Biomedical subjects

J Miyakoshi

Publications and source records attributed to J Miyakoshi.

At least 19 recordsLinked to original sources

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↗

PubMed1995

Enhancement of beta-galactosidase gene expression in rat pheochromocytoma cells by exposure to extremely low frequency magnetic fields.

Exposure of PC12-VG cells to an extremely low frequency magnetic field (ELFMF) enhanced the beta-galactosidase gene expression stimulated by treatment of the cells with forskolin. The enhancing effect of the ELFMF was inhibited by treatment of the cells with a specific inhibitor of PKC, calphostin C, as well as with the Ca2+ entry blockers nifedipin and dantrolen. Enhancement appeared within the first hour of a 4h forskolin treatment when the ELFMF was given at different times during culture. We speculate that exposure of PC12-VG cells to an ELFMF during the early response to forskolin treatment affects cell signal transduction, resulting in enhanced gene expression.

Adrenal Gland Neoplasms↗

PubMed1995

Changes in radiation sensitivity of human osteosarcoma cells after p53 introduction.

Human osteosarcoma SAOS-2 cells, which have a deletion in p53 gene, were transfected with plasmid pMSVneop53 containing human p53 cDNA and neomycin-resistance gene. Three clones (SAOS-MC10, SAOS-MC11 and SAOS-MC43) among 60 clones expressed p53 mRNA. No p53 protein was observed in SAOS-MC10, while SAOS-MC11 and SAOS-MC43 produced p53 protein. The molecular weight of p53 protein in SAOS-MC43 was lower than that in SAOS-MC11, SAOS-MC11 and SAOS-MC43 were more sensitive and more resistant, respectively, to ionizing radiation than the parental SAOS-2. We suggest that exogenous p53 protein might be one of the factors determining cellular radiosensitivity.

Bone Neoplasms↗

PubMed1995

Modification of the radiosensitivity of human cells to which simian virus 40 T-antigen was transfected.

Effects of the introduction of the Simian virus 40 T-antigen (SV40 T-Ag) gene to cultured human cells were examined in relation to radiosensitivity. Two relatively radioresistant tumor cell lines (T98 and G361) became significantly radiosensitive after the introduction of SV40 T-Ag, whereas radiosensitive tumor cell lines did not show a change in radiosensitivity. In contrast, a human fibroblast cell line became radioresistant after SV40 T-Ag introduction. T98 cells which have a mutation at codon 237 in the p53 gene were unable to form a complex between p53 protein and SV40 T-Ag, whereas G361, which became radiosensitive by a SV40 T-Ag introduction, formed the complex. This indicates that the status of p53 is independent of the change in radiosensitivity in the cell lines studied.

Antigens, Polyomavirus Transforming↗

PubMed1994

Mutations in ras genes in cells cultured from mouse skin tumors induced by ultraviolet irradiation.

Mutations in ras oncogenes were detected in cultured cells of mouse skin tumors induced by near-UV irradiation. DNA extracted from the UV-induced tumor cells was transfected to golden hamster embryo cells, and focus-forming ability was confirmed in 22 of 26 cell strains, 15 of which had the repetitive mouse sequence. Mouse ras genes were detected in 10 of these 22 cell strains. Point mutations in the ras genes were at Ha-ras codon 13 (GGC-->GTC in two strains, GGC-->AGC in one strain), Ki-ras codon 61 (CAA-->GAA in two strains), and N-ras codon 61 (CAA-->CAT in two strains, CAA-->AAA in two strains). In one tumor cell strain no base change was directed. Most mutations occurred at dipyrimidine sites. Pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproducts are the likely cause of the skin cancers. The base change occurred preferentially at G.C base pairs, and transversions predominated.

Animals↗

PubMed1994

X-ray-induced transcriptional activation of c-myc and XRCC1 genes in ataxia telangiectasia cells.

The transcriptional level of c-myc, c-jun and XRCC1 genes after X-irradiation was compared in human cells originating from subjects presumably with different DNA repair abilities. The mRNA amount of the beta-actin gene was used as an internal standard of transcription. The relative mRNA level of c-myc and XRCC1 genes was significantly increased 15 min after X-irradiation with doses of 2-8 Gy in ataxia telangiectasia (AT) cells (AT5BIVA and TAT2SF), in contrast to little change in xeroderma pigmentosum (XP2OS(SV) and XP2YO(SV)) and normal cells (WI38VA13 and GM0637). The increased mRNA level of the XRCC1 gene in AT5BIVA and of the c-myc and XRCC1 genes in TAT2SF cells was maintained for up to 8 h after X-irradiation with 2 Gy. For the c-jun mRNA level after X-irradiation with 2-8 Gy, no significant change was observed in all cell lines tested. These results indicate that AT cells show a high transcriptional response of certain genes in response to X-irradiation, and suggest that the transcriptional activation of c-myc and XRCC1 genes after X-irradiation may be related to the hyper-radiosensitivity of AT cells.

Actins↗

PubMed1994

A newly designed experimental system for exposure of mammalian cells to extremely low frequency magnetic fields.

To examine the biological effects of extremely low frequency magnetic field (ELFMF), we have designed and manufactured a new equipment for long-term and high-density exposure of cells to ELFMF. The ELFMF exposure system consists of a generator of magnets with a built-in CO2 incubator, an alternating current (AC) power supply, a gas compressor and a thermocontroller for the incubator, and a cooling unit for the magnets. The CO2 incubator made of acrylic resin is inserted into the inner-space of the silicon steel strip-cores. In this system, the temperature of the incubator is maintained at 37 +/- 0.5 degrees C. The maximum magnetic flux density on the exposure area of the incubator is 500 mT (T; tesla) at a current of 556 Arms (rms; root mean square) at 50 Hz. The long-term (up to 120 hr) exposure of 400 mT ELFMF did not affect the growth of both HL60RG and CCRF-CEM cells originated from human leukemia. The post-X-irradiation exposure of 400 mT ELFMF for 2 hr also did not affect the radiation sensitivity of GM0637 and TAT2SF cells originated from a normal human and an ataxia telangiectasia patient.

Cell Division↗