The Journal of Cell Biology, Vol 109, 1993-2002, Copyright © 1989 by The Rockefeller University Press
Cellular and adenovirus dl312 DNA metabolism in cycling or mitotic human cultures exposed to supralethal gamma radiation
PM Ross
Rockefeller University, New York 10021.
Cellular repair of DNA damage due to lethal gamma irradiation was studied
to reveal differences between strains and cell cycle stages that are
otherwise difficult to detect. Cycling and metaphase-blocked cultures of
normal fibroblasts and carcinoma cells were compared for repair of gamma
sites (gamma radiation-induced nicks, breaks, and alkalilabile sites in
DNA) at supralethal exposures ranging from 7 to 150 krad 137Cs radiation
and at postirradiation incubations of 20-180 min. Fibroblasts from normal
human skin or lung repaired gamma sites efficiently when cycling but did
not repair them when blocked at mitosis. Bladder (253J) or lung (A549)
carcinoma cells, unlike normal fibroblasts, repaired gamma sites
efficiently even when blocked at mitosis. HeLa cells degraded their DNA
soon after exposure at all doses tested, regardless of mitotic arrest.
Whether the above differences in DNA repair between cell cycle stages and
between strains result from differences in chromatin structure (cis
effects) or from differences in the nuclear enzymatic environment (trans
effects) could be resolved by placing an inert, extrachromosomal DNA
molecule in the cell nucleus. Specifically, cis effects should be confined
to the host chromosomes and would not be detected in the inert probe
whereas trans effects should be detected in host chromosomes and inert
probe DNA alike. Indeed, we found a suitable DNA molecule in the adenovirus
deletion mutant dl312, which does not proliferate in the absence of E1A
complementation. Gamma sites in 32P-labeled adenovirus dl312 DNA were
repaired efficiently in all hosts, regardless of mitotic arrest. Failure of
mitosis-arrested fibroblasts to repair gamma sites was therefore due to a
cis effect of chromatin organization rather than to a trans effect such as
repair enzyme insufficiency. In sharp contrast, chromosomes of mitotic
carcinoma cells remained accessible to repair enzymes and nucleases alike.
By means of these new tools, we should get a better understanding of
higher-order chromatin management in normal and cancer cells.
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