Biomedical subjects

H Takebe

Publications and source records attributed to H Takebe.

At least 55 records · Page 3Linked to original sources

PubMed1995

Vasoactive intestinal peptide gene expression in the rat pheochromocytoma cell line PC12.

Vasoactive intestinal peptide (VIP) gene expression was analyzed in PC12 cells. VIP mRNA was detected in PC12 cells treated with VIP or forskolin whereas no VIP mRNA was detected in the untreated cells. The induction of the VIP mRNA was enhanced by the simultaneous treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). PC12 cells stimulated with forskolin plus TPA released immunoreactive VIP. Sephadex G-50 column chromatography revealed that the immunoreactive VIP secreted from PC12 cells is comprised of multiple forms, one of which was indistinguishable from the authentic VIP. PC12 cells supported an efficient transcription from the human VIP gene promoter in a cell-specific as well as cAMP-dependent manner. These results definitely demonstrated the expression of the VIP gene in PC12 cells. VIP biosynthesis may be positively regulated by VIP in an autocrine fashion in PC12 cells.

Animals↗

PubMed1995

Molecular analysis of mutations induced by 2-chloroacetaldehyde, the ultimate carcinogenic form of vinyl chloride, in human cells using shuttle vectors.

Vinyl chloride (VC) is a carcinogen associated with human and animal cancers. The ultimate carcinogenic form of VC, 2-chloroacetaldehyde (CAA), has been suspected to be mutagenic and we confirmed the mutagenicity of CAA using a modified shuttle vector plasmid. Base sequence analyses of 109 mutant plasmids with mutations in the supF gene, which were treated with CAA and propagated in the cultured human cells, revealed that more than half of the single base substitutions were G:C to A:T transitions with eight hotspots. The majority of the mutations involving G:C base pairs were in 5'-AAGG-3' or 5'-CCTT-3' sequences suggesting that these sequences are the main targets of mutagenesis caused by CAA.

Acetaldehyde↗

PubMed1995

Correlation of (6-4)photoproduct formation with transforming mutations in UV-irradiated Ha-ras.

Sites and types of mutations in relation to the amount of cyclobutane pyrimidine dimers (CPDs) and (6-4)-photoproducts in UV-irradiated normal Ha-ras sequences were investigated. Mouse BALB/C 3T3 cells were transfected with UV-irradiated pYN-mHras plasmids containing mouse normal Ha-ras sequences, and transformed foci developed. Direct DNA sequencing of the Ha-ras retrieved from the foci revealed that most mutations (23/24, 96%) took place at dipyrimidine sequences, and the C-->T transition at the 3'-cytosine in 5'-TC or 5'-CC sequences was predominant (17/24, 71%) in codons 12, 13 and 60. In codon 61, where 5'-TC or 5'-CC is absent, two mutations were found at the 5'-TT sequence. More (6-4)photoproducts were produced than CPDs in codons 12, 13 and 60, and more CPDs were produced than (6-4)photoproducts in codon 61. These results suggest that (6-4)photoproducts are the major lesion leading to the mutations in the mouse Ha-ras sequence and subsequent transformation of BALB/C 3T3 cells.

3T3 Cells↗

PubMed1995

High prevalence of mutations in the p53 gene in poorly differentiated squamous cell carcinomas in xeroderma pigmentosum patients.

Mutations in the p53 gene were analyzed in 23 squamous cell carcinomas (SCCs) and five basal cell carcinomas from 10 xeroderma pigmentosum patients in Tunisia. Fourteen mutations were detected. Most occurred at the dipyrimidine sequences of DNA, suggesting that they were caused by ultraviolet light. A strong correlation was noted between the presence of the p53 mutations and clinical characteristics such as histology and growth of SCC. In well-differentiated grade 1 SCCs, three (27.3%) of 11 tumors had the p53 gene mutations, whereas in grade 2 and grade 3 SCCs, six (85.7%) of seven tumors had the p53 gene mutations (p < 0.05). Tumors less than 8.0 mm in diameter showed a relatively low frequency of mutation (two of 10 tumors, 20.0%), whereas most of the tumors larger than 8.1 mm (seven of eight tumors, 87.5%) had mutations of the p53 gene (p < 0.025). Multiple tumors in the same xeroderma pigmentosum patients also showed this relation. These results suggest that mutations in the p53 gene lead to the invasive and rapid-growing character of SCC.

Adolescent↗

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↗

PubMed1995

[Mechanisms of hereditary tumorigenesis].

Hereditary cancers have been shown to develop through at least two mechanisms. Development of retinoblastoma and Wilms' tumor is usually not accompanied by other hereditary symptoms. Although they are inherited autosomal dominantly, gene mutations in recessive tumor suppressor genes were found to be responsible for these tumors. The other mechanism is the enhanced mutation in the cancer-prone hereditary diseases, such as xeroderma pigmentosum, Bloom's syndrome, etc. In xeroderma pigmentosum, for example, defects in DNA repair has been shown to enhance the frequency of mutation in the oncogenes and tumor suppressor genes. Recently, another repair-related mechanism, involving the mismatch repair, was found to be involved in the hereditary nonpolyposis colon cancer.

DNA Damage↗

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

Far less frequent mutations in ras genes than in the p53 gene in skin tumors of xeroderma pigmentosum patients.

Mutations in Ha-ras, Ki-ras, and N-ras genes in squamous and basal cell carcinomas in patients with xeroderma pigmentosum (XP) were examined by the polymerase chain reaction followed by single-strand conformation polymorphism analysis and direct base sequencing. No mutation was detected in codons 12, 13, and 61 of the ras genes in XP skin tumors. This was in contrast with previous findings of a high frequency of mutation in the p53 gene in skin tumors in XP patients. A novel mutation in codon 6 of the Ki-ras gene was detected in a squamous cell carcinoma. The mutation was a C-->T transition at a dipyrimidine (5'-CT) sequence and could have been produced by solar ultraviolet light. The mutated ras gene did not have the ability to transform NIH/3T3 cells. In three tumors, multiple base substitutions were detected in exon 1 of the Ki-ras and N-ras genes. These results and our previous work on p53 gene mutations suggest that mutations in ras genes are far less frequent than in the p53 gene in the skin tumors in XP patients and that ras genes are less important in skin tumorigenesis in XP patients than is the p53 gene.

3T3 Cells↗

PubMed1994

Functional analysis of the cell-specific enhancer in the human proopiomelanocortin gene by beta-galactosidase histochemical staining.

Nucleotide sequences responsible for the cell-specific expression of the human proopiomelanocortin (POMC) gene were analyzed by histochemical staining of beta-galactosidase in culture cells transfected with chimeric genes containing the 5'-flanking regions of the human POMC gene fused to the Escherichia coli lacZ gene. The chimeric genes were stably introduced into various culture cells, including AtT-20 cells, which express the endogenous mouse POMC gene. Whereas the control gene containing the cytomegalovirus enhancer was expressed in all cell lines tested, only AtT-20 cells supported the efficient transcription of the gene containing 2.9 kb of the human POMC 5'-flanking region. These results indicate that the stable transfection-expression system utilizing the histochemical detection of the gene expression is a useful method for the analysis of cell-specific gene expression. These results have also confirmed that the trans-acting factors in mouse AtT-20 cells interact with the human POMC gene promoter region and activate the transcription of the gene. Deletion analysis has demonstrated that the profiles of the transcriptional activity of the various human POMC-lacZ fusion genes are similar to those of the rat POMC gene described previously. Comparison of the human and the rat 5'-flanking sequences revealed close homology in several regions, which might be involved in the efficient transcription of the POMC gene in AtT-20 cells.

Animals↗

PubMed1994

Similarity in the molecular profile of mutations induced by UV light in shuttle vector plasmids propagated in mouse and human cells.

Shuttle vector plasmids pYZ289 were irradiated with UV and transfected to mouse cells to permit repair of damage, mutation and replication in the cells. The frequency and types of mutations were compared with those of UV-irradiated shuttle vector plasmids pZ189 which were propagated in normal human and xeroderma pigmentosum (XP) patient cells defective in DNA repair. Both shuttle vector plasmids contain a bacterial suppressor tRNA gene supF as a common mutation target. pYZ289 propagated in the mouse cells showed survival and mutation frequency similar to pZ189 propagated in the normal human cells. All single base substitution mutations were induced in dipyrimidine sequences and G:C to A:T transition was most frequently observed (47%) in the mouse cells; however, the frequency was significantly lower than that in the XP cells. The frequency of the base substitution mutations at A:T base pairs was significantly higher in the mouse cells (29%) than in the normal human (12%) and the XP cells (6%). These results show that similar types of mutations are induced by UV in mouse and normal human cells, and that the A:T base pair is relatively more mutable in mouse than in normal human and XP cells.

Animals↗

PubMed1994

Increased rate of spontaneous mitotic recombination in T lymphocytes from a Bloom's syndrome patient using a flow-cytometric assay at HLA-A locus.

Bloom's syndrome (BS) is an autosomal recessive disorder conferring high propensity for cancer and displaying a high degree of genetic instability; the frequency of sister chromatid exchange is characteristically 10 times above background. The symmetrical four-armed chromatid interchanges are much more readily detected in peripheral blood lymphocytes of BS patients, suggesting that the frequency of somatic recombination is also increased. In the present study, the rate of spontaneous loss of HLA-A allele expression was estimated following fluctuation analysis in cultured T lymphocytes using a flow-cytometric assay. It was found to be 10 times or more higher than normal in lymphocytes from a BS patient. Molecular and chromosome analyses showed that all 13 independent variants from the patient were most likely derived from somatic recombinations. Further tests for loss of heterozygosity at a closely linked proximal locus, HLA-DQA1, showed that as many as half of the recombinants retained heterozygosity irrespective of the donor. The results suggest that the HLA region is hyperrecombinogenic in somatic cells and that the elevated recombination rate in BS cells results from the general increase at ordinary sites and not from random creation of unusual sites for recombination.

Adult↗

PubMed1994

An androgen receptor mutation causing androgen resistance in undervirilized male syndrome.

The molecular basis of androgen resistance was investigated in a patient with undervirilized male syndrome. Binding studies of the androgen receptors in the patient's genital skin fibroblasts revealed a normal binding capacity of 5 alpha-dihydrotestosterone, although the affinity to androgen was slightly lower than the normal control value. The androgen binding of the patient's receptor showed a moderate thermal instability when the assay temperature was raised from 30 to 41 C. Nucleotide sequencing analysis of the androgen receptor gene revealed a single nucleotide substitution in exon F, resulting in an amino acid alteration from leucine (CTC) to phenylalanine (TTC) at position 789 within the steroid-binding domain of androgen receptor. When expressed in COS-7 cells, the mutant androgen receptor harboring phenylalanine at position 789 showed thermolabile androgen-binding properties similar to those observed in the patient's genital skin fibroblasts. Cotransfection experiments with an androgen-inducible reporter gene demonstrated a decreased transactivational capability of the mutant receptor. These results indicate that this point mutation modified the receptor function and caused androgen resistance in this patient. This mutation caused the mildest form of all androgen insensitivity syndromes ever examined for mutations in the androgen receptor gene.

Adult↗

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↗

PubMed1994

Gene alterations and clinical characteristics of xeroderma pigmentosum group A patients in Japan.

BACKGROUND AND DESIGN: The responsible gene for xeroderma pigmentosum group A was recently identified. This study was performed to detect the gene alteration of xeroderma pigmentosum group A complementing gene in 29 xeroderma pigmentosum group A patients in Japan and to analyze whether genetic alterations in the xeroderma pigmentosum group A complementing gene are related to the clinical features. RESULTS: Of 29 patients, 25 (86%) had a mutation at the splicing junction of intron 3 and exon 4 in the homozygous state that was detected by the polymerase chain reaction and the AlwN I-restriction fragment length polymorphism. Patients having a splicing mutation at intron 3 in the homozygous state develop severe skin manifestations from early infancy and severe progressive neurologic abnormalities, including sensorineural hearing impairment, brain atrophy, mental retardation, areflexia, and ataxia. Four patients were heterozygous for splicing mutation in intron 3. Among them, at least three patients showed milder skin symptoms and milder neurologic abnormalities than patients with the homozygous splicing mutation. Two patients (single-ovum twins) revealed a compound heterozygote of splicing mutation of intron 3 and a nonsense mutation of exon 6. One patient had the splicing mutation and another mutation at the last codon of exon 5, a type of mutation that has never been reported. CONCLUSION: These data indicate that different genetic alterations in the xeroderma pigmentosum group A complementing gene may induce different clinical features.

Adolescent↗

PubMed1993

Ultraviolet-specific mutations in p53 gene in skin tumors in xeroderma pigmentosum patients.

Mutations in the p53 gene were identified in five of eight non-melanoma skin tumors in the sun-exposed areas of xeroderma pigmentosum patients by the polymerase chain reaction and single strand conformation polymorphism analysis followed by sequencing of the DNA. All mutations occurred at the dipyrimidine sites, indicating that they were caused by UV irradiation. Two tumors had multiple mutations, and four tumors had nonsense mutations. Since xeroderma pigmentosum patients are extremely sensitive to UV, the solar UV should have caused the mutations in the p53 gene and the mutations must have played a significant role in UV tumorigenesis.

Adolescent↗

PubMed1993

Analysis of mutations caused by DNA double-strand breaks produced by a restriction enzyme in shuttle vector plasmids propagated in ataxia telangiectasia cells.

Rejoining of DNA double-strand breaks (DSB) produced by a restriction endonuclease AvaI in the supF gene in a plasmid pZ189Ava, and mutations presumably due to the altered rejoinings were analyzed. After allowing the rejoining and replication of the plasmids in human cells originating from normal subjects and ataxia telangiectasia (AT) patients, the plasmids were retrieved and those containing mutated supF were screened in an indicator strain of Escherichia coli. The proportion of correctly rejoined plasmids was significantly lower in AT cells than in normal cells, suggesting that AT cells have lower fidelity in rejoining DSB. DNA sequencing of the mutated supF genes revealed that all mutations were deletions or insertions occurring exactly or closely at the rejoining site in both normal and AT cells. In AT cells, the majority of mutations were deletions, while deletions and insertions were evenly formed in normal cells. AT cells may be deficient in the mechanism to protect the broken ends of DNA strands from the exonucleolytic digestion.

Ataxia Telangiectasia↗