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Differential Response of Elutriated 9L Cells to Treatment with 1,3-Bis(2-chloroethyl)1-nitrosourea¹

Brain Tumor Research Center of the Department of Neurological Surgery, School of Medicine, University of California, San Francisco, California 94143

Earlier cytokinetic studies using 9L monolayer culture cells showed that cells began accumulating in the G₂ phase (4C position in the DNA distribution) within 30 hr after administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (3 µg/ml). Most of these cells reached the G₂ phase at 48 hr and remained there for another 24 hr. Similar studies using 9L spheroid culture cells show that 9L cells also began to accumulate at the 4C DNA position 30 hr after administration of 1,3-bis(2-chloroethyl)-1-nitrosourea (3 µg/ml); however, only one-half of the cells accumulated in the 4C peak during the first 48 hr after treatment, while the other half remained at the 2C position. Those that remained at the 2C peak might correspond to the G₀ cells that exist in 9L spheroids. When one-half the cells were in 2C and one-half were in 4C, the spheroids were dissociated using an enzyme cocktail and sorted using centrifugal elutriation. Cells from each elutriated fraction were analyzed for DNA content using flow cytometry and for viability using a colony-forming efficiency assay. Results indicated that in spheroids, 1,3-bis(2-chloroethyl)-1-nitrosourea (3µg/ml) is more effective against noncycling cells and that repopulation is more vigorous in G₂-arrested cells, which presumably were cycling at the time of treatment.

¹ Supported by National Cancer Institute Grants CA-13525 and CA-19992 and a gift from The Morris Stulsaft Foundation.

² To whom requests for reprints should be addressed.

Received 10/ 6/80. Accepted 7/29/81.