Inhibition of [³H]Thymidine Transport Is a Nonspecific Effect of PDMP in Primary Cultures of Mouse Epidermal Keratinocytes1

Abstract

Inhibitors of sphingolipid metabolism are frequently used to investigate the role of ceramide and other sphingolipids as intracellular signaling molecules. For example, the inhibitor of glucosylceramide synthased-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) is commonly used to deplete glycosphingolipids and increase ceramide levels. Ceramide is known to induce growth arrest and differentiation of keratinocytes, and we hypothesized that PDMP would increase ceramide levels and induce growth arrest of primary cultures of mouse epidermal keratinocytes. As expected, PDMP increased ceramide levels and decreased the incorporation of [³H]thymidine into DNA, but surprisingly, PDMP was found to rapidly inhibit the intracellular transport of [³H]thymidine. This is likely due to a direct effect on nucleoside transport by PDMP and not due to elevations in ceramide levels because increasing ceramide levels by the addition of exogenous sphingomyelinase had no effect on [³H]thymidine transport. Furthermore, it is unlikely that the decreased [³H]thymidine transport is in response to growth arrest because PDMP had no effect on the cell cycle profile of keratinocytes. These results reveal that PDMP inhibits nucleoside transport independent of effects on ceramide levels or cell growth but probably by a direct effect on the nucleoside transport apparatus. Thus, this compound may be unsuitable for investigating the role of ceramide or other sphingolipids in some cellular processes.