The Effect of Growth Conditions on NAD+ and NADH Concentrations and the NAD+:NADH Ratio in Normal and Transformed Fibroblasts

Abstract

Concentrations of total NAD+ and NADH have been measured and the NAD+:NADH ratio calculated for 3T3 fibroblasts and SV40 transformed 3T3 cells and for normal rat kidney fibroblasts and murine sarcoma virus (Kirsten strain)-transformed normal rat kidney fibroblasts. The absolute levels were found to decrease 2- to 3-fold with transformation. The NAD+:NADH ratio in the normal cells ranged from 3.1 to 3.6, and increased 3- to 4-fold as they grew from logarithmic to stationary phase. The final ratio was found to be a direct function of the final cell density achieved for the 3T3 cells. In contrast, the NAD+:NADH ratio of transformed cells was low, (1.2 to 3.2) and did not rise when the cells reached a heavy density. Comparable results were obtained for the ratio of free NAD+:NADH. This ratio was determined by analysis of cellular pyruvate and lactate concentrations and calculated using the Keq of lactate dehydrogenase. Attempts to alter the ratio by treatment of the cells with agents capable of elevating intracellular cyclic adenosine 3′ : 5′-monophosphate levels, such as N6, O2′-dibutyryl cyclic AMP or prostaglandin E1, were unsuccessful. Nor did serum deprivation or agents capable of lowering cyclic adenosine 3′ : 5′-monophosphate levels affect the NAD+:NADH ratio. We suggest that the inability of cells to elevate their NAD+: NADH ratio at confluency is a characteristic of transformed cells and related to their defective growth control.