Expression Of cMOAT Research Articles

Abscisic acid and hydrogen peroxide regulate proline homeostasis in melon seedlings under cold stress by forming a bidirectional closed loop

Low temperatures severely limit melon cultivation and production in winter and spring. Cold stress can induce proline accumulation in melon seedlings. However, the accumulation mechanism is unclear and the way by which abscisic acid (ABA) and hydrogen peroxide (H 2 O 2 ) act as signaling molecules in the process remains unknown. We first identified key enzyme genes in the proline metabolism pathway, and identified six genes, namely, two CmP5CSs and one each of CmP5CR , CmOAT , ProDH , and CmP5CDH . Low temperature induced proline accumulation by up-regulating three synthase genes, CmP5CS1 , CmP5CR , and CmOAT , while suppressing the expressions of two catabolic genes, namely, CmProDH and CmP5CDH . Cold stress triggered ABA and H 2 O 2 synthase genes to activate their accumulation. Both H 2 O 2 and ABA pretreatments enhanced melon seedling’s cold tolerance, but the two behaved divergently in proline metabolism. Exogenous H 2 O 2 enhanced or maintained the aforementioned induction efficiency of low temperature. In contrast, ABA only induced CmP5CR and CmOAT expression, with no effect on CmP5CS1 , and synchronously induced CmProDH and CmP5CDH expressions, leading to a slight reduction in proline content. Alternatively, ABA controlled proline homeostasis under cold stress, at least in part, by inhibiting H 2 O 2 generation. Overall, the results suggest that ABA and H 2 O 2 formed a fine bidirectional closed loop to regulate proline homeostasis in melon seedlings. • Proline metabolism genes in melon were identified for the first time. • Cold induced proline accumulation by promoting CmP5CS1 , CmP5CR , CmOAT expression, and inhibiting CmProDH , CmP5CDH expression. • Exogenous H 2 O 2 induced proline and ABA accumulation for cold tolerance improvement. • ABA inhibited H 2 O 2 generation to keep proline homeostasis under cold stress.

Expression of basolateral and canalicular transporters in rat liver and cultures of primary hepatocytes

1. Basolateral and canalicular proteins are expressed in the liver and besides their role in the transport of bilirubin, glutathione, hormones and various glucuronides, they also function as transporters of a wide range of drugs.2. Despite their frequent use in drug research, little is known about the expression of genes coding for transporters in cultures of primary sandwiched hepatocytes.3. The kinetics of gene expression of canalicular and basolateral membrane transporters in cultures of primary rat sandwiched hepatocytes were investigated, and the expression of cMOAT, spgp, mdr1a and mdr2 were shown to be comparable with transcript levels observed in vivo.4. Strikingly, expression of the basolateral membrane transporter ntcp and oatp1 and 2 were dramatically reduced, the level being <10 and <5%, respectively, of those found in vivo in rat liver tissue.5. Notably, mRNA expression of the canalicular membrane transporter mdr1b was increased up to 13-fold.6. The findings point to a dramatic change in the expression of basolateral and canalicular transporters in cultured hepatocytes, and this should be considered when hepatocytes are used for drug profiling studies.

Expression of drug resistance genes in VP-16 and mAMSA-selected human carcinoma cells.

The cell lines described in the present study were isolated as part of an effort to understand resistance to topoisomerase (topo) II inhibitors. To that end, 50 sublines were isolated from four human breast cancer cell lines, i.e., MCF‐7, T47D, MDA‐MB‐231, and ZR‐75B. As an initial step, a concentration that would be lethal to the majority of cells (IC99) was selected for both VP‐16 and mAMSA, for each cell line. The identification of an increasing number of putative drug resistance‐related proteins provided the opportunity to examine expression of the corresponding genes in the selected cell lines. Northern blot analysis revealed different responses to the selecting agents in the different cell lines. Previous studies examining expression of multidrug resistance (MDR)‐l in resistant cell lines had found undetectable levels in all cells. In the ZR‐75B sublines, increased expression of MDR‐associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) was observed, and when the relative levels of overexpression were compared, a high correlation was found. In contrast, increased expression of MRP was observed in some of the MDA‐MB‐231 sublines, without a concomitant increase in cMOAT expression. Finally, in both T47D and MCF‐7 sublines, increased expression of cMOAT or MRP was observed infrequently, and where it occurred, was of a much smaller magnitude. In the analysis of expression of MRP, the highest levels were found in the ZR‐75B and MDA‐MB‐231 sublines, with lower levels in the MCF‐7 and T47D clones. Similarly, differences in the expression of topo IIα were observed among the sublines. Although the differences in expression appear to depend on the parental cell line from which the resistant sublines were derived, a strong correlation was observed between the expression of MRP and the levels of topo IIα. Cell lines with low levels of MRP had lower levels of topo IIα, while those with high levels of MRP maintained higher levels of topo IIα. While a reduced topo IIα level was common, there did not appear to be a compensating increase in the expression of topo IIβ or topo I or casein kinase (CK) IIα in any of the cell lines. While the possibility that such compensation could occur has been discussed and even reported in some cell lines, such an adaptation was not observed in the present study, suggesting that it is not common.

Cisplatin induces renal expression of P-glycoprotein and canalicular multispecific organic anion transporter.

The expression of two members of the ATP-binding cassette family of transport proteins, P-glycoprotein (P-gp) and the canalicular multispecific organic anion transporter (cMOAT or Mrp2), was evaluated in renal brush-border membranes (BBM) and various rat tissues after cisplatin treatment. One administration of cisplatin (5 mg/kg) increased P-gp expression by >200-300% in renal BBM and in crude membranes from liver and intestine. The increase in P-gp expression in the kidney was also detected in photolabeling experiments, suggesting the induction of functional P-gp. cMOAT expression was increased by >10-fold in renal BBM after cisplatin administration, although it had no effect on liver cMOAT expression. The increase in the levels of both proteins was maximal at 2 days after cisplatin treatment and lasted for at least 8 days. These results indicate that a single administration of cisplatin induces overexpression of P-gp and cMOAT in specific tissues. This may be of significant relevance to the design of clinical trials using cisplatin as a single chemotherapeutic agent or in combination with other drugs.

In vivo cisplatin resistance depending upon canalicular multispecific organic anion transporter (cMOAT).

The in vitro sensitivities to cisplatin of AH66 and AH66F cells, a variant obtained from AH66 cells, were very similar, when assayed in a medium containing 5% fetal bovine serum (FBS), whereas in the in vivo experiments AH66F cells were sensitive and AH66 cells were highly resistant to cisplatin. In this study, we examined the mechanism of the in vivo cisplatin resistance of AH66 cells. The in vitro cisplatin sensitivity of AH66 cells was lowered by changing FBS to 5% ascites fluid (ASF) in the assay medium and the sensitivity in FBS by treatment with buthioninesulfoximine (BSO). The sensitivity of AH66F cells was not changed by these treatments. Moreover, after culture in 5% ASF for 48 h, the accumulation of cisplatin in AH66 cells was decreased and the efflux of cisplatin from the cells was accelerated. The accumulation of cisplatin in AH66 cells in ASF was increased by pretreatment with BSO, sodium azide or probenecid. Then, we examined the expression of the glutathione (GSH) conjugate efflux pump family. Among them, only the expression of canalicular multispecific organic anion transporter (cMOAT) in AH66 cells was decreased by culture in FBS and enhanced by ASF. These results suggest that some substances contained in ASF enhanced the expression of cMOAT in the plasma membrane of AH66 cells and this transporter actively extruded cisplatin‐GSH conjugate from the cells. Consequently, AH66 cells afford a cisplatin‐resistant tumor in the host.

Overexpression of multidrug resistance genes MDR1 and cMOAT in human hepatocellular carcinoma and hepatoblastoma cell lines.

Overexpression of either of the multidrug resistance genes MDR1 and MRP is associated with resistance of tumors to multiple chemotherapeutic agents. Overexpression of MDR1 has been reported in some cell lines derived from human hepatocellular carcinomas (HCC) and hepatoblastomas (HB). The human gene for cMOAT (<canalicular multispecific organic anion transporter>), a homologue of MRP, is thought to mediate hepatobiliary excretion of organic anions and to be associated with cisplatin resistance. In this study, expression levels of MDR1 and cMOAT were examined in 9 human HCC and HB cell lines and 10 other human cancer cell lines. Overexpression of the cMOAT gene was observed in all 9 HCC and HB cell lines and 3 of 10 other cancer cell lines. Co-overexpression of the cMOAT and MDR1 genes was observed in 7 of 9 HCC and HB cell lines, but in none of the 10 other cancer cell lines. Seven of the HCC and HB cell lines that had overexpression of the cMOAT gene were shown to be highly resistant to cisplatin compared to 2 HCC cell lines with low levels of cMOAT expression. These findings suggest that overexpression of cMOAT could contribute to cisplatin resistance in HCC and HB.

Dexamethasone modulation of multidrug transporters in normal tissues

The expression of P-glycoprotein (P-gp) and canalicular multispecific organic anion transporter (cMOAT or Mrp2) was evaluated by Western blotting analysis of rat tissues isolated following daily administration (1 mg kg −1 day −1) of dexamethasone over 4 days. Dexamethasone rapidly increased P-gp expression more than 4.5- and 2-fold in liver and lung, respectively, while it was decreased 40% in kidney. cMOAT expression was increased 2-fold in liver and kidney following dexamethasone treatment. The levels of both proteins returned to control values by 6 days after the conclusion of dexamethasone administration. These results indicate that dexamethasone can modulate P-gp and cMOAT expression in specific rat tissues and may have significant relevance for patients treated with dexamethasone as a single agent or in combination therapy with other drugs.

Expression of gamma-glutamylcysteine synthetase (gamma-GCS) and multidrug resistance-associated protein (MRP), but not human canalicular multispecific organic anion transporter (cMOAT), genes correlates with exposure of human lung cancers to platinum drugs.

We examined the steady-state levels of mRNA for gamma-glutamylcysteine synthetase (gamma-GCS), multidrug resistance-associated protein (MRP) and human canalicular multispecific organic anion transporter (cMOAT) in human lung cancer specimens to elucidate their roles in relation to platinum drug resistance in vivo. Seventy-six autopsy samples (38 primary tumours and their corresponding normal lung tissues) obtained from 38 patients were analysed using the quantitative reverse transcription polymerase chain reaction (RT-PCR) method. Both subunits (heavy and light subunits) of gamma-GCS expression levels of normal lung and tumour tissues exposed to platinum drugs during life were significantly higher than those of non-exposed tissues, whereas only the MRP expression levels of tumours were elevated in association with ante-mortem platinum drug exposure. The gamma-GCS and MRP expression levels correlated significantly. The cMOAT expression levels did not correlate with ante-mortem platinum drug exposure. Next, we monitored gamma-GCS heavy subunit expression levels in peripheral mononuclear cells of eight previously untreated lung cancer patients after platinum drug administration, which revealed that these drugs induced gamma-GCS expression in vivo. These results suggest that gamma-GCS expression is induced by platinum drugs in vivo and/or the physiological stress response to xenobiotics.

Transfected rat cMOAT is functionally expressed on the apical membrane in Madin-Darby canine kidney (MDCK) cells.

The purpose of the present study is to investigate the expression of canalicular multispecific organic anion transporter (cMOAT) by its cDNA transfection in polarized Madin-Darby canine kidney cells (MDCK). MDCK cells were transfected with an expression vector (pCXN2) containing the rat cMOAT cDNA with lipofectamine to obtain the stable transfectant under G418. Cells from a single colony whose cMOAT expression was the highest were seeded to form a tight epithelial monolayer on microporous membrane filters. Export of glutathione S-bimane (GS-B) from monolayers was determined after preloading its precursor, monochloro bimane (MCB). A comparable amount of GS-B was excreted to the apical and basal compartments in the vector-transfected cells. In contrast, in cMOAT-transfected cells, the amount apically excreted was approximately twice that excreted into the basal compartment. Cyclosporin A (CsA) (30 microM), an inhibitor of cMOAT at higher concentrations, inhibited the preferential apical export of GS-B from cMOAT-transfected cells. Rat cMOAT is functionally expressed on the apical membrane of MDCK cells after transfection.

Molecular cloning of canalicular multispecific organic anion transporter defective in EHBR.

Several organic anions are excreted into the bile via a canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats, such as the Eisai hyperbilirubinemic rat (EHBR) and TR- rat. In the present study, we cloned cMOAT from the Sprague-Dawley rat liver cDNA library based on the homology with human multidrug resistance-associated protein (hMRP). cMOAT was encoded by 4,623-base pair (bp) cDNA with a homology of 53.0 and 46.3% with hMRP at the cDNA and deduced amino acid level, respectively. The deduced amino acid sequence was the same as that cloned in Wistar rats (C. C. Paulusma, P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Sceffer, P. Borst, and R. P. Oude Elferink. Science Wash. DC 271: 1126, 1996) except for four amino acid substitutions. By screening the library, three kinds of cDNA species for cMOAT with the same open reading frame and different 3'-untranslated region lengths (0.2, 1.5, and 3.5 kbp) were isolated. The Northern blot analysis of poly(A)+ RNA from the liver revealed that the expression of plural bands (approximately 5, 6, and 8 kb) was defective in EHBR, and this may be due to the presence of these cDNA species. Expression of cMOAT was observed almost exclusively in the liver and to a lesser extent in the duodenum, kidney, and jejunum. Reverse transcription-polymerase chain reaction (RT-PCR) and subsequent sequence analysis of EHBR liver, kidney, duodenum, and jejunum revealed that 1-bp replacement from G to A at nucleotide 2564 resulted in the introduction of the premature stop codon in all tissues examined. This mutation was different from that observed in TR (C. C. Paulusma, P. J. Bosma, G. J. Zaman, C. T. Bakker, M. Otter, G. L. Sceffer, P. Borst, and R. P. Oude Elferink. Science Wash. DC 271: 1126, 1996). Because EHBR and TR- are allelic mutants and both strains exhibit an autosomal recessive inheritance in the biliary excretion of organic anions it was concluded that the impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant animals.

Role of ABC transporters for the biliary excretion of organic anions

Several organic anions, including the glutathione- and glucuronide-conjugates, are excreted into the bile via a canalicular multispecific organic anion transporter (cMOAT), which is hereditarily defective in mutant rats such as Eisai hyperbilirubinemic rat (EHBR). In the present study, we cloned cMOAT from the SD rat liver cDNA library using the ATP-binding cassette region as a probe which was amplified by PCR with the degenerated primers for human multidrug resistance associated protein (hMRP). cMOAT was encoded by 4623 by cDNA with a homology of 53.0 % and 46.3 % with hMRP at the cDNA and deduced amino acid level, respectively. By screening the library, three kinds of cDNA species for cMOAT with the same open reading frame and different 3′-untranslated region lengths were isolated. The Northern blot analysis of poly A+ RNA from the liver with the entire open reading frame of cMOAT as a probe revealed that the expression of plural bands (approximately 5, 6 and 8 kb) was defective in EHBR and this may be due to the presence of these cDNA species. RT-PCR and subsequent sequence analysis were performed using RNA specimens from EHBR liver. The analysis revealed that one base pair replacement within the open reading frame resulted in the introduction of the premature stop codon. The impaired expression of this particular protein is related to the pathogenesis of hyperbilirubinemia in the mutant rats. Although the expression of cMOAT is defective in EHBR, our recent kinetic studies suggested the presence of ATP-dependent organic anion transporters other than cMOAT in EHBR. Using the degenerated PCR primers described previously, we could amplify two kinds of PCR product (MRP like protein (MLP-1 and MLP-2)) from EHBR liver. The nucleotide homology of ABC region between MLP-1, 2, MRP and cMOAT was approximately 60-70%. Northern blot analysis indicated that MLP-1 hybridized with a single 6 kb transcript in the liver of SD rats and EHBR. In contrast, MLP-2 hybridized to a single 5 kb transcript in the liver and the expression was rather induced in EHBR. These results may suggest the possibility that MLP-1 and MLP-2 are novel transporters for organic anions.