Response of 9L rat brain tumor multicellular spheroids to single and fractionated doses of 1,3-bis(2-chloroethyl)-1-nitrosourea.

This study was designed to examine the relative effect of each of four fractions of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) against 9L rat brain tumor multicellular spheroids and to compare the results of the cell survival and growth delay assays. Similar levels of cell kill resulted when BCNU was administered either as single fractions of 1.5, 3.0, 4.5, or 6.0 micrograms/ml for 1 hr or as one to four fractions of 1.5 micrograms/ml that were administered sequentially for 1 hr each. Survival was increased if the assay was delayed until 24 hr after drug treatment, which indicates that 9L cells in spheroids recover from BCNU-induced potentially lethal damage. When BCNU was administered in 1.5-micrograms/ml fractions, plating efficiencies depended markedly on the interval between the fractions. The 12-hr protocol produced an overall higher cell kill. Fractionation schedules of 24 and 36 hr produced less cell kill than did the other schedules. Survival plateaued for the last three treatments with BCNU in the 36-hr schedule. Cells in S phase at the time of administration of the initial 1.5-micrograms/ml fraction of BCNU moved into G1- and G2-M phases by 12 hr after treatment. For time periods longer than 12 hr, cells began to appear in the BCNU-resistant S phase. Thus, the movement of cells into the drug-sensitive and -resistant phases after the first fraction correlates well with the corresponding overall cytotoxic effect produced by treatment with the combined BCNU (1.5 micrograms/ml) fractions. For a higher concentration (3.0 micrograms/ml for 1 hr), maximum cell kill was reached within the 12- to 18-hr interval, after which cell kill plateaued. Cells were not found in the S-phase fraction 12 to 36 hr after the first treatment with 3.0 micrograms/ml; maximum cell kill for the fractionated protocols resulted at these times. Therefore, BCNU, which is classified as a cell cycle-nonspecific drug, can induce a partial synchrony in 9L spheroid cells, which determines the overall cytotoxicity produced by fractionated BCNU protocols. Although spheroids did not shrink during or after exposure to BCNU, growth was retarded by treatment with all doses and schedules. An optimum time point for growth delay measurement could not be determined from the data. However, correlations between cell survival and growth delay were obtained with arbitrarily chosen end point volumes of four and ten times the volume at the time of treatment.