The long-term objectives are to gain an understanding of the mechanisms controlling: i) progression through the cell cycle (with emphasis on the G1-S-phase transition); ii) heat-induced cell cycle arrest; and iii) thermotolerance in mammalian cells.
The specific aim of this project is to test the hypothesis that heat shock of mammalian cells stimulates the production of heat shock proteins (HSP), some of which prevent the G1-S-phase transition and other different HSP which control the phenomenon of thermotolerance. An understanding of those mechanisms controlling progression through the cell cycle and thermotolerance are intrinsically important both to cell biology and cancer research, as well as to the treatment of cancer. The proposed studies will use Chinese hamster ovary cells in exponential growth, in plateau phase and synchronized by mitotic selection and allowed to progress into G1- and S-phase. Flow cytometry, labeling indices, and incorporation of TCA precipitable 3HTdR will be used to monitor cell cycle distribution and progression. The temporal synthesis and decay of specific proteins associated with (1) heat-induced G1-arrest and (2) thermotolerance will be monitored by one-dimensional and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) of mixtures of control and experimentally treated cells pulse labeled with 3H- and 14C-amino acids. The modification of existing proteins (possible activator proteins) will be screened for using 2-D PAGE of pre-labeled cells. The bioassay for examining the repressive/stabilizing/stimulatory ability of exogenous cytoplasm on DNA synthesis in nuclei in vitro will use cytoplasmic and nuclear components derived from control and heated G1- and S-phase cells. Rates and extents of DNA synthesis will be measured by incorporation of TCA precipitable 3H-TTP; the percent of labeled nuclei in the reaction mixtures will be determined by autoradiography. The temporal appearance of the repressor(s) of DNA synthesis in heated cytoplasm will be determined by adding heated cytoplasm to S-phase nuclei at varying times starting immediately after heat shock. Cytoplasm from cells most active in repressing DNA synthesis and stimulating DNA synthesis will be analyzed by 2-D PAGE. The cytoplasm will be fractionated by column chromatography for further physical-chemical characterization. The bioassay will be used to monitor the biological activity of the fractions.
| Coss, R A (1986) Decay of thermal resistance following acute heating is independent of the G1- to S-phase transition. Radiat Res 107:143-6 |
