Cell cycle kinetics in human lymphocyte cultures

Abstract

Short-term cultures of phytohaentagglutinin (PHA)-stimulated human lymphocytes are widely used to detect chromosome-damaging agents^1,2, possible human exposure to mutagenic carcinogens^1,2 and the immune response of blood³. Because the results are affected by the number of cell divisions before sampling^1,2,4,5, an accurate knowledge of lymphocyte proliferation in culture is essential for these studies. Unfortunately, the information available on the lymphocyte proliferative characteristics is quite conflicting. For instance, although after stimulation of blood lymphocytes with PHA the cultures soon contain cells that have divided different numbers of times^6,7: this heterogeneity has been explained variously as a difference in cell-cycle times^8,9 or in the times when the cells start blasto-genesis by responding to PHA^10–12. Prolonged treatment with high concentrations of ³H-thymidine (TdR) have often been used to investigate lymphocyte proliferation^8,10,13,14. Incorporated ³H-TdR can, however, affect cell kinetics^15–19. The differential staining of sister chromatids²⁰ in cells dividing for different numbers of times in the presence of bromodeoxy-uridine (BUdR) can be used to study cell kinetics^7,8,12. In experiments combining sister chromatid differential staining and autoradiography, we show here that ³H-TdR labelling at more than 0.1 µCi ml⁻¹ slows lymphocyte cycling, and that the heterogeneity of different generations of cells is caused by a difference in the times when they start their first DNA synthesis in response to PHA.