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Biomedical subjects

M Ikenaga

Publications and source records attributed to M Ikenaga.

At least 19 recordsLinked to original sources
PubMed1992

Accumulation of allelic loss on arms of chromosomes 13q, 16q and 17p in the advanced stages of human hepatocellular carcinoma.

We examined loss of heterozygosity at 13 loci on 5 chromosomes in hepatocellular carcinomas (HCCs) from 56 patients. In 42 of these cases, regenerative nodules of liver cirrhosis were also analyzed. High frequencies of allelic losses were detected on chromosomes 13q (47%), 16q (40%) and 17p (64%), whereas losses on chromosome 4p and 11p were observed in less than 22% of cases in HCCs. In contrast, LOH was not detected on any loci in cirrhotic nodules. On chromosome 13q, the common region of allelic loss was mapped to the region including the retinoblastoma (RB) locus, by using 8 polymorphic probes. Furthermore, one case with 13q loss had an interstitial deletion of the RB gene, indicating the involvement of inactivation of the RB gene in hepatotumorigenesis. Losses were associated with portal-vein thrombosis or intrahepatic metastasis, increased tumor size, a poorly differentiated phenotype and clinical stage. Losses occurring together on 13q, 16q and 17p were significantly higher in patients in clinical stage IV or histologically poorly differentiated tumors, suggesting that the accumulation of allelic loss occurs in advanced tumors and that patients with multiple allelic losses may have a worse prognosis than those with a single loss.

Alleles↗
PubMed1992

Infrequent mutation of the ras genes in skin tumors of xeroderma pigmentosum patients in Japan.

By using PCR amplification and oligonucleotide mismatch hybridization, base-substitution mutations of the ras genes in 26 skin tumors of Japanese xeroderma pigmentosum (XP) patients were studied. Thin sections of tumor tissues which were fixed and embedded in paraffin blocks were used in this study. After analyzing codons 12, 13 and 61 of the H-, K- and N-ras genes by using 66 oligomer probes, we detected only one mutation of the K-ras gene at codon 61 in one tumor sample. All the other tumors were therefore considered not to have a mutation in the ras genes. These results suggest that mutations of the ras genes are not particularly associated with skin tumors of Japanese XP patients.

Base Sequence↗
PubMed1992

Correlation between DNA methylation and expression of O6-methylguanine-DNA methyltransferase gene in cultured human tumor cells.

Approximately 20% of human tumor cell strains are deficient in a DNA repair protein, O6-methylguanine-DNA methyltransferase (MGMT), and are called Mer- strains. In an attempt to determine the molecular basis for the extinction of MGMT expression in Mer- human cells, the distribution of DNA methylation sites in and around the exon sequences of the repair gene was compared in 6 Mer+ (repair-proficient) and 12 Mer- cell lines. Southern blot analysis of the genomic DNA digested with isoschizomeric restriction endonucleases MspI and HpaII to detect 5-methylcytosine in CCGG sequences indicated that the DNA of all the Mer+ cells but of none of the Mer- cells is heavily methylated in the exon-containing regions. The methylation pattern contradicts the general belief that inactive genes are hypermethylated compared to hypomethylation of transcriptionally active genes. It appears that the regulation of the MGMT gene in human cells is much more complex than simply dictated by its methylation level.

Blotting, Northern↗
PubMed1992

O6-methylguanine-DNA methyltransferase activity in human tumors.

The distribution of O6-methylguanine-DNA methyltransferase (MGMT) activity in extracts of tumors from 74 patients was measured. The results demonstrated that there was considerable variation of MGMT activity in different human tumor tissues as well as in different individuals. The mean values (X +/- SD, pmol/mg of protein) in breast cancer, stomach cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, esophageal carcinoma, brain tumors, colon carcinoma and malignant melanoma were 1.071 +/- 0.374 (9), 0.515 +/- 0.107 (5), 0.509 +/- 0.251 (5), 0.461 +/- 0.227 (24), 0.329 +/- 0.246 (5), 0.273 +/- 0.376 (5), 0.244 +/- 0.175 (14), 0.242 +/- 0.308 (5) and 0.201 +/- 0.161 (2) respectively. It was notable that six samples (1/24 non-small cell lung cancer, 3/5 esophageal carcinoma, 1/14 brain tumors and 1/5 colon carcinoma) did not have any detectable level of MGMT activity. Activity of glutamine pyruvic transaminase (GPT) was also measured in the same extracts used for the assay of MGMT activity. The activity of GPT in these samples with undetectable level of MGMT activity was similar to those with significant MGMT activity. These results further strengthen the assumption that a certain fraction of human tumors are Mer-.

Adult↗
PubMed1992

Repair of thymine dimers and (6-4) photoproducts in group A xeroderma pigmentosum cell lines harboring a transferred normal chromosome 9.

Transfer of a normal chromosome 9 into a xeroderma pigmentosum (XP)-A cell line partially restored its DNA repair activity. XP-A cell lines harboring a transferred chromosome were much more UV-resistant than parental XP-A cells but still more UV-sensitive than normal cells. The amount of UV-induced unscheduled DNA synthesis was only one-third of that in normal cells. The repair of thymine dimers and (6-4) photoproducts in these cell lines was analyzed by using monoclonal antibodies raised against them. Although these XP-A cell lines carrying a normal chromosome 9 could repair (6-4) photoproduct with a little lower efficiency than normal cells, the repair of thymine dimers was completely absent in these cells. The present results suggest a gene-dosage effect in DNA excision repair mechanisms in human cells or a rather complicated mechanism which involves two or more pathways.

Cell Line↗
PubMed1992

A mutant endonuclease IV of Escherichia coli loses the ability to repair lethal DNA damage induced by hydrogen peroxide but not that induced by methyl methanesulfonate.

A mutant allele of the Escherichia coli nfo gene encoding endonuclease IV, nfo-186, was cloned into plasmid pUC18. When introduced into an E. coli xthA nfo mutant, the gene product of nfo-186 complemented the hypersensitivity of the mutant to methyl methanesulfonate (MMS) but not to hydrogen peroxide (H2O2) and bleomycin. These results suggest that the mutant endonuclease IV has normal activity for repairing DNA damages induced by MMS but not those induced by H2O2 and bleomycin. A missense mutation in the cloned nfo-186 gene, in which the wild-type glycine 149 was replaced by aspartic acid, was detected by DNA sequencing. The wild-type and mutant endonuclease IV were purified to near homogeneity, and their apurinic (AP) endonuclease and 3'-phosphatase activities were determined. No difference was observed in the AP endonuclease activities of the wild-type and mutant proteins. However, 3'-phosphatase activity was dramatically reduced in the mutant protein. From these results, it is concluded that the endonuclease IV186 protein is specifically deficient in the ability to remove 3'-terminus-blocking damage, which is required for DNA repair synthesis, and it is possible that the lethal DNA damage by H2O2 is 3'-blocking damage and not AP-site damage.

Amino Acid Sequence↗
PubMed1991

Deletion of 17p and amplification of the int-2 gene in esophageal carcinomas.

We have analyzed allelic deletion at 23 loci on 18 different chromosomes in 35 esophageal squamous cell carcinoma tissues by using restriction fragment length polymorphism markers. Loss of heterozygosity was detected on chromosomes 2, 3, 6, 7, 11-14, 16-18, 21, and 22, while no loss was detected on chromosomes 1, 4, and 8-10. Only the loss of chromosome 17p was detected with high frequency (45%), and losses on other chromosomes had frequencies of less than 22%. These losses with low frequencies might be random losses caused by chromosomal rearrangement during the course of tumor development and progression. On the contrary, the loss of 17p might play an important role in the development of esophageal squamous cell carcinoma, such as inactivation of a tumor suppressor gene. Amplification of the int-2 gene was observed in 39% of the tumors. However, no significant relationship between int-2 amplification and the deletion of any chromosome was detected.

Aged↗
PubMed1991

Duct endoscopy and endoscopic biopsy in the evaluation of nipple discharge.

Microdochectomy is usually performed on patients with nipple discharge caused by intraductal proliferative lesions, such as intraductal papilloma and carcinoma. But this operation often sacrifices large amounts of normal mammary gland even when the lesion is a benign intraductal papilloma a few millimeters in diameter. We have developed duct endoscopy for the mammary duct system, and have reliably performed biopsies for intraductal proliferative lesions intraductally. From June 1989 to April 1990, we examined 22 cases by duct endoscopy, and performed endoscopic biopsy in 16 cases. The method of endoscopic biopsy is as follows. First, a bougie is inserted, without anesthesia other than Xylocaine jelly, into the orifice of the duct to enlarge it. Second, the outer cylinder and the inner needle are inserted; then the inner needle is removed, and the endoscope is inserted. After examination, the outer cylinder is moved up to the lesion to be biopsied and the endoscope is taken out. Then a sample is taken into the outer cylinder by aspiration. We diagnosed 10 cases of benign lesion and 5 cases of malignant lesion by cytological and/or histological examination. In conclusion, endoscopic biopsy, aided by duct endoscopy, is a useful and harmless diagnostic procedure in the evaluation of nipple discharge.

Biopsy↗
PubMed1991

Specificity of mutational DNA sequence changes induced by X-rays in the cloned Escherichia coli crp gene.

Plasmid DNA carrying the adenosine 3',5'-cyclic monophosphate receptor protein (crp) gene of Escherichia coli was irradiated, in solution, with X-rays, and the mutations produced in the crp gene were assayed by transforming the recipient E. coli cells. Ninety-six mutant clones were isolated, and mutational changes were determined by DNA sequencing. Of the 92 mutations thus detected, 74 represented base substitution mutations and the remaining 18 were frameshifts. The base substitutions included 56 G:C to A:T transitions, 10 G:C to T:A transversions and 7 G:C to C:G transversions. An A:T to G:C transition was found only once, and neither an A:T to T:A nor an A:T to C:G transversion was detected. The frameshift mutations consisted of 11 one-base deletions and 7 one-base insertions. Accordingly, G:C to A:T transition was the predominant type of mutation, which constituted 76% (56/74) of the total base substitutions and 60% (56/92) of all detected mutations. Furthermore, of the 56 transitions, about three-quarters (41 clones) clustered at an identical site, a cytosine residue at the 706 position, demonstrating that this site is a distinct hot spot for X-ray mutagenesis. These results raise the possibility that radiation-induced mutations may not necessarily occur randomly, at least in certain cases.

Bacterial Proteins↗
PubMed1991

O6-methylguanine-DNA methyltransferase activity in human liver tumors.

Previous studies have shown that in about one-fifth of human tumor cell strains, the activity of O6-methylguanine-DNA methyltransferase (MGMT), which can repair O6-alkylguanine in DNA produced by alkylating agents, is deficient. These strains are termed Mer- cells. To see if there is any human tumor lacking MGMT activity, we measured the MGMT activity in extracts from liver tumors of 21 patients, and compared it to the activity in normal peritumoral tissues derived from the same patients. The MGMT activity was assayed by measuring the 3H radioactivity transferred from the substrate DNA containing [methyl-3H]-labeled O6-methylguanine to an acid-insoluble protein fraction. There was considerable variation in MGMT activity among individual extracts; the interindividual variation was approximately 6-fold in normal liver tissue and much larger in liver tumors. Although in many cases similar high levels of MGMT activity were found both in liver tumors and in the normal counterpart, six tumors had greater than 3-fold less activity compared with the normal liver tissue from the same patient. Liver tumors from two patients did not have any detectable level of MGMT activity by the present method used, in spite of the fact that the corresponding normal liver samples demonstrated significant activities. We also measured in the same tissue extracts the activities of two common enzymes, glutamic pyruvic transaminase (GPT) and lactic dehydrogenase (LDH). The activities of GPT and LDH in the liver tumor samples that showed undetectable levels of MGMT activity were similar to those in the surrounding normal liver tissues. These results may suggest the existence of human Mer- tumors, deficient or very low MGMT activity.

Aged↗
PubMed1991

Assignment of a Chinese xeroderma pigmentosum patient from Taiwan to complementation group C.

A 2 years and 7 months-old Chinese boy with severe skin symptoms was diagnosed as xeroderma pigmentosum (XP) at Chang Gung Memorial Hospital in Taipei, Taiwan. Skin fibroblasts derived from the patient (patient identification number, XP1CTA) were used for genetic complementation analysis by the conventional cell-fusion technique followed by measurement of ultraviolet light (UV)-induced unscheduled DNA synthesis (UDS). The level of UDS in XP1CTA cells measured by autoradiography was about 20% of that in normal cells. When XP1CTA cells were fused with cells of a representative strain from each of the complementation groups A, D, E, F, G, and H, binuclear cells showed UDS levels in the range of normal cells, demonstrating a clear complementation between XP1CTA strain and either one of these strains. XP1CTA cells failed to complement with all the five reference strains belonging to group C. From these results, the XP1CTA was unambiguously assigned to complementation group C. Sensitivity of XP1CTA cells to UV, as measured by colony-forming ability, also fell within a range of variation in UV sensitivities of these group C XP cell strains.

Asian People↗
PubMed1990

Elevated frequency of a specific allele of the L-myc gene in male patients with bone and soft-tissue sarcomas.

Restriction fragment length polymorphisms (RFLPs) of 2 oncogenes, c-Ha-ras and L-myc, have been analyzed in 101 patients with bone and soft-tissue sarcoma and in 98 normal individuals. The c-Ha-ras gene was highly polymorphic both in sarcoma patients and in normal individuals. In contrast to our previous observation in breast cancer patients (Honda et al., 1988), no significant difference in allele frequencies between normal and sarcoma populations was observed. The L-myc locus revealed 2-allele polymorphism, allele L (10-kb) and S (6.6-kb), after digestion with Eco RI. The allele frequencies of L and S in sarcoma patients were 0.381 and 0.619, respectively, and those in normal individuals were 0.485 and 0.515, respectively. While the distributions of alleles in both populations fitted well with the Hardy-Weinberg equilibrium, the frequency of the S allele was significantly higher in sarcoma patients than in normal individuals (p less than 0.05). When sarcoma patients were divided into male and female subpopulations, this difference was highly significant only in males (p less than 0.01) but not in females. Our results suggest that males carrying the S allele may be prone to develop sarcoma.

Alleles↗
PubMed1990

Linkage between O6-methylguanine-DNA methyltransferase (O6-MT) activity and cellular resistance to antitumour nitrosoureas in cultured rat brain tumour cell strains.

We have examined O6-methylguanine-DNA methyltransferase (O6-MT) activity of rat brain tumour cell strains with reference to cellular resistance to antitumour nitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (nimustine, ACNU) and methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy-alpha-D-glucopyrano side (ramustine, MCNU). The values of O6-MT activity were 52 and 160 fmol/mg protein extract in 9L and C6 rat brain tumour cells, respectively; while HeLa S3 cells, as a methyl excision repair positive (Mer+) cell strain, revealed a rather high value of 488 fmol/mg. 9L cells indicative of a low O6-MT activity showed 13 microM for ACNU and 18 microM for MCNU at a 10% survival dose (SD10), determined by a clonogenic cell assay as an index of cellular resistance. In contrast to this, C6 cells revealed a SD10 value of 67 microM and 36 microM for ACNU and MCNU, respectively, indicating higher resistance than 9L cells. HeLa S3 cells showed the highest SD10 value as follows: 84 microM for ACNU and 73 microM for MCNU. The relationship between the O6-MT activity and the cellular resistance was almost linear, with relatively resistant cell lines exhibiting the higher levels of the O6-MT activity. This correlation between the O6-MT activity and the cellular resistance to nitrosoureas as ACNU and MCNU was not observed among other antitumour drugs, which included bleomycin (BLM), neocarzinostatin (NCS), cis-diamminedichloroplatinum (II) (CDDP), and etoposide (VP-16) in clinical use for brain tumour chemotherapy. This indicates that O6-MT activity can be an indicator of cellular resistance to antitumour nitrosoureas in the chemotherapy of brain tumours.

Animals↗
PubMed1990

Human chromosome 9 can complement UV sensitivity of xeroderma pigmentosum group A cells.

A single human chromosome derived from normal human fibroblasts and tagged with the G418 resistance gene was transferred into SV40-transformed xeroderma pigmentosum group A (XP-A) cells via microcell fusion. When chromosome 1 or 12 was transferred, UV sensitivity of microcell hybrid cells was not changed. By contrast, after transferring chromosome 9, 7 of 11 recipient clones were as UV-resistant as normal human cells. Four other clones were still as UV-sensitive as the parental XP-A cells. Southern hybridization analysis using a polymorphic probe, pEKZ19.3, which is homologous to a sequence of the D9S17 locus on chromosome 9, has confirmed that at least a part of normal human chromosome 9 was transferred into the recipient clones. However, amounts of UV-induced unscheduled DNA synthesis in the UV-resistant clones were only one-third of those in normal human cells. These results indicate that a gene on chromosome 9 can confer complementation of high UV sensitivity of XP-A cells although it is still possible that 2 or more genes might be involved in the defective-repair phenotypes of XP-A.

Blotting, Southern↗
PubMed1990

[A case of xeroderma pigmentosum treated by oral etretinate].

A case of xeroderma pigmentosum (XP) with neurological disorders is reported. The patient is a 16 year old male who presented a marked photosensitivity from his early infancy. The family history is negative for XP with no consanguinity being observed. He displayed neurological symptoms such as a low IQ and audiological disturbances. He developed six basal cell epitheliomas (BECEs) on his face at age of 14. We tried oral etretinate for 4 years at a initial dosage of 30 mg per day for the possible prevention of new skin cancers. Efficacy of the drug for prevention of cancers has not been confirmed, but evidence is obtained that the drug might prevent the growth of tumors. We also reviewed the chemoprevention of skin cancers with retinoids, in patients with xeroderma pigmentosum.

Administration, Oral↗
PubMed1989

Assignment of common allele loss in osteosarcoma to the subregion 17p13.

Human osteosarcomas frequently show loss of alleles on chromosome 17 as well as those on chromosome 13 that harbors the retinoblastoma gene, indicating concerted operation of another tumor-suppressing gene on chromosome 17. To assign the affected gene to a defined region of chromosome 17, we performed mitotic recombination/deletion mapping by the use of 10 polymorphic loci on chromosome 17. Of 37 tumors studied, 28 (75.7%) showed loss of heterozygosity on chromosome 17. The affected regions varied among tumors, ranging in extent from a whole chromosome to a distal segment of the short arm. However, allele loss in one region, notably in 17p13 between D17S1 and D17S30, was common to all 28 tumors, suggesting the presence of a tumor-suppressing gene in this defined region.

Alleles↗