An in vitro colony formation assay was modified to determine the effects of in vivo 1,3-bis(2-chloroethyl)-1-nitrosourea therapy on tumor cell kill and subsequent clonogenic cell kinetics. The measured surviving fraction must be multiplied by the relative total number of tumor cells for each posttreatment interval in order to eliminate inaccuracies caused by dead cell removal in vivo and the lysis of damaged cells by the disaggregation procedure. The assumptions, limitations, and applications of the technique are discussed.

1,3-Bis(2-chloroethyl)-1-nitrosourea doses of 0.25, 0.50, and 1.00 × dose lethal to 10% of animals resulted in approximately a 1-, 2-, and 3-log cell kill, respectively. Significant proliferation of surviving clonogenic cells was observed after a latency period of approximately 2 days, and the rate of tumor regrowth was dose dependent. The cell-doubling times following treatment with 0.25, 0.50, and 1.00 × doses lethal to 10% of animals were 15, 21, and 38 hr, respectively. The interval to complete repopulation of the clonogenic pool corresponds to the observed increase in animal lifespan for the 2 larger doses and further validates the assay as a true measure of in vivo chemotherapeutic efficacy.


This work was supported by NIH Grant CA-13525, The National Phi Beta Psi Sorority, The Joe Gheen Medical Foundation, and the Association for Brain Tumor Research.

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