P-glycoproteins encoded by mdr 1b in murine gravid uterus and multidrug resistant tumor cell lines are differentially glycosylated

There are 3 members of the multidrug-resistance gene family expressed in mouse. Only one of these, mdr 1b, and its gene product P-glycoprotein are induced to high levels in the mouse endometrium during pregnancy. It is shown here that P-glycoprotein in the gravid uterus is significantly larger (Mr 155,000) compared to P-glycoprotein encoded by mdr 1b in a murine multidrug-resistant cell line (Mr 140,000). However, both species co-migrate after enzymatic removal of N-linked sugars (Mr 125,000). These results demonstrate that differential glycosylation of the mdr 1b gene product contributes to molecular heterogeneity found in P-glycoprotein from normal and multidrug-resistant cells.     

The multidrug resistance protein 1: a functionally important activation marker for murine Th1 cells

Previously, we described the expression of an energy-dependent pump in resting murine Th2 (but not resting Th1) cells which extruded the fluorescent dye Fluo-3. After stimulation with Ag and APCs, Th1 cells also expressed this pump. Furthermore, expression of the murine multidrug resistance protein 1 (mrp1) correlated with the presence of the pump. In this study, we report that Fluo-3 is indeed transported by murine mrp1 or its human ortholog MRP1, as revealed by transfection of HEK 293 cells with mrp1 or MRP1 cDNA. Like antigenic activation, IL-2 dose-dependently enhanced the Fluo-3-extruding activity in murine Th1 cells. Although TNF-alpha and IL-12 by themselves only weakly enhanced Fluo-3 extrusion, each of them did so in strong synergism with IL-2. An Ab directed against mrp1 was used to quantify the expression of mrp1 protein in T cells at the single-cell level. Like the Fluo-3 pump, mrp1 protein expression was enhanced by IL-2. Immunohistochemical studies using confocal laser microscopy indicated that mrp1 is localized mainly at the plasma membrane. In addition, protein expression of mrp1 was induced in Vbeta8+CD4+ T cells 12 h after in vivo application of Staphylococcal enterotoxin B. Finally, mrp1 was functionally relevant during the activation process of Th1 cells, because T cell activation could be suppressed by exposure of cells to the mrp1 inhibitor MK571. Thus, we present mrp1 as a novel, functionally important activation marker for Th1 cells and short-term in vivo activated CD4+ T cells, whereas its expression seems to be constitutive in Th2 cells.     

Decreased hepatic accumulation and enhanced esterification of cholesterol in mice deficient in mdr1a and mdr1b P-glycoproteins

Class I P-glycoproteins [Pgp; MDR1 (ABCB1) in humans, mdr1a and mdr1b in mice] confer resistance to structurally diverse chemotherapeutic drugs in cultured cells and intact animals, but the function of these proteins in normal physiology remains poorly characterized. Based on studies in cell culture, a putative role for class I Pgp in absorption and intracellular trafficking of sterols has been proposed. We examined wild-type and mdr1a(-/)-/1b(-/)- mice to determine whether class I Pgp affects cholesterol absorption and esterification in vivo. Using a dual-isotope protocol, absorption of orally administered radiolabeled cholesterol into serum did not differ between wild-type and mdr1a(-/)-/1b(-/)- mice, demonstrating that class I Pgp is not essential for overall absorption of cholesterol through the intestine. However, the ratio of oral to intravenous labeled cholesterol in liver was decreased significantly in mdr1a(-/)-/1b(-/)- mice. In the liver, but not other tested organs, deletion of class I Pgp enhanced kinetics of esterification of an oral bolus of radiolabeled cholesterol without affecting esterification of cholesterol administered intravenously. Steady-state hepatic content of cholesterol and esterified cholesterol also were unaffected by absence of mdr1a and mdr1b.Thus, in normal animals, class I Pgp functions to kinetically increase hepatic accumulation and decrease esterification of orally administered cholesterol in vivo.     

Expression of mdr49 and mdr65 multidrug resistance genes in larval tissues of Drosophila melanogaster under normal and stress conditions

In situ expression of 2 multidrug resistance genes, mdr49 and mdr65, of Drosophila melanogaster was examined in wild-type third instar larval tissues under physiological conditions and after heat shock or colchicine feeding. Expression of these 2 genes was also examined in tumorous tissues of lethal (2) giant larvae I(2)gl4 mutant larvae. These 2 mdr genes show similar constitutive expression in different larval tissues under physiological conditions. However, they are induced differentially by endogenous (tumorous growth) and exogenous stresses (colchcine feeding or heat shock): whereas heat shock and colchicine feeding induce mdr49, tumorous condition is accompanied by enhanced expression of mdr49 and mdr65 genes.     

Functional analysis of chimeric genes obtained by exchanging homologous domains of the mouse mdr1 and mdr2 genes.

A full-length cDNA clone for the mouse mdr1 gene can confer multidrug resistance when introduced by transfection into otherwise drug-sensitive cells. In the same assay, a full-length cDNA clone for a closely related member of the mouse mdr gene family, mdr2, fails to confer multidrug resistance. To identify the domains of mdr1 which are essential for multidrug resistance and which may be functionally distinct in mdr2, we have constructed chimeric cDNA molecules in which discrete domains of mdr2 have been introduced into the homologous region of mdr1 and analyzed these chimeras for their capacity to transfer drug resistance. The two predicted ATP-binding domains of mdr2 were found to be functional, as either could complement the biological activity of mdr1. Likewise, a chimeric molecule in which the highly sequence divergent linker domain of mdr2 had been introduced in mdr1 could also confer drug resistance. However, the replacement of either the amino- or carboxy-terminus transmembrane (TM) domain regions of mdr1 by the homologous segments of mdr2 resulted in inactive chimeras. The replacement of as few as two TM domains from either the amino (TM5-6) or the carboxy (TM7-8) half of mdr1 by the homologous mdr2 regions was sufficient to destroy the activity of mdr1. These results suggest that the functional differences detected between mdr1 and mdr2 in our transfection assay reside within the predicted TM domains.     

The multidrug resistance and cystic fibrosis genes have complementary patterns of epithelial expression.

The cystic fibrosis gene product, CFTR, and the multidrug resistance P-glycoprotein (encoded by the MDR1 gene) are structurally related proteins and both are associated with epithelial chloride channel activities. We have compared their cell-specific expression in the rat by in situ hybridization. In all tissues examined the two genes were found to have complementary patterns of expression, demonstrating exquisite regulation in both cell-specific and temporal fashions. Additionally, a switch in expression from one gene to the other was observed in certain tissues. For example, expression in the intestine switches from CFTR to MDR1 as the cells migrate across the crypt-villus boundary. A switch from CFTR to MDR1 expression was also observed in the uterine epithelium upon pregnancy. These data suggest that CFTR and P-glycoprotein serve analogous roles in epithelial cells and provide additional evidence that P-glycoprotein has a physiological role in regulating epithelial cell volume. The patterns of expression suggest that the regulation of these two genes is coordinately controlled.     

The reversion effect of the RNAi-silencing mdr1 gene on multidrug resistance of the leukemia cell HT9

Overexpression of P-glycoprotein (P-gp), the mdr1 gene product, confers multidrug resistance (MDR) to tumor cells and often limits the efficacy of chemotherapy. This study evaluated RNAi for specific silencing of the mdr1 gene and reversion of multidrug resistance. Three different short hairpin RNAs (shRNAs) were designed and constructed in a pSilencer 3.1-H1 neo plasmid. The shRNA recombinant plasmids were transfected into HT9 leukemia cells. The RNAi effect was evaluated by real-time PCR, Western blotting and cell cytotoxicity assay. In the cell, shRNAs can specifically down-regulate the expression of mdr1, mRNA and P-gp. Resistance against harringtonine, doxorubicin and curcumin was decreased. The study indicated that shRNA recombinant plasmids could modulate MDR in vitro.     

Treatment of multidrug resistant (MDR1) murine leukemia with P-glycoprotein substrates accelerates the course of the disease

The prolactin (PRL) surge in cycling rats during the proestrous afternoon is an inducer of apoptotic cell death in luteal cells. This luteolytic action of PRL is peculiar, because PRL may be categorized as a survival factor, if other known physiological functions of PRL are taken into account. Here we analyzed the underlying molecular/cellular mechanisms of this PRL-induced apoptosis. Corpora lutea (CL) were prepared from the ovary on the proestrous day and cultured with or without PRL (2 microg/ml). An addition of PRL to the culture medium induced DNA breakdown in the nuclei of cells mostly identified as steroidogenic by 3beta-HSD activity staining, and the number of 3beta-HSD-positive cells were significantly decreased, indicating the induction of apoptotic cell death by PRL among luteal cells in culture. Next, the expression of membrane form-Fas ligand (mFasL) in the luteal cell lysate was quantified, because Fas receptor is known to have an exact physiological role in luteolysis. An addition of PRL increased the expression of mFasL. Immunostaining and TUNEL assay on regressing CL revealed that both CD3-positive cells and FasL-positive cells were co-localized in the regions where apoptosis convergently occurred. Moreover, an addition of concanavalin A (ConA), a T-cell specific activator, to the culture mimicked the PRL action by inducing apoptosis in luteal cells and enhancing the expression of mFasL. These data suggest that the CD3-positive T lymphocyte in the CL is at least one of the PRL-effector cell species during the process of luteolysis in rats, and that FasL expression of these cells is upregulated by PRL.     

Identification of a 170 kDa membrane kinase with increased activity in KB-V1 multidrug resistant cells

Using an in situ kinase assay we have identified kinases that are elevated in some multidrug resistant cells. Kinases were detected by measurement of ³²P incorporation in proteins that were renatured after being subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes [Ferrell and Martin: J Biol Chem 264:20723–20729, 1989; Mol Cell Biol 10:3020–3026, 1990]. Kinases at 79, 84, and 92 kDa showed increased activity in the multidrug resistant human KB-V1 cells as compared to the sensitive parental KB-3-1 cells. The KB-V1 multidrug resistant cell line exhibited a 170 kDa membrane associated kinase activity that was not present in the parental drug sensitive line. The 170 kDa kinase activity was not affected by Ca⁺⁺, phosphatidylserine, or cAMP, but was diminished after incubation in the presence of the kinase inhibitors staurosporine, K252a and KT5720. The 170 kDa kinase activity phosphorylated mainly threonine, with no evidence of tyrosine phosphorylation, and was not identical to either the multidrug resistance associated P-glycoprotein or the EGF receptor. Other multidrug resistant cell lines also showed elevated 170 kDa kinase activity, such as the human breast cancer MCF-7/Adr^R and murine melanoma B16/Adr^R. cells, but the activity was not present in murine leukemia P-388 sensitive or multidrug resistant cells.     

The multidrug resistance gene mdr1a influences resistance to ectromelia virus infection by mechanisms other than conventional immunity

P-glycoprotein (P-gp), an ATP-dependent membrane pump encoded by mdr, plays, in addition to its ability to efflux toxins, a role in the resistance to pathogens. We employed mdr1a gene knock out (mdr1a-/-) mice and ectromelia virus (EV) to elucidate the role of P-gp in resistance to EV. Mdr1a-/- mice are more susceptible to EV infection than wild type (wt) mice, showing increased mortality and morbidity. Unexpectedly, virus titres in liver, and in vitro in macrophages and splenocytes were significantly lower in the more susceptible mdr1a-/- mice than wt littermates. Analysis of immunological mechanisms known to influence resistance to EV infection, such as NK and cytotoxic T cell responses, EV specific antibody and cytokine levels did not reveal significant differences between the two strains of mice. Only dendritic cells from mdr1a-/- mice showed impaired migration to the draining lymph nodes compared to wt mice. Our data show that P-gp plays an important role in EV infection by as yet undefined mechanisms.     

Detection of P-glycoprotein in the nuclear envelope of multidrug resistant cells

P-glycoprotein is a plasma membrane efflux pump which is responsible for multidrug resistance of many cancer cell lines. A number of studies have demonstrated the presence of P-glycoprotein molecules, besides on the plasma membrane, also in intracellular sites, such as the Golgi apparatus and the nucleus. In this study, the presence and function of P-glycoprotein in the nuclear membranes of human breast cancer cells (MCF-7 WT) and their multidrug resistant variants (MCF-7 DX) were investigated. Electron and confocal microscopy immunolabelling experiments demonstrated the presence of P-glycoprotein molecules in the nuclear membranes of MCF-7 DX cells. Moreover, the labelling pattern was strongly dependent on pH values of the incubation buffer. At physiological pH (7.2), a strong labelling was detected in the cytoplasm and the nuclear matrix in both sensitive and resistant MCF-7 cells. By raising the pH to 8.0, the P-glycoprotein molecules were easily detected in the cytoplasm (transport vesicles and Golgi apparatus), plasma and nuclear membranes exclusively in MCF-7 DX cells. Furthermore, drug uptake and efflux studies, performed by flow cytometry on isolated nuclei in the presence of the P-glycoprotein inhibitor cyclosporin A, suggested the presence of a functional P-glycoprotein in the nuclear membrane, but not in the nuclear matrix, of drug resistant cells. Therefore, P-glycoprotein in the nuclear envelope seems to represent a further defense mechanism developed by resistant cells against antineoplastic agents.