Biomedical subjects

H Takebe

Publications and source records attributed to H Takebe.

At least 73 records · Page 4Linked to original sources

PubMed1993

Absence of DNA repair deficiency in the confirmed heterozygotes of xeroderma pigmentosum group A.

This study was performed to elucidate whether xeroderma pigmentosum complementation group A (XPA) carrier has DNA repair abnormality against sun-exposure and ultraviolet (UV)-mimetic chemical carcinogen 4-nitroquinoline 1-oxide (4NQO). Here we report three sporadic cases of XP that were defined as group A by genetic complementation test as well as polymerase chain reaction (PCR) analysis to detect the point mutation in the responsible gene for XPA. DNA repair analyses in the skin fibroblasts revealed that the cells from the patients were much more sensitive to UV and 4NQO and had extremely low UV-induced unscheduled DNA synthesis (UDS) than control cells, whereas the cells from the carriers (heterozygotes of XP) had sensitivity to UV and 4NQO and levels of UV-induced UDS similar to normal cells. These results indicate that the obligate heterozygotes, despite having a mutated allele in XPA complementing gene demonstrated by PCR, have no DNA repair abnormality after UV irradiation and UV-mimetic 4NQO treatment. Our observations imply that XPA heterozygotes do not have higher risk of skin cancers than normal subjects based on their DNA repair abnormality.

4-Nitroquinoline-1-oxide↗

PubMed1993

A case of xeroderma pigmentosum complementation group F with neurological abnormalities.

We report a 48-year-old Japanese man suffering from xeroderma pigmentosum associated with mental retardation, cerebral atrophy and cerebellar ataxia. Cultured fibroblasts from an unexposed area of skin had reduced DNA repair capacity after UV irradiation, with higher sensitivity to UV than normal cells in colony-forming ability and host cell reactivation using herpes simplex virus. Genetic complementation tests by cell fusion with polyethylene glycol revealed that the patient belonged to group F. He died of bile duct cancer at the age of 50. This is the first report of an XP-F patient with neurological abnormalities.

Brain Diseases↗

PubMed1993

High prevalence of the point mutation in exon 6 of the xeroderma pigmentosum group A-complementing (XPAC) gene in xeroderma pigmentosum group A patients in Tunisia.

Xeroderma pigmentosum (XP) patients in Tunisia who belong to the genetic complementation group A (XPA) have milder skin symptoms than do Japanese XPA patients. Such difference in the clinical features might be caused by the difference in the site of mutation in the XP A-complementing (XPAC) gene. The purpose of this study is to identify the genetic alterations in the XPAC gene in the Tunisian XPA patients and to investigate the relationship between the clinical symptoms and the genetic alterations. Three sites of mutation in the XPAC gene have been identified in the Japanese XPA patients, and about 85% of them have a G-->C point mutation at the splicing acceptor site of intron 3. We found that six (86%) of seven Tunisian XPA patients had a nonsense mutation in codon 228 in exon 6, because of a CGA-->TGA point mutation, which can be detected by the HphI RFLP. This type of mutation is the same as those found in two Japanese XPA patients with mild clinical symptoms. Milder skin symptoms in the XPA patients in Tunisia than in those in Japan, despite mostly sunny weather and the unsatisfactory sun protection in Tunisia, should be due to the difference in the mutation site.

Adolescent↗

PubMed1993

A case of Rothmund-Thomson syndrome with reduced DNA repair capacity.

BACKGROUND: Rothmund-Thomson syndrome is an autosomal recessively inherited disease with multiple skin disorders, and little has been known about the cause of the clinical features. We cultured the cells from a patient with Rothmund-Thomson syndrome and examined the ultraviolet repair characteristics. OBSERVATIONS: A 5-year-old boy with Rothmund-Thomson syndrome is presented. He has had reticular pigmentation and hypopigmentation on his cheeks, upper aspect of the trunk, palms, and soles since 6 months of age. Cells originating from the patient had reduced unscheduled DNA synthesis, 37% of normal, after exposure to ultraviolet C (predominantly at 254 nm), and they were slightly more sensitive to ultraviolet C than were normal cells in cell ultraviolet survival. CONCLUSION: Such repair deficiency might account for the mild sun sensitivity in early childhood. Heterogeneity in the repair mechanism as well as in clinical features in this syndrome was suggested.

Cells, Cultured↗

PubMed1993

Activation of c-mos oncogene by integration of an endogenous long terminal repeat element during transfection of genomic DNA from mouse skin tumor cells.

An activated c-mos oncogene was identified in a transformed clone of golden hamster embryo cells transfected with DNA extracted from cells cultured from a UV-induced mouse skin tumor. Southern blot hybridization with a v-mos oncogene probe showed that the mos oncogene was amplified in the primary and secondary transformed cells but not in the original tumor cells. Expression of the mos oncogene was very high in the primary and secondary transformants, but mos mRNA was undetectable in the original tumor cells. A genomic DNA fragment containing the activated mos oncogene was cloned and sequenced. The upstream mouse sequence of the mos oncogene, which functions as the transcription terminator, was lost and replaced by a mouse endogenous long terminal repeat (LTR) element that provides the promoter sequence, resulting in high expression of the gene. The rearrangement apparently occurred during transfection, since the polymerase chain reaction (PCR) product encompassing the junction region was present in the primary and secondary transformants but not in the original tumor cells. The LTR element is likely to have been amplified during the skin tumor development caused by UV irradiation. Southern blot hybridization showed that the copy number of LTR in the tumor cells was significantly higher than that in normal skin cells. The amplification of the LTR in the cells may have increased the chance of recombination between the LTR and c-mos gene during the DNA transfection.

Animals↗

PubMed1992

Induction of c-fos gene expression by exposure to a static magnetic field in HeLaS3 cells.

Effects of a static magnetic field on cell growth and c-fos oncogene expression were investigated. HeLaS3 cells exposed to the magnetic field of 0.18-0.2 T for 1-6 days were not affected in the cell growth. Exposure to the magnetic field did not enhance the effects of X-rays or heat treatment which caused the transient cell growth delay. c-fos mRNA in the HeLaS3 cells was undetectable in untreated cells, but the expression was induced in cells by the magnetic field exposure for 2-24 h. The amounts of the mRNA expression changed time dependently with a peak at a 6-h exposure. c-fos was expressed following the heat treatment at 45 degrees C for 10 and 15 min, and the expression was further enhanced by the treatment of cells with heating followed by 4 h of the magnetic field exposure. Exposure of the cells to a static magnetic field may affect some cellular metabolic events leading to the c-fos gene expression.

Cell Division↗

PubMed1992

Amyloidosis cutis dyschromica. DNA repair reduction in the cellular response to UV light.

BACKGROUND: Amyloidosis cutis dyschromica, a special type of primary cutaneous amyloidosis, is assumed to be a congenital disorder and sun exposure is thought to be the major causal factor. Herein we report a case of this rare disease and DNA repair characteristics of UV damages in the fibroblasts derived from the patient. OBSERVATIONS: A 24-year-old Japanese woman showed hyperpigmented and hypopigmented xerotic lesions in sun-exposed skin since she was 10 years old; deposits of amyloid material were detected in the papillary dermis. The fibroblasts were hypersensitive to UV-B, but not so sensitive to UV-C. Unscheduled DNA synthesis of the patient's cells after UV-C exposure was lower than that of normal cells at 3 hours and both reached the same level at 6 hours. After UV-B exposure, unscheduled DNA synthesis of the patient's cells was lower than that of normal cells at least until 6 hours after UV exposure. CONCLUSION: Although the origin of amyloidosis cutis dyschromica is unknown, hypersensitivity to UV-B with possible DNA repair defects is suggested to be the cause of this disease.

Adult↗

PubMed1992

UV-induced base substitution mutations in a shuttle vector plasmid propagated in group C xeroderma pigmentosum cells.

To assess the contribution to mutagenesis of human DNA repair defects, the UV-irradiated shuttle vector plasmid pZ189 was propagated in fibroblasts derived from a xeroderma pigmentosum (XP) patient in DNA repair complementation group C. In comparison to results with DNA repair-proficient human cells (WI-38 VA13), UV-irradiated pZ189 propagated in the XP-C (XP4PA(SV)) cells showed fewer surviving plasmids and a higher frequency of mutated plasmids. Base sequence analysis of 67 mutated plasmids recovered from the XP-C cells revealed similar classes of point mutations and mutation spectrum, and a higher frequency of G:C to A:T transitions along with a lower frequency of transversions among plasmids with single or tandem mutations compared to plasmids recovered from the normal line. Most single-base substitution mutations (83%) occurred at G:C base pairs in which the 5'-adjacent base of the cytosine was thymine or cytosine. These results indicate that the DNA repair defects in XP-C, in comparison to data previously reported for XP-A, XP-D and XP-F, result in different UV survival and mutation frequency but in similar types of base substitution mutations.

Base Sequence↗

PubMed1992

O6-alkylguanine-DNA alkyltransferase activity in human malignant melanoma.

O6-alkylguanine has been known to be the major lesion in DNA for the cytotoxicity of alkylating agents and it is repaired by O6-Alkylguanine-DNA alkyltransferase (O6-AGT). To examine the relation of O6-AGT to the clinical characteristics in malignant melanoma (MM), O6-AGT activity in 13 human MM tissues was measured. The activity in tumor tissues varied widely from 0 to 0.11 pmol/mg protein. The activity in normal skin tissues was lower and less variable than in the tumors. The activity was not related to the tumor size or clinical stage of melanomas, but it was higher in tumors after chemotherapy with alkylating agents than in those without chemotherapy. In metastatic tissues, in primary tumors of the patients with metastases and in tumors of the patients with bad prognosis, the activity was also high. Two of the tumors, having the highest O6-AGT activity, were both transplantable to nude mice. These results suggest two possibilities; melanomas exposed to the alkylating agents may change to have high O6-AGT activity, followed by the resistance to such agents, or O6-AGT in melanomas may be originally diverse. O6-AGT activity in the tumour tissue may represent the effect of alkylating agents and can be used in selecting the methods of therapy.

Adult↗

PubMed1992

Accelerated appearance of skin tumors in hairless mice by repeated UV irradiation with initial intense exposure and characterization of the tumors.

Skin tumors were produced on the back of hairless mice, HOS (HR/De), by exposure to ultraviolet B light (UVB, 290-320 nm) with 4 different protocols. The first tumors appeared earlier (in 10 weeks in group I and 7 weeks in group III) when initial intense exposure was given, followed by repeated lower-level exposures, than when the mice were exposed to the repeated UV only (in 16 weeks both in group II and group IV). All mice developed skin tumors earlier in the groups given the repeated UV exposures three times a week than in the groups given the exposures twice a week. Most of the skin tumors produced by the UVB exposure were histologically malignant, being transplantable to nude mice, and the cultured cells grown from the tumors were capable of producing tumors when injected into nude mice. The accelerated development of skin tumors by initial intense exposure and short intervals of repeated exposure observed in this study may have implications for humans who expose themselves to intense sunbathing and UV tanning (burning) by fluorescent sun lamps.

Animals↗

PubMed1992

Lack of expression of tumor-suppressor genes in human malignant glioma cell lines.

Human malignant gliomas (glioblastomas and anaplastic astrocytomas) are the most frequent brain tumors and are associated with a variety of genetic alterations including retinoblastoma (RB) and p53 gene mutations, loss of interferon alpha and beta (IFNA, IFNB) genes and lack of O6-methylguanine-DNA methyltransferase (MGMT) expression. Yet, in the studies performed to date, the relationship between these alterations has not been addressed. In this report, we have studied gene expression in 29 malignant glioma cell lines and have determined that, although loss of the interferon genes and loss of RB, p53 and MGMT mRNAs are frequent events, combinations of genetic alterations involving these four proven or putative tumor-suppressor genes are relatively infrequent. The exception was loss of RB mRNA, which may be associated with lack of MGMT mRNA.

Chromosome Deletion↗

PubMed1991

Analysis of N-methyl-N-nitrosourea-induced mutations in a shuttle vector plasmid propagated in mouse O6-methylguanine-DNA methyltransferase-deficient cells in comparison with proficient cells.

A shuttle vector plasmid, pYZ289, was constructed from the pZ189 plasmid and polyoma virus DNA. The plasmid contains a supF gene as a marker of mutation and can replicate in both Escherichia coli and mouse cells. The pYZ289 plasmids treated with N-methyl-N-nitrosourea were passed through mouse cells originating from skin tumors, which are either proficient (HL18) or deficient (HL8) in O6-methylguanine-DNA methyltransferase activity, and mutations in the supF gene were analyzed. In the repair-deficient HL8 cells, N-methyl-N-nitrosourea-treated pYZ289 showed lower plasmid survival and higher mutation frequency than in the repair-proficient HL18 cells. DNA sequence analysis in the mutated supF gene revealed that most mutations occurred in G:C base pairs (86% for HL8, 76% for HL18), and the frequency of G:C----A:T transition was higher in HL8 cells (69%) than in HL18 cells (31%). G:C----T:A transversions occurred more frequently in HL18 cells (31%) than in HL8 cells (12%). Mutations occurred frequently at the base pair positions of 123 and 159 of supF gene in HL18 cells and at 169 in HL8 cells. Analysis of the bases neighboring the mutations appeared to be related to the mutability of the base pairs with the sequence of 5'-purine-G-G-3' being the most frequently mutated. These results show that the new pYZ289 plasmid is useful for the analysis of mutations and that a deficiency in O6-methylguanine-DNA methyltransferase enhances the N-methyl-N-nitrosourea-induced mutation with significant specificity.

Animals↗

PubMed1991

Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F.

To assess the contribution to mutagenesis by human DNA repair defects, a UV-treated shuttle vector plasmid, pZ189, was passed through fibroblasts derived from Japanese xeroderma pigmentosum (XP) patients in two different DNA repair complementation groups (A and F). Patients with XP have clinical and cellular UV hypersensitivity, increased frequency of skin cancer, and defects in DNA repair. The XP DNA repair defects represented by complementation groups A (XP-A) and F (XP-F) are more common in Japan than in Europe or the United States. In comparison to results with DNA repair-proficient human cells (W138-VA13), UV-treated pZ189 passed through the XP-A [XP2OS(SV)] or XP-F [XP2YO(SV)] cells showed fewer surviving plasmids (XP-A less than XP-F) and a higher frequency of mutated plasmids (XP-A greater than XP-F). Base sequence analysis of more than 200 mutated plasmids showed the major type of base substitution mutation to be the G:C----A:T transition with all three cell lines. The XP-A and XP-F cells revealed a higher frequency of G:C----A:T transitions and a lower frequency of transversions among plasmids with single or tandem mutations and a lower frequency of plasmids with multiple point mutations compared to the normal line. The spectrum of mutations in pZ189 with the XP-A cells was similar to that with the XP-F cells. Seventy-six to 91% of the single base substitution mutations occurred at G:C base pairs in which the 5'-neighboring base of the cytosine was thymine or cytosine. These studies indicate that the DNA repair defects in Japanese XP patients in complementation groups A and F result in different frequencies of plasmid survival and mutagenesis but in similar types of mutagenic abnormalities despite marked differences in clinical features. These results, together with comparable studies from United States patients in XP complementation groups A and D, suggest that G:C----A:T somatic mutations may be important in the generation of human skin cancer by UV radiation.

Base Sequence↗

PubMed1991

Altered O6-alkylguanine-DNA alkyltransferase activity in cell strains originating from mouse skin tumors induced by UV irradiation.

O6-Alkylguanine-DNA alkyltransferase (AGT) activity was assayed in the extracts of 47 cell strains originating from mouse skin tumors induced by UV irradiation. They were also examined for the sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride by colony formation. The AGT activity (fmol/mg protein) of the tumor cell strains varied widely and the mean +/- SE was 72.5 +/- 9.37, while the AGT activity of the nontumor cell strains was 134 +/- 17. Among 47 strains, 6 strains showed extremely low or no AGT activity, about 5 fmol/mg protein or less, and were hypersensitive to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride. Long-term culture of the tumor cells did not change the AGT activity except in some strains which might have had coexisting normal cells in the population in early passages. All strains showed similar UV sensitivity regardless of AGT activity. This is the first report which demonstrates that about 13% of newly induced tumor cell strains are deficient in AGT activity similar to Mer-/Mex- phenotype that was found in approximately 20% of the established human tumor cell strains.

Animals↗

PubMed1991

Reduced DNA repair in progeria cells and effects of gamma-ray irradiation on UV-induced unscheduled DNA synthesis in normal and progeria cells.

A reduction in the amount of UV-induced unscheduled DNA synthesis (UDS), and reduced cell survival and host-cell reactivation against UV exposure in Hutchinson-Gilford progeria syndrome cell strains were shown. UV-induced UDS in 4 progeria cell strains was 33-50% of the normal level. A similar reduction in the UV-induced UDS in normal cells was caused by gamma-ray irradiation to the cells before UV irradiation. The dose of gamma-rays required to cause a reduction in UDS of normal cells to the level of progeria cells was 40 Gy and the reduction was reversible after 2 days. In progeria cells, gamma-ray irradiation further reduced UDS with a lower gamma-ray dose required than in normal cells, and the reduction was also reversible but with less relative recovery than in normal cells. The presence of a 'built-in' defect in progeria cells responsible for the reduced DNA-repair capacity was suggested, and such defect may share a common mechanism with the reduction of UV-induced UDS in normal cells caused by gamma-ray irradiation.

Adolescent↗

PubMed1991

Two types of DNA ligase I activity in lymphoblastoid cells from patients with Bloom's syndrome.

DNA ligases I and II were separated by hydroxylapatite (HA) column chromatography in cell-free extracts of lymphoblastoid cell lines (LCLs) derived from two unrelated patients with Bloom's syndrome (BS) and two healthy individuals. The specific activity of ligase I from the crude extract was consistently lower in GM3403, a BS LCL from an Ashkenazi Jewish patient, than in normal control LCLs. By contrast, the level of ligase I activity in BSL-2KA, another BS LCL derived from a Japanese patient, was equivalent to those in normal LCLs, although GM3403 and BSL-2KA shared the feature of exceedingly high frequency of spontaneous sister-chromatid exchange. The levels of total ligase activity in crude extracts without the separation into the two forms, however, were approximately two-fold higher for the two BS LCLs than for the normal LCLs. Partial purification by chromatography on a DEAE-cellulose 23 column and a phosphocellulose column did not affect the superiority of the two BS LCLs over the normal LCLs in the specific activity of the total ligases. Nonetheless, subsequent application to an HA column again resulted in much less elevation of the specific activity of ligase I for GM3403 than for BSL-2KA and control LCLs. The levels of ligase II activity, accounting for 4-13% of total ligase activity, were similar among the LCLs examined. Irrespective of the extent of purification, essentially no difference in the heat lability of DNA ligase I was detected among the four LCLs. These findings suggest that there may exist among BS LCLs at least two types of subtle abnormality of DNA ligase I itself and/or a putative substance modulating the enzyme function.

Bloom Syndrome↗

PubMed1991

Xeroderma pigmentosum patients belonging to complementation group F and efficient liquid-holding recovery of ultraviolet damage.

A 73-year-old woman with xeroderma pigmentosum (XP), XP107TO, was assigned to complementation group F. The fibroblast of this patient showed a reduced level (19%) of unscheduled DNA synthesis (UDS) compared with normal cells. XP107TO cells were 2.3 times more hypersensitive to ultraviolet C (UVC) than normal cells and in the same range of other XPF cells. A clinical case report of 2 patients belonging to XP group F is also described; together with these clinical findings, the characteristics of XP group F are discussed. These patients manifested mild clinical symptoms, developing skin cancer in later age, although acute sun sensitivity was observed in early age (usually a few months after birth). Fibroblasts derived from these patients showed a substantially reduced level of UDS, considering its mild clinical features. To analyze the discrepancy of low UDS level and late onset of skin cancer in XPF cells, we examined the UV sensitivity under density-inhibited condition using XPF cells. XP cells belonging to complementation group F showed marked enhancement of UV survival when they were held in a density-inhibited condition for 1-4 d after UV irradiation. The enhancement was also observed in normal and XP group A cells, but the recovery in these cell lines was less than that in XP group F cells. XP group F cells have been shown to possess slow and long-lasting excision repair capacity and this type of repair might be stimulated by holding through time of the repair process within one cell cycle.

Adult↗

PubMed1990

Molecular changes in UV-induced and gamma-ray-induced mutations in human lymphoblastoid cells.

We have characterized the structural changes in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of 14 UV-induced, 15 gamma-ray-induced and 17 spontaneous mutants of human lymphoblastoid cells selected for 6-thioguanine (6TG) resistance. Southern blot analysis using the full-length HPRT cDNA as a probe revealed that 29% (5/17) of the spontaneous mutants contained detectable alterations in their restriction fragment patterns. Among the 15 mutants induced by gamma rays, 7 (47%) had such alterations indicative of large deletions in the HPRT gene. In contrast, all 14 UV-induced mutants exhibited hybridization patterns indistinguishable from those of the wild-type cells. These results suggest that UV is likely to induce point mutations at the HPRT locus on the human chromosome and that the molecular mechanism of UV-induced mutation is quite different from that of ionizing radiation-induced mutation or spontaneous mutation in human cells.

Blotting, Southern↗