Biomedical subjects

J Miyakoshi

Publications and source records attributed to J Miyakoshi.

At least 37 records · Page 2Linked to original sources

PubMed2000

Inhibition of I kappaB-alpha phosphorylation at serine and tyrosine acts independently on sensitization to DNA damaging agents in human glioma cells.

Molecular mechanisms and/or intrinsic factors controlling cellular radiosensitivity are not fully understood in mammalian cells. The recent studies have suggested that nuclear factor kappaB (NF-kappaB) is one of such factors. The activation and regulation of NF-kappaB are tightly controlled by IkappaB-alpha, a cellular inhibitory protein of NF-kappaB. Most importantly, phosphorylation regulates activity of the inhibitor IkappaB-alpha, which sequesters NF-kappaB in the cytosol. Two different pathways for the phosphorylation of IkappaB-alpha are demonstrated, such as serine (at residues 32 and 36) and tyrosine (at residue 42) phosphorylations. To assess a role of the transcription factor, NF-kappaB, on cellular sensitivity to DNA damaging agents, we constructed three different types of expression plasmids, i.e. S-IkappaB (mutations at residues 32 and 36), Y-IkappaB (mutation at residue 42) and SY-IkappaB (mutations at residues 32, 36 and 42). The cell clones expressing S-IkappaB and Y-IkappaB proteins became sensitive to X-rays as compared with the parental and vector-transfected cells. The cell clones expressing SY-IkappaB were further radiosensitive. By the treatment with herbimycin A, an inhibitor of phosphorylation, the X-ray sensitivity of cells expressing SY-IkappaB did not change, while that of the cells expressing S-IkappaB and Y-IkappaB and the parental cells was enhanced. Change in the sensitivity to adriamycin and UV in those clones was very similar to that in the X-ray sensitivity. The inhibition of IkappaB-alpha phosphorylation at serine and tyrosine acts independently on the sensitization to X-rays, adriamycin and UV. These findings suggest that the transcriptional activation induced by NF-kappaB may play a role in the DNA damage repair. The present study proposes a possibility that the inactivation of NF-kappaB by inhibition of both serine and tyrosine phosphorylations may be useful for the treatment of cancer in radio- and chemotherapies.

Antineoplastic Agents↗

PubMed2000

Increased chromatid-type chromosomal aberrations in mouse m5S cells exposed to power-line frequency magnetic fields.

PURPOSE: To investigate the induction of chromosomal aberrations in mouse m5S cells after exposure to power-line frequency magnetic fields (extremely low frequency magnetic fields; ELFMF) at high-flux densities. MATERIAL AND METHOD: m5S cells were either untreated or pretreated during the G1 phase with mitomycin C (MMC, 1 microM) for 1 h or 3 Gy X-rays, and then exposed to ELFMF at three different flux densities (5 and 50 mT at 60 Hz, 400 mT at 50 Hz) for 40 h. Unexposed control cells were incubated for the same period in a conventional CO2 incubator. Chromosomal aberrations were analysed in the first post-treatment metaphases. Cell kinetics were assessed by DNA flow cytometry and the mitotic index. RESULTS AND CONCLUSIONS: ELFMF enhanced the formation of spontaneous and MMC- or X-ray-induced chromosomal aberrations, in a flux-density-dependent manner. Statistically significant increases in the frequency of chromosomal aberrations were observed in cells exposed to 400 mT ELFMF with respect to unexposed controls. The aberrations induced by ELFMF were mostly chromatid-type, not chromosome-type. The cells exposed to 400 mT ELFMF exhibited a three-fold higher level of chromatid-type aberrations than did the unexposed cells. Flow cytometric and mitotic index analyses revealed that the S or G2 arrest following MMC or X-irradiation was more profound in ELFMF-exposed cells than in unexposed cells. Our results suggest that ELFMF can interfere with post-replication repair, resulting in increased levels of chromatid-type chromosomal aberrations induced spontaneously and by DNA damaging agents.

Animals↗

PubMed2000

Exposure to strong magnetic fields at power frequency potentiates X-ray-induced DNA strand breaks.

We examined the effect of an extremely low-frequency magnetic field (ELFMF) at 5, 50 and 400 mT on DNA strand breaks in human glioma MO54 cells. A DNA damage analysis was performed using the method of alkaline comet assay. The cells were exposed to X-rays alone (5 Gy), ELFMF alone, or X-rays followed by ELFMF at 4 degrees C or on ice. No significant difference in the tail moment was observed between control and ELFMF exposures up to 400 mT. X-ray irradiation increased DNA strand breaks. When cells were exposed to X-rays followed by ELFMF at 50 and 400 mT, the tail moment increased significantly compared with that for X-rays alone. When the exposure of cells was performed at 37 degrees C, no significant change was observed between X-rays alone and X-rays plus 400 mT. We previously observed that exposure to 400 mT ELFMF for 2 h increased X-ray-induced mutations (Miyakoshi et al, Mutat. Res., 349: 109-114, 1996). Additionally, an increase in the mutation by exposure to the ELFMF was observed in cells during DNA-synthesizing phase (Miyakoshi et al., Int. J. Radiat. Biol., 71: 75-79, 1997). From these results, it appears that exposure to the high density ELFMF at more than 50 mT may potentiate X-ray-induced DNA strand breaks.

Cell Death↗

PubMed1999

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells.

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.

Animals↗

PubMed1999

Long-term exposure to a magnetic field (5 mT at 60 Hz) increases X-ray-induced mutations.

Exposure to extremely low frequency magnetic field (ELFMF) at 400 mT has been shown to induce mutations (Mutat. Res., 349: 109-114, 1996; Int. J. Radiat. Biol., 71: 75-79, 1997; and Biochem. Biophys. Res. Commun., 243: 579-584, 1998). However, whether ELFMF at low flux densities (under 1 mT) induces mutations is debatable. We investigated the effect of long-term exposure to 5 mT ELFMF at 60 Hz on mutant frequency. Chinese hamster ovary K1 (CHO-K1) cells were exposed or sham-exposed to 5 mT ELFMF for up to 6 weeks with or without X-irradiation (3 Gy), and the mutant frequency of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene was analyzed. Long-term exposure to 5 mT ELFMF did not increase mutations, suggesting a threshold for mutation induction greater than 115 mA/m2 or a magnetic density of 5 mT. However, enhancement of the X-ray-induced mutation rate was observed after treatment with X-irradiation followed by long-term exposure to 5 mT ELFMF. At little as a 1-week exposure to ELFMF after X-irradiation enhanced the mutation rate. We also found that 400 mT exposure enhanced the mutation rate induced by X-irradiation (Mutat. Res., 349: 109-114, 1996). These results suggest that exposure to more than 5 mT ELFMF may promote X-ray-induced mutations.

Animals↗

PubMed1999

Decreased host-cell reactivation of UV-irradiated adenovirus in human colon tumor cell lines that have normal post-UV survival.

An ongoing study in our laboratories is to examine the relationship of DNA repair defects to human cancer. Our underlying hypothesis has been that human tumors may arise that lack interesting DNA repair pathways if these pathways are important in preventing cancer. In this study, we found that the UV-irradiated adenoviruses showed hypersensitivity when assayed on monolayers of certain human colon tumor cell lines, including three that are reported to have defects in long patch DNA mismatch repair genes and one with no reported defect in mismatch repair. The survival curves showed two components. The first sensitive component was characteristic of 77-95% of the infections depending upon the cell line and the experiment and had an average slope indicating 4.8-fold hypersensitivity to UV. The average of the second-component slopes indicated that the remainder of the infections was accompanied by near-normal repair. Although the value of the first component indicated that the colon tumor lines supported the growth of UV-damaged adenoviruses poorly, the cell lines themselves showed the same post-UV colony-forming ability as did normal human fibroblasts, and their ability to support the growth of N-methyl-N'-nitro-N-nitrosoguanidine-damaged adenoviruses was normal, i.e. it parallelled their ability to repair O6-methylguanine in vitro. We previously observed two-component survival curves when assaying UV-irradiated adenovirus on monolayers of all of seven strains of fibroblasts from Cockayne's syndrome patients. By contrast, single-component curves have been obtained using 21 strains of normal human fibroblasts and seven other tumor lines. We interpret the two-component survival curves in terms of the defective transcription-coupled repair of UV-induced DNA damage that is characteristic both of Cockayne's and certain colon tumor cell lines. In addition, four mismatch repair-deficient colon tumor lines were resistant to killing by elevated levels of dG.

Adenoviruses, Human↗

PubMed1998

Suppression of high-density magnetic field (400 mT at 50 Hz)-induced mutations by wild-type p53 expression in human osteosarcoma cells.

Exposure of cultured human osteosarcoma cells (Saos-LP-12) to high-density (400 mT at 50 Hz) extremely low frequency magnetic fields (ELFMF) induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene. Saos-LP-12 cells, which are isolated from parental Saos-2 cells and have a deletion in the coding region of the p53 gene, are introduced to the wild-type (wt) p53 expression plasmid (pOPRSVp53). The mutation in Saos-LP-12 cells was suppressed by expression of the introduced wt p53 gene during 400 mT ELFMF exposure. No marked difference in the mutation spectrum was observed among the treatments of ELFMF [p53 (-)], ELFMF [p53 (+)], and sham exposures. Our findings suggest that wt p53 has a function in suppression of DNA replication errors and/or in maintenance of genomic stability after high-density ELFMF exposure.

DNA Mutational Analysis↗

PubMed1998

Reduced UV-induced mutations in human osteosarcoma cells stably expressing transfected wild-type p53 cDNA.

We constructed the plasmid which can express human wild-type p53 cDNA and introduced it into the human osteosarcoma cell line SAOS-2 that lacks the chromosomal p53 gene. A cell clone stably expressing p53 protein was isolated and UV sensitivity and UV-induced mutation frequencies of the clone were examined. The UV sensitivity of the clone was slightly higher and UV-induced hprt mutation frequencies of the clone were markedly lower than those of parental SAOS-2 cells. The capability to repair UV-induced DNA damage assessed by the amount of unscheduled DNA synthesis or DNA single strand breaks as well as cell cycle progression after UV irradiation were not different between the clone and SAOS-2 cells. These results indicate that wild-type p53 protein would be involved in the human DNA damage-processing pathway other than the genome-overall excision repair.

Cell Survival↗

PubMed1998

Enhanced NOR-1 gene expression by exposure of Chinese hamster cells to high-density 50 Hz magnetic fields.

Enhanced expression of neuron derived orphan receptor (NOR-1) gene was observed by exposure of Chinese hamster ovary K1 (CHO-K1) cells to an extremely low frequency magnetic field (ELFMF) of 50 Hz at 400 mT, but not at 5 mT. The enhanced expression, reaching the maximum at 6 h, was transient and reduced to the control level after exposure to 400 mT ELFMF for 24 h. The NOR-1 expression induced by treatment with forskolin and TPA was further enhanced by the simultaneous treatment with 400 mT ELFMF, in which the maximum response was at 3 h. The NOR-1 expression by these treatments was induced more earlier than that by 400 mT ELFMF alone. When cells were treated with an inhibitor of the protein kinase C (calphostin C or crocetin) and Ca2+ entry blockers (nifedipin and dantrolen) during the 400 mT ELFMF exposure, the enhanced NOR-1 expression was not observed. Exposure of CHO-K1 cells to the high-density 400 mT ELFMF may affect the signal transduction in the cells, resulting in the enhanced NOR-1 gene expression.

Animals↗

PubMed1997

Increase in radiation sensitivity of human malignant melanoma cells by expression of wild-type p16 gene.

The influence of wild-type p16 expression on the radiation sensitivity of human melanoma cell lines was investigated. MeWo cells, which alone expressed intrinsic wild-type p16 among six melanoma cell lines examined, showed higher radiosensitivity in comparison with the other five melanoma cells. The introduction of human wild-type p16 cDNA into A875 cells, which homozygously lost p16 genes, and AKI cells, which retained p16 gene but did not express p16 mRNA, led to increased sensitivity of those cells to X-ray irradiation. The radiosensitizing effect by the p16 introduction to those cells was prominent after rather higher doses of X-rays (8 and 10 Gy). In both A875 and AKI, no significant difference in sensitivities to UVC and cisplatin was observed between the parental and p16-transfectant cells. These results suggest that the loss or dysfunction of p16 gives melanoma cells the radioresistant characteristics.

Chromosomes, Human, Pair 9↗

PubMed1997

Enhanced radiosensitivity by inhibition of nuclear factor kappa B activation in human malignant glioma cells.

To clarify the relationship between cellular radiosensitivity and nuclear factor kappa B (NF-kappa B) activation, an expression plasmid was constructed for I kappa B-alpha, a cellular inhibitory protein of NF-kappa B, and transfected it into two human malignant glioma cell lines. Cells overexpressing the I kappa B-alpha protein were more radiosensitive than the parental cells and one transfected clone with low expression. In the parental cell lines and one transfected clone with low expression, the sequence specific DNA-binding activity of NF-kappa B was considerably increased between 1 and 2 h after irradiation. In contrast, no increase in the DNA-binding activity was observed in the transfected clone overexpressing I kappa B-alpha protein. These results suggest that the activation of NF-kappa B may be one of the intrinsic responses determining cellular radiosensitivity.

Clone Cells↗

PubMed1997

Mutation induction by high-density, 50-Hz magnetic fields in human MeWo cells exposed in the DNA synthesis phase.

Exposure of cultured human MeWo cells to high-density (400 mT at 50 Hz) extremely low frequency magnetic fields (ELF-MF) induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene. Mutation induced by the ELF-MF increased during the DNA-synthesis phase in synchronously growing cells. DNA replication errors and/or disturbance of the mismatch repair systems caused by exposure to ELF-MF may be involved in the mutagenic effect.

DNA↗

PubMed1997

Decrease in the frequency of X-ray-induced mutation by wild-type p53 protein in human osteosarcoma cells.

Tumor suppressor p53 protein acts as a checkpoint factor following DNA damage. Inactivation of checkpoint control may increase the frequency of mutation following DNA damage, resulting in tumor progression. Here we examine whether wild-type (wt) p53 protein suppresses X-ray-induced mutations using an isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated p53 expression system in human osteosarcoma Saos-2 cells. Frequency of X-ray-induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene was enhanced about 10 and 20 times by 1 and 2 Gy respectively in cells without expression of wt p53 protein, while enhancement of mutations by X-rays was slight in cells with expression of wt p53 protein. Furthermore, arrest at the G/S boundary was induced by X-ray irradiation when p53 protein was expressed by treatment with IPTG. These findings suggest that wt p53 protein has a function in maintaining genomic stability after X-ray irradiation through the G1 checkpoint and loss of p53 function(s) may lead to tumor progression in multi-step tumorigenesis.

Apoptosis↗

PubMed1997

Suppression of UV-induced mutations by wild-type p53 protein in human osteosarcoma cells.

We have examined whether the tumour suppressor p53 protein suppressed UV-induced mutations in the hypoxathine-guanine phosphoribosyl transferase (HPRT) gene and in the supF gene of the shuttle vector plasmid pMY189. We used human osteosarcoma Saos-LP12 cells, in which wild type (wt) p53 protein was induced by treatment with isopopyl-beta-D-thiogalactopyranoside. The induction of wt p53 protein suppressed UV-induced mutations but not spontaneous mutations in the HPRT gene. The frequency of UV-induced mutations induced by UV-irradiation of the plasmid was also significantly lower in cells with induced wt p53 protein than in the uninduced cells. In addition, we found that frequency of G : C to A : T transition mutations which occurred at the 3' base pair of dipyrimidine sites were significantly lower in the cells with induced wt p53 protein than in the uninduced cells. These findings suggest that wt p53 protein may play roles in modulating DNA repair pathway, resulting in the suppression of UV-induced mutations.

Base Sequence↗

PubMed1997

Increased radiosensitivity of p16 gene-deleted human glioma cells after transfection with wild-type p16 gene.

The A1235 and T98 cell lines derived from human gliomas have homozygous deletions in their p16 genes and are radiosensitive and radioresistant, respectively, with respect to other established glioma cell lines. These differences in radiosensitivity may be due to variations to some extent among cell lines, rather than genetically defined resistance or sensitivity. We examined the effect on radiation sensitivity of introducing a wild-type p16 gene into both p16-deficient glioma cell lines. The plasmid pOPMTS containing human wild-type p16 cDNA and a neomycin resistance gene, or the control plasmid pOPRSV1, were transfected into these cells. Clones from both cell lines, which expressed wild-type p16 mRNA constitutively after transfection with pOPMTS, were more radiosensitive than the parental cells and clones obtained after transfection with the negative control plasmid.

Carrier Proteins↗

PubMed1996

Increase in hypoxanthine-guanine phosphoribosyl transferase gene mutations by exposure to high-density 50-Hz magnetic fields.

Exposure to extremely low frequency magnetic field (ELFMF) of 50 Hz and 400 mT induced mutations in the hypoxanthine-guanine phosphoribosyl transferase gene of human melanoma MeWo cells. The mutant frequency was enhanced both by increasing the exposure period and the induced current intensity. Mutations induced by X-rays were enhanced by ELFMF exposure. No significant increase in mutant frequency occurred when DNA replication was inhibited during ELFMF exposure. DNA replication error is suspected of causing the mutations produced by ELFMF exposure.

Cell Division↗

PubMed1996

Sites and types of UV-induced mutations leading to inactivation of the growth-arresting activity in p21 (sdi1/cip1/waf1) cDNA.

The p53-regulated gene product p21 (sdi1,cip1,waf1) negatively regulates cell growth and has been suggested to be a potential tumour-suppressor gene. To determine the sites and types of mutations which inactivate the growth-arresting activity in sdi1 cDNA, plasmids containing sdi1 cDNA and the neomycin-resistant gene were irradiated with UV light and transfected into CHO cells. The UV irradiation increased number of the geneticin-resistant colonies which should have the UV-mutated sdi1 cDNA. Sdi1 mRNA was expressed in 23 out of 36 colonies (64%). In 13 sdi1 cDNA sequences analysed, mutations were found at codon 46 in nine cDNAs, and one each at codons 34, 54, 66 and 73. All the mutation sites are in the CDK-binding region. Ten mutations (77%) (codons 46 and 66) are C to T transition mutation at the dipyrimidine sequences, which is the major type of the UV-induced mutation.

Animals↗

PubMed1996

Exposure to magnetic field (5 mT at 60 Hz) does not affect cell growth and c-myc gene expression.

We designed and manufactured equipment for long-term and low-density (0 to 9 mT) exposures of cultured cells to extremely low frequency magnetic fields (ELF-MF), and examined the effects of ELF-MF on cell growth and c-myc mRNA expression in Chinese hamster ovary (CHO) cells. The ELF-MF equipment consists of a CO2 incubator with a built-in magnet generator using Helmholtz coils being 250 mm in inner diameter, 160 mm in distance and 128 turns, a slide regulator and a thermocontroller. No significant difference in the growth rate and the c-myc expression of CHO cells was observed with 5 mT ELF-MF exposure, sham-exposure and incubation in a conventional incubator.

Actins↗