The purpose of this work was to determine the role of metabolic energy [adenosine triphosphate (ATP)] in the expression of cytotoxicity of the antitumor drug, Adriamycin (ADRM), in Chinese hamster fibroblasts. When ADRM-sensitive V79 cells or an ADRM-resistant variant, 77A, were pretreated with either 6 mm 2-deoxy-d-glucose or 0.2 mm 2,4-dinitrophenol for 18 hr and then exposed to ADRM for 1 hr at 37°, we observed a substantial increase in cellular survival. In the case of V79 cells pretreated with deoxyglucose, as much as a 1000-fold increase in survival was measured. The maximum dose of ADRM for V79 cells was 3 µg/ml, and for 77A cells it was 15 µg/ml.
The effects of metabolic inhibitors on intracellular ATP content, ADRM uptake, mitotic indices, cellular multiplication, and plating efficiency were measured in untreated as well as in inhibitor-treated cells. The same concentrations of deoxyglucose (6 mm) and dinitrophenol (0.2 mm) that were used for cytotoxicity studies were also used in the biochemical studies. Under conditions of pretreatment with metabolic inhibitors which increased the survival of cells, a lowering of ATP content and a concomitant slight enhancement of drug uptake were observed. Plating efficiency was not affected. We also have observed a partial inhibition of growth after pretreatment with metabolic inhibitors as shown by lowered mitotic indices and by cell counts. We conclude that the increase in survival of both ADRM-sensitive and resistant cells is not due to decreased intracellular accumulation of the drug. Flow microfluorimetric analysis of V79 cells pretreated with deoxyglucose showed no reduction in the numbers of cells in the S phase of the cell cycle, the most sensitive compartment for ADRM, while pretreatment with dinitrophenol, on the other hand, significantly reduced the number of cells in S phase. When inhibitors were removed, and the cells were rinsed, refed complete growth medium, and then exposed to a single dose of drug (1.5 µg/ml for V79; 10 µg/ml for 77A) at various times up to 7 hr, cellular survival decreased while ATP content increased as a function of time. We conclude that the effects of the metabolic inhibitors are reversible and that cells slowly regain their sensitivity to ADRM. As intracellular levels of ATP increase, sensitivity to ADRM increases. Thus, metabolic energy may be important for cytotoxic damage by ADRM. However, the enhancement in cellular survival may also be partly related to slower cell cycle progression.
This work was supported by NIH Grants RO1 CA 25333 and CA12662.