Induction of mdr1b mRNA and P-glycoprotein expression by tumor necrosis factor alpha in primary rat hepatocyte cultures

Mammalian liver exhibits expression of members of the family of multidrug resistance (mdr) transporters (P-glycoproteins). P-glycoprotein isoforms encoded by mdr1 genes participate in extrusion of an array of xenobiotics into the bile. Induction of mdr1b mRNA expression has been shown to occur in rat hepatocytes in response to hepatotrophic growth factors. As the cytokine tumor necrosis factor alpha (TNF-α) is known to exert a direct mitogenic effect on hepatocytes, its influence on mdr1b expression was investigated. In primary rat hepatocytes cultured in the absence of TNF-α, a time-dependent increase in basal expression of mdr1b mRNA and in immunodetectable P-glycoprotein was observed. In cells treated with TNF-α (4,000 U/ml) for 3 days, expression of mdr1b mRNA and of immunodetectable P-glycoprotein was induced approximately twofold. Moreover, intracellular steady-state levels of the mdr1 substrate rhodamine 123 were decreased in cells pretreated with TNF-α in comparison to controls, indicating an increase in functional transporter(s) mediating dye extrusion. Treatment of hepatocytes with antioxidants (1 mM ascorbic acid and 2% dimethyl sulfoxide) for 3 days markedly suppressed mdr1b mRNA and P-glycoprotein expression both in cells cultured in the presence of TNF-α and in the absence of the cytokine, but did not fully abolish mdr1b mRNA induction by TNF-α, supporting the notion that reactive oxygen species participate in regulation of basal mdr1b gene expression during hepatocyte culture. In conclusion, the present data indicate that by inducing mdr1b expression in hepatocytes, TNF-α may affect the capacity of the liver for extrusion or detoxification of endogenous or xenobiotic mdr1 substrates. J. Cell. Physiol. 176:506–515, 1998. © 1998 Wiley-Liss, Inc.     

Puromycin selectively increases mdr1a expression in immortalized rat brain endothelial cell lines

The blood-brain barrier (BBB) plays an important role in controlling the passage of molecules from blood to brain extracellular fluid. The multidrug efflux pump P-glycoprotein (P-gp) is highly expressed in the luminal membrane of brain endothelium and contributes to the formation of a functional barrier to lipid-soluble drugs such as anticancer agents. The mdr1a P-gp-encoding gene is exclusively expressed in the rodent BBB. Primary cultures of rat brain endothelial cells and GP8.3 cells showed a dramatic decrease in mdr1a mRNA level and some expression of mdr1b mRNA. GPNT cells, derived from GP8.3 cells after transfection with a puromycin resistance gene, were chronically treated with 5 microg/mL puromycin, a P-gp substrate. Compared with rat brain endothelial cells and GP8.3 cells, GPNT cells exhibited a very high level of expression of mdr1a mRNA together with a moderate level of mdr1b mRNA expression. Accordingly, P-gp expression and activity were strongly increased. When GP8.3 and puromycin-starved GPNT cells were treated with puromycin, mdr1a expression was selectively increased. High expression of mdr1a mRNA in GPNT cells may thus be related to the chronic treatment with puromycin. We conclude that GPNT cells may be used as a valuable rat in vitro model for studying the regulation of mdr1a expression at the BBB level.     

Identification of a Cryptic Bacterial Promoter in Mouse (mdr1a) P-Glycoprotein cDNA

The efflux transporter P-glycoprotein (P-gp) is an important mediator of various pharmacokinetic parameters, being expressed at numerous physiological barriers and also in multidrug-resistant cancer cells. Molecular cloning of homologous cDNAs is an important tool for the characterization of functional differences in P-gp between species. However, plasmids containing mouse mdr1a cDNA display significant genetic instability during cloning in bacteria, indicating that mdr1a cDNA may be somehow toxic to bacteria, allowing only clones containing mutations that abrogate this toxicity to survive transformation. We demonstrate here the presence of a cryptic promoter in mouse mdr1a cDNA that causes mouse P-gp expression in bacteria. This expression may account for the observed toxicity of mdr1a DNA to bacteria. Sigma 70 binding site analysis and GFP reporter plasmids were used to identify sequences in the first 321 bps of mdr1a cDNA capable of initiating bacterial protein expression. An mdr1a M107L cDNA containing a single residue mutation at the proposed translational start site was shown to allow sub-cloning of mdr1a in E. coli while retaining transport properties similar to wild-type P-gp. This mutant mdr1a cDNA may prove useful for efficient cloning of mdr1a in E. coli.     

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.     

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.     

Effect of hemorrhagic shock on apoptosis and energy-dependent efflux system in the brain

Recent findings suggest that apoptosis, which contributes to neuronal damage after ischemic injury, may play a role in sequelae associated with severe blood loss. This study examined the effect of hemorrhage and resuscitation on the expression (in situ hybridization and computerized image analysis) of bcl-2 mRNA, which codes for a protein that inhibits apoptosis, and mdr1 mRNA, which codes for a glycoprotein marker for drug efflux from the brain. Anaesthetized rats were subjected to volume-controlled (15 mL/kg) hemorrhage followed by resuscitation with shed blood (BR) or nonresuscitated (NR); control animals had femoral artery cannulation only (SHAM). Following 24 hr blood loss, distinctly lower levels of bcl-2 gene expression were observed in dentate gyrus of NR rats (0.25 ± 0.04) as compared to SHAM rats (0.52 ± 0.07); suscitation with shed blood prevented this reduction (0.58 ± 0.05). Similar results were observed in cortex, striatum, and hypothalamus. Also, mdr1 mRNA levels were significantly reduced in all brain areas of the NR group as compared to the BR and SHAM groups. The findings suggest that blood resuscitation suppressed apoptosis and protected against loss of energy-dependent efflux system in the brain in response to hemorrhage.     

Co-amplification and over-expression of two mdr genes in a multidrug-resistant human colon carcinoma cell line.

The human P-glycoprotein gene family contains the mdr1 and the mdr3 gene. The mdr1 P-glycoprotein is over-expressed in multidrug resistant (MDR) tumor cells and is believed to play a role in the elimination of certain cytotoxic drugs used in the chemotherapy of cancer. The mdr3 gene has not been found to be amplified or over-expressed in MDR cells. In this study, gene-specific mdr gene probes were developed for the detection of the gene and the total mRNA level. Southern and Northern hybridization analyses showed that the mdr genes and the mRNA levels were increased 30--40-fold in a MDR human colon cancer cell line. In addition, this MDR cell line had an altered growth rate and morphology and detectable double minute chromosomes.     

Effect of Food Restriction on Ghrelin in Normal‐Cycling Female Rats and in Pregnancy

Objective: Ghrelin is a 28‐amino‐acid acylated peptide that was recently identified as the endogenous ligand for the growth hormone secretagogue receptor. Previous studies have shown that ghrelin potently increases growth hormone release and food intake. The aim of this study was to clarify the physiological implications of ghrelin in the regulation of energy balance, by assessing the effect of undernutrition throughout 21 days in normal‐cycling and pregnant rats on ghrelin. Research Methods and Procedures: We have determined ghrelin levels by radioimmunoassay and gastric ghrelin mRNA expression by Northern blot analysis during 21 days of chronic food restriction (30% of ad libitum available diet) in normal‐cycling female rats and in pregnancy. Results: Our results show that chronic food restriction led to an increase in plasmatic ghrelin levels in normal‐cycling female rats. In pregnancy, ghrelin plasmatic levels were enhanced particularly during the latter part of gestation (19 and 21 days) compared with pregnant rats with free access to food. Gastric ghrelin mRNA expression showed a similar expression pattern, being higher in the food‐restricted group than in the group fed ad libitum, in normal‐cycling as well as in pregnant rats. Discussion: These observations indicate that ghrelin plasmatic levels and ghrelin gastric mRNA are up‐modulated during undernutrition in normal‐cycling rats and in pregnancy. These findings suggest that increased ghrelin levels may have a role in mediating the physiological responses to undernutrition and could represent an adaptative response to prevent long‐lasting alterations in energy balance and body weight homeostasis.     

Expression of mdr1 is required for efficient long term regeneration of dystrophic muscle

The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their precise role in muscle is unknown. Dystrophic muscle is characterized by repeated cycles of degeneration and regeneration. To explore the role of the mdr1 genes during muscle regeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and the dystrophin genes. The resulting ReX mice developed normally and were fertile. However, as adults, ReX had a higher proportion of degenerating muscle fibers and greater long-term loss of muscle mass than mdx. ReX muscles were also characterized by a reduced proportion of muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscle regeneration. We found too that mSP cells derived from dystrophic muscle are more myogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene plays an important role in the preservation of the mSP and of the myogenic regenerative potential. Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells of regenerating tissue; they therefore provide an important clue to the physiological significance of mdr1 expression in stem cells.     

Repetitive DNA sequences located in the central region of the human mdr1 (multidrug resistance) gene may account for a gene fusion event during its evolution

The mdr1 gene, first member of the human multidrug-resistance gene family, is a major gene involved in cellular resistance to several drugs used in anticancer chemotherapy. Its product, the drug-excreting P-glycoprotein, shows a bipartite structure formed by two similar adjacent halves. According to one hypothesis, the fusion of two related ancestral genes during evolution could have resulted in this structure. The DNA sequence analysis of the introns located in the region connecting the two halves of the human mdr1 gene revealed a highly conserved poly(CA) · poly (TG) sequence in intron 15 and repeated sequences of the Alu family in introns 14 and 17. These repeated sequences most likely represent molecular fossils of ancient DNA elements which were involved in such a recombination event. Correspondence to: M. Pauly     

Multidrug resistance of DNA-mediated transformants is linked to transfer of the human mdr1 gene.

Mouse NIH 3T3 cells were transformed to multidrug resistance with high-molecular-weight DNA from multidrug-resistant human KB carcinoma cells. The patterns of cross resistance to colchicine, vinblastine, and doxorubicin hydrochloride (Adriamycin; Adria Laboratories Inc.) of the human donor cell line and mouse recipients were similar. The multidrug-resistant human donor cell line contains amplified sequences of the mdr1 gene which are expressed at high levels. Both primary and secondary NIH 3T3 transformants contained and expressed these amplified human mdr1 sequences. Amplification and expression of the human mdr1 sequences and amplification of cotransferred human Alu sequences in the mouse cells correlated with the degree of multidrug resistance. These data suggest that the mdr1 gene is likely to be responsible for multidrug resistance in cultured cells.     

Stimulation of the side population fraction of ATDC5 chondroprogenitors by hypoxia

The influence of oxygen tension on the side population (SP) fraction sorted from ATDC5 chondroprogenitor cells was investigated. ATDC5 cells cultured in normoxia (20%) or hypoxia (1% oxygen) were compared. The SP fraction was significantly higher in the cells cultured in hypoxia. The gene expression of 3 ABC transporters, abcb1a/b (mdr1a/b) and abcg2 (bcrp1) was quantified by RT-PCR. SP cells were characterized by the higher expression of abcb1a. The expression levels of abcb1b and abcg2 were higher than abcb1a. However, there was no significant difference between SP and non-SP fractions in the expression of abcb1b and abcg2. The telomeric repeat amplification protocol assay showed that SP cells tended to show lower telomerase activity than non-SP cells. Chondrogenic properties of ATDC5 cells derived from SP or non-SP were assessed by micromass culture. There were not significant differences between SP and non-SP derived cells in Alcian blue staining and sox9, Aggrecan, Col2a1 and SZP mRNA expression. The results demonstrate that the SP fraction was stimulated by hypoxia and chondrogenic properties of SP and non-SP fraction of ATDC5 cells were similar.