Sadayuki Ban

HUman MicroNucleus project: international database comparison for results with the cytokinesis-block micronucleus assay in human lymphocytes: I. Effect of laboratory protocol, scoring criteria, and host factors on the frequency of micronuclei

Micronucleus (MN) expression in peripheral blood lymphocytes is well established as a standard method for monitoring chromosome damage in human populations. The first results of an analysis of pooled data from laboratories using the cytokinesis‐block micronucleus (CBMN) assay and participating in the HUMN (HUman MicroNucleus project) international collaborative study are presented. The effects of laboratory protocol, scoring criteria, and host factors on baseline micronucleated binucleate cell (MNC) frequency are evaluated, and a reference range of “normal” values against which future studies may be compared is provided. Primary data from historical records were submitted by 25 laboratories distributed in 16 countries. This resulted in a database of nearly 7000 subjects. Potentially significant differences were present in the methods used by participating laboratories, such as in the type of culture medium, the concentration of cytochalasin‐B, the percentage of fetal calf serum, and in the culture method. Differences in criteria for scoring micronuclei were also evident. The overall median MNC frequency in nonexposed (i.e., normal) subjects was 6.5‰ and the interquartile range was between 3 and 12‰. An increase in MNC frequency with age was evident in all but two laboratories. The effect of gender, although not so evident in all databases, was also present, with females having a 19% higher level of MNC frequency (95% confidence interval: 14–24%). Statistical analyses were performed using random‐effects models for correlated data. Our best model, which included exposure to genotoxic factors, host factors, methods, and scoring criteria, explained 75% of the total variance, with the largest contribution attributable to laboratory methods. Environ. Mol. Mutagen. 37:31–45, 2001

Chromosomal instability in BRCA1- or BRCA2-defective human cancer cells detected by spontaneous micronucleus assay

The BRCA1 and BRCA2 gene products are believed to play an important part in the onset and/or development of many sporadic mammary cancers. Recently, it has been reported that these two proteins contribute to a centrosome function which is believed to help maintain the integrity of the chromosome segregation process. This may mean a reduced level of the BRCA1 or BRCA2 protein in mammary cells will occasionally lead to nondisjunctional chromosomal loss or gain. We now report that spontaneous micronuclei arising from chromosome(s) which fail to be incorporated into the relevant daughter nuclei during mitosis tend to occur more frequently in BRCA1- or BRCA2-defective human cancer cells than in BRCA-positive cancer cells. Some cases of mammary carcinogenesis may therefore stem from the loss of integrity of chromosome segregation in cells which have a reduced capacity to express either BRCA1 or BRCA2.

Effect of radiation and cigarette smoking on expression of FUdR-inducible common fragile sites in human peripheral lymphocytes

In vitro X-irradiation of human peripheral blood lymphocytes increased the frequencies of fluorodeoxyuridine-induced fragile sites in a dose-related manner. However, the cells from 30 atomic bomb survivors exposed to either high or low radiation doses 47 years earlier showed no demonstrable difference in fragile site expression, indicating that fragile site induction was ephemeral in nature. When fragile sites were analyzed on the basis of tobacco smoking habits, an elevated number was observed in the smokers. The results confirm that fragile sites can be affected by recent exposure to exogenous agents, but the effect is probably of limited duration, based on the atomic bomb survivor experience.

X-ray induction of micronuclei in human lymphocyte subpopulations differentiated by immunoperoxidase staining.

In this study we sought to confirm the radiosensitivity of human peripheral blood lymphocyte subpopulations using a micronucleus assay. Mononucleated cells isolated from peripheral blood were irradiated with X rays. After being cultured for 3 days, cells were fixed and stained using the immunoperoxidase staining technique. Lymphocyte subpopulations were characterized by means of the monoclonal antibodies Leu4 (CD3), Leu2a (CD8) and Leu19 (CD56). Dose-response curves were obtained by scoring the number of micronuclei in binucleated cells that reacted with a specific antibody and were then stained. The dose response of CD8+ (suppressor/cytotoxic) cells was quite similar to that of CD3+ (pan T) cells. In comparison, CD56+ (natural killer) cells were significantly less sensitive, although scorable binucleated CD56+ cells made up less than 4% of the total number of binucleated cells.

Application of Generalized Estimating Equations to a Study of In vitro Radiation Sensitivity

We describe an application of the generalized estimating equation (GEE) method (Liang and Zeger, 1986, Biometrika 73, 13-22) for regression analysis of correlated Poisson data from a split-plot design with a small number of experimental units. As an alternative to the use of an arbitrarily chosen working correlation matrix, we demonstrate the use of GEE with a reasonable model for the true covariance structure among repeated observations within individuals. We show that, under such a split-plot design with large clusters, the asymptotic relative efficiency of GEE with simple (independence or exchangeable) working correlation matrices is rather low. We conclude by summarizing issues and needs for further work concerning efficiency of the GEE parameter estimates in practice.

Radiation sensitivities of 31 human oesophageal squamous cell carcinoma cell lines.

The purpose of this study was to determine the radiosensitivities of 31 human oesophageal squamous cell carcinoma cell lines with a colony‐formation assay. A large variation in radiosensitivity existed among 31 cell lines. Such a large variation may partly explain the poor result of radiotherapy for this cancer. One cell line (KYSE190) demonstrated an unusual radiosensitivity. Ataxia‐telangiectasia‐mutated (ATM) gene in these cells had five missense mutations, and ATM protein was truncated or degraded. Inability to phosphorylate Chk2 in the irradiated KYSE190 cells suggests that the ATM protein in these cells had lost its function. The dysfunctional ATM protein may be a main cause of unusual radiosensitivity of KYSE190 cells. Because the donor of these cells was not diagnosed with ataxia telangiectasia, mutations in ATM gene might have occurred during the initiation and progression of cancer. Radiosensitive cancer developed in non‐hereditary diseased patients must be a good target for radiotherapy.

Lethal and Mutagenic Effects of 252Cf Radiation in Cultured Human Cells

HeLa MR cells were exposed to radiation emitted from a man-made spontaneously fissioning isotope, californium-252. The neutron to gamma-ray ratio in the radiation dose was measured to be 2.0. The extrapolation number of the dose-survival curve was 1.3 and the Do was 200 cGy. A dose-dependent increase in mutation to 6-TGr (6-thioguanine resistant) was observed. The relative biological effectiveness (r.b.e.) for cell killing of the neutrons from 252Cf, calculated relative to high-dose-rate X-rays, was 2.6 at 50 per cent survival. The r.b.e. for mutation induction was 2.7 at a mutation frequency of 5 X 10(-5) per surviving cell.

Radiosensitivity and expression of nucleotide excision repair genes in peripheral blood mononuclear cells of myelodysplastic syndrome patients

Abstract Myelodysplastic syndrome (MDS) is the only other radiogenic blood disease apart from leukemia. Clinically, MDS involves dysplastic hematopoisis and an increased risk of leukemic transformation. We compared the micronucleus (MN) frequency in the peripheral T lymphocytes of atomic bomb survivors with MDS and normal individuals. The spontaneous and X-ray-induced MN frequencies were significantly higher in MDS patients than in normal individuals. To explain the cause of unusual radiosensitivity, we measured the expression levels of nucleotide excision repair (NER) genes in peripheral blood mononuclear cells using an RT-PCR method. The reduction of NER genes was expressed in only 1 of the 10 patients with mild symptoms, but in 4 of the 11 patients with severe symptoms. Our data suggest that chromosomal instability and DNA repair defects may be involved in the pathophysiology of disease progression.

Tissue-specificity of reactivity to concanavalin-A of human cells in culture.

Concanavalin-A (Con-A) reactivity was studied to identify the tissue-specificity of cells established from various normal human tissues. Cells were treated with Con-A-labelled human red blood cells (C-RBC). C-RBC was not absorbed on the cells derived from the bone marrow, skin and liver. Lung-derived fibroblast cells showed weak C-RBC adsorption. Kidney-derived cells showed epithelial morphology and easily adsorbed C-RBC. These suggest that a large number of Con-A receptors exists on the membrane surface of kidney cells.

Induction of endoreduplication in Chinese hamsters V79 cells by cytosine arabinoside

Endoreduplication (ER) could be induced very effectively in Chinese hamster V79 cells exposed to cytosine arabinoside (1-beta-D-arabinofuranosylcytosine; Ara-C). Cells were cultured for 48 hours in Ara-C containing medium. ER frequency increases rapidly after Ara-C release. About 60% of metaphase cells were endoreduplicated at 8-10 hours after release from Ara-C (5 micrograms/ml). Induction of ER also depends on Ara-C concentrations.