A method for measuring the generation time and length of DNA synthesizing phase of clonogenic cells in a heterogenous population

Abstract

Information on the cell cycle of progenitor cells in haemopoietic tissue is useful for understanding population control under physiological and abnormal conditions. Unfortunately, methods that have been developed for measuring cell cycle parameters are applicable only to cells of homogenous populations and not to morphologically non-recognizable progenitor cells such as colony forming units (CFU) that are present at low frequency in a heterogenous population. To circumvent this difficulty, a method was developed to measure CFU cell cycle parameters based on specific killing of cells in S phase by [3H]thymidine ([3H]TdR). This was done by estimating the number of CFU killed following exposure of the cell suspension to [3H]TdR for various time periods. since cycling CFU are continuously entering S phase, a linear curve relating the percentage CFU-kill to the length of exposure of the cells to [3H]TdR in culture can be obtained. The slope of the curve (percentage kill/hr) indicates the rate that CFU enter the S phase and travel through the cell cycle. The inverse of this value will then represent a time period for CFU to move through a complete cell cycle (generation time). The length of S phase can then be obtained by multiplying generation time by the fraction of cells in S phase at time zero. This method has been used to measure generation time and length of S phase of three kinds of haemopoietic progenitor cells: mouse granulocyte-macrophage CFU, human T lymphocyte CFU and CFU from regenerating mouse spleens. This method should be applicable to any normal or neoplastic clonogenic cell populations and the latter could be either of haematological or of solid tumour origin.