Abstract Reactive oxygen species generated in living cells during normal cellular metabolism and in response to exogenous inducers cause the formation of oxidatively damaged bases in DNA. These oxidized base lesions are primarily removed by the base excision repair (BER) pathway. A BER enzyme, NTH1, has activity that is capable of removing oxidized pyrimidines, such as thymine glycol, from DNA. To clarify whether the NTH1 gene is involved in gastric carcinogenesis, we first examined the NTH1 expression level in eight gastric cancer cell lines, and the results showed that NTH1 expression was downregulated in all of them, including cell line AGS. Next, a comparison of excisional repair activity against thymine glycol by empty-vector-transfected AGS clones and FLAG-NTH1-expressing AGS clones showed that a low NTH1 expression level led to low capacity to repair the damaged base in the gastric epithelial cells. Reduced mRNA expression of NTH1 was also detected in 36% (18/50) of primary gastric cancers. Moreover, immunohistochemical analysis revealed that NTH1 was predominantly localized in the cytoplasm in 24% (12/50) of the primary gastric cancers in contrast to the nuclear localization in non-cancerous tissue, suggesting impaired excisional repair ability for nuclear DNA. Next, we found two novel genetic polymorphisms, i.e., c. −163C>G and c. −241_-221del, in the NTH1 promoter region, and a luciferase assay showed that both were associated with reduced promoter activity. However, there were no associations between the polymorphisms and risk of gastric cancer in a gastric cancer case-control study. Next, all the coding exons of the NTH1 gene and their boundary regions in DNAs of 23 Japanese patients with gastric cancer was searched for genetic polymorphisms by direct sequencing. A novel variant, c. −31A>G, and a single nucleotide polymorphism, c.98G>A (Arg33Lys, rs2302172) were detected at an allele frequency of 2.2%, and 4.3%, respectively. However, the c. −31A>G polymorphism did not affect reduction of promoter activity. We are going to reveal functional characterization of c.98G>A and to examine excisional repair activity against new substrates, such as oligonucleotide containing etheno adducts. Our data suggested that downregulation of NTH1 expression, abnormal localization of NTH1 and the missense polymorphism may be involved in the pathogenesis of a subset of gastric cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2802.
Sequences of human endogenous retroviruses (HERVs) are members of the long terminal repeat (LTR) retrotransposon family. Although the expression of HERV has long been a topic of investigation, HERV-insertion polymorphisms are not well known, and a genetic association between HERV-insertion polymorphisms and cancer has never been reported. To identify novel HERV loci in the genome from cancer tissues, we carried out the inverse PCR method targeting a conserved LTR region of HML-2, which is the most recently acquired HERV group. Novel two insertions, HML-2_sLTR ( 1p13.2 ) and HML-2_sLTR ( 19q12 ), were identified as insertionally polymorphic solo LTRs. Furthermore, a significant prevalence of HML-2_sLTR ( 1p13.2 ) homozygosity was detected in female never-smoking patients aged 60 years and over who had lung adenocarcinoma [versus the other genotyping; odds ratio (OR): 1.97; 95% confidence interval (CI): 1.01–3.81]. In another cohort consisting of female never-smoking patients with lung adenocarcinoma, a prevalence of HML-2_sLTR ( 1p13.2 ) homozygosity tended to be high in patients aged 60 years and over (versus the other genotyping; OR: 2.03; 95% CI: 0.96–4.29), whereas a low prevalence of HML-2_sLTR ( 1p13.2 ) homozygosity was detected in patients <60 years old (versus the other genotyping; OR: 0.31; 95% CI: 0.11–0.94). Our results suggest that HML-2_sLTR ( 1p13.2 ) is involved with the susceptibility to lung adenocarcinoma in female never-smokers in an age-dependent manner and that other HERV polymorphisms related to human diseases might remain to be identified in the human genome.
Mitosis is the most conspicuous cell cycle phase and Shugoshin-like 1 (SGOL1) is a key protein in protecting sister chromatids from precocious separation during mitosis. We studied the role of SGOL1 and its splice variants in non-small cell lung cancer (NSCLC) using 82 frozen NSCLC tissue samples. SGOL1-B expression was prevalent in smokers, in cases with a wild-type (WT) EGFR status and in cases with the focal copy number amplification of genes that are known to be important for defining the biological behaviors of NSCLC. The overexpression of SGOL1-B1 in an NSCLC cell line induced aberrant chromosome missegregation, precociously separated chromatids and delayed mitotic progression. A higher level of SGOL1-B mRNA was related to taxane resistance, while the forced downregulation of SGOL1-B increased the sensitivity to taxane. These results suggest that the expression of SGOL1-B causes abnormal mitosis and taxane resistance in NSCLC cells.
To test the feasibility of using bacterial artificial chromosomes (BAC) containing kinases for pathological diagnosis using fluorescence in situ hybridization (FISH), 10 BAC probes containing a gene amplified in 5% or more of a pilot cohort were selected from a previous survey using arbitrarily selected BAC clones harboring 100 kinases. In this report, we describe the prevalence and association with the clinicopathological profile of these selected 10 BAC probes in 365 gastric cancer tissues. FISH analyses using these 10 BAC probes containing loci encoding EGFR, ERBB2(HER2), EPHB3, PIK3CA, MET, PTK7, ACK1, STK15, SRC, and HCK showed detectable amplifications in paraffin-embedded tissue in 2.83% to 13.6% of the gastric cancer tissues. Considerable numbers of the cases showed the co-amplification of two or more of the probes that were tested. BAC probes located within a genome neighborhood, such as PIK3CA, EPHB3, and ACK1 at 3q26-29 or HCK, SRC, and STK15 at 20q11-13.1, were often co-amplified in the same cases, but non-random co-amplifications of genes at distant genomic loci were also observed. These findings provide basic information regarding the creation of a strategy for personalizing gastric cancer therapy, especially when using multiple kinase inhibitors.
