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Biomedical subjects

M Ikenaga

Publications and source records attributed to M Ikenaga.

At least 73 records · Page 4Linked to original sources
PubMed1994

Allelotype analysis of esophageal squamous cell carcinoma.

In previous studies, we have shown that allelic loss on chromosome 17p, on which the p53 gene is located, is very frequent, and loss-of-function mutations of the p53 gene are closely associated with the tumorigenesis of esophageal cancer. In this study, we performed allelotype analysis to investigate whether other tumor suppressor genes are also involved in esophageal cancer. Using 55 polymorphic DNA markers covering every autosomal arm except 13p, 21p, and 22p, restriction fragment length polymorphism analysis was performed on 36 esophageal squamous cell carcinomas (ESCs) and their adjacent normal tissue samples. Frequent loss of heterozygosity (LOH) of > 30% of the informative cases was observed on chromosomes 3p (41.1%), 5q (52.6%), 6p (30.4%), 8p (33.3%), 9p (35.7%), 9q (30.8%), 11p (32.4%), 13q (52.7%), 17p (55.2%), 17q (33.3%), 18q (45.7%), and 19q (30.4%). Among these, LOH on 5q, 13q, 17p, and 18q was previously reported in ESC and is considered to involve the APC, RB, p53, and DCC genes, respectively. However, our deletion analysis of chromosome 18q revealed that the region commonly lost did not include the DCC locus, suggesting that a possible tumor suppressor gene on 18q other than the DCC gene is involved in ESC. We screened 60 primary ESC tumors and 20 cultured ESC cell lines for the mutation of the APC gene within a mutation cluster region in exon 15, where the "hot spot" of somatic mutation for colorectal and pancreatic cancers is thought to be. We could not find any mutation despite the high frequency of LOH on chromosome 5q. We also analyzed the relationship between the clinicopathological data and the allelic loss and found that LOH on chromosomes 6p and 13q was associated with poor prognosis.

Alleles↗
PubMed1994

O6-alkylguanine-DNA alkyltransferase activity of human malignant glioma and its clinical implications.

Activity of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) is an important determinant of responsiveness of tumor cells to chloroethylnitrosoureas (CENUs), representative chemotherapeutic agents for primary malignant gliomas. In order to assess the real states of this repair protein in human malignant gliomas, we assayed AGT activity in surgically extirpated 42 malignant glioma samples and studied the distribution of the activity under certain clinical conditions. There were wide variations in AGT activity between individuals. No significant difference in AGT activity on average was seen either between glioblastoma and anaplastic astrocytoma, nor between primary and recurrent tumors. Among 42 malignant gliomas, 7 samples (16.7%) had low AGT activity less than 0.1 pmoles/mg protein. In the case of glioblastoma, tumors possessing higher AGT activity tended to be less responsive to post-operation remission-induction therapy including CENUs. The result of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) chemosensitivity assay by using the corresponding surgical specimens suggested a close relationship between cellular resistance to CENUs and AGT activity. It was found to be unlikely that a short term administration of CENUs had a significant effect on AGT activity of brain tumors in human body. We could detect a bit of definite evidences of the relevance of AGT to resistance to CENUs and need to conduct further investigations for other resistance factors.

Astrocytoma↗
PubMed1994

Potential of O6-methylguanine or O6-benzylguanine in the enhancement of chloroethylnitrosourea cytotoxicity on brain tumours.

The purine analogues O6-methylguanine and O6-benzylguanine are well-known as a chemical modulator of the DNA repair enzyme O6-methylguanine-DNA methyltransferase. Inactivation of the enzyme by O6-methylguanine or O6-benzylguanine is expected to enhance sensitivity of tumours to chloroethylnitrosoureas. We studied the effect of O6-methylguanine or O6-benzylguanine pretreatment on cytotoxicity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3- (2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in brain tumour cells and transplanted brain tumours. Two-hour exposure of O6-methylguanine at higher concentrations (500 microM, 1,000 microM) increased ACNU cytotoxicity by only 2 times in ACNU-resistant C6-1 brain tumour cells. O6-Benzylguanine at concentrations between 10 and 100 microM markedly enhanced the cytotoxic effect. The ACNU sensitivity of the tumour cels pretreated with O6-benzylguanine was 5-40 times that of the cells without O6-benzylguanine. Neither O6-methylguanine nor O6-benzylguanine appreciably enhanced ACNU cytotoxicity of 9 L cells, which were originally sensitive to ACNU. Intracarotid ACNU with O6-methylguanine or O6-benzylguanine decreased proliferating activity of transplanted C6-1 brain tumours significantly during 48 hours. O6-Benzylguanine pretreatment resulted in a greater degree of suppression for a long time. The C6-1 tumours treated only with intracarotid ACNU showed a transient inhibition and a rapid regrowth during 24 hours after the treatment. These results indicate that O6-methylguanine or O6-benzylguanine increases ACNU cytotoxicity and may be feasible for effective combination therapy with chloroethylnitrosourea in the chemotherapy of malignant brain tumours.

Animals↗
PubMed1994

Cockayne syndrome in two adult siblings.

Although survival beyond the second decade is unusual in patients who have Cockayne syndrome, we describe two brothers with the syndrome who are 42 and 55 years of age. Their cultured skin fibroblasts showed extreme UV sensitivity but had almost normal UV-induced unscheduled DNA synthesis. The patients were classified as genetic complementation group B after study of the recovery of RNA synthesis after UV irradiation of fused cells. Clinical phototesting revealed a reduced threshold for UVB erythema.

Adult↗
PubMed1994

Assignment of six patients with xeroderma pigmentosum in Hokkaido area to a variant form.

Six Japanese patients in Hokkaido area were diagnosed as xeroderma pigmentosum (XP) at two University Hospital; one patient, a 29 years old female (XP2AS) at Asahikawa Medical College, and five patients, namely, a 53 years old male (XP1SA), a 45 years old female (XP2SA), a 46 years old female (XP3SA), a 47 years old male (XP4SA) and a 34 years old male (XP5SA) at Hokkaido University School of Medicine. All these XP patients showed mild skin symptoms and had no apparent neurological abnormalities. To identify genetic complementation groups of these patients, DNA repair capacities in skin fibroblasts derived from the patients were analyzed by ultraviolet light (UV)-induced unscheduled DNA synthesis (UDS), and also by colony-forming abilities of UV-irradiated cells incubated with or without caffeine added in post-UV culture medium. The levels of UDS measured by autoradiography in these XP cells irradiated with 20 J/m2 of UV were 67 to 84% of those of normal human fibroblasts. Although one XP cell strain (XP2AS) showed about 2-fold greater sensitivity to killing by UV than normal cells, UV sensitivities of the rest of 5 XP strains were similar or only slightly higher than those of normal cells. However, UV sensitivities of all the 6 XP cell strains markedly increased by addition of 1 mM caffeine in the post-UV incubation medium, a phenomenon characteristic of XP variant cells. We also analyzed post-replication repair in XP1SA cells by sedimentation of newly-synthesized DNA in alkaline sucrose gradient, and found that this strain is defective in this repair system. From these results, we concluded that all the 6 XP strains studied here belong to XP variants.

Adult↗
PubMed1994

[A case of advanced gastric cancer successfully treated with resection after FEP (5-FU, epirubicin, CDDP) combined chemotherapy].

We reported an experience with an advanced gastric cancer patient with direct invasion into liver and metastases to liver, who had responded extremely well to an FEP combined chemotherapy. He had curatively undergone total gastrectomy with partial hepatectomy and enlarged lymph node dissection. The patient received three courses of 5-FU (750 mg/m2/day, for four days, continuous infusion), epirubicin (30 mg/m2, on day 1, i.v.), CDDP (17.5 mg/m2/day, on days 1, 2, 3, 4 i.v.) every 3 weeks in our hospital. No remarkable side effect was encountered. Partial response in the primary and invasive lesions was observed by X-P, endoscopy and CTscan. Accordingly, we could perform curative resection of the stomach with lymph nodes and parts of liver. The effect (partial response) of neoadjuvant chemotherapy was confirmed by histological examinations. FEP combined chemotherapy appears useful as a neoadjuvant approach to advanced gastric cancer.

Adenocarcinoma↗
PubMed1994

[Cytotoxic action of alkylating agents in human tumor cells and its relationship to apoptosis].

Various anticancer agents have been known to induce apoptosis in certain types of human tumor cells. The fact that a variety of agents, which attack different cellular targets, induce common apoptotic cell death suggests that the nature of initial damage is not directly involved in apoptosis. The mechanism by which a damage leads to apoptosis is not known. However, modulation of this process may affect the outcome of anticancer drug treatment. This article briefly reviewed the studies of endogenous as well as exogenous factors which modulate apoptosis, and then described the characteristics of cell death induced by alkylating agents. O6-Alkylguanine, a major cytotoxic DNA damage produced by simple alkylating agents, can be repaired by the cellular enzyme O6-methylguanine-DNA methyltransferase (MGMT). About one-fifth of human tumor cell strains lack the MGMT activity and termed as Mer- cells. Mer- cells are hypersensitive to alkylating agents like chloroethyl nitrosoureas (CNUs), compared with repair-proficient Mer+ cells. It is suggested that identification of a factor which suppresses the MGMT gene expression in CNU-resistant Mer+ cells, may enable us to convert these Mer+ cells to Mer- phenotype, thus resulting in much higher sensitivity of Mer+ cells to CNUs.

Alkylating Agents↗
PubMed1993

Loss of 17p, mutation of the p53 gene, and overexpression of p53 protein in esophageal squamous cell carcinomas.

We analyzed mutations of the p53 gene by single-strand conformation polymorphism analysis of polymerase chain reaction products and direct sequencing through all coding exons and exon-intron junctions in 32 cases with esophageal squamous cell carcinoma. Mutations were detected in 15 of 32 (47%) tumor samples, in which G:C to T:A transversions were rather frequent (33%). Previously, we reported deletion of chromosome 17p where the p53 gene is located in 45% of Japanese esophageal squamous cell carcinoma, and here the relationship between mutation of the p53 gene and loss of 17p was analyzed. Mutations were observed in 12 of 16 patients with loss of 17p, whereas only 2 of 11 without loss were positive for mutations, suggesting that mutations of the p53 gene were closely associated with a 17p deletion. Furthermore, we immunohistochemically analyzed the expression of p53 protein in esophageal squamous cell carcinoma tumor tissues using a monoclonal antibody. Five of 6 tumors with missense mutations of the p53 gene were positively stained, while in tumors with nonsense mutations or without mutations of the p53 gene staining was very weak or negative. These results suggest a good correlation between mutations and abnormal expression of the p53 gene.

Aged↗
PubMed1993

Localized hyperthermic treatment of experimental bone tumors with ferromagnetic ceramics.

Localized hyperthermic treatment was carried out with use of a metastatic bone tumor model in rabbits. The experimental bone tumor was created by transplantation of pieces of tumor line VX2 into the medullary canal of rabbit tibiae. Two weeks after the transplantation, a ferromagnetic ceramic pin was inserted in the medullary canal. Then, hyperthermia (HT) of the tumor was accomplished with use of an alternating magnetic field for 50 min. All the rabbits were killed 5 weeks after tumor transplantation, and the therapeutic effect was evaluated histologically and roentgenographically. Almost all the tumor cells within the bone marrow were killed by this procedure. The area of tumor necrosis in the HT group was significantly larger than in the control group. The pathological fracture rate and displacement rate were reduced significantly by this treatment (38.5 and 0%) compared with the controls (92.3 and 92.3%). Therefore, HT with the use of ferromagnetic ceramics was effective for local control of malignant bone tumors and seems to be a promising new method of treatment.

Animals↗
PubMed1993

Interrelationship between O6-alkylguanine-DNA alkyltransferase activity and susceptibility to chloroethylnitrosoureas in several glioma cell lines.

In order to study the dynamic relationship in glioma cells between O6-alkylguanine-DNA alkyltransferase (AGT) activity and resistance to the cytotoxic effect of chloroethylnitrosoureas (CENUs), we investigated the changes in sensitivity to 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) after modulation of AGT activity. In ACNU-resistant rat glioma cell lines (9LR1, 9LR3, and 9LR12) and a human glioma cell (HNG-1), O6-methylguanine enhanced cytotoxicity to ACNU following a depletion of AGT activity. But no enhancement of cytotoxicity was seen in an ACNU-sensitive rat glioma cell line (9L). In the 9L and 9LR12 cells, equivalently sublethal doses of ACNU similarly depleted AGT activity but the regeneration rates of this repair protein were different. In the case of a 7-day pretreatment with human recombinant interferon-beta (HuIFN-beta), although it could modulate AGT activity in HNG-1 cells, no definite influence on cellular sensitivity to CENUs was observed. However, a 50-day pretreatment with HuIFN-beta conferred resistance to CENUs on them despite its effect to reduce AGT activity. Thus, diversity was seen in the relation between AGT activity and resistance to CENUs when AGT activity was modulated by HuIFN-beta. The results of this study suggest that AGT activity is one of factors affecting cellular sensitivity to CENUs but that alternative mechanisms of tolerance may be induced depending upon some environmental effects.

Animals↗
PubMed1993

Depletion of O6-methylguanine-DNA methyltransferase and potentiation of 1,3-bis(2-chloroethyl)-1-nitrosourea antitumor activity by O6-benzylguanine in vitro.

The overcoming effect of O6-benzylguanine on O6-methylguanine-DNA methyltransferase (MGMT)-mediated 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro was evaluated. Depletion of MGMT activity in Mer+ HeLa S3 cells by O6-benzylguanine was dose-dependent and a complete loss of MGMT activity was achieved at a concentration of 0.5 microM. The cytotoxic potential of BCNU on MGMT proficient HeLa S3 (1.10 pmol/mg of protein), SMMC-7721 (0.72 pmol/mg of protein) and Cc801 (0.39 pmol/mg of protein) was greatly enhanced when cells were exposed to 10 microM O6-benzylguanine for 1 h, but there was a lack of potentiation of BCNU sensitivity in Mer- HeLa MR cells due to its nearly undetectable level of MGMT. There existed a correlation between the extent of enhancement and the amount of MGMT activity. The intensity of enhancement expressed as dose modifying factor = IC50 (BCNU alone)/IC50 (10 microM O6-benzylguanine + BCNU) was 4.56, 3.89, 3.67 and 0.97 in HeLa S3, SMMC-7721, Cc801 and HeLa MR cells respectively. The results further demonstrated that O6-benzylguanine may have potential utility as an adjuvant in combination chemotherapy with chloroethylnitrosourea agents.

Antineoplastic Agents↗
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↗