/10, 80%), lung (7/10, 70%) and pancreatic (10/10, 100%) cancers. These results showed that silencing of 3-OST-2 was present in a wide range of human cancers. The 3-OST-2 gene encodes an enzyme involved in the final modification step of heparan sulfate proteoglycans (HSPGs), and its silencing is expected to result in abnormal modification of HSPGs and abnormal signal transduction. From the high incidence, silencing of the 3-OST-2 gene is expected to have high diagnostic, and potentially therapeutic, values.