The combined effect of 1-(4-amino 2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (AC NU) and 5-fluorouracil (5-Fu) on cells grown exponentially as a monolayer and multicell spheroids of rat glioma clone-6 (RG cells) was analyzed by the colony forming assay in order to obtain a more effective way of the combination.
Mature spheroids (500-600 μm in diameter) with central necrosis resembled the solid tumors in vivo in histological appearance and in the distribution of cells labeled by 3H-thymidine. Both mature spheroids and cells in monolayer were exposed to ACNU for 2 hours and to 5-Fu for 24 hours. After the drug exposure, spheroids and cells in the monolayer were trypsinized and single cells were plated for the colony forming assay. The dose survival curves for 5-Fu, for ACNU and for the combination of ACNU and 5-Fu were obtained for both spheroids and cells in the monolayer. The dose survival curve for ACNU showed an exponential cell killing following a shoulder for both cells in the monolayer and spheroids. However, the terminal slope of the curve expressed as the reciprocal of Co (concentration of drug to reduce the surviving fraction to e-1) was steeper for spheroids than for cells in the monolayer. The dose survival curve for cells in monolayer treated by 5-Fu again showed an exponential cell killing following a shoulder. On the other hand, the dose survival curve for spheroids treated by 5-Fu showed a biphasic curve. This suggested that a proportion of cells in these spheroids was resistant to treatment by 5-Fu.
The cytotoxic effect of ACNU was enhanced by the combined treatment with 5-Fu for cells both in monolayer and spheroids. The enhancement was more marked when 5-Fu was administered before ACNU. The combined treatment also reduced the fraction of cells in spheroids resistant to 5Fu, suggesting that ACNU may be more effective for quiescent cells. The enhancement by the combined treatment on spheroids was compared with that on cells in the monolayer, based on the ratio of the surviving fraction to the concentration of the drug. The results indicated that there was a concentration range, which was more effective for spheroids than for cells in the monolayer. That was 10-35 μg/ml of ACNU and 2-15 μg/ml of 5-Fu.
