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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.

Qualitative and quantitative disorders of the tear film were far more common than recognized in this population of elderly subjects, meibomian gland dysfunction being the most common associate of the tear film disorder and dry eye status. Conjunctivochalasis (conjunctival laxity), although commonly associated with dry eye disease in the elderly, was observed not to be related to age or gender in this study.

Warm moist air device use provided symptomatic relief of ocular fatigue and improvement of tear stability in patients with MGD. The new warm moist air device seems to be a safe and promising alternative in the treatment of MGD.

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The SFRP1 gene on chromosome 8p11.2 encodes a Wnt signaling antagonist, and was recently demonstrated to be a new tumor suppressor that is inactivated by promoter methylation in human colon cancers. Here, we analyzed promoter methylation of the SFRP1 gene in human ovarian cancers, in which loss of heterozygosity in 8p is frequently observed and involvement of the Wnt signaling pathway has been suggested. Methylation-specific PCR (MSP) analysis showed that four of 13 ovarian cancer cell lines and two of 17 primary ovarian cancers had methylated SFRP1, while an immortalized ovarian epithelial cell line, HOSE, and seven ovarian endometrial cyst samples did not. In the four ovarian cancer cell lines with the methylation, SFRP1 was not expressed at all as determined by quantitative RT-PCR analysis. A cell line with SFRP1 methylation, MCAS, was treated with a demethylating agent, 5-aza-2'-deoxycytidine, and demethylation of the promoter and re-expression of SFRP1 were observed. These results show that SFRP1 is inactivated by promoter methylation in human ovarian cancers, as well as colon cancers. varian cancer is the most lethal gynecological malignancy, and is characterized by the diversity of its histology.1) The four major histological subtypes, serous, clear cell, mucinous, and endometrioid, constitute approximately 36, 19, 14, and 15% of the total, respectively, in Japan, 2) and all of these are considered to be derived from ovarian surface epithelium.3) As for genetic alterations, only limited information is available, such as mutations of β-catenin in 31% of endometrioid type 4,5) and c-erbB-2 amplification in serous type with poor prognosis.6) Due to promoter methylation, BRCA1, hMLH1, and RASSF1A are inactivated (silenced) in 10%, 7) 12.5%, 8) and 10-40% of ovarian cancers, 9, 10) respectively. Clarification of molecular alterations is important to identify novel diagnostic and therapeutic targets, and further investigations in ovarian cancers are necessary.Secreted frizzled-related proteins (SFRPs) are extracellular signaling molecules that antagonize the Wnt signaling pathway.11, 12) SFRP1 (8p11.2) is inactivated in colorectal cancers by promoter methylation [13][14][15] and also by infrequent mutations.15) Other members, SFRP2 (4q31.3) and SFRP5 (10q24.1), are also inactivated by promoter methylation in colorectal cancers.14) Strikingly, the inactivation of SFRP genes enhanced the constitutive Wnt signaling even in the presence of APC or β-catenin mutations.14, 15) In ovarian cancers, SFRP inactivation could also potentially be present for the following reasons. First, the Wnt signaling pathway can be affected in ovarian cancers, as indicated by the presence of β-catenin mutations in cancers of the endometrioid type. 4,5) Second, in the serous type, in spite of increased β-catenin accumulation, 16) β-catenin mutations are infrequent, 17) and abnormality in other genes in the Wnt signaling pathway is suggested.In this study, we analyzed methylation-associated silencing of the SFRP1 gene on chromosome 8p11.2...