The purpose of the proposed study is to determine the heat- and x-ray-induced changes that occur in nuclei and how such changes inhibit nuclear function. Following hyperthermia increases in nuclear protein content appear to correlate with cell killing, disruption of DNA synthesis and DNA repair. The results of these proposed studies will determine if heat-induced excess nuclear proteins disrupt these nuclear functions by alteration of enzymatic access to DNA or the DNA available for supercoiling changes. Additional experiments will correlate the presence and removal of excess nuclear proteins with the onset of heat-shock protein accumulation and the resumption of cell progression through the cell cycle. Proteins will be separated for nuclei from heated and control cells and analyzed by polyacrylamide gel electrophoresis. DNA supercoiling will be detected from the response of DNA in increasing concentrations of intercalating agents. Changes in the distribution of cells throughout the cell cycle will be measured using flow cytometry. These same techniques will be used to determine the role of nuclear protein changes during the X-ray-induced G2 block. The synthesis of nuclear proteins in G2 cells will be monitored by labeling with radioactive amino acids at various time intervals after irradiation. In addition changes in DNA supercoiling following x-irradiation will be monitored. These studies will delineate the mechanisms by which hyperthermia and x-irradiation disrupt cellular function. Since both of these agents are used in cancer therapy, the understanding of the above phenomena will contribute to the biological rationale for the clinical use of heat and radiation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA041102-03
Application #
3181421
Study Section
Radiation Study Section (RAD)
Project Start
1985-04-01
Project End
1991-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
3
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Washington University
Department
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Goswami, P C; Roti Roti, J L; Hunt, C R (1996) The cell cycle-coupled expression of topoisomerase IIalpha during S phase is regulated by mRNA stability and is disrupted by heat shock or ionizing radiation. Mol Cell Biol 16:1500-8
Higashikubo, R; Roti Roti, J L (1993) Alterations in nuclear protein mass and macromolecular synthesis following heat shock. Radiat Res 134:193-201
Goswami, P C; Hill, M; Higashikubo, R et al. (1992) The suppression of the synthesis of a nuclear protein in cells blocked in G2 phase: identification of NP-170 as topoisomerase II. Radiat Res 132:162-7
Wright, W D; Roti Roti, J L (1992) Resolution of DNA topoisomerase II by two-dimensional polyacrylamide gel electrophoresis and western blotting. Anal Biochem 204:124-30
Roti Roti, J L; Mackey, M A; Higashikubo, R (1992) The effects of heat shock on cell proliferation. Cell Prolif 25:89-99
Laszlo, A; Wright, W; Roti Roti, J L (1992) Initial characterization of heat-induced excess nuclear proteins in HeLa cells. J Cell Physiol 151:519-32
Mackey, M A; Roti Roti, J L (1992) A model of heat-induced clonogenic cell death. J Theor Biol 156:133-46
Taylor, Y C; Duncan, P G; Zhang, X et al. (1991) Differences in the DNA supercoiling response of irradiated cell lines from ataxia-telangiectasia versus unaffected individuals. Int J Radiat Biol 59:359-71
Higashikubo, R; Wright, W D; Roti Roti, J L (1990) Flow cytometric methods for studying isolated nuclei: DNA accessibility to DNase I and protein-DNA content. Methods Cell Biol 33:325-36
Wright, W D; Higashikubo, R; Roti Roti, J L (1990) Fluorescent methods for studying subnuclear particles. Methods Cell Biol 33:353-62

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