Changes in phospholipase D isoform activity and expression in multidrug-resistant human cancer cells

Abstract

Multidrug resistance (MDR) is a major cause of failure of cancer chemotherapy and is often associated with elevated expression of drug transporters such as P-glycoprotein (P-gp) in the cancer cells. MDR is, however, accompanied by additional biochemical changes including modifications of membrane composition and properties. We have shown that MDR is associated with a massive up-regulation of caveolin expression and an elevated surface density of caveolae. We report that phospholipase D (PLD), a constituent enzyme of caveolae and detergent-insoluble glycolipid-rich membranes (DIGs), is up-regulated in human MDR cancer cells. Lysates of HT-29-MDR human colon adenocarcinoma cells, MCF-7 AdrR human breast adenocarcinoma cells and the corresponding parental drug-sensitive cells, were fractionated on discontinuous sucrose density gradients. PLD activity was found to be enriched in low density fractions that contain DIGs and caveolar membranes, and the activity in these fractions was 4- to 6-fold higher in the MDR cells compared with the parental drug- sensitive cells. Utilizing specific antibodies to PLD1 and PLD2, the distribution of PLD isoforms along the gradient was determined and the PLD localized in DIGs and caveolar membranes has been identified as PLD2. Northern blot analysis of PLD1 and PLD2 mRNA levels has indicated that PLD2 mRNA is elevated in both HT-29-MDR and MCF-7 AdrR cells. PLD1 mRNA levels were either unchanged or reduced in the MDR cells. Finally, in vivo experiments have confirmed previous results showing that activation of PLD by phorbol esters is markedly potentiated in the MDR cells. We conclude that MDR is accompanied by an increase in PLD2 activity in DIGs and caveolar membranes.