Cancer chemotherapy often results in the development of multidrug resistance (MDR), which is commonly associated with overexpression of P-glycoprotein (P-gp), a plasma membrane drug efflux ATPase. It was shown recently that glycosphingolipids are elevated in MDR cells. Sphingolipids are major constituents of caveolae and of detergent-insoluble, glycosphingolipid-rich membrane domains. Here we report that multidrug-resistant HT-29 human colon adenocarcinoma cells exhibit a 12-fold overexpression of caveolin-1, a 21-kDa coat/adaptor protein of caveolae. Similar observations were made in adriamycin-resistant MCF-7 human breast adenocarcinoma cells. Caveolin-2 expression is also up-regulated in MCF-7-AdrR cells, but neither caveolin-1 nor caveolin-2 were detected in MCF-7 cells stably transfected with P-gp. The up-regulation of caveolins is associated with a 5-fold increase in the number of caveolae-like structures observed in plasma membrane profiles of HT-29-MDR cells and with the appearance of a comparable number of caveolae in MCF-7-AdrR cells. A significant fraction (ϳ40%) of cellular P-gp is localized in low density detergent-insoluble membrane fractions derived from either HT-29-MDR or MCF-7-AdrR cells. The distribution of recombinant P-gp in stably transfected MCF-7 cells was similar, even though these cells do not express caveolins and are devoid of caveolae. The possibility that caveolae contribute to the multidrug resistance and thus are coselected with P-gp during the acquisition of this phenotype is discussed.Although chemotherapy improves long term survival in cancer patients, the treatment often results in the development of tumors that are resistant to most cytotoxic drugs commonly used in chemotherapy, leading to an untreatable and incurable disease (1). Known as multidrug resistance (MDR), 1 this phenomenon may be defined as the ability of cancer cells exposed to a given drug to resist the cytotoxic actions of a broad range of structurally and functionally unrelated drugs. MDR is often caused by overexpression of a plasma membrane ATPase called P-glycoprotein (P-gp) (2). P-gp acts as an energy-dependent drug efflux pump, increasing outward transport of active drugs and thereby decreasing their intracellular concentration and reducing their cytotoxic efficacy. However, additional mechanisms that contribute to MDR have been described (see Ref. 3 for review). Recent studies have indicated that glucosylceramide accumulates to a major extent in various types of MDR cells (4). Glucosylceramide and other glycosphingolipids are important constituents of detergent-insoluble membrane domains termed DIGs (5) that are enriched also in sphingomyelin and cholesterol (6). DIGs are related in their lipid composition and their insolubility in cold non-ionic detergents to nonclathrin-coated, plasma membrane vesicular invaginations termed caveolae (reviewed in Ref. 7). Caveolin-1, a 21-kDa integral membrane protein, is a major caveolar coat protein (8) that has the ability to engage in complex interactions with other...
The activation of cellular phospholipase D (PLD) is implicated in vesicular trafficking and signal transduction. Two mammalian PLD forms, designated PLD1 and PLD2, have been cloned, but their cellular localization and function are not fully understood. Here, we report that in HaCaT human keratinocytes, as well as other cell lines, PLD activity is highly enriched in low density, Triton X-100-insoluble membrane domains that contain the caveolar marker protein caveolin-1. Similar to other PLDs, the PLD activity in these membrane domains is stimulated by phosphatidylinositol 4,5-bisphosphate and is inhibited by neomycin. Immunoblot analysis indicated that caveolin-rich membrane domains do not contain the PLD1 isoform. Stable transfection of mouse PLD2 in Chinese hamster ovary cells greatly increased PLD activity in these domains compared with PLD activity in control Chinese hamster ovary cells transfected with vector alone. PLD activity is enriched in low density Triton-insoluble membrane domains also in U937 promonocytes, even though these cells do not express caveolin-1. In U937 cells, also, PLD1 is largely excluded from low density Triton-insoluble membrane domains. Expression of recombinant caveolin-1 in v-Src-transformed NIH-3T3 cells resulted in up-regulation of PLD activity in the caveolin-containing membrane domains. The caveolin scaffolding peptide (caveolin-1 82-101 ) modulated the caveolar PLD activity, causing stimulation at concentration of 1-10 M and inhibition at concentrations >10 M. We conclude that a PLD activity, which is likely to represent PLD2, is enriched in low density Triton-insoluble membrane domains. The effects of caveolin-1 expression and of the caveolin scaffolding peptide suggest that in cells that express caveolin-1, PLD may be targeted to caveolae. The possible functions of PLD in the dynamics of caveolae and related domains and in signal transduction processes are discussed.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.