Experimental Exploration for Genes Related to Susceptibility and Resistance to Irinotecan.

BACKGROUND/AIM: Anticancer drug resistance is an important issue in cancer treatment. Multiple genes are thought to be involved in resistance to anticancer drugs; however, this is still not fully understood. This study aimed to identify the genes involved in irinotecan resistance and their functional characteristics. MATERIALS AND METHODS: Gene trap insertion mutant Chinese hamster ovary (CHO) cells were used in the experiments, and next-generation sequencing, gene-ontology enrichment, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were used to evaluate the biological functions of differentially expressed genes (DEGs). RESULTS: In total, 2,134 DEGs were identified, including 1,216 up-regulated and 918 down-regulated genes. In KEGG pathways, microRNAs in cancer were significantly associated with up-regulated DEGs, while spliceosome and p53 signaling pathways were significantly associated with down-regulated DEGs. The pathway analysis identified several genes that might be involved in irinotecan resistance. CONCLUSION: Using CHO cells, the genes involved in irinotecan resistance and functional characteristics were predicted. These results provide new clues for predicting irinotecan resistance.

Gene disruption of ribosomal protein L5 (RPL5) decreased the sensitivity of CHO-K1 cells to uncoupler carbonylcyanide-3-chlorophenylhydrazone.

Protonophoric uncoupler carbonylcyanide-3-chlorophenylhydrazone (CCCP) decreases the proton motive force (ΔP) of the mitochondrial inner membrane and results in inhibition of oxidative phosphorylation. In this study, a CCCP-resistant clone was isolated from a random gene trap insertional mutant library of Chinese hamster ovary (CHO)-K1 cells which was constructed by infecting a retrovirus vector, ROSAßgeo. Although we expected the isolation of the mutants defective in nuclear genes responsible for mitochondrial functions, the disrupted gene of the isolated mutant that we named R1 cells was identified as one of the alleles for ribosomal protein 5 of large subunit (RPL5). The R1 cells express as much as 80% RPL5 protein compared with the parental CHO-K1 cells, possibly due to enhanced transcription from a remaining wild-type RPL5 allele in R1 cells. Furthermore, the protein amount is not decreased by CCCP in R1 cells, in contrast to its clear reduction by CCCP in parental cells. Since mutations of RPL5 and other ribosomal proteins are responsible for the ribosomopathies and cancer, the present mutant may be a useful cellular model of such human diseases from a viewpoint of energy metabolism as well as a tool for the study of ribosome biogenesis and extra-ribosomal function of the RPL5 protein.

Genotypes and haplotypes of CCR2 and CCR3 genes in Japanese cedar pollinosis.

Whole genome scan analyses have revealed that the chromosomal region 3p21.3, which contains a gene cluster of the CC chemokine receptor, is possibly critical for the pathogenesis of allergic inflammation. Japanese cedar pollinosis is mediated by a type I allergy and induces seasonal rhinitis and conjunctivitis in humans as the most common form of hay fever in spring in Japan, although the candidate genes for cedar pollinosis remain to be elucidated. We sequenced CCR1, CCR2, CCR3, CCR5, and CCXCR1 using the PCR restriction fragment length polymorphism method in subjects with cedar pollinosis and controls. We found 8 polymorphisms of A111G, Arg127Cys and Arg252Gln in CCXCR1, T885C in CCR1, Val64Ile and T780C in CCR2, T51C in CCR3 and Arg223Gln in CCR5. The transmission disequilibrium test using 60 children with pollinosis and their parents and an association study using unrelated adult subjects (151 patients and 157 controls) showed a significant association of 64Ile in CCR2 and 51C in CCR3 with cedar pollinosis. The frequency of haplotype 64Ile/780C/51C in pollinosis was significantly higher than in controls. Our results suggest that CCR2 and CCR3 genes are candidate genes for Japanese cedar pollinosis.

[Hyperlipidemia: complex pathophysiology caused by multiple genetic and environmental factors--in considering the approaches to preventive medicine].

In Japan, over the last 40 years, the change in lifestyle, particularly the westernization of the diet, has led to increased frequency of "lifestyle-related disorders" such as, hyperlipidemia, diabetes mellitus, and atherosclerosis. Consequently, the morbidity and mortality due to coronary heart disease have increased, and the prevention of this disorder is now one of the major concerns when considering the quality of life of individuals and the public health policy. Hyperlipidemia, particularly hypercholesterolemia, is one of the major risk factors of atherosclerosis. Therefore, the clarification of the mechanisms of the development of hyperlipidemia is important in the consideration of the prevention and management of atherosclerotic disorders. Genetic and epidemiological studies have provided compelling evidence that genetic factors, environmental influences and the interaction between them all contribute to the development of this complex disorder. Various unexpected mechanisms of the development of hyperlipidemia have been elucidated by analyzing some hereditary disorders. Furthermore, the cloning of the genes involved in the maintenance of cholesterol and/or lipid homeostasis has made it possible to investigate the molecular mechanisms of the development of hyperlipidemia in detail. Elucidation of the genetic and environmental factors and detailed understanding of the molecular pathophysiology of hyperlipidemia are essential in the pursuit of evidence-based preventive medicine and health policy.

Gene trap mutagenesis-based forward genetic approach reveals that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2.

OVCA1 is a tumor suppressor identified by positional cloning from chromosome 17p13.3, a hot spot for chromosomal aberration in breast and ovarian cancers. It has been shown that expression of OVCA1 is reduced in some tumors and that it regulates cell proliferation, embryonic development, and tumorigenesis. However, the biochemical function of OVCA1 has remained unknown. Recently, we isolated a novel mutant resistant to diphtheria toxin and Pseudomonas exotoxin A from the gene trap insertional mutants library of Chinese hamster ovary cells. In this mutant, the Ovca1 gene was disrupted by gene trap mutagenesis, and this disruption well correlated with the toxin-resistant phenotype. We demonstrated direct evidence that the tumor suppressor OVCA1 is a component of the biosynthetic pathway of diphthamide on elongation factor 2, the target of bacterial ADP-ribosylating toxins. A functional genetic approach utilizing the random gene trap mutants library of mammalian cells should become a useful strategy to identify the genes responsible for specific phenotypes.

Association of single nucleotide polymorphisms in the eosinophil peroxidase gene with Japanese cedar pollinosis.

BACKGROUND: Japanese cedar pollinosis is the most common form of hay fever in spring in Japan. We have previously demonstrated that single nucleotide polymorphism Pro358Leu of exon 7 in the eosinophil peroxidase (EPO) gene is associated with cedar pollinosis, although the association has not been confirmed by analysis of the whole gene in a different population. METHODS: We sequenced all exons of the EPO gene in 60 children with pollinosis and their parents using the PCR-restriction fragment length polymorphism method. RESULTS: We found 8 polymorphisms, Ile40Met, Gln122His, Arg202Arg (A660G), Asn303Asn (C909T), Arg326Pro, Arg326His, Pro358Leu, and Asn572Ty, in the EPO gene. As a result of the transmission disequilibrium test, we recognized significant transmissions of 202Arg (660G) in exon 6 in addition to 358Leu of exon 7 in the EPO gene of affected children. CONCLUSIONS: Our results might indicate that polymorphisms of the EPO gene are associated with Japanese cedar pollinosis.

Induction of myeloperoxidase and nitrotyrosine formation in a human eosinophilic leukemia cell line, EoL-1.

The human eosinophilic leukemia cell line, EoL-1, differentiated with butyrate as an eosinophilic cellular model was evaluated for peroxidase-dependent tyrosine nitration. Butyrate suppressed cell growth and induced eosinophilic granules in EoL-1 cells after 9 days of culture. Peroxidase activity was detected biochemically and histochemically from 3-day cultures and it increased in a time dependent manner. This peroxidase activity was inhibited by cyanide. Nitrotyrosine formation catalysed by peroxidase using hydrogen peroxide and nitrite was detected at a high level similar to that of mature eosinophils. However, no expression of eosinophil peroxidase (EPO) was detected by RT-PCR or immunocytochemistry. In contrast, the induction of myeloperoxidase (MPO) by butyrate was clearly detected by RT-PCR, Northern blot, and immunocytochemical staining. These results suggest that butyrate induces MPO rather than EPO in EoL-1 cells and that the formation of nitrotyrosine in butyrate-induced cells is dependent on MPO.

Enhancement of a sense of coherence and natural killer cell activity which occurred in subjects who improved their exercise habits through health education in the workplace.

We have previously demonstrated that a sense of coherence (SOC), a candidate for a mediating stress factor, is involved in natural killer cell activity (NKCA) reduced in smokers, whereas the relationship among exercise, NKCA and SOC is unclear. To clarify the effects of exercise on SOC and NKCA, we examined the changes in SOC and NKCA before and after health education to encourage exercise. Of one-hundred and one male office workers who received the health education for one year, 27 improved, 65 were unchanged and 9 had deteriorated exercise habits. The repeated measures analysis of variance showed that SOC in workers with improvement in the exercise habit were increased more significantly by the health education than those in workers without improvement (p<0.05). Although the change in NKCA produced by health education was recognized to be significantly different in those who had never smoked from that in current smokers (p<0.05), multiple regression analysis demonstrated that improvement in health practice significantly contributed to increases in both SOC (p<0.01) and NKCA (p<0.05) in never smokers, independently of other psychological factors. These results suggest that subjects with improvement in exercise enhance NKCA through increased SOC in never smokers.

Nonthermal effects of mobile-phone frequency microwaves on uteroplacental functions in pregnant rats.

Exposure to high-density microwaves can cause detrimental effects on the testis, eye, and other tissues, and induce significant biologic changes through thermal actions. To examine nonthermal effect of continuous wave (CW) 915MHz microwaves used in cellular phones, we compared the effects of microwaves with those of heat. Thirty-six pregnant rats were assigned to six groups: rats exposed to microwaves at 0.6 or 3mW/cm(2) incident power density at 915MHz for 90min, rats immersed in water at 38 or 40 degrees C, which induces about the same increase in colonic temperature of 1.0 or 3.5 degrees C as 0.6 or 3mW/cm(2) microwaves, respectively; rats immersed in water at 34 degrees C, which is considered to be thermoneutral; and control rats. We identified significant differences in the uteroplacental circulation, and in placental endocrine and immune functions between pregnant rats immersed in water at 34 and 38 degrees C, but not between rats immersed at 38 degrees C and those exposed to microwaves at 0.6mW/cm(2). By contrast, we observed significant decreases in uteroplacental blood flow and estradiol in rats exposed to microwaves at 3mW/cm(2) as compared with those immersed in water at 40 degrees C. These results suggest microwaves at 0.6mW/cm(2) at 915MHz, equal to a specific absorption rate (SAR) of 0.4W/kg, which is the maximum permissible exposure level recommended by the American National Standards Institute (ANSI), do not exert nonthermal effects on blood estradiol and progesterone, on splenic natural killer cell activity, on the uteroplacental circulation.

Antitumor agents 220. Antitumor-promoting effects of cimigenol and related compounds on Epstein-Barr virus activation and two-stage mouse skin carcinogenesis.

Cimigenol (1) and 39 related compounds were screened as potential antitumor promoters by examining the ability of the compounds to inhibit Epstein-Barr virus early antigen (EBV-EA) activation (induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells. Structure-activity relationship analysis indicated that compound 1 showed the highest activity and also exhibited significant inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test. These data suggest that 1 and the related compounds might be valuable anti-tumor promoters.

Interiotherins C and D, two new lignans from Kadsura interior and antitumor-promoting effects of related neolignans on Epstein-Barr virus activation.

Two new lignans, interiotherins C (1) and D (2), together with the known compounds interiorin (3), heteroclitin F (4), neokadsuranin (5), heteroclitin D (6), kadsurin (7), gomisin A (8), schisandrin C (9), interiotherin A (10), angeloylgomisin R (11), gomisin G (12), interiotherin B (13), and gomisin C (14), were isolated from the stems of Kadsura interior. The structures and stereochemistries of the new compounds were determined from mass, CD, and NMR spectral data. Fourteen neolignans were screened as potential antitumor promoters by examining their ability to inhibit Epstein-Barr virus early antigen (EBV-EA) activation (induced by 12-O-tetradecanoylphorbol-13-acetate) in Raji cells. Neokadsuranin (5) and schisandrin C (9) were the most potent compounds. These data suggest that some neolignans might be valuable antitumor promoters or chemopreventors.

Chemopreventive effects of emodin and cassiamin B in mouse skin carcinogenesis.

In continuation of our works of natural and synthetic products as cancer chemopreventive agents, we have examined emodin and cassiamin B, which were isolated from Cassia siamea. These compounds exhibited the remarkable anti-tumor promoting effect on two-stage carcinogenesis test of mouse skin tumors induced by 7,12-dimethylbenz[a]anthracene as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter by both topical application. Furthermore, emodin exhibited potent inhibitory activity on two-stage carcinogenesis test of mouse skin tumors induced by nitric oxide donor, (+/-)-(E)-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexeneamide as an initiator and TPA as a promoter.