Frequency of CD59 mutations induced in human-hamster hybrid A(L) cells by low-dose X-irradiation.

Determination of the genotoxic effects of ionizing radiation, especially at low-doses, is of great importance for risk assessment, e.g. in radiological diagnostics. The human-hamster hybrid A(L) cell line has been shown previously to be a well-suited in vitro model for the study of mutations induced by various mutagens. The A(L) cells contain a standard set of hamster chromosomes and a single human chromosome 11, which confers the expression of the human cell surface protein CD59. Using CD59 specific antibodies, cells mutated in the CD59 gene can be detected and quantified by the loss of the cell surface marker. In contrast to previous studies, prior to irradiation we removed spontaneous mutants by magnetic cell separation (MACS) which allows analysis of radiation-induced mutation events only. We exposed A(L) cells to 100kV X-rays at 0.1 to 5Gy. The proportions of X-irradiation-induced CD59(-) mutants were quantified by flow cytometry after immunofluorescence labeling. Between 0.2 and 5Gy the yield of CD59 mutants was a linear function of dose. The molecular analysis of individual CD59-negative clones induced after exposure of 1, 3 and 5Gy of X-ray revealed a dose-dependent linear increase of large deletions (>6Mbp), whereas, point mutations could be seen only in spontaneous CD59 mutants or after low-dose exposure (< or =1Gy). We conclude that the modified A(L) assay presented here is appropriate for detection and quantification of non-lethal DNA lesions induced by low-dose ionizing radiation.

[Effect of neocarzinostatin on E. coli mutants deficient in DNA repair]. / Wirkung von Neocarzinostatin auf E. coli-Mutanten mit defekter DNA-Reparatur.

The colony forming ability of E. coli mutants defective in DNA repair was compared to that of the parent strain AB 1157 after neocarzinostatin treatment. A recA and a recB mutant were most sensitive. The suppression of the recB mutation in the recBC sbcBC mutant, which is as sensitive as the parent strain, indicates that recB is not the primary pathway by which lesions after NCS treatment are repaired. The survival curve of the recBC recF sbcBC mutant, corresponding to that of the recF mutant, further supports this interpretation. The relative resistance of the recBC recF sbcBC mutant suggests that NCS lesions are not only repaired by the recF and recB pathway. An alternative pathway could be the SOS induction, as a lexA mutant also is sensitive to NCS. The sensitivity of the uvrA and polA xthA mutants, however is explained by the involvement of the uvrA and polA gen products in rec repair.

Isolation and fast purification of neocarzinostatin by FPLC-ion exchange chromatography.

Neocarzinostatin, a highly toxic antitumor protein containing an essential nonprotein chromophore, can be isolated and purified from culture filtrates of Streptomyces carcinostaticus. Usually a lengthy procedure of up to 60 h is necessary for the isolation, including several chromatographic steps partly under conditions which favour inactivation of the drug by release of chromophore. We describe a new method yielding practically clinical grade Neocarzinostatin from crude extracts in 20 min. This very fast and reproducible method was made possible by using a Mono Q anion exchange column filled with monodisperse gel material which has been recently developed.