X-irradiation of splenic lymphocytes. I. Thymidine pools and radiothymidine incorporation into DNA.

Abstract

An immediate suppression of radiothymidine incorporation into DNA is found in splenic lymphocytes of the golden hamster when they are irradiated in vitro in the kilorad range (Manasek, Adelstein and Lyman 1966). Although the mechanism of this suppression has not been elucidated, Smets (1966 a) has put forth the hypothesis that at least part of the apparent depression in DNA synthesis is due to changes in the precursor-pool size. A number of investigators, working with a wide range of biological systems, have suggested that the decreased uptake of radiothymidine into DNA often observed after irradiation is due to an expansion of nucleoside and nucleotide pools consequent on DNA degradation and not to a net decrease in DNA synthesis (Hell, Berry and Lajtha 1960, Kelly 1961, Nygaard 1962, Smets 1966b). Several experimental approaches have been utilized to test for such amplification of intracellular pools. In one (Hell et al. 1960, Smets 1966b), the precursor is varied over a wide concentration range in the hope of eliminating pool effects by flooding. In the experiments to be described, the method is applied to x-irradiated splenic lymphocytes. Cell suspensions were prepared from the spleens of mature, male, golden hamsters (Manasek et al. 1966). From radioautographs we have determined that the predominant nucleated cell which is synthesizing DNA is a medium-sized lymphocyte. The minced tissue was passed through stainless-steel screens for dispersion, and the cells were suspended in Hanks' salt solution at pH 7-25. They were subsequently exposed to unfiltered 250kvp x-rays generated at a dose-rate of 300 rads per minute as determined with ferrous ammonium sulphate. The suspensions were maintained at a controlled temperature throughout their irradiation. Immediately thereafter, the cells were incubated with tritiated thymidine (specific activity 2 Ci/mmole) at 37°c for 30 min. DNA was isolated by a modification of Schmidt and Thannhauser's method (Munro and Fleck 1966). In this assay the incorporation of thymidine into DNA is linear for 4 hours in control and up to 1 hours in irradiated suspensions. In figure 1, the uptake of radiothymidine into the DNA of unirradiated splenic lymphocytes is shown for total thymidine concentrations varied from 1 x 10 - 7 M to 35 x 10- M. The 350-fold increase in concentration results in a ten-fold increase in radiothymidine incorporation. The ' S ' shape of the uptake curve suggests that the overall incorporation obeys Michaelis-Menten kinetics. Under