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MOLECULAR MECHANISMS OF RADIATION DAMAGE TO CHROMOSOMES OF HUMAN AND RODENT CELLS

Objective


The question arises as to whether the kinetics of disappearance of chromatid breaks represents a repair process. It might be argued that the kinetics of chromatid breaks reflect a changing sensitivity of cells to X-rays as they progress through the G2 phase. This hypothesis appears however not to be supported by 2 strong lines of evidence. Firstly, the disappearance of chromatid breaks can be inhibited in human cells by known inhibitors of deoxyribonucleic acid (DNA) double strand break (DSB) repair 9-B-D-arabinofuranosyladenine (ara A) and 1-B-D-arabinofuranosyladenine (ara C), and secondly that in Chinese hamster ovary (CHO) cells, lowering temperature from 37 C to 33 C increased the length of the G2 phase (from 2.8 h to 5.7 h) but does not significantly alter the rate of disappearance of breaks. An alteration in frequencies of breaks would be expected, based on a changing sensitivity through the cell cycle. It is therefore concluded that the kinetics of breaks in both human and CHO cells may reflect an underlying repair of a DNA lesion which we presume to be DSBs. It then seems to be paradoxical that the rate of repair of chromatid breaks observed in xrs-5 cells is essentially the same as that in CHO K1 cells, since the xrs 5 line is known to be DSB repair deficient. We cannot at this stage explain this paradox, however 1 possibility is that the results indicate the existence of a subclass of DSB which gives rise to chromatid aberrations and the repair of which is not altered in xrs 5. There is however clearly a higher frequency of conversion of DSB into visible chromatid breaks in xrs 5 as compared to that in CHO K1 cells. A similar high frequency of breaks is also observed in ataxia telangiectasia (AT) cells which may in part contribute to the hypersensitivity to X-rays of these mutant cell lines. However in this case the (AT) cells are known to be proficient in DSB repair. There must therefore be other reasons than the ability to repair DSB for the high freque ncy of conversion of DSB into chromatid breaks.

Our results showing that a different kinetic of exchange aberration induction from that of deletions appear to add further support for the notion that the misjoining mechanism leading to chromosomal exchanges is independent of that for joining breaks (presumed to be the same mechanism as that for repair of DSB or subclass of DSB). The separate nature of the chromosome joining and misjoining mechanisms is also strongly indicated by an earlier result in which we show that the frequency of exchanges increased 3-fold while the rejoining of breaks was inhibited.

Our results for X-irradiated human fibroblasts treated with ara C after X-ray exposure suggest that this agent induces a type of lesion, not in itself manifest as a visible chromosomal aberration at metaphase, but nevertheless able to interact with X-ray induces lesions to increase the number of these giving rise to chromosomal aberrations. This result is supported by data shown for human peripheral blood lymphocytes. In this respect the action of ara C appears to differ from that of ara A which seems to act solely as an inhibitor of repair.

These results throw some further light on the kinetics and mechanisms of induction of chromosomal aberrations and suggest future directions. Although we are as yet still far from understanding either the biochemical basis of repair of DSB, or the kinetics of chromatid breaks and exchanges, our results suggest that we should concentrate our effort into understanding the basis and mechanism of the conversion of DSB into chromosomal aberrations. Further experiments which seek to identify the enzymes involved in this pathway are planned.

Chromosomal aberrations appear to occupy a central role in radiation effects such as cell killing, and perhaps more importantly from a radiation protection point of view, genetic mutations and radiation induced oncogenic transformation. In order to appreciate and evaluate risks of low le vel radiation to man it will be necessary to understand how these alterations in the genome occur. Mechanisms of chromosomal aberration induction appear to be the key to understanding these effects.
CYTOGENETIC STUDIES
THE PROJECT WILL INVOLVE A STUDY OF THE FORMATION OF EXCHANGE TYPE CHROMOSOMAL ABERRATIONS IN GO AND G2 HUMAN AND RODENT CELLS TREATED WITH X-RAYS. TRANSFORMED HUMAN AND CHINESE HAMSTER FIBROBLASTS WILL BE USED FOR THIS PURPOSE. IN PHASE I CONDITIONS WILL BE SOUGHT FOR OPTIMUM MAINTENANCE OF THESE LINES FOR PROLONGED PERIODS IN GO PHASE AND IN G2 USING ALTERED GROWTH CONDITIONS. LENGTH OF G2 WILL BE MEASURED USING THYMIDINE LABELLING. IN PHASE II OF THE WORK KINETICS OF REJOINING OF CHROMOSOME BREAKS IN X-IRRADIATED GO CELLS WILL BE CARRIED OUT USING THE TECHNIQUE OF PREMATURE CHROMOSOME CONDENSATION (PCC).

THIS WORK WILL INVOLVE THE FUSION OF SELECTED MITOTIC CELLS WITH TEST CELLS USING SENDAI VIRUS AND EXAMINING THE PREMATURELY CONDENSED INTERPHASE CHROMOSOMES. FREQUENCIES OF EXCHANGES IN GO CELLS WILL BE DETERMINED IN PCC CELLS AND IN CELLS ALLOWED TO PROGRESS TO FIRST MITOSIS. SCORING WILL BE CARRIED OUT ON GIEMSA STAINED PREPARATIONS USING LIGHT MICROSCOPY. IN PHASE III OF THE WORK, THE KINETICS OF BREAK REJOINING AND EXCHANGE FORMATION WILL BE STUDIED IN DETAIL IN IRRADIATED G2 CELLS. THIS WILL BE CARRIED OUT BY THE USE OF SHORT MULTIPLE SAMPLING TIMES AFTER X-IRRADIATION. SOME WORK WILL ALSO BE CARRIED OUT IN SENDAI VIRUS PERMEABILISED GO AND G2 CELLS TREATED WITH RESTRICTION ENDONUCLEASES IN ORDER TO ESTABLISH KINETICS OF EXCHANGE FORMATION FROM SELECTED PURE DOUBLE-STRAND BREAKS AS A COMPARISON WITH X-RAYS. IN PHASE IV THE EFFECTS OF THE INHIBITOR 9-B-D-ARABINOFURANOSYLADENINE (ARA A) WILL BE EXAMINED ON KINETICS OF GO AND G2 BREAK REJOINING AND FREQUENCIES OF EXCHANGES FORMED.

DNA REPAIR STUDIES
IN PARALLEL WITH CYTOGENETIC STUDIES, MEASUREMENTS WILL BE MADE OF THE REPAIR OF DNA DSB USING THE METHOD OF NEUTRAL FILTER ELUTION AND DNA UNWINDING. CELLS WILL BE RADIOACTIVELY LABELLED USING TRITIATED THYMIDINE.
DSB WILL BE QUANTITATED BY THE RELATIVE AMOUNT OF RADIOACTIVITY ELUTED FROM FILTERS AFTER A GIVEN DOSE OR TIME. DNA UNWINDING IN WEAK ALKALI WILL BE ASSAYED USING HYDROXYLAPATITE CHROMATOGRAPHY AND COLLECTION OF SINGLE- AND DOUBLE-STRANDED FRACTIONS. THE RELATIVE MASS OF SINGLE- AND DOUBLE-STRANDED DNA WILL BE DETERMINED AFTER LIQUID SCINTILLATION COUNTING OF THESE FRACTIONS. KINETICS OF DSB REJOINING WILL BE MEASURED UNDER THE ALTERED GROWTH CONDITIONS EMPLOYED IN THE CYTOGENETIC EXPERIMENTS. THE EFFECTS OF ARA A ON DSB REJOINING WILL ALSO BE INVESTIGATED.

DNA LIGASE I ASSAY
A DNA LIGASE ASSAY WILL BE DEVELOPED FROM ESTABLISHED PROCEDURES. THIS WILL INVOLVE THE PARTIAL PURIFICATION OF THE ENZYME FROM LARGE POPULATIONS OF HUMAN AND CHINESE HAMSTER CELLS HELD UNDER VARIOUS GROWTH CONDITIONS. ASSAY OF PARTIALLY PURIFIED CELL EXTRACTS WILL BE CARRIED OUT USING LIGATION OF RESTRICTION ENDONUCLEASE CUT PLASMID DNA AND QUANTITATIVE ANALYSIS OF LIGATED DNA USING AGAROSE GEL ELECTROPHORESIS.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

UNIVERSITY OF ST ANDREWS
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KY16 9AH St Andrews
United Kingdom