Retinol-induced mdr1 and mdr3 modulation in cultured rat Sertoli cells is attenuated by free radical scavengers

The effects of retinol on modulation of mdr genes in Sertoli cells were investigated. The hypothesis that free radical scavengers may attenuate the effect of retinol was also tested. Sertoli cells isolated from 15-day-old Wistar rats were cultured for 48 h and then treated with retinol for 24 h with or without free radical scavengers (1 mM mannitol, 0.1 mM Trolox or superoxide dismutase [200 U/ml]). Expression of mdr1, mdr2 and mdr3 genes was monitored by RT-PCR. Mitochondrial superoxide production was used as an index of ROS production. Expression of mdr1 and mdr3 was inhibited by retinol treatment (7 microM, 24 h), while mdr2 was not detected in response to any of the treatments. We also observed that retinol supplementation (7 microM, 24 h) increased superoxide production. The observed inhibition of mdr genes was attenuated by all co-treatments, suggesting that retinol-induced ROS are required for inhibition of mdr1 and mdr3 expression. The results suggest that retinol may play an important role in the modulation of the mdr gene family in cultured rat Sertoli cells and that these effects appear to be mediated by ROS.     

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.     

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.     

Interaction of LNA oligonucleotides with mdr1 promoter

LNA oligonucleotides [1] can be used for targeting to double stranded DNA by the "strand invasion" mechanism. We used affinity modification by reactive oligonucleotide conjugates for investigation of oligonucleotides interaction with structured DNA. The tested LNAs and oligonucleotides of the same sequence were assayed as anti-mdr1 drugs in different cell cultures. One of the oligos, LNA79 strongly inhibited mdr1 induction in Hela cells and totally prevented activation of mdr1 in K-562.     

The products of the mdr1a and mdr1b genes from multidrug resistant murine cells have similar degradation rates

Two vinblastine-resistant sublines of the murine macrophage-like cell line J774.2, J7.V1-1 and J7.V3-1, overproduce unique forms of P-glycoprotein that are encoded by distinct mdr genes, mdr1b and mdr1a, respectively. Degradation rates of the two P-glycoprotein isoforms were measured by immunoprecipitation of P-glycoprotein. The half-life of immunoprecipitable P-glycoprotein from J7.V1-1 cells was 16.8 +/- 0.5 hours and from J7.V3-1 cells, 17.4 +/- 0.5 hours. This rate was not influenced by the presence of vinblastine in the growth medium. The data indicate that P-glycoproteins derived from distinct genes have similar degradation rates.     

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.     

Sequence of mdr3 cDNA encoding a human P-glycoprotein

We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.     

多药耐药基因分型芯片的制备及优化

目的:建立多药耐药基因(mdr1)分型芯片,以检测患者的单核苷酸多态性(SNPs)。方法:设计并合成探针和引物,制备芯片;构建野生型和突变型质粒,以其为模板经PCR仪扩增后,与芯片上的探针杂交,并用扫描仪分析结果。结果:构建了野生型和突变型质粒,与芯片杂交能很好地区分基因型;优化了制备条件,建立了分型标准。结论:该基因芯片是一种快速特异的基因分型方法。     

Targeting the human mdr1 gene by 125I-labeled triplex-forming oligonucleotides

Antigene radiotherapy is our approach to targeting specific sites in the genome by combining the highly localized DNA damage produced by the decay of Auger electron emitters, such as 125I, with the sequence-specific action of triplex-forming oligonucleotides (TFO). As a model, we used the multidrug resistance gene (mdr1) overexpressed and amplified nearly 100 times in the human KB-V1 carcinoma cell line. Phosphodiester pyrrazolopyrimidine dG (PPG)-modified TFO complementary to the polypurine-polypyrimidine region of the mdr1 gene were synthesized and labeled with 125I-dCTP at the C5 position of two cytosines by the primer extension method. 125I-TFO were delivered into KB-V1 cells with several delivery systems. DNA from the 125I-TFO-treated cells was recovered and analyzed for sequence-specific cleavage in the mdr1 target by Southern hybridization. Experiments with plasmid DNA containing the mdr1 polypurine-polypyrimidine region and with purified genomic DNA confirmed the ability of the designed 125I-TFO to bind to and introduce double-strand breaks into the target sequence. We showed that 125I-TFO in nanomolar concentrations can recognize and cleave a target sequence in the mdr1 gene in situ, that is, within isolated nuclei and intact digitonin-permeabilized cells. Our results demonstrate the ability of 125I-TFO to target specific sequences in their natural environment, that is, within the eukaryotic nucleus. The nearly 100-fold amplification of the mdr1 gene in KB-V1 cells affords a very useful cell culture model for evaluation of methods to produce sequence-specific DNA double-strand breaks for gene-specific radiotherapy.     

Psychological stress induces chemoresistance in breast cancer by upregulating mdr1

Psychological distress reduces the efficacy of chemotherapy in breast cancer patients. The mechanism may be related to the altered neuronal or hormonal secretions during stress. Here, we reported that adrenaline, a hormone mediating the biological activities of stress, upregulates mdr1 gene expression in MCF-7 breast cancer cells via alpha(2)-adrenergic receptors in a dose-dependent manner. Mdr1 upregulation can be specifically inhibited by pretreatment with mdr1-siRNA. Consequently, adrenergic stimulation enhances the pump function of P-glycoprotein and confers resistance of MCF-7 cells to paclitaxel. In vivo, restraint stress increases mdr1 gene expression in the MCF-7 cancers that are inoculated subcutaneously into the SCID mice and provokes resistance to doxorubicin in the implanted tumors. The effect can be blocked by injection of yohimbine, an alpha(2)-adrenergic inhibitor, but not by metyrapone, a corticosterone synthesis blocker. Therefore, we conclude that breast cancers may develop resistance against chemotherapeutic drugs under psychological distress by over-expressing mdr1 via adrenergic stimulation.     

The mdr1 gene, responsible for multidrug-resistance, codes for P-glycoprotein

The development of simultaneous resistance to multiple drugs in cultured cells occurs after selection for resistance to single agents. This multidrug-resistance phenotype is thought to mimic multidrug-resistance in human tumors treated with chemotherapy. Both the expression of a membrane protein, termed P170 or P-glycoprotein, and the expression of a cloned DNA fragment, termed mdr1, have been shown independently to be associated with multidrug-resistance in cultured cells. In this work, we show that human KB carcinoma cells which express the mdr1 gene also express P-glycoprotein, and that cDNAs encoding P-glycoprotein cross-hybridize with mdr1 cDNAs. Thus, the mdr1 gene codes for P-glycoprotein.     

Differential transport properties of two mdr gene products are distinguished by progesterone

P-glycoprotein is an integral membrane protein that is overproduced in multidrug-resistant cells. It is likely to function as an energy-dependent drug efflux pump to maintain intracellular drug concentrations below cytotoxic levels. Individually isolated multidrug-resistant murine cell lines, J7.V1-1 and J7.V3-1, overproduce P-glycoproteins encoded by the mdr1b and mdr1a genes, respectively. The transport properties of these cell lines and the drug binding characteristics of their P-glycoproteins have been compared. It is concluded that 1) the mdr1a gene product is a more efficient efflux pump than the mdr1b gene product, and 2) whereas a single class of vinblastine binding sites is present in J7.V1-1 membrane vesicles, there appears to be two classes of such sites in J7.V3-1 membrane vesicles. The effects of verapamil and progesterone, two compounds that are known to interact with P-glycoprotein, have been analyzed in the two cell lines. Progesterone inhibited drug binding and efflux and increased drug sensitivity to vinblastine with more potency in J7.V1-1 cells than in J7.V3-1 cells. It is concluded that progesterone, but not verapamil, can be used to differentiate the two mdr gene products in the mouse.