Ionizing Radiation-Induced DNA Damage and Its Repair in Human Cells
DNA damage in mammalian chromatin in vitro and in cultured mammalian cells including human cells was studied. In the first phase of these studies, a cell culture laboratory was established. Necessary equipment including an incubator, a sterile laminar flow hood and several centrifuges was purchased. We have successfully grown several cell lines such as murine hybridoma cells, V79 cells and human K562 leukemia cells. This was followed by the establishment of a methodology for the isolation of chromatin from cells. This was a very important step, because a routine and successful isolation of chromatin was a prerequisite for the success of the further studies in this project, the aim of which was the measurement of DNA darnage in mammalian chromatin in vitro and in cultured cells. Chromatin isolation was accomplished using a slightly modified procedure of the one described by Mee & Adelstein (1981). For identification and quantitation of DNA damage in cells, analysis of chromatin was preferred over the analysis of "naked DNA" for the following reasons: i. DNA may not be extracted efficiently from nucleoprotein in exposed cells, due to formation of DNA-protein cross-links, ii. the extractability of DNA is well known to decrease with increasing doses of radiation, iii. portions of DNA may not be extracted due to fragmentation, iv. unextracted DNA may contain a significant portion of damaged DNA bases and DNA-protein cross-links. The technique of gas chromatography/mass spectrometry (GC/MS), which was used in the present project, permits the identification and quantitation of modified DNA bases in chromatin in the presence of proteins without the necessity of first isolating DNA from chromatin. This has been demonstrated previously by the results from our laboratory and by the results obtained during the course of the present project. The quality of isolated chromatin was tested by measurement of its content of DNA, proteins, and RNA, by analysis of its protein components using gel electrophoresis, and by absorption spectral analysis. GeneraUy, the RNA content was <5% of the amount of DNA, and the ratio of the amount of protein to that of DNA was =1. 8-2 (w/w). Having developed a suitable methodology for routine isolation of chromatin from mammalian cells, studies of DNA damage in chromatin in vitro and in cultured human cells were pursued.
- Research Organization:
- National Institute of Standards and Technology
- Sponsoring Organization:
- USDOE Office of Science (SC)
- DOE Contract Number:
- AI05-89ER60826
- OSTI ID:
- 7163
- Country of Publication:
- United States
- Language:
- English
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