Inactivation of the dna template in HeLa cells treated with chlorambucil

The effect of chlorambucil on the colony‐forming ability of HeLa cells following either treatment for 1 h or continuous treatment has been measured. Concentrations of chlorambucil which had only minimal effects on cell survival inhibited the rate of DNA but not RNA and protein synthesis within 1 h of treatment. Nevertheless, cells continued to synthesize DNA for many hours after this treatment in the absence of cell division. Synchronous populations of HeLa cells treated prior to DNA synthesis, in the G 1 phase of the cell cycle, were not delayed in their progression into the S phase where they exhibited a marked dose‐dependent inhibition of the rate of DNA synthesis. Cells in which DNA synthesis had been depressed showed a prolongation of the S phase and this was accompanied by a corresponding dose‐dependent mitotic delay. Treatment during the G 2 phase of the cell cycle did not induce any delay or block in the next mitosis, but did inhibit the rate of DNA synthesis in the following cell cycle in a dosedependent manner; this depression of DNA synthesis was followed by a delay in the next mitosis. Cross‐linking of either isolated DNA or DNA present in treated HeLa cells was demonstrated, and in the latter case calculated to be of the same order as that which occurred with other difunctional agents at comparable toxic concentrations.