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

Multidrug resistance (MDR) is a major cause of failure of cancer chemothera py and is often associated with elevated expression of drug transporters su ch as P-glycoprotein (P-gp) in the cancer cells. MDR is, however, accompani ed by additional biochemical changes including modifications of membrane co mposition and properties. We have shown that MDR is associated with a massi ve up-regulation of caveolin expression and an elevated surface density of caveolae, We report that phospholipase D (PLD), a constituent enzyme of cav eolae and detergent-insoluble glycolipid-rich membranes (DIGs), is up-regul ated in human MDR cancer cells. Lysates of HT-29-MDR human colon adenocarci noma cells, MCF-7 AdrR human breast adenocarcinoma cells and the correspond ing parental drug-sensitive cells, were fractionated on discontinuous sucro se density gradients. PLD activity was found to be enriched in low density fractions that contain DIGs and caveolar membranes, and the activity in the se fractions was 4- to 6-fold higher in the MDR cells compared with the par ental drug- sensitive cells. Utilizing specific antibodies to PLD I and PLD 2, the distribution of PLD isoforms along the gradient was determined and t he PLD localized in DIGs and caveolar membranes has been identified as PLD2 . Northern blot analysis of PLD1 and PLD2 mRNA revels has indicated that PL D2 mRNA is elevated in both HT-29-MDR and MCF-7 AdrR cells. PLD1 mRNA level s were either unchanged or reduced in the MDR cells. Finally, in vivo exper iments have confirmed previous results showing that activation of PLD by ph orbol esters is markedly potentiated in the MDR cells, We conclude that MDR is accompanied by an increase in PLD2 activity in DIGs and caveolar membra nes. (C) 2000 Wiley-Liss, Inc.