We examined the effects of 18 h of incubation of Chinese hamster ovary (CHO K1) cells with cycloheximide, hydroxyurea, and aphidicolin. Treatment of cells with cycloheximide alone at a concentration adequate to inhibit DNA synthesis to <10% of control was significantly less cytotoxic and clastogenic than treatment with hydroxyurea or aphidicolin, did not induce unbalanced cellular growth, and had no effect on the frequency of resistant cells in methotrexate selections compared with control cells. When combined with hydroxyurea or aphidicolin and compared with the effects of either drug alone, cycloheximide blocked the induction of unbalanced growth during drug treatment, reduced the frequency of chromosomal aberrations in recovering cell populations, and decreased cell killing. In addition, the increased frequency of methotrexate-resistant cells observed after treatment with hydroxyurea or aphidicolin was eliminated when cycloheximide was present during drug treatment.The frequency with which cultured mammalian cells become resistant to the antifolate methotrexate (MTX) can be increased if cells are treated with any of a number of agents before drug selection. Included in these treatments are hydroxyurea (HU) (5, 14, 21), MTX (24), UV irradiation (15, 33), hypoxia (5), tumor promoters (2, 35), and carcinogens (17,18,35). Several features are common to most of these treatments including transient inhibition of DNA synthesis (S-phase block) and the induction of unbalanced cellular growth (increase in cell size without increase in DNA content), chromosomal damage, and cytotoxicity.Transient inhibition of DNA synthesis induced by these treatments has been suggested to be the crucial event leading to the facilitation of MTX resistance during subsequent selections (28). While the mechanism of this effect is not understood, dihydrofolate reductase (DHFR) and several other unidentified proteins accumulate during the cell cycle block induced by HU and aphidicolin (APC) (16). These proteins have been proposed to include proteins involved in DNA synthesis which contribute after release of the cell cycle block to the overreplication of DNA and facilitate the development of MTX resistance (16,28). While the relative frequency of cells with amplified genes is increased by these treatments, this effect is not specific for gene amplification and presumably the frequency of cells with altered DHFR enzyme and transport alterations is also increased (5).In this study, we explored the role of continued protein synthesis during inhibition of DNA synthesis by examining the effect of treating cells with cycloheximide alone or in combination with HU and APC. Our data showed that continued protein synthesis during inhibition of DNA synthesis is required for the induction of chromosomal aberrations and cytotoxicity by HU and APC and that the increased frequency of MTX-resistant cells observed after pretreatment with HU and APC is eliminated if cycloheximide is present during inhibition of DNA synthesis. passaged twice per week and tested...