Abnormal accumulation and toxicity of polyamines in a difluoromethylornithine-resistant HTC cell variant

Abstract

Mammalian cells possess an inducible, active polyamine transport system that is stringently regulated by feedback controls. This study provides evidence that DH23b cells, which were initially selected from the rat hepatoma HTC line for overproduction of ornithine decar☐ylase, demonstrate an abnormality in the regulation of polyamine transport. Exposure of these cells to micromolar levels of spermidine or spermine resulted in inhibition of protein synthesis and eventual cell lysis. These effects were not due to by-products of polyamine oxidation by serum oxidases as neither inhibition of protein synthesis nor cell lysis was mitigated by aminoguanidine, reduced glutathione, dithiothreitol, or catalase. Although the polyamine transport system in the DH23b cells has the same K m and V max as that in the parental HTC line, the variant cells accumulated abnormally high levels of both spermidine (8-times normal) and spermine (4-times normal). In the HTC line, however, transport of both polyamines as well as putrescine was feedback inhibited within approx. 3 h, while in the variant cells uptake was not diminished by 12 h and terminated only with cell lysis. The DH23b cells appear to lack the normal mechanism responsible for feedback control of active polyamine incorporation. This defect provided the opportunity to manipulate intracellular levels of spermidine from 30 to approx. 800% of normal, allowing the demonstration that cellular protein synthesis is as sensitive to spermidine levels as previous in-vitro studies had suggested.