Biomedical subjects

H Takebe

Publications and source records attributed to H Takebe.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1998

Complete restoration of normal DNA repair characteristics in group F xeroderma pigmentosum cells by over-expression of transfected XPF cDNA.

XP-F cDNA was cloned into a mammalian expression vector plasmid, and introduced into group F xeroderma pigmentosum (XP-F) cells. Several cell clones possessing transfected XPF cDNA were randomly isolated, and DNA repair characteristics of a clone, XP-FR2, were extensively analyzed. The XP-FR2 cells expressed high level of XPF protein as well as ERCC1 protein, although their parental XP-F cells expressed extremely low level of both proteins. The XP-FR2 cells showed UV resistance comparable to normal human cells, and had normal levels of UV-induced unscheduled DNA synthesis and normal capability to remove cyclobutane pyrimidine dimers and (6-4) photoproducts. Frequencies and types of UV-induced mutations examined by shuttle vector plasmids in XP-FR2 cells were similar to those in normal human cells. These results demonstrate that excision repair defect in XP-F cells is fully corrected by over-expression of XPF cDNA alone, although only partial correction of the cells by XPF cDNA has been reported before.

Base Sequence↗

PubMed1998

A spectrum of mutations induced by crotonaldehyde in shuttle vector plasmids propagated in human cells.

A spectrum of crotonaldehyde-induced mutations in the supF gene of the shuttle vector plasmid pMY189 replicated in human fibroblast cells was examined. Base sequence analysis of 104 plasmids with mutations in the supF gene revealed that the majority of the mutations were base substitutions (85%) and the rest were frameshifts (15%). A single base substitution was most frequently found (47%), while 25% had multiple base substitutions and interestingly 13% had tandem (adjacent two) base substitutions. Of the base substitution mutations, 50% were G:C-->T:A transversions and 23% were G:C-->A:T transitions. The mutations were not distributed randomly but were located at several hotspots, most of which were G:C base pairs in 5'-AAGG-3' (or 5'-CCTT-3') sequences. Production of propanodeoxyguanosine adducts may be related to such specificity in the mutation spectrum.

Aldehydes↗

PubMed1998

Characterization of molecular defects in xeroderma pigmentosum group F in relation to its clinically mild symptoms.

Xeroderma pigmentosum (XP) complementation group F was first reported in Japan and most XP-F patients reported to date are Japanese. The clinical features of XP-F patients are rather mild, including late onset of skin cancer. Recently a cDNA that corrects the repair deficiency of cultured XP-F cells was isolated. The XPF protein forms a tight complex with ERCC1 and this complex functions as a structure-specific endonuclease responsible for the 5' incision during DNA excision repair. Here we have identified XPF mRNA mutations and examined levels of the mRNA and protein expression in seven primary cell strains from Japanese XP-F patients. The XP-F cell strains were classified into three types in terms of the effect of the mutation on the predicted protein; (i) XPF proteins with amino acid substitutions; (ii) amino acid substituted and truncated XPF proteins; and (iii) truncated XPF protein only. A normal level of expression of XPF mRNA was observed in XP-F cells but XPF protein was extremely low. These results indicate that the detected mutations lead to unstable XPF protein, resulting in a decrease in formation of the ERCC1-XPF endonuclease complex. Slow excision repair of UV-induced DNA damage due to low residual endonuclease activity provides a plausible explanation for the typical mild phenotype of XP-F patients.

Adult↗

PubMed1998

Sensitivity of group F xeroderma pigmentosum cells to UV and mitomycin C relative to levels of XPF and ERCC1 overexpression.

The XPF and ERCC1 proteins form a tight complex and function as an endonuclease to incise on the 5'-side of pyrimidine dimers in DNA. Levels of both proteins are extremely low in group F xeroderma pigmentosum (XP-F) cells. We transfected XP-F cells with the plasmids expressing XPF or ERCC1 and examined levels of both proteins in the cells. Although XP-F cells are sensitive to UV and mitomycin C (MMC), cells overexpressing XPF expressed ERCC1 as well and resistance to UV and MMC was restored to the normal level. In contrast, cells overexpressing ERCC1 did not express XPF and were still sensitive to UV and MMC. These results indicate that both the XPF and ERCC1 proteins are required to repair UV- and MMC-induced DNA damage. Even though a high level of ERCC1, which has been presumed to be a catalytic subunit of the endonuclease, is stably present in XP-F cells, ERCC1 protein alone cannot carry out excision repair completely.

Clone Cells↗

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

A low content of ERCC1 and a 120 kDa protein is a frequent feature of group F xeroderma pigmentosum fibroblast cells.

The ERCC1 protein has been predicted to form part of a tight complex with a protein partner, the yet-unidentified XPF/ERCC4 protein, in normal human cells. We used an anti-ERCC1 antibody to detect the complex by immunoprecipitation and immunoblotting. The amount of ERCC1 protein expressed in five different XP-F cell strains was 1/ 5-1/34 of that of the protein in normal and XP cell strains representing other complementation groups. A 120 kDa protein was co-immunoprecipitated with ERCC1 by the anti-ERCC1 antibody, and the amount of the 120 kDa protein in XP-F cell strains was 1/5-1/8 of that of the protein in normal and XP cell strains representing other complementation groups. The XPA protein was not co-immunoprecipitated with ERCC1 in any cell strain. These results demonstrate that a low level of ERCC1 and the 120 kDa protein is a frequent feature of XP-F cell extracts and that a lower amount of a complex between these proteins occurs in XP-F cells than in normal cells.

Antibodies↗

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

Characterization of p53 gene mutations in basal-cell carcinomas: comparison between sun-exposed and less-exposed skin areas.

Mutations in the p53 gene in 32 basal-cell carcinomas (BCC) developed in Japanese patients were identified by the polymerase chain reaction and single-strand-conformation polymorphism analysis, followed by sequencing the DNA. Among 16 BCC developed in continuously sun-exposed areas, 6 tumors showed 7 base substitutions, most of which were G:C to A:T transitions, mainly at the dipyrimidine sites. Seven out of 16 BCC developed in less-exposed areas showed 8 base substitutions, but the majority (75.0%) of them were transversions. These results suggest that the mutation in the p53 gene plays a significant role in the tumorigenesis of BCC developed in less-exposed areas as well as those in sun-exposed areas in Japanese patients. There must be therefore causative factors other than UV irradiation for BCC in less-exposed areas.

Aged↗

PubMed1996

Aberrant splicing and truncated-protein expression due to a newly identified XPA gene mutation.

A group A xeroderma pigmentosum (XPA) patient, XP2NI, is a compound heterozygote with a newly identified G to C transversion at the last nucleotide in exon 5 in one chromosome, and with the known splicing mutation in intron 3 in another chromosome in the XPA gene. XP2NI had mild skin symptoms and the cells were slightly less sensitive to UV radiation than the cells of typical severe XPA patients who have the splicing mutation in intron 3 homozygously. Reverse transcriptase (RT)-PCR and sequencing of the PCR products revealed that the mutation in exon 5 resulted in producing three types of aberrant mRNA, lacking 7 nucleotides at the end of exon 5, lacking entire exon 5, and lacking exons 3, 4 and 5. A significant amount of a truncated type of protein was produced in XP2NI cells, and the size of the protein indicated that it should have been translated from the mRNA, lacking the 7 nucleotides and retained one of the zinc-finger domains required for the DNA repair activity. The clinical mildness of XP2NI may be due to the residual DNA repair activity of the truncated XPA protein, while no XPA protein was detected in the XPA cells with the homozygous intron 3 splicing mutation.

Base Sequence↗

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↗