Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Nov 1;109(5):1993–2002. doi: 10.1083/jcb.109.5.1993

Cellular and adenovirus dl312 DNA metabolism in cycling or mitotic human cultures exposed to supralethal gamma radiation

PMCID: PMC2115870  PMID: 2808517

Abstract

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.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aiello L., Guilfoyle R., Huebner K., Weinmann R. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology. 1979 Apr 30;94(2):460–469. doi: 10.1016/0042-6822(79)90476-8. [DOI] [PubMed] [Google Scholar]
  2. Andreu J. M., Timasheff S. N. Tubulin-colchicine interactions and polymerization of the complex. Ann N Y Acad Sci. 1986;466:676–689. doi: 10.1111/j.1749-6632.1986.tb38451.x. [DOI] [PubMed] [Google Scholar]
  3. Berk A. J., Lee F., Harrison T., Williams J., Sharp P. A. Pre-early adenovirus 5 gene product regulates synthesis of early viral messenger RNAs. Cell. 1979 Aug;17(4):935–944. doi: 10.1016/0092-8674(79)90333-7. [DOI] [PubMed] [Google Scholar]
  4. Bohr V. A., Smith C. A., Okumoto D. S., Hanawalt P. C. DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell. 1985 Feb;40(2):359–369. doi: 10.1016/0092-8674(85)90150-3. [DOI] [PubMed] [Google Scholar]
  5. Chiu S. M., Oleinick N. L., Friedman L. R., Stambrook P. J. Hypersensitivity of DNA in transcriptionally active chromatin to ionizing radiation. Biochim Biophys Acta. 1982 Oct 29;699(1):15–21. doi: 10.1016/0167-4781(82)90166-x. [DOI] [PubMed] [Google Scholar]
  6. Chiu S. M., Oleinick N. L. The role of DNA damage and repair in the function of eukaryotic genes: radiation-induced single-strand breaks and their rejoining in chromosomal and extrachromosomal ribosomal DNA of tetrahymena. Radiat Res. 1980 Apr;82(1):146–161. [PubMed] [Google Scholar]
  7. Chiu S. M., Oleinick N. L. The sensitivity of active and inactive chromatin to ionizing radiation-induced DNA strand breakage. Int J Radiat Biol Relat Stud Phys Chem Med. 1982 Jan;41(1):71–77. doi: 10.1080/09553008214550061. [DOI] [PubMed] [Google Scholar]
  8. Collins A. R., Downes C. S., Johnson R. T. Cell cycle-related variations in UV damage and repair capacity in Chinese hamster (CHO-K1) cells. J Cell Physiol. 1980 May;103(2):179–191. doi: 10.1002/jcp.1041030203. [DOI] [PubMed] [Google Scholar]
  9. Collins A. R., Ord M. J., Johnson R. T. Correlations of DNA damage and repair with nuclear and chromosomal damage in HeLa cells caused by methylnitrosamides. Cancer Res. 1981 Dec;41(12 Pt 1):5176–5187. [PubMed] [Google Scholar]
  10. Collins A., Johnson R. Novobiocin; an inhibitor of the repair of UV-induced but not X-ray-induced damage in mammalian cells. Nucleic Acids Res. 1979 Nov 10;7(5):1311–1320. doi: 10.1093/nar/7.5.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Day R. S., 3rd, Scudiero D., Dimattina M. Excision repair by human fibroblasts of DNA damaged by r-7, t-8-dihyroxy-t-9,10-oxy-7,8,9,10- tetrahydrobenzo(a)pyrene. Mutat Res. 1978 Jun;50(3):383–394. doi: 10.1016/0027-5107(78)90043-x. [DOI] [PubMed] [Google Scholar]
  12. Day R. S., 3rd Studies on repair of adenovirus 2 by human fibroblasts using normal, xeroderma pigmentosum, and xeroderma pigmentosum heterozygous strains. Cancer Res. 1974 Aug;34(8):1965–1970. [PubMed] [Google Scholar]
  13. Elliott A. Y., Cleveland P., Cervenka J., Castro A. E., Stein N., Hakala T. R., Fraley E. E. Characterization of a cell line from human transitional cell cancer of the urinary tract. J Natl Cancer Inst. 1974 Nov;53(5):1341–1349. doi: 10.1093/jnci/53.5.1341. [DOI] [PubMed] [Google Scholar]
  14. Engin A. Glutathione content of human skin carcinomas. Arch Dermatol Res. 1976 Nov 26;257(1):53–55. doi: 10.1007/BF00569113. [DOI] [PubMed] [Google Scholar]
  15. Fogh J., Fogh J. M., Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst. 1977 Jul;59(1):221–226. doi: 10.1093/jnci/59.1.221. [DOI] [PubMed] [Google Scholar]
  16. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  17. Hoar D. I., Davis F. Host-cell reactivation of UV-irradiated adenovirus in Cockayne syndrome. Mutat Res. 1979 Oct;62(3):401–405. doi: 10.1016/0027-5107(79)90035-6. [DOI] [PubMed] [Google Scholar]
  18. Jeeves W. P., Rainbow A. J. An aberration in gamma-ray-enhanced reactivation of irradiated adenovirus in ataxia telangiectasia fibroblasts. Carcinogenesis. 1986 Mar;7(3):381–387. doi: 10.1093/carcin/7.3.381. [DOI] [PubMed] [Google Scholar]
  19. Jones N., Shenk T. Isolation of adenovirus type 5 host range deletion mutants defective for transformation of rat embryo cells. Cell. 1979 Jul;17(3):683–689. doi: 10.1016/0092-8674(79)90275-7. [DOI] [PubMed] [Google Scholar]
  20. Liu S. C., Meagher K., Hanawalt P. C. Role of solar conditioning in DNA repair response and survival of human epidermal keratinocytes following UV irradiation. J Invest Dermatol. 1985 Aug;85(2):93–97. doi: 10.1111/1523-1747.ep12276441. [DOI] [PubMed] [Google Scholar]
  21. Liu S. C., Parsons C. S., Hanawalt P. C. DNA repair response in human epidermal keratinocytes from donors of different age. J Invest Dermatol. 1982 Nov;79(5):330–335. doi: 10.1111/1523-1747.ep12500087. [DOI] [PubMed] [Google Scholar]
  22. Matsuo N., Ross P. M. Measurement of interstrand cross-link frequency and distance between interruptions in DNA exposed to 4,5',8-trimethylpsoralen and near-ultraviolet light. Biochemistry. 1987 Apr 7;26(7):2001–2009. doi: 10.1021/bi00381a033. [DOI] [PubMed] [Google Scholar]
  23. Mee L. K., Adelstein S. J. Formation of strand breaks in the DNA of gamma-irradiated chromatin. Radiat Environ Biophys. 1987;26(1):13–22. doi: 10.1007/BF01211361. [DOI] [PubMed] [Google Scholar]
  24. Nevins J. R. Control of cellular and viral transcription during adenovirus infection. CRC Crit Rev Biochem. 1986;19(4):307–322. doi: 10.3109/10409238609082543. [DOI] [PubMed] [Google Scholar]
  25. Nose K., Nikaido O. Transcriptionally active and inactive genes are similarly modified by chemical carcinogens or X-ray in normal human fibroblasts. Biochim Biophys Acta. 1984 Apr 5;781(3):273–278. doi: 10.1016/0167-4781(84)90093-9. [DOI] [PubMed] [Google Scholar]
  26. Oleinick N. L., Chiu S. M., Friedman L. R. Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome. Radiat Res. 1984 Jun;98(3):629–641. [PubMed] [Google Scholar]
  27. Sergeant A., Tigges M. A., Raskas H. J. Nucleosome-like structural subunits of intranuclear parental adenovirus type 2 DNA. J Virol. 1979 Mar;29(3):888–898. doi: 10.1128/jvi.29.3.888-898.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Smith C. A., Hanawalt P. C. Repair replication in cultured normal and transformed human fibroblasts. Biochim Biophys Acta. 1976 Oct 4;447(2):121–132. doi: 10.1016/0005-2787(76)90335-x. [DOI] [PubMed] [Google Scholar]
  29. Taichman L. B., Setlow R. B. Repair of ultraviolet light damage to the DNA of cultured human epidermal keratinocytes and fibroblasts. J Invest Dermatol. 1979 Sep;73(3):217–219. doi: 10.1111/1523-1747.ep12514242. [DOI] [PubMed] [Google Scholar]
  30. Téoule R. Radiation-induced DNA damage and its repair. Int J Radiat Biol Relat Stud Phys Chem Med. 1987 Apr;51(4):573–589. doi: 10.1080/09553008414552111. [DOI] [PubMed] [Google Scholar]
  31. Wheeler K. T., Wierowski J. V. DNA accessibility: a determinant of mammalian cell differentiation? Radiat Res. 1983 Feb;93(2):312–318. [PubMed] [Google Scholar]
  32. White E., Grodzicker T., Stillman B. W. Mutations in the gene encoding the adenovirus early region 1B 19,000-molecular-weight tumor antigen cause the degradation of chromosomal DNA. J Virol. 1984 Nov;52(2):410–419. doi: 10.1128/jvi.52.2.410-419.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Yaniv M., Cereghini S. Structure of transcriptionally active chromatin. CRC Crit Rev Biochem. 1986;21(1):1–26. doi: 10.3109/10409238609113607. [DOI] [PubMed] [Google Scholar]
  34. Young C. S., Cachianes G., Munz P., Silverstein S. Replication and recombination in adenovirus-infected cells are temporally and functionally related. J Virol. 1984 Sep;51(3):571–577. doi: 10.1128/jvi.51.3.571-577.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Youngs D. A., Smith K. C. Single-strand breaks in the DNA of the uvrA and uvrB strains of Escherichia coli K-12 after ultraviolet irradiation. Photochem Photobiol. 1976 Dec;24(6):533–541. doi: 10.1111/j.1751-1097.1976.tb06870.x. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES