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Effects of Certain Nitrogen Mustards upon the Progression of Cultured H.Ep. No. 2 Cells through the Cell Cycle¹

Kettering-Meyer Laboratory, Southern Research Institute², Birmingham, Alabama 35205

The Colcemid block method with some modifications has been used to analyze the cell cycle of cultured H.Ep. No. 2 cells and to determine the effects of certain nitrogen mustards upon the progression of cells through the cycle. The results and conclusions are as follows:

Analysis of the cell cycle of H.Ep. No. 2 cells under the described experimental conditions yielded the following values: T_C, 26.1 hr; T_G1, 14.4 hr; T_S, 6.9 hr; T_G2, 3.9 hr; and T_M, 0.9 hr.

Treatment with 2,2'-dichloro-N-methyldiethylamine did not interfere with the gross fixation of thymidine-methyl-³H into the acid-insoluble residue of cell nuclei, and hence supposedly not with the gross synthesis of DNA, during the concurrent cell cycle.

Treatment with low but toxic doses of 2,2'-dichloro-N-methyldiethylamine did not interfere with the progression of cells through G₁ and into S during the concurrent cell cycle.

Continuous treatment with low but toxic doses of 2,2'-dichloro-N-methyldiethylamine had little effect upon the progression to mitosis of cells initially in G₂ at the time of addition of the agent, but it retarded or inhibited the progression to mitosis of cells initially in G₁ or S.

Pulse treatment of cells in G₁ and in S interfered with the subsequent progression of these cells from S to mitosis.

The fact that cells treated with low doses of 2,2'-dichloro-N-methyldiethylamine eventually do progress to mitosis is consistent with the possibility of repair of sublethal damage.

Most cells treated with low concentrations of 2,2'-dichloro-N-methyldiethylamine that reach metaphase are probably capable of dividing and initiating colonies. Upon treatment with higher concentrations the cells that reach metaphase are apparently nonviable, as determined by the cloning technique.

Continuous treatment with two monofunctional nitrogen mustards (2-chloro-N, N-dimethylethylamine and 2-chloro-N, N-diethylethylamine) affect the progression of cells through the cycle in a manner similar to that of 2,2'-dichloro-N-methyldiethylamine, but higher concentrations of the agents are required. The results suggest that although cross-linking of DNA by polyfunctional alkylating agents might contribute to the toxic effects of these agents, cross-linking is not a prerequisite for toxicity by alkylating agents.

¹ This investigation was supported by Contract PH43-66-29, Chemotherapy, National Cancer Institute, NIH, and by grants from the Charles F. Kettering Foundation and the Alfred P. Sloan Foundation, Inc.

² Affiliated with the Sloan-Kettering Institute for Cancer Research, New York, N. Y.

Received 3/20/69. Accepted 5/20/69.