[Radiation sensitivity of lymphocyte stimulation. 1. Experiments on in-vitro sensitivity of cell number and mitosis...

Radiation-induced transformations of stimulated lymphocytes are not to be regarded as characteristical responses of lymphocyte populations as such, but represent general radiobiologic principles of the stimulated proliferation. Thus, basic problems of stimulated proliferation in comparison with a spontaneous quick proliferation of normal cellular systems in vitro have chiefly been studied by means of two stimulation models in vivo, the model of liver regeneration following partial hepatectomy and the model of beta stimulation of the salivary glands. The radiosensitivity of the lymphocyte stimulation in vitro as well as in vivo has been analyzed especially with regard to its quality as a model. The task was approached methodically by: a) Determination in vitro of the number of cells (66 cultures), b) morphological analysis of the mitotic activity in vitro (270 cultures), c) measurements in vitro with 3h-thymidine of the DNA synthesis using liquid scintillation counts (more than 3000 cultures), d) measurements in vitro with 3H-thymidine of the DNA synthesis using autoradiography (66 cultures), e) determination in vivo of the DNA synthesis by means of the incorporation of 131I-desoxyuridine into the mouse spleen (investigation with 100 mice). For a model of lymphocyte stimulation mainly that of PHA stimulation in vitro and in vivo of T-lymphocytes was chosen, supplemented by the one of PWM stimulation of T- and B- lymphocytes for measuring in vitro the DNA synthesis. In particular, influence of in vitro irradiation upon several variables of the stimulation model was investigated in order to examine the methodics critically: a) During the determination of the mitotic index too short an acbition by partial synchronization effects within the dose range of 10 to 500 R. The chronological analysis of the process during determination of the mitotic activity is indispensable in order to differentiate the mitotic inhibition from a mitotic delay in the case of high doses, b) For the determination of the DNA synthesis the concentration of stimulative PHA or PWM has no substantial influence on the radiation-induced inhibition effect; the constancy of serum percentage in the culture system, however, is an important condition for comparability of different experiments. Moreover, only a short period of labeling with 3H-thymidine, lasting 1.5 to 12 hours, interferes with radiation-induced inhibition of the DNA synthesis, this probably being also due to partial synchronization effects within the dose range of 10 to 500 R. c) In order to determine the DNA synthesis wether in vitro or in vivo, a chronological analysis is a condition indispensable for differentiation between the three factors involved: Delay of DNA synthesis, blockage of DNA synthesis, and inhibition of the proliferation...