Jan Willem Kok

ACTIN MICROFILAMENTS ARE ESSENTIAL FOR THE CYTOLOGICAL POSITIONING AND MORPHOLOGY OF THE GOLGI COMPLEX

The organization and function of the Golgi complex was studied in normal rat kidney cells following disruption of the actin cytoskeleton induced by cytochalasin D. In cells treated with these reagents, the reticular and perinuclear Golgi morphology acquired a cluster shape restricted to the centrosome region. Golgi complex alteration affected all Golgi subcompartments as revealed by double fluorescence staining with antibodies to the cis/middle Mannosidase II and the trans-Golgi network TGN38 proteins or vital staining with the lipid derivate C6-NBD-ceramide. The ultrastructural and stereological analysis showed that the Golgi cisternae remained attached in a stacked conformation, but they were swollen and contained electron-dense intra-cisternal bodies. The Golgi complex cluster remained linked to microtubules since it was fragmented and dispersed after treatment with nocodazole. Moreover, the reassembly of Golgi fragments after the disruption of the microtubuli with nocodazole does not utilize the actin microfilaments. The actin microfilament requirement for the disassembly and reassembly of the Golgi complex and for the ER-Golgi vesicular transport were also studied. The results show that actin microfilaments are not needed for either the retrograde fusion of the Golgi complex with the endoplasmic reticulum promoted by brefeldin A or the anterograde reassembly after the removal of the drug, or the ER-Golgi transport of VSV-G glycoprotein. However, actin microfilaments are directly involved in the subcellular localization and the morphology of the Golgi complex.

Altered sphingolipid metabolism in multidrug-resistant ovarian cancer cells is due to uncoupling of glycolipid biosynthesis in the Golgi apparatus.

Multidrug‐resistant tumor cells display enhanced levels of glucosylceramide. In this study, we investigated how this relates to the overall sphingolipid composition of multidrug‐resistant ovarian carcinoma cells and which mechanisms are responsible for adapted sphingolipid metabolism. We found in multidrug‐resistant cells substantially lower levels of lactosylceramide and gangliosides in sharp contrast to glucosylceramide, galactosylceramide, and sphingomyelin levels. This indicates a block in the glycolipid biosynthetic pathway at the level of lactosylceramide formation, with concomitant accumulation of glucosylceramide. A series of observations exclude regulation at the enzyme level as the underlying mechanism. First, reduced lactosylceramide formation occurred only in intact resistant cells whereas cell‐free activity of lactosylceramide synthase was higher compared with the parental cells. Second, the level of lactosylceramide synthase gene expression was equal in both phenotypes. Third, glucosylceramide synthase (mRNA and protein) expression and activity were equal or lower in resistant cells. Based on the kinetics of sphingolipid metabolism, the observation that brefeldin A does not restore lactosylceramide synthesis, and altered localization of lactosylceramide synthase fused to green fluorescent protein, we conclude that lactosylceramide biosynthesis is highly uncoupled from glucosylceramide biosynthesis in the Golgi apparatus of resistant cells.

DIFFERENTIAL EXPRESSION OF SPHINGOLIPIDS IN MRP1 OVEREXPRESSING HT29 CELLS

We have obtained a novel multidrug resistant cell line, derived from HT29 G+ human colon carcinoma cells, by selection with gradually increasing concentrations of the anti‐mitotic, microtubule‐disrupting agent colchicine. This HT29col cell line displayed a 25‐fold increase in colchicine resistance and exhibited cross‐resistance to doxorubicin, VP16, vincristine and taxol. Immunoblotting, combined with RT‐PCR showed that the multidrug resistance phenotype was conferred by specific overexpression of the multidrug resistance protein 1. Confocal scanning laser microscopy revealed that multidrug resistance protein 1 specifically localized in the plasma membrane of HT29col cells. In a functional assay, using the fluorescent multidrug resistance protein 1 substrate 5‐carboxyfluorescein, an increased efflux activity of HT29col cells was measured, as compared to the wild‐type HT29 G+ cells. MK571, a specific inhibitor of multidrug resistance protein 1, blocked the 5‐carboxyfluorescein efflux, but only partially reversed resistance to colchicine, indicating that additional multidrug resistance mechanisms operate in HT29col cells. In conclusion, these results show for the first time overexpression of a functional multidrug resistance protein 1 under colchicine pressure, indicating that colchicine is not a P‐glycoprotein‐specific substrate. Colchicine‐induced overexpression of multidrug resistance protein 1 is accompanied by a changed sphingolipid composition, i.e., enhanced levels of glucosylceramide and galactosylceramide. In addition, ceramide, a lipid messenger molecule involved in apoptosis‐related signal transduction processes, was much more abundant in HT29col cells, which is indicative of a stress response. Int. J. Cancer 87:172–178, 2000.

Lipid dependence of ABC transporter localization and function

Lipid rafts have been implicated in many cellular functions, including protein and lipid transport and signal transduction. ATP-binding cassette (ABC) transporters have also been localized in these membrane domains. In this review the evidence for this specific localization will be evaluated and discussed in terms of relevance to ABC transporter function. We will focus on three ABC transporters of the A, B and C subfamily, respectively. Two of these transporters are relevant to multidrug resistance in tumor cells (Pgp/ABCB1 and MRP1/ABCC1), while the third (ABCA1) is extensively studied in relation to the reverse cholesterol pathway and cellular cholesterol homeostasis. We will attempt to derive a generalized model of lipid rafts to which they associate based on the use of various different lipid raft isolation procedures. In the context of lipid rafts, modulation of ABC transporter localization and function by two relevant lipid classes, i.e. sphingolipids and cholesterol, will be discussed.

Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes

Enveloped viruses infect cells by a mechanism involving membrane fusion. This process is mediated and triggered by specific viral membrane glycoproteins. Evidence is accumulating that fusion of intracellular membranes, as occurs during endocytosis and transport between intracellular organelles, also requires the presence of specific proteins. The relevance of elucidating the mechanisms of virus fusion for a better understanding of fusion of intracellular membranes is discussed.

Fas receptor clustering and involvement of the death receptor pathway in rituximab-mediated apoptosis with concomitant sensitization of lymphoma B cells to Fas-induced apoptosis

Ab binding to CD20 has been shown to induce apoptosis in B cells. In this study, we demonstrate that rituximab sensitizes lymphoma B cells to Fas-induced apoptosis in a caspase-8-dependent manner. To elucidate the mechanism by which Rituximab affects Fas-mediated cell death, we investigated rituximab-induced signaling and apoptosis pathways. Rituximab-induced apoptosis involved the death receptor pathway and proceeded in a caspase-8-dependent manner. Ectopic overexpression of FLIP (the physiological inhibitor of the death receptor pathway) or application of zIETD-fmk (specific inhibitor of caspase-8, the initiator-caspase of the death receptor pathway) both specifically reduced rituximab-induced apoptosis in Ramos B cells. Blocking the death receptor ligands Fas ligand or TRAIL, using neutralizing Abs, did not inhibit apoptosis, implying that a direct death receptor/ligand interaction is not involved in CD20-mediated cell death. Instead, we hypothesized that rituximab-induced apoptosis involves membrane clustering of Fas molecules that leads to formation of the death-inducing signaling complex (DISC) and downstream activation of the death receptor pathway. Indeed, Fas coimmune precipitation experiments showed that, upon CD20-cross-linking, Fas-associated death domain protein (FADD) and caspase-8 were recruited into the DISC. Additionally, rituximab induced CD20 and Fas translocation to raft-like domains on the cell surface. Further analysis revealed that, upon stimulation with rituximab, Fas, caspase-8, and FADD were found in sucrose-gradient raft fractions together with CD20. In conclusion, in this study, we present evidence for the involvement of the death receptor pathway in rituximab-induced apoptosis of Ramos B cells with concomitant sensitization of these cells to Fas-mediated apoptosis via Fas multimerization and recruitment of caspase-8 and FADD to the DISC.

ATP-binding cassette transporters are enriched in non-caveolar detergent-insoluble glycosphingolipid-enriched membrane domains (DIGs) in human multidrug-resistant cancer cells

In this study we show that P-glycoprotein in multidrug-resistant 2780AD human ovarian carcinoma cells and multidrug resistance-associated protein 1 in multidrug-resistant HT29col human colon carcinoma cells are predominantly located in Lubrol-based detergent-insoluble glycosphingolipid-enriched membrane domains. This localization is independent of caveolae, since 2780AD cells do not express caveolin-1. Although HT29col cells do express caveolin-1, the ATP-binding cassette transporter and caveolin-1 were dissociated on the basis of differential solubility in Triton X-100 and absence of microscopical colocalization. While both the multidrug resistance-associated protein 1 and caveolin-1 are located in Lubrol-based membrane domains, they occupy different regions of these domains.

Low body temperature governs the decline of circulating lymphocytes during hibernation through sphingosine-1-phosphate

Hibernation is an energy-conserving behavior consisting of periods of inhibited metabolism (‘torpor’) with lowered body temperature. Torpor bouts are interspersed by arousal periods, in which metabolism increases and body temperature returns to euthermia. In deep torpor, the body temperature typically decreases to 2–10 °C, and major physiological and immunological changes occur. One of these alterations constitutes an almost complete depletion of circulating lymphocytes that is reversed rapidly upon arousal. Here we show that torpor induces the storage of lymphocytes in secondary lymphoid organs in response to a temperature-dependent drop in plasma levels of sphingosine-1-phosphate (S1P). Regulation of lymphocyte numbers was mediated through the type 1 S1P receptor (S1P1), because administration of a specific antagonist (W146) during torpor (in a Syrian hamster at ∼8 °C) precluded restoration of lymphocyte numbers upon subsequent arousal. Furthermore, S1P release from erythrocytes via ATP-binding cassette (ABC)-transporters was significantly inhibited at low body temperature (4 °C) but was restored upon rewarming. Reversible lymphopenia also was observed during daily torpor (in a Djungarian hamster at ± 25 °C), during forced hypothermia in anesthetized (summer-active) hamsters (at ± 9 °C), and in a nonhibernator (rat at ∼19 °C). Our results demonstrate that lymphopenia during hibernation in small mammals is driven by body temperature, via altered plasma S1P levels. S1P is recognized as an important bioactive lipid involved in regulating several other physiological processes as well and may be an important factor regulating additional physiological processes in hibernation as well as in mediating the effects of therapeutic hypothermia in patients.

MRP1 and glucosylceramide are coordinately over expressed and enriched in rafts during multidrug resistance acquisition in colon cancer cells

Previously we have described a novel multidrug‐resistant cell line, HT29col, which displayed over expression of the multidrug‐resistance protein 1 (MRP1) and an altered sphingolipid composition, including enhanced levels of glucosylceramide (GlcCer; Kok JW, Veldman RJ, Klappe K, Koning H, Filipeanu C, Muller M. Int J Cancer 2000;87:172–8). In our study, long‐term screening revealed that, during colchicine‐induced acquisition of multidrug resistance in a new HT29col cell line, increases in GlcCer occurred concomitantly with upregulation of MRP1 expression. Both MRP1 and GlcCer were found enriched in Lubrol‐insoluble membrane domains. The expression of MRP1 and GlcCer were tightly correlated, as indicated also by a reversal of both at the later stage of colchicine consolidation. Resistance to colchicine was determined by MRP1, while glucosylceramide synthase (GCS) did not contribute: 1) Resistance was fully inhibited by MK571. 2) GCS expression and activity were not upregulated in HT29col cells. 3) Inhibition of GCS did not affect MRP1‐mediated efflux function or sensitivity to colchicine. Instead, overall sphingolipid metabolism was upregulated through an increased rate of ceramide biosynthesis. In conclusion, upregulation of MRP1 occurs in concert with upregulation of GlcCer during multidrug‐resistance acquisition, and both are enriched in rafts. The increased GlcCer pool does not directly modulate MRP1 function and cell survival.

Sphingolipid metabolism enzymes as targets for anticancer therapy

Treatment with anti-cancer agents in most cases ultimately results in apoptotic cell death of the target tumor cells. Unfortunately, tumor cells can develop multidrug resistance, e.g., by a reduced propensity to engage in apoptosis by which they become insensitive to multiple chemotherapeutics. Ceramide. the central molecule in cellular sphingolipid metabolism, has been recognized as an important mediator of apoptosis. Moreover, an increased cellular capacity for ceramide glycosylation has been identified as a novel multidrug resistance mechanism. Indeed, virtually all multidrug resistant cell types exhibit a deviating sphingolipid composition, most typically an increased level of glucosylceramide. Thus, the enzyme glucosylceramide synthase, which converts ceramide into glucosylceramide, has emerged as a potential target to increase apoptosis and decrease drug resistance of tumor cells. In addition, several other steps in the pathways of sphingolipid metabolism arc altered in multidrug resistant cells, opening a perspective on additional sphingolipid metabolism enzymes as targets for anti-cancer therapy. In this article, we present an overview of the current understanding concerning drug resistance-related changes in sphingolipid metabolism and how interference with this metabolism can be exploited to over come multidrug resistance.