BackgroundThe Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.MethodsWe used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the KEAP1 promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, NQO-1 and AKR1C1.ResultsDNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the KEAP1 promoter region. HT29 cells with a hypermethylated KEAP1 promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased KEAP1 mRNA expression. These result suggested that methylation of the KEAP1 promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of NQO-1 and AKR1C1 mRNA than Colo320DM cells. Aberrant promoter methylation of KEAP1 was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the KEAP1 promoter region, conferring a protective effect against cytotoxic anticancer drugs.ConclusionHypermethylation of the KEAP1 promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.

We previously showed that plasmids containing a mammalian replication initiation region and a matrix attachment region were efficiently amplified to few thousand copies per cell, and that they formed extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). In these structures, the plasmid sequence was arranged as a tandem repeats, and we suggested a mechanism of plasmid amplification. Since amplification was very efficient, easy, and convenient, it might be adapted to a novel method for protein production. In the current study, we found that gene expression from the tandem plasmid repeat was suppressed. We identified several strategies to overcome this suppression, including: (1) use of higher concentrations of antibiotic during cell selection; (2) treatment of cells with agents that influence DNA methylation (5-azacytidine) or histone acetylation (butyrate); (3) co-amplification of an insulator sequence; and (4) co-amplification of sequences that encode a transcriptional activator. Expression from the plasmid repeat was always higher at DMs compared to HSRs. We found that continuous activation of a plasmid-encoded inducible promoter prevented the generation of long HSRs, and favored amplification at DMs. Consistent with this finding, there was a synergistic effect of transcriptional activation and inhibition of DNA methylation on the fragmentation of long HSRs and the generation of DMs and short HSRs. Our results indicate that both transcriptional activation and DNA methylation regulate the interconversion between extra- and intra-chromosomal gene amplification. These results have important implications for both protein production technology, and the generation of chromosomal abnormalities found in human cancer cells.

Galectin-9 is an eosinophil chemoattractant produced by activated T lymphocytes. We have addressed expression of galectin-9 in normal human astrocytes in culture. Expression of galectin-9 mRNA and protein were examined by reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunofluorescent staining. Interleukin-1beta (IL-1beta) was found to enhance the galectin-9 expression in time- and concentration-dependent manners. Galectin-9 protein was detected in the membrane fraction, 105 000 x g precipitate, and immunofluorescent staining revealed diffuse cellular and perinuclear distributions. Dexamethasone pretreatment almost completely suppressed the production. We conclude that astrocytes produce galectin-9 in response to the stimulation with IL-1beta, and this may contribute to inflammatory reactions in the CNS.

Background: The turning point in the deterioration of physical function seems to occur between the ages of 70 and 80 years. In particular, muscle strength may decline even more in subjects older than 75. A recent study found that the angiotensin-converting enzyme (ACE) genotype also affects physiological left ventricular hypertrophy. A very limited number of papers have examined genetic differences in resistance and endurance forms of a single sporting discipline. Objective: The purpose of this study was to evaluate the relationship between ACE genotype and physical function by controlling the known confounding factors including dental status. Methods:We selected 431 subjects who were aged 76 years and did not require special care for their daily activities. We conducted a medical examination, followed by 5 physical function tests, as follows: (1) maximum hand grip strength, (2) maximal isometric knee extensor strength, (3) maximal stepping rate for 10 s, (4) one-leg standing time with eyes open and (5) 10-meter maximum walking speed. Subjects were genotyped for the ACE intron 16 Alu insertion. In addition, serum concentrations of total cholesterol, total protein, IgA and IgG were measured at a commercial laboratory. The Eichner index was used as an indicator of occlusal condition. Multiple linear regression analysis was performed to evaluate the relationship between the ACE gene insertion/deletion (I/D) polymorphism and physical function considering confounding factors. Results: The ACE gene I/D polymorphism was positively associated with hand grip strength and 10-meter maximum walking speed. Betas of hand grip strength were 0.09 for I/D (p = 0.022) and 0.12 for insertion/insertion (I/I; p = 0.004). Betas of 10-meter walking speed were –0.11 for I/D (p = 0.093) and –0.14 for I/I (p = 0.039). Dental status such as Eichner index class C was significantly associated with one-leg standing time with eyes open (beta –0.11; p = 0.028). Conclusion: This study suggests that there is a significant relationship between ACE genotype and physical function. In particular, subjects with the ACE deletion/deletion genotype were associated with upper extremities.

From these preliminary data, these polymorphisms would be candidates predicting the clinicopathological features of colorectal cancer, but further more systematic gene analyses are warranted.