An investigation of factors influencing mitotic G2 delay in synchronous cultures of human kidney cells after X-irradiation

Abstract

Factors affecting mitosis have been studied in human kidney T-cells synchronized by two exposures to 7.5 mM thymidine. Biochemical measurements were correlated with the position of the cells in the cell cycle from S to mitosis. Anaerobic conditions during S, although not inhibition of respiration, prevented cells from progressing to mitosis. ATP production is largely anaerobic and glycolytic metabolism is sufficient for a cell to complete mitosis once it has entered G2 or prophase. Both RNA and protein synthesis are required in G2 for the initiation of cell division, while treatment with carbon monoxide or thiol blocking agents also prevents mitosis.Postirradiation mitotic delay is greater for cells irradiated in G2 than for cells irradiated in S. This G2 delay is reversible if the cells are blocked in S by addition of excess thymidine. Pretreatment with 5–15 mM cysteamine largely protected irradiated cells from mitotic delay. Cysteine at a concentration of 20 mM and AET at 10 mM were only weakly protective, while 20 mM mercaptoethanol was ineffective. All these compounds added after irradiation were ineffective. Anoxia during irradiation had a slight protective effect. Mitotic delay was enhanced by lowered concentrations but not reduced by elevated concentrations of calcium, while agmatine before or after irradiation was without protective effect.The protein thiol content of cells increases from S to a maximum at metaphase, while the nonprotein thiol content is unchanged. X-irradiation of up to 500 R has no effect on the protein or nonprotein thiol content of the cell up to mitosis. The intracellular ATP content is maximal for cells in G2, then falls subsequently to mitosis. Irradiation slows this premitotic accumulation of ATP. The respiration rate per cell is constant up to mitosis then drops sharply following division. Irradiation has no effect on respiration. Decreased specific activities of DNA from cells irradiated in S and incorporating 3H-TdR post-irradiation over a 2-h period suggest a pool effect as the mechanism of reduced thymidine incorporation. No evidence could be obtained for any significant breakdown of DNA following irradiation. Prolonged DNA synthesis following irradiation is not considered to be the primary reason for mitotic delay. The radiation block is located less than 30 min before the onset of prophase and is possibly correlated with a condensation mechanism of the chromatin strands.