Involvement of the polyamine transport system in cellular uptake of the radioprotectants WR-1065 and WR-33278.

High levels of the aminothiol WR-1065 protect cells from ionizing radiation, while much lower levels of this compound or its disulfide, WR-33278, impart anti-mutagenic effects. In view of the structural similarity of these agents to the essential cellular polyamines putrescine, spermidine and spermine we investigated the possibility transport system. WR-33278 appears to be a very close analog of spermidine or spermine in that it not only inhibits spermidine incorporation, but is also transported at the same velocity as spermidine, with a Kt of approximately 0.8 microM compared with 0.4 microM for the polyamine. Further, repression of the activity of the polyamine transporter by antizyme or its elimination by selected mutation affected both transport of WR-33278 and spermidine equally. In contrast, WR-1065 is not a good substrate for the polyamine transporter and appears to enter cells predominantly by non-mediated passive diffusion. There appears to be no uptake of either WR-33278 or the polyamines by this non-mediated diffusion. Thus both the form of the aminothiol and the activity of the polyamine transport system need to be considered in evaluating the efficacy of low exogenous levels of this drug on mutagenesis or carcinogenesis.

Recovery of ornithine decarboxylase activity after inhibition with alpha-difluoromethylornithine.

alpha-Difluoromethylornithine is an effective inhibitor of polyamine biosynthesis because of its specificity for ornithine decarboxylase and the fact that its attachment to this enzyme is considered to be irreversible. We have found, however, that ornithine decarboxylase inactivated with this inhibitor in intact cells, as well as purified enzyme inactivated in vitro, both are capable of releasing this inhibitor and recovering enzyme activity. This reactivation can be initiated by freezing of inactivated enzyme samples in the presence of reducing agents at -7 or -20 degrees C and can be partially induced at 37 degrees C. These results reveal an unexpected lability of this enzyme-inhibitor complex that needs to be considered in future experimental designs.

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

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 decarboxylase, 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 Km and Vmax 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.

Feedback repression of polyamine uptake into mammalian cells requires active protein synthesis.

Two mammalian cell lines, rat hepatoma (HTC) and Chinese hamster ovary (CHO), were fed 10 to 50 microM spermidine while changes were monitored in intracellular polyamine levels and spermidine uptake activity. Normal feedback control preventing excessive polyamine uptake was found to be completely blocked by the addition of inhibitors of protein synthesis at the time of polyamine exposure. Under these conditions the cells accumulated abnormally high, toxic concentrations of spermidine. Further, continuous protein synthesis was needed to maintain repression of polyamine transporter proteins that had been inhibited previously by normal or elevated intracellular polyamines. These results suggest that a major factor in the regulation of polyamine uptake is the rapid, reversible inactivation of existing polyamine carrier molecules by an unstable protein whose synthesis is stimulated by intracellular polyamines.