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Biomedical subjects

M Ikenaga

Publications and source records attributed to M Ikenaga.

At least 109 records · Page 6Linked to original sources
PubMed1989

Somatic mosaicism for DNA repair capacity in fibroblasts derived from a group A xeroderma pigmentosum patient.

A female Japanese xeroderma pigmentosum (XP) patient with severe skin lesions and various neurologic abnormalities was assigned to complementation group A by conventional cell fusion studies. Ultraviolet (UV)-irradiated skin fibroblasts showed a biphasic survival curve, as measured by colony-forming ability. The surviving fraction decreased rapidly up to 2 J/m2 of UV, with a steep slope of D(O) (mean lethal dose) = 0.95 J/m2. At much higher doses it decreased more slowly, with D(O) = 3.5 J/m2. To èlucidate the cause of this unique survival response, we isolated a large number of independent clones from single colonies and measured their responses to UV. Of 81 clones analyzed, ten showed a marked resistance to killing by UV, which was only slightly more sensitive than normal cells, and these clones had a rate of unscheduled DNA synthesis (UDS) that was about 45% of normal cells. By contrast, the remaining 71 clones were extremely sensitive to UV, typical of XP group A strains, and had a UDS level 1%-3% of normals. Analysis of restriction fragment length polymorphism using seven polymorphic DNA probes indicated that the UV-resistant clones were derived from the same individual as the UV-sensitive clones. These results clearly demonstrate that this patient's fibroblast cells consist of two types with differing responses to UV, and provide direct evidence of somatic mosaicism for DNA repair capacity in an XP patient.

Cell Survival↗
PubMed1989

High voltage electric pulses efficiently induce fusion of cells in monolayer culture.

High voltage electric pulses, 1000 V/cm, were found to induce cell fusion efficiently when delivered to cells growing in a monolayer culture. The maximum yield of fused cells was about 30% of cells remaining after treatment. Colonies of interspecies hybrid cells between mouse and others also appeared with a frequency of 3 X 10(-4) - 2 X 10(-5) after culturing fused cells in a selection medium that permits the growth of only hybrid cells. This method of electrofusion was applied for complementation analysis of DNA repair-deficient xeroderma pigmentosum cells. Efficient cell fusion was also observed with these human diploid fibroblasts and the resulting heterodikaryons showed a recovery of ultraviolet light-induced unscheduled DNA synthesis to the same extent as in normal cells.

Animals↗
PubMed1989

Effects of X-ray irradiation on natural killer (NK) cell system. I. Elevation of sensitivity of tumor cells and lytic function of NK cells.

The in vitro effect of X-ray irradiation on the human natural killer (NK) system was studied. When K562 cells were irradiated with X-rays and cultured for 18 hours, they showed increased sensitivity to lysis by blood lymphocytes and purified large granular lymphocytes (LGL). The X-ray-induced augmentation was observed as little as 2 Gy irradiation, reaching maximum at 5 to 20 Gy. The doses of X-rays did not influence the viability and spontaneous release of the target cells. On the other hand, irradiation with X-rays of NK cells at 5 to 15 Gy resulted in a transient increase in NK activity at 1 hour, and then the activity declined and was completely lost after 24 hours. However, when LGL were cultured with interferon immediately after irradiation, they maintained elevated NK activity. These results suggest the possible use of low doses of X-ray irradiation in combination with biological response modifiers for treatment of cancer.

Cytotoxicity, Immunologic↗
PubMed1989

Effects of X-ray irradiation on natural killer (NK) cell system. II. Increased sensitivity to natural killer cytotoxic factor (NKCF).

Irradiation with low-doses of X-rays of tumor cells elevated their susceptibility to lysis by natural killer (NK) cells in an accompanying paper. Cytotoxicity assays conducted at the single cell level revealed that X-ray irradiation of K562 cells did not affect the number of effector-target conjugates but increased the frequency of dead conjugated target cells. During interaction with K562 cells large granular lymphocytes released a soluble cytotoxic factor (NKCF) that killed the target cells. X-ray irradiation did not affect the NKCF stimulatory ability of K562 cells, while it elevated their sensitivity to the lytic effect of NKCF. In contrast to X-rays, exposure to ultraviolet (UV) radiation of K562 cells did not elevate their NK sensitivity but rather reduced it. Treatment with mitomycin C produced no effect on NK sensitivity. These results indicate that X-ray irradiation elevates the target sensitivity to NKCF, which may be involved in the increased NK sensitivity, and that the X-ray effect may be different from that of UV radiation or DNA synthesis inhibition.

Cytotoxicity Tests, Immunologic↗
PubMed1989

[Role of O6-methylguanine-DNA methyltransferase in repair of DNA damaged by alkylating agents].

Some human tumor cell lines show the Mer- (Mex-) phenotype characterized by defects in repair of O6-methylguanine. Since the Mer- cells are deficient in O6-methylguanine-DNA methyltransferase activity, we examined whether introduction of the cloned bacterial gene, ada, encoding methyltransferase would make Mer- cells resistant to alkylating agents. When a DNA fragment carrying the ada gene of Escherichia coli on pSV2 neo vector was introduced into HeLa Mer- cells, transformants which are resistant to alkylating agents were obtained. The cells produced a large amount of methyltransferase enzyme. By expressing foreign genes in E. coli cells, it may be possible to clone DNA repair genes of various organisms. In this manner, we cloned the Bacillus subtilis gene that encodes a constitutive O6-methylguanine-DNA methyltransferase. Based on the nucleotide sequence of the gene, it was deduced that the protein comprises 165 amino acids and that the molecular weight is about 18,800. In principle, this method is applicable for cloning of cDNA for methyltransferase from higher organisms. The sequence homology evidenced in the present experiments also provides a useful tool for screening the gene or cDNA for methyltransferase. We developed a new vector which can replicate autonomously in EB-transformed lymphoblasts as well as in fibroblast cell lines such as HeLa. We found that the cloned ada gene on the vector is effective for increasing survival rates of Mer- cells treated with alkylating agents. This system can be used for cloning cDNA for human methyltransferase.

Alkylating Agents↗
PubMed1988

Chromosomal reorganization for the expression of recessive mutation of retinoblastoma susceptibility gene in the development of osteosarcoma.

Recent evidence indicates that the mutation of retinoblastoma susceptibility (RB) gene is also involved in the development of osteosarcoma. We studied 30 cases of osteosarcoma for the structural anomalies of the RB gene by Southern hybridization analysis with cDNA probes of the RB gene. Thirteen cases (43%) showed structural anomalies of the RB gene. They included the total or partial deletion, or rearrangement of the RB gene; seven with homozygous deletions and six with hemizygous deletions or rearrangements. By the use of restriction fragment length polymorphism fragments as chromosome markers, those seven tumors having homozygous deletions and four of six tumors having hemizygous anomalies showed the loss of heterozygosity at other loci on chromosome 13. Among those tumors with no apparent structural changes of the RB gene, seven cases showed the loss of heterozygosity on chromosome 13, and altogether the loss of heterozygosity by either homozygosity or hemizygosity was found in 18 (64%) of 28 informative cases. The loss of heterozygosity was also found for nine of 10 other chromosomes, of which chromosome 17 showed the highest frequency (77%). The tumors with loss of chromosome 13 alleles also showed additional losses of alleles on other chromosomes, while tumors retaining heterozygosity of chromosome 13 also retained heterozygosity at the informative loci on other chromosomes. Southern hybridization and karyotype analysis in some selected cases suggest that the concerted loss of heterozygosity at multiple loci may be a consequence of the polyploidization-segregation process.

Adolescent↗
PubMed1988

Increased frequency of specific alleles of the c-Ha-ras gene in Japanese cancer patients.

We have examined the c-Ha-ras locus in 145 cancer patients of a mixed group and 164 normal individuals in Japan for restriction fragment length polymorphisms and compared the allele distributions in normal and cancer populations. The c-Ha-ras gene is highly polymorphic in Japanese as previously reported in Caucasians. Two rare alleles were found to be present with increased frequencies in Japanese cancer patients. These results suggest that genotype analysis of the c-Ha-ras gene could be used to detect cancer-prone individuals.

Alleles↗
PubMed1987

Expression of the truncated E. coli O6-methylguanine methyltransferase gene in repair-deficient human cells and restoration of cellular resistance to alkylating agents.

We have constructed a truncated E. coli O6-methylguanine methyltransferase (MT) gene (ada gene) to express the MT activity for O6-methylguanine and O4-methylthymine but not for methylphosphotriester in human cells and transferred it into Mer- HeLa MR cells. The transfectant cells expressed the truncated E. coli MT were resistant to alkylating agents as same as the transfectant cells with the intact ada gene in cell killing, sister-chromatid exchange induction and host-cell reactivation of adenovirus 5. These results strongly suggest that methylphosphotriester may not contribute to the biological effect of alkylating agents in human cells.

Alkylating Agents↗
PubMed1987

Comparative analysis of O6-methylguanine methyltransferase activity and cellular sensitivity to alkylating agents in cell strains derived from a variety of animal species.

Using 26 cultured cell lines derived from 17 different animal species, we have measured both the activity of O6-methylguanine (O6-MeG) methyltransferase (MT) in cell extracts and the sensitivity of the strains to the lethal effects of the alkylating agents, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU). The MT activity was assayed by measuring the amount of 3H radioactivity transferred from methyl-[3H]-labeled O6-MeG in DNA to acceptor protein molecules in the extracts. In all the 21 mammalian cell strains, lethal sensitivity to ACNU as measured by colony-forming ability correlated well with cellular MT activity, indicating that the major lethal ACNU damage is reparable by the MT. On the other hand, MNNG sensitivity did not necessarily correlate with the MT activity.

Alkylating Agents↗
PubMed1987

Sensitivity to UV radiation of fibroblasts from a Japanese group A xeroderma pigmentosum patient with mild neurological abnormalities.

A 12-year-old girl, suffering from xeroderma pigmentosum (XP), had mild cutaneous and neurological abnormalities. She showed no neurological abnormalities at the age of seven, but areflexia of the patellar tendons at II. She had no malignant tumours. The skin fibroblasts from the patient were about twice as sensitive to the lethal effects of 254 nm ultraviolet (UV) radiation as those of Group C XP patients, and about twice as resistant as those of typical Group A XP patients. The ability of these fibroblasts to reactivate UV-damage adenovirus 5 was intermediate between those of Group C and typical Group A patients. The patient's cells were assigned to genetic complementation Group A by use of the cell-fusion technique. This is the first case in Japan of a Group A XP patient with mild neurological abnormalities. The mild cutaneous manifestations of this patient may be explained by the residual ability of the cells to repair UV-damaged DNA.

Adenoviruses, Human↗
PubMed1987

O6-methylguanine methyltransferase activity and sensitivity of Japanese tumor cell strains to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)- 3-nitrosourea hydrochloride.

Using 40 tumor cell strains derived from various organs of Japanese tumor patients and also 12 normal cell strains, we have measured the activity of O6-methylguanine-DNA methyltransferase (MT), which can repair O6-methylguanine produced in DNA by alkylating agents. Then, the lethal sensitivities of the strains to the anti-tumor drug 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-)2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) were measured. The MT activity was assayed by measuring the 3H radioactivity transferred from the substrate DNA containing [methyl-3H]-O6-methylguanine to acceptor molecules in the cell extracts. Extracts from the majority of tumor strains and all normal cell strains contained substantial MT activity of varying degree, while the extracts of 6 tumor strains showed virtually undetectable MT activity. Hence these 6 strains were assigned as Mer-, the phenotype which is characterized by the inability to repair O6-methylguanine in DNA due to the lack of MT. The Mer- tumor strains were much more sensitive to ACNU than the rest of Mer+ strains, as measured by colony-forming ability. Furthermore, with all the tumor and normal strains tested, a good correlation was observed between MT activity and cellular resistance to ACNU. These results indicate that the frequency of Mer- strain is about 15% among Japanese tumor cell strains so far analyzed, and further suggest that MT may be the only system able to repair lethal DNA damage induced by ACNU.

Cell Survival↗
PubMed1986

Transfer of the E. coli O6-methylguanine methyltransferase gene into repair-deficient human cells and restoration of cellular resistance to N-methyl-N'-nitro-N-nitrosoguanidine.

We have constructed a plasmid on which the E. coli O6-methylguanine-DNA methyltransferase (MT) gene (ada gene) was linked with an SV40 promoter sequence and a poly(A) site. After transferring this plasmid into Mer- HeLa MR cells by DNA transfection, effective expression of E. coli MT was observed. Isolated stable transformant clones showed higher resistance to N-methyl-N'-nitro-N-nitrosoguanidine in colony formation and sister-chromatid exchange induction than HeLa MR cells.

Cell Survival↗
PubMed1985

Restriction fragment length polymorphism detected by human salivary amylase cDNA.

The plasmid clone which contains human salivary amylase cDNA was used to detect restriction fragment length polymorphisms (RFLPs). After double digestion with Pst 1 and Bam H1, a polymorphism with two alleles was observed. In Japanese, frequencies of these alleles, tentatively called 5.7 kb and 6.5 kb fragment alleles, are 0.55 and 0.45, respectively.

Alleles↗
PubMed1985

Analysis of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage in tumor cell strains from Japanese patients and demonstration of MNNG hypersensitivity of Mer xenografts in athymic nude mice.

Among 15 human tumor cell strains from Japanese patients, one strain derived from a patient with thyroid cancer showed inability to support the growth of adenovirus 5 treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). When plated on this Mer- strain, adenovirus 5 showed 3-4 times higher sensitivity to MNNG-induced killing than when plated on any of the other 14 Mer+ tumor cell strains. Biochemical analysis showed that the Mer- strain was defective in demethylation repair of O6-methylguanine produced by MNNG treatment. The sensitivities of 12 of the 15 human tumor strains, including the Mer- strain, to MNNG were compared by measuring their colony-forming abilities. All the strains tested showed the Rem- phenotype (having higher sensitivity to MNNG-produced cell killing than normal fibroblasts). The differential killing effects of MNNG on Mer- and Mer+ tumor cells under in vivo conditions were tested using the Mer+ HeLa S3 strain and its Mer- variant. Mer+ cells and Mer- cells were implanted subcutaneously into the left and right flanks, respectively, of 10 nude mice and the next day, MNNG solution (0.25 ml at 1 mg/ml) was injected into the implantation sites of eight mice. Mer- tumor cells in six of eight treated mice showed no growth and those in the other two mice did grow, but regressed after approximately 3 weeks. In contrast, Mer+ tumor cells continued to grow in all the eight mice treated, indicating that Mer- tumor cells may be selectively inactivated by suitable therapeutic regimens with appropriate methylating drugs.

Adenoviruses, Human↗
PubMed1985

[Automated autoradiographic grain counting of DNA repair in cultured human fibroblasts after ultraviolet irradiation].

Measurement of autoradiographic grains produced by the decay of incorporated radioisotopes is often used for a quantitative assay of the rate of DNA replication and DNA repair in cells or tissues. However, visual grain counting by microscopic observation is time-consuming and tedious process. Recently, Kraemer et al. reported that automated measurement of grains in cultured human cells may be facilitated by using appropriate grain counting instruments. Under their experimental conditions using Kodak NTB-3 emulsion, instrument-determined grain number per nucleus was proportional to visual counts up to 30 grains, and then leveled off at much larger visual counts. The saturation phenomenon was due to counting-loss by the instrument caused by overlapping of neighboring grains. To prevent the counting-loss, we have used in the present study Japanese Sakura NR-M2 emulsion which is less sensitive to radiation exposure than Kodak NTB-3, thereby yielding smaller size of grains per radioactive decay. Samples were prepared from cultured skin fibroblasts derived from normal individuals and xeroderma pigmentosum (XP) patients defective in DNA repair. These cells were irradiated with 254 nm UV incubated for 3 h with culture medium containing 3H-thymidine, and autoradiograms were made by dipping in Sakura NR-M2 emulsion. The number of grains as well as grain surface area per nucleus was measured by using ARTEK CYTO TALLY MODEL 900 counting instrument, and compared with visual counts. The results showed that, under our optimum condition, the instrument-determined number of grains was directly proportional to visual counts, at least up to 150 grains per nucleus, with a correlation coefficient of 0.971.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoradiography↗