It is proposed to investigate whether synthesis of poly(adenosine diphosphoribose) acts as a signal to induce G2-arrest in response to radiation-induced DNA damage. This hyphothesis will be tested in G2-phase Chinese hamster ovary (CHO) cells in vitro, using irradiation or 125IUdR incorporation to induce G2-arrest. Mitotic cell selection will be used to monitor cell progression, a permeabilized cell assay to measure poly(adenosine diphosphoribose) polymerase activity, and filter elution to measure DNA damage. Correlations will be sought between poly(adenosime diphosphoribose) polymerase activity, the duration of G2-arrest and DNA damage, as functions of radiation dose. Known inhibitors of poly(adenosine diphosphoribose) polymerase and their inactive analogues will be used over a range of concentrations to perturb expression of these endpoints. The timing, magnitude and direction of these perturbations will be examined to determine whether causal relationships exist. Preliminary data indicate the existence of an additional, non-poly(adenosine diphosphoribose) requiring step in the induction of G2-arrest by radiation, which is susceptible to cordycepin. The effects of combined drug treatments (polymerase inhibitors and cordycepin) will therefore be tested on G2-arrest induced by radiation and on polymerase activity with the purpose establishing if: 1) Agents believed to act solely through poly(adenosine diphosphoribose) inhibition do indeed share a common pathway, 2) Caffeine and related methyl xanthines affect both pathways, 3) The cordycepin susceptible step occurs before, after or in parallel with the poly(adenosine diphosphoribose) requiring step. For this purpose iso-effect data will be interpreted with the aid of drug uptake and competition binding studies. The association of a DNA damage recognition process with radiation-induced G2-arrest will help to provide an understanding, not only of molecular repair processes relevant to survival, but also of the factors governing the rate and probability of cell progression to mitosis. This knowledge may ultimately allow repair processes and cell progression to be manipulated for the construction of improved radiation therapeutic protocols, especially hyperfractionation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040245-02
Application #
3179938
Study Section
Radiation Study Section (RAD)
Project Start
1985-01-15
Project End
1987-12-31
Budget Start
1986-01-01
Budget End
1986-12-31
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
Rowley, R; Zhang, J (1999) Caffeine-mediated override of checkpoint controls. A requirement for rhp6 (Schizosaccharomyces pombe). Genetics 152:61-71
Jimenez, G; Yucel, J; Rowley, R et al. (1992) The rad3+ gene of Schizosaccharomyces pombe is involved in multiple checkpoint functions and in DNA repair. Proc Natl Acad Sci U S A 89:4952-6
Rowley, R (1992) Radiation-induced mitotic delay: a genetic characterization in the fission yeast. Radiat Res 132:144-52
Rowley, R (1992) Reduction of radiation-induced G2 arrest by caffeine. Radiat Res 129:224-7
Rowley, R; Subramani, S; Young, P G (1992) Checkpoint controls in Schizosaccharomyces pombe: rad1. EMBO J 11:1335-42
Feilotter, H; Lingner, C; Rowley, R et al. (1992) Regulation of the G2-mitosis transition. Biochem Cell Biol 70:954-71
Hudson, J D; Feilotter, H; Lingner, C et al. (1991) stf1: a new suppressor of the mitotic control gene, cdc25, in Schizosaccharomyces pombe. Cold Spring Harb Symp Quant Biol 56:599-604
Rowley, R (1990) Repair of radiation-induced chromatid aberrations: relationship to G2 arrest in CHO cells. Int J Radiat Biol 58:489-98
Rowley, R; Kort, L (1989) Novobiocin, nalidixic acid, etoposide, and 4'-(9-acridinylamino)methanesulfon-m-anisidide effects on G2 and mitotic Chinese hamster ovary cell progression. Cancer Res 49:4752-7
Rowley, R; Egger, M J (1988) Method for probing cells in radiation-induced G2 arrest: demonstration of potentially lethal damage repair. Cell Tissue Kinet 21:395-403

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