Different mutations are responsible for the elevated sister-chromatid exchange frequencies characteristic of Bloom's syndrome and hamster EM9 cells.

Abstract

Experimental hybridization of cultured cells was employed to determine whether the strikingly elevated rates of sister-chromatid exchange (SCE) exhibited by Bloom's syndrome (BS) and hamster cell line EM9 have the same or different bases. Seventeen cell lines were developed from polyethylene glycol-treated mixtures of BS and EM9 cells. Cytogenetic analysis proved the hybrid nature of 12 of the lines; 9 of those 12 exhibited low (normal) numbers of SCEs, signifying complementation. The parental BS and EM9 cells, although resembling each other in exhibiting very high SCE frequencies in BrdUrd-containing medium, differ from one another with respect to their proliferative abilities in such medium, the EM9 cells but not the BS cells being exquisitely hypersensitive to BrdUrd. In the low-SCE hybrid lines, hypersensitivity to growth in BrdUrd-containing medium was restored to normal whereas the hypersensitivity was retained by the high-SCE hybrids. It is concluded, first, that the mutations in BS and EM9 cells are different and, second, that both the elevated SCE frequency and the excessive BrdUrd hypersensitivity of EM9 cells are due to the same mutation.Three striking phenotypic features are shared by cultured cells from persons with Bloom's syndrome (BS) and Chinese hamster cell line EM9: (i) an elevated sister-chromatid exchange (SCE) frequency following growth for one or two cycles of DNA replication in bromodeoxyuridine (BrdUrd)-containing medium (1-3), (it) a markedly elevated number of SCEs following exposure to the mutagen ethyl methanesulfonate (4, 5), and (iii) a retarded rate of DNA-chain maturation (4, 6-10). BS is a recessively transmitted growth disorder that predisposes to cancer (11); EM9 is an experimentally induced mutant derivative of Chinese hamster ovary (CHO) cells (1). The similar phenotypic features of cultured BS and EM9 cells suggested the possibility that the molecular defects in the two cell types were the same, in which case EM9 cells might serve as a valuable in vitro model for BS. To determine whether the mutation in BS and the mutation in EM9 were, in fact, the same, complementation studies were performed by fusing BS lymphoblastoid cells with EM9 cells. The SCE frequency was found to have been restored to normal in a large proportion of the cell lines derived from BS/EM9 hybrids-i.e., complementation occurred. fetal bovine serum, 2 mM L-glutamine, 100 international units of penicillin per ml, and 100 tkg of streptomycin per ml. EM9 cells were grown as monolayers in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum, 100 ,uM nonessential amino acids, 100 international units of penicillin per ml, and 100 ug of streptomycin per ml (complete MEM). MATERIALS AND METHODSCell-Fusion and Hybrid-Selection Techniques. The cellfusion method of Yoshida was employed (14). In preparation for each fusion, equal numbers of BS and EM9 cells (1 x 106 of each) were added to a 25-ml flat-bottomed specimen vial (Wheaton Scientific, Millville, NJ) and pelleted by centrif...

An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

“…EM9 is phenotypically similar to cells derived from individuals with Bloom's syndrome (BS), a cancer-prone autosomal recessive disorder characterized by high SCEs (10-fold) and sensitivity to alkylating agents (4,14,15). Although chromosome localization and somatic cell hybrid complementation studies indicate that the EM9 and BS defects are genetically distinct (18,21,23), the two gene products most likely function in the same, or a closely related, biochemical pathway. The phenotype of both EM9 and BS cells might be explained by a defect in one or more of the three DNA ligases so far identified in mammalian cells, which are designated DNA ligases I, II, and III, respectively (24,31).…”

Involvement of Hexose transport in myogenic differentiation

“…In other words, they retained the ability to take up D-glucose, albeit with much reduced efficiency. Type I1 mutants were reduced that HAHT may be required f or myogenesis, and it is Ray et al, 1987). More definitive r l iochemical port may conceivably be invo f ved in myogenesis.…”

“…1A). Unfortunately, CB and BrdUrd can also act on other proteins with similar affinity Sanwal, 1979;Pearson, 1981;Ray et al, 1987). Thus they cannot be used to examine the relationshi between hexose transport and myogenesis.…”

Coordination of DNA single strand break repair

“…BrdU can itself influence the rate of crossover events, because incorporation is recognized and excised by uracil-DNA glycosylase [187, 188]. Impaired downstream repair of the resultant nicked intermediate would increase replication fork stalling, which has been observed in XRCC1-deficient cells [183, 189, 190]. RAD51-mediated homologous recombination (HR) to facilitate replication fork restart is a major mechanism of crossover events [191].…”