Takayuki Kurihara

Increased rate of spontaneous mitotic recombination in T lymphocytes from a Bloom's syndrome patient using a flow-cytometric assay at HLA-A locus.

Blooms syndrome (BS) is an autosomal recessive disorder conferring high propensity for cancer and displaying a high degree of genetic instability; the frequency of sister chromatid exchange is characteristically 10 times above background. The symmetrical four‐armed chromatid interchanges are much more readily detected in peripheral blood lymphocytes of BS patients, suggesting that the frequency of somatic recombination is also increased. In the present study, the rate of spontaneous loss of HLA‐A allele expression was estimated following fluctuation analysis in cultured T lymphocytes using a flow‐cytometric assay. It was found to be 10 times or more higher than normal in lymphocytes from a BS patient. Molecular and chromosome analyses showed that all 13 independent variants from the patient were most likely derived from somatic recombinations. Further tests for loss of heterozygosity at a closely linked proximal locus, HLA‐DQA1, showed that as many as half of the recombinants retained heterozygosity irrespective of the donor. The results suggest that the HLA region is hyperrecombinogenic in somatic cells and that the elevated recombination rate in BS cells results from the general increase at ordinary sites and not from random creation of unusual sites for recombination.

Hypersensitivity of Bloom's syndrome fibroblasts to N-ethyl-N-nitrosourea

Fibroblast cells from two Japanese patients with Blooms syndrome (BS) and normal donors were studied for the inactivation of colony-forming ability and the induction of sister-chromatid exchanges (SCEs) after N-ethyl-N-nitrosourea (ENU) treatment. The reduction of ENU-induced SCEs as a function of post-treatment incubation time was also compared between BS and normal fibroblasts. BS cells were approximately 4 times more sensitive than normal cells to the lethal effect of ENU and remarkably hypersensitive to the SCE induction by ENU. The post-treatment incubation of ENU-treated normal cells in the fresh medium resulted in a time-dependent decrease of the SCE level until 6 h after which time the SCE level remained the plateau of about 50% of the initial level. In contrast, the ENU-induced SCEs in BS cells decreased much more slowly with post-treatment incubation time and its half life was 24 h. These results collectively support the view that BS cells may be defective in the rapid repair of certain type(s) of DNA damages induced by ENU.

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 Blooms 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.

Transition of Phenotypic Dimorphism with Regard to Spontaneous Sister Chromatid Exchange in Epstein‐Barr Virus‐transformed Bloom's Syndrome Lymphoblastoid Cell Lines

We recently established four lymphoblastoid cell lines (LCLs) by infecting the peripheral blood of four Japanese patients suffering from Blooms syndrome (BS) with Epstein‐Barr virus (EBV). During the course of propagating these cell lines, two of them exhibited dimorphism regarding spontaneous sister chromatid exchange (SCE), i.e., a mixed population consisted of cells with extremely high SCE levels characteristic of BS and cells with low SCE levels indistinguishable from that of normal control cells. On the other hand, the other two cell lines maintained a monomorphic population with high SCE levels at least until 30 weeks after EBV infection. The proportion of the cells with high SCE levels in the cell lines with dual phenotype declined as the population doubling numbers (PDN) increased with time and they became ultimately undetectable. The proportion of cells with low SCE levels at the time of EBV infection was estimated in one of these LCLs as 0.075% by extrapolating the linear regression of the logit for the proportion plotted against PDN. In view of the well‐known stability of the monomorphic phenotype in representative BS LCLs during extended cultivation, together with the present observations on the dual phenotype, we conclude that the frequent establishment of BS LCLs exclusively with low spontaneous SCE levels is attributable to the various proportions of low‐SCE cells existing in vivo in the B‐lymphocytes pool of BS individuals and to the selective pressure against the high‐SCE cells in in vitro cultures.