Mdr1b facilitates p53-mediated cell death and p53 is required for Mdr1b upregulation in vivo.

The mdr1b gene is thought to be a "stress-responsive" gene, however it is unknown if this gene is regulated by p53 in the whole animal. Moreover, it is unknown if overexpression of mdr1b affects cell survival. The dependence of mdr1b upon p53 for upregulation was evaluated in p53 knockout mice. Wild-type (wt) or p53-/- mice were treated singly or in combination with gamma irradiation (IR) and/or the potent DNA damaging agent, diethylnitrosoamine (DEN). Both IR and DEN induced mdr1b in wild-type animals, but not in the p53-/- mice. IR also upregulated endogenous mdr1b in the H35 liver cell line, and the mdr1b promoter was activated by IR and activation correlated with p53 levels; moreover activation required an intact p53 binding site. Colony survival studies revealed that co-transfection of both mdr1b and p53 dramatically reduced colony numbers compared to cells transfected with either p53 or mdr1b alone and cells microinjected with both mdr1b and p53 had a more dramatic loss in viability compared to cells injected with either expression vector alone. Further studies using acridine orange and ethidium bromide to measure apoptosis revealed that mdr1b caused apoptosis and this was enhanced by p53, however the increased apoptosis required a functional p53 transactivation domain. These studies indicate that mdr1b is a downstream target of p53 in the whole animal and expression of mdr1b facilitates p53-mediated cell death.

Alterations of intracellular pH homeostasis in apoptosis: origins and roles.

Intracellular pH (pHi) has an important role in the maintenance of normal cell function, and hence this parameter has to be tightly controlled within a narrow range, largely through the activity of transporters located at the plasma membrane. These transporters can be modulated by endogenous or exogenous molecules as well as, in some pathological situations, leading to pHi changes that have been implicated in both cell proliferation and cell death. Whereas intracellular alkalinization seems to be a common feature of proliferative processes, the precise role of pHi in apoptosis is still unclear. The present review gathers the most recent advances along with previous data on both the origin and the role of pHi alterations in apoptosis and highlights the major concerns that merit further research in the future. Special attention is given to the possible role played by pHi-regulating transporters.

The antiprogestatin drug RU 486 potentiates doxorubicin cytotoxicity in multidrug resistant cells through inhibition of P-glycoprotein function.

The antiprogestatin drug RU 486 was examined for its effect on doxorubicin cellular retention and cytotoxicity in multidrug resistant cells overexpressing P-glycoprotein (P-gp). RU 486 was shown to strongly enhance intracellular accumulation of doxorubicin in both rat hepatoma RHC1 and human leukemia K562 R7 drug-resistant cells but had no action in SDVI drug-sensitive liver cells. The antiprogestatin drug when used at 10 microM, a concentration close to plasma concentrations achievable in humans, was able to hugely increase the sensitivity of RHC1 cells to doxorubicin. RU 486 appeared to prevent the P-gp-mediated doxorubicin efflux out of RHC1 cells and was demonstrated to interfere directly with P-gp drug binding sites since it blocked P-gp labelling by the photoactivable P-gp ligand azidopine. These results thus demonstrate that RU 486 can downmodulate anticancer drug resistance through inhibition of P-gp function.

Regulation by dexamethasone of P-glycoprotein expression in cultured rat hepatocytes.

We have examined P-glycoprotein (P-gp) expression and function in cultured rat hepatocytes in response to dexamethasone (DEX), which is known to modulate various liver functions. Northern blot analyses revealed high levels of P-gp mRNAs in cultured untreated liver cells in comparison to those found in freshly isolated hepatocytes, while DEX-treated hepatocytes also displayed elevated, although weaker, P-gp levels. Similarly, Western blotting analysis indicated high levels of P-gp in liver cells maintained in the absence of DEX. The use of mdr gene-specific probes allowed us to show that DEX-modulated P-gp induction in cultured hepatocytes involved mostly, if not specifically, mdr1 gene regulation. Doxorubicin P-gp-mediated efflux analyses revealed lower intracellular doxorubicin accumulation in DEX-untreated liver cells than in DEX-treated cells, thus indicating that over-expressed P-gp was functional. These data clearly show that DEX treatment strongly modulates P-gp expression in primary rat hepatocyte cultures through a specific effect on the mdr1 gene.

Differential expression of the polyspecific drug transporter OCT1 in rat hepatocarcinoma cells.

The polyspecific drug transporter OCT1 is a plasma transmembrane protein involved in the uptake of cationic drugs into hepatocytes. In order to determine whether hepatic OCT1 levels, like those of the other cationic drug transporter P-glycoprotein, may be altered during hepatocarcinogenesis, we have investigated OCT1 expression and activity in rat liver carcinoma cells. Similar levels of OCT1 mRNAs were evident in both normal liver and diethylnitrosamine-induced hepatocarcinomas by Northern blot analysis. In contrast, five hepatoma cell lines (Fao, Faza, H5, HTC and RHC1) showed either a decrease or an absence of OCT1 expression compared to normal hepatocytes; these hepatoma cells also displayed lower intracellular accumulation of tetraethylammonium (TEA), a well-known substrate for OCT1. However, among the hepatoma cell lines, the well-differentiated Fao cell line was found to retain substantial levels of OCT1 expression and of intracellular TEA uptake. Therefore, these data provide the first evidence that OCT1 expression is well-preserved in chemically-induced rat malignant neoplastic liver lesions, whereas it is either decreased or undetectable in hepatoma cell lines, which may be related to the loss of various liver functions usually occurring in these cell lines.

Rifampicin enhances anti-cancer drug accumulation and activity in multidrug-resistant cells.

Rifampicin, a semi-synthetic antibiotic used in the treatment of tuberculosis and belonging to the chemical class of rifamycins, was examined for its effect on anti-cancer drug accumulation and activity in multidrug resistant cells overexpressing P-glycoprotein (P-gp). Rifampicin was shown to strongly enhance vinblastine accumulation in both rat hepatoma RHC1 and human leukemia K562 R7 multidrug resistant cells, but had no effect in rat SDVI drug-sensitive liver cells. By contrast, two other rifamycins, rifamycins B and SV, had no or only minor effect on vinblastine accumulation in RHC1 cells. Efflux experiments revealed that rifampicin was able, like the well-known chemosensitizer agent verapamil, to decrease export of vinblastine out of resistant cells. Rifampicin, when used at a concentration close to plasma concentrations achievable in humans (25 microM), was able to increase sensitivity of RHC1 cells to both vinblastine and doxorubicin. Rifampicin was also demonstrated to inhibit P-gp radiolabeling by the photoactivable P-gp ligand azidopine, thereby suggesting that the antituberculosis compound can interfere directly with P-gp drug binding sites. These results thus indicate that rifampicin was able to down-modulate P-gp-associated resistance through inhibition of P-gp function.

P-glycoprotein induction in rat liver epithelial cells in response to acute 3-methylcholanthrene treatment.

Expression of P-glycoprotein (P-gp), a plasma membrane glycoprotein involved in multidrug resistance and encoded by mdr genes, was investigated in nonparenchymal rat liver epithelial (RLE) cells in response to acute exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). High levels of mdr mRNAs were evidenced by Northern blotting in two independent RLE cell lines after treatment by either 3-methylcholanthrene (MC) or benzo-(a)pyrene. MC-mediated mdr mRNA induction was demonstrated to be dose-dependent; it occurred through enhanced expression of the mdr 1 gene, as indicated by reverse transcriptase-polymerase chain reaction analysis using rat mdr gene-specific primers and paralleled an induction of a 140 kDa P-gp as demonstrated by Western blotting. In addition, MC-induced P-gp appeared to be fully functional because RLE cells exposed to MC displayed enhanced cellular efflux of rhodamine 123, a known P-gp substrate, compared to their untreated counterparts. Analysis of time-course induction revealed that mdr mRNA levels were maximally increased when RLE cells were treated for 48 to 96 hr and returned to low levels after the PAH was removed. In contrast to P-gp, both cytochrome P-450 1A1 and cytochrome P-450 1A2 were not detected after exposure to MC, thus indicating that these liver detoxification pathways are not coordinately regulated with P-gp in RLE cells. In addition, MC-mediated P-gp regulation was not associated with major cellular disturbances such as alteration of protein synthesis and, thereby, differed from the known mdr mRNA induction occurring in response to cycloheximide. Moreover, cotreatment with MC and cycloheximide led to a superinduction of mdr mRNAs, thus suggesting that the effects of the two xenobiotics were, at least partly, additive. In contrast to MC and benzo(a)pyrene, 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(e)pyrene were unable to increase P-gp expression. These results indicate that some PAHs can act as potent inducers of P-gp in RLE cells and may be interpreted as an adaptive reaction of these cells in lowering cellular accumulation of toxic drugs, including carcinogens transported by P-gp and, therefore, conferring protection on these compounds.

Involvement of P-glycoprotein in an in vitro blood-brain barrier model.

The P-glycoprotein (P-gp) multidrug transporter is present at the luminal face of the brain capillary endothelial cells that contribute to the blood-brain barrier. To study its role in transendothelial anticancer drug transport, we made use of a co-culture system formed of bovine brain capillary endothelial cells and astrocytes which allows the in vitro maintenance of specialized properties of the brain endothelial cells, including expression of P-gp as assessed by Northern and Western blot analyses. Vinblastine, an anticancer drug substrate for P-gp and known not to enter the brain, was found to be poorly transferred across the endothelial cell monolayer. This low vinblastine transport was however strongly increased in the presence of verapamil, a well known P-gp blocker. Moreover, verapamil was shown to increase the accumulation of the anticancer drug in the brain endothelial cells through inhibition of drug efflux. These results suggest that P-gp activity evidenced in the co-culture model is involved in the low transendothelial transport of vinblastine, thus supporting the conclusion that P-gp expressed at the blood-brain barrier level may prevent xenobiotics, including anticancer drugs, from entering the central nervous system.

Regulation of biliary drug efflux pump expression by hormones and xenobiotics.

Biliary elimination of endogenous compounds and xenobiotics usually requires carrier-mediated systems allowing movement across the canalicular membrane of hepatocytes. The major systems implicated belong to the ATP binding cassette transporter family: P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2), principally mediate the passage into the bile of cationic and anionic compounds, respectively, whereas the bile salt export pump (BSEP) handles biliary acids and also some anticancer drugs. Expression of these canalicular proteins can be altered in response to various hormones and structurally unrelated xenobiotics. Indeed, glucocorticoids up-regulate expression of both MRP2 and BSEP in rat hepatocytes, whereas insulin induces P-gp. P-gp expression is also up-regulated by numerous chemical carcinogens, such as polycyclic aromatic hydrocarbons and 2-acetylaminofluorene and by some anticancer drugs, such as anthracyclins. 2-Acetylaminofluorene also induces MRP2; in addition, expression of this transporter in liver cells is increased in response to various drugs, such as the barbiturate phenobarbital, the chemopreventive agent, oltipraz and the anticancer drug, cisplatin. Most of the chemical inducers acting on canalicular transporter levels are well-known to up-regulate some hepatic drug metabolizing enzymes, suggesting a coordinate regulation of liver detoxifying proteins in response to these compounds.

Expression and regulation of hepatic drug and bile acid transporters.

Transport across hepatocyte plasma membranes is a key parameter in hepatic clearance and usually occurs through different carrier-mediated systems. Sinusoidal uptake of compounds is thus mediated by distinct transporters, such as Na(+)-dependent or Na(+)-independent anionic transporters and by some cationic transporters. Similarly, several membrane proteins located at the apical pole of hepatocytes have been incriminated in the excretion of compounds into the bile. Indeed, biliary elimination of anionic compounds, including glutathione S-conjugates, is mediated by MRP2, whereas bile salts are excreted by a bile salt export pump (BSEP) and Class I-P-glycoprotein (P-gp) is involved in the secretion of amphiphilic cationic drugs, whereas class II-P-gp is a phospholipid transporter. The expression of hepatic transporters and their activity are regulated in various situations, such as ontogenesis, carcinogenesis, cholestasis, cellular stress and after treatment by hormones and xenobiotics. Moreover, a direct correlation between a defect and the absence of transporter with hepatic disease has been demonstrated for BSEP, MDR3-P-gp and MRP2.

Differentiation of the P-gp and MRP1 multidrug resistance systems by mobile lipid 1H-NMR spectroscopy and phosphatidylserine externalization.

We have previously demonstrated that proton NMR spectra of fatty acid chains in erythroleukemia K562 wild-type cells and their MDR1 counterparts show variations related to the phenotype over-expressing the P-glycoprotein (P-gp). Human lung cancer cells whose multidrug resistance (MDR) counterparts over-express the multidrug resistance-associated protein MRP1 have not yet been studied by NMR. Both P-gp and MRP1 belong to the same ATP-binding cassette transporter superfamily. A comparison of NMR spectra from both these multidrug-resistance phenotypes showed that the results previously obtained on the MDR1 family are not valid for MRP1. Furthermore, flow cytofluorimetry studies with external phosphatidylserine labelling showed that P-gp and MRP1 overexpressions have strong but differentiated effects on cell lipid pools.

[Drug membrane transporters in the liver: regulation of their expression and activity]. / Les transporteurs membranaires de xénobiotiques à travers les barrières hépatiques: modulation de leur expression et de leur activité

Membrane transport proteins play a major role in hepato-biliary secretion of xenobiotics. Some of them, especially OATPs and OCT1, are present at the vascular pole of hepatocytes and mediate uptake of xenobiotics into parenchymal liver cells from blood whereas others, such as P-glycoprotein and MRP2, are ABC transporters present at the canalicular domain of hepatocytes and responsible for the transmembrane passage into bile of drugs or their metabolites. Many endogenous or exogenous factors, including drug metabolizing enzyme inducers, alter expression of hepatic transporters whose activity can moreover be inhibited by various structurally-unrelated compounds. Such changes of expression and/or activity of membrane transport proteins may contribute to some drug interactions.

Mechanisms involved in lipid accumulation and apoptosis induced by 1-nitropyrene in Hepa1c1c7 cells.

1-Nitropyrene (1-NP) is a nitro-polycyclic aromatic hydrocarbon (nitro-PAH) present in diesel exhaust and bound to particular matter in urban air. We show that 1-NP and the referent PAH benzo(a)pyrene (BP) induce apoptosis and a lipid accumulation dependent on cytochrome P450 1A1-metabolites in mouse hepatoma cells, whereas 1-amino-pyrene had no effect. The caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk), inhibits 1-NP-induced apoptosis, but failed to alter 1-NP-triggered lipid accumulation determined by Nile red staining. We further show that cholesterol and fatty acid contents are modified after nitro-PAH exposure and that 1-NP-induced cholesterol level is partially involved in related apoptosis. In parallel, the activity of the stearoyl-CoA desaturase 1 (SCD1), determined by fatty acid analysis, and its expression are reduced by 1-NP. The role of SCD1 in 1-NP-induced apoptosis is demonstrated in cells down-expressing SCD1, in which an increased apoptosis is observed, whereas the SCD1 overexpression elicits the opposite effects. In contrast, changes in SCD1 gene expression have no effect on the induced lipid accumulation. Moreover, 1-NP increases the activity of the AMP-dependent protein kinase (AMPK) leading to a caspase-independent apoptosis. Overall, our study demonstrates that the 1-NP-induced apoptosis is caspase- and AMPK-dependent, and is associated to a decrease of SCD1 expression which results in an alteration of lipid homeostasis.

Differential regulation of mdr genes in response to 2-acetylaminofluorene treatment in cultured rat and human hepatocytes.

Expression of P-glycoprotein (P-gp), the mdr gene product, was investigated in primary cultures of rat and human hepatocytes exposed to 2-acetylaminofluorene (2-AAF). Increased levels of mdr1 mRNAs were evident in 2-AAF-treated rat hepatocytes by Northern blot analysis using rat mdr gene-specific probes, while transcripts of the mdr2 and mdr3 genes were decreased and unaffected respectively. Rat hepatocytes exposed to 2-AAF were also found to accumulate doxorubicin, an anticancer drug known to be transported by P-gp, poorly, thereby demonstrating that 2-AAF-mediated mdr1 induction resulted in increased P-gp activity. In contrast to their rat counterparts, human hepatocytes obtained from 10 individuals exhibited no change in both MDR1 and MDR2 mRNA levels, as well as in doxorubicin intracellular retention, in response to 2-AAF treatment, while cytochromes P-450 CYP1A1 and CYP1A2 were induced in both human and rat hepatocyte cultures. These data provide strong evidence that regulation of expression of mdr genes in liver cells in response to carcinogens such as 2-AAF is gene- and species-specific.

The P-glycoprotein multidrug transporter.

1. P-glycoprotein (P-gp) is a transmembrane protein involved in ATP-dependent efflux of various structurally unrelated anticancer drugs. Its overexpression in cancer cells decreases intracellular drug concentrations and, thus, confers a multidrug resistance phenotype. 2. P-gp is encoded by MDR genes, which constitute a small gene family comprising two genes in humans and three genes in rodents. Only the MDR1 gene in humans and mdr1 and mdr3 genes in rodents have been demonstrated to be involved in drug resistance. 3. P-gp encoded by the human MDR1 gene is a phosphorylated and glycosylated protein 1289 amino acids long, and consists of 2 halves that share a high degree of similarity. 4. A wide variety of cancers have been shown to express P-gp, including solid tumors and hematological malignancies. This P-gp positivity can be evidenced at the time of diagnosis prior to chemotherapy or at relapse after treatment, and has been correlated with treatment failure and poor prognosis in several types of cancer. In addition, P-gp is also expressed by some normal tissues, such as liver and kidney. 5. P-gp expression is regulated by various factors, including xenobiotics and hormones. 6. P-gp-mediated multidrug resistance can be reversed by various unrelated compounds called chemosensitizers or reversing agents. These drugs act through inhibition of P-gp function and have entered clinical trials.

Constitutive expression of functional P-glycoprotein in rat hepatoma cells.

P-glycoprotein is a plasma-membrane glycoprotein involved in multidrug resistance. P-glycoprotein overexpression has been demonstrated to occur in tumor cells after cytotoxic drug exposure, but also in some cancers including hepatocellular carcinomas before any chemotherapeutic treatment. In order to better analyze this constitutive type of tumoral drug resistance, we have investigated P-glycoprotein expression and function in rat liver tumors induced experimentally by administration of diethylnitrosamine and in two cell clones derived from one of these tumors designated as RHC1 and RHC2. High levels of P-glycoprotein mRNAs were found in both liver tumor samples and the two hepatoma cell clones as assessed by Northern blotting; both RHC1 and RHC2 cells displayed altered liver functions commonly observed in rat hepatoma cells, particularly the decreased expression of albumin and overexpression of the fetal glutathione S-transferase 7-7. The use of specific multidrug resistance (mdr) probes revealed a major induction of the mdr1 gene in liver tumor samples while RHC1 and RHC2 cells expressed both mdr1 and mdr3 genes without displaying a major alteration in the number of mdr gene copies as assessed by Southern blotting. High amounts of P-glycoprotein were also demonstrated in RHC1 and RHC2 cells by Western blotting. These cells were strongly resistant to doxorubicin and vinblastine, two anticancer drugs transported by P-glycoprotein. Doxorubicin intracellular retention was low in RHC1 and RHC2 cells, but was strongly enhanced in the presence of verapamil, a known modulator agent of P-glycoprotein; low retention appeared to occur via a drug efflux mechanism, indicating that P-glycoprotein was fully active. These results show that rat hepatoma cells can display elevated levels of functional P-glycoprotein without any prior cytotoxic drug selection and suggest that these cells represent a useful model for analyzing P-glycoprotein regulation in intrinsically clinical drug-resistant cancers.

Up-regulation of P-glycoprotein expression in rat liver cells by acute doxorubicin treatment.

Expression of P-glycoprotein, a plasma-membrane glycoprotein involved in multidrug resistance and encoded by mdr genes, was investigated in cultured rat liver cells acutely exposed to doxorubicin. This anticancer drug was shown to increase mdr mRNA levels in a dose-dependent manner in both rat liver epithelial (RLE) cells and primary rat hepatocytes. This induction of mdr transcripts was detected as early as a 4-h exposure to doxorubicin used at 0.5 microg/ml. It occurred through increased expression of the mdr1 gene as assessed by northern blot analysis using rat mdr-gene-specific probes. In addition, RLE cells exposed to doxorubicin displayed an overexpression of a 140-kDa P-glycoprotein as demonstrated by western blotting. Moreover, doxorubicin-treated RLE cells displayed enhanced cellular efflux of the P-glycoprotein substrate rhodamine 123 that was inhibited by the P-glycoprotein blocker verapamil, thus providing evidence that doxorubicin-induced P-glycoprotein was functional in liver cells. Doxorubicin-mediated mdr mRNA induction was found to be fully inhibited by actinomycin D, thus indicating its dependence on RNA synthesis; it was demonstrated to be not associated with alteration of protein synthesis, suggesting it differed from the known mdr mRNA overexpression occurring in response to cycloheximide. In contrast to P-glycoprotein, other liver detoxification pathways such as cytochromes P-450 1A were not induced by doxorubicin treatment. These data indicate that doxorubicin can act as a potent acute inducer of functional P-glycoprotein in rat liver cells and therefore may modulate both chemosensitivity of hepatic cells and P-glycoprotein-mediated biliary secretion of xenobiotics.

Induction of multidrug resistance gene expression in rat liver cells in response to acute treatment by the DNA-damaging agent methyl methanesulfonate.

Expression of multidrug resistance (mdr) genes encoding the P-glycoprotein (P-gp) drug efflux pump was analysed in cultured rat liver epithelial cells acutely treated by the DNA-damaging agent methyl methanesulfonate (MMS). Exposure to this alkylating agent used at 30 microg/ml for 12 or 24 h was shown to enhance mdr mRNA levels in rat liver cells without alteration of cell viability. Induction of mdr transcripts occurred through increased expression of the mdr1b gene as indicated by reverse transcriptase-polymerase chain reaction analysis using rat mdr gene-specific primers and was not associated with up-regulation of cytochrome P-450 1A1, thereby suggesting that this detoxifying enzyme and P-gp were not coordinately regulated by MMS. In addition, the DNA-damaging agent was found to enhance in a dose-dependent manner cellular efflux of the P-gp substrate rhodamine 123, which was inhibited by the P-gp inhibitor verapamil, thus providing evidence that exposure to MMS led to increased P-gp-related drug transport in rat liver cells. The up-regulation of functional P-gp expression occurring in MMS-treated liver cells may be interpreted as a part of the cellular response to DNA damage.