ATP-dependent transport of bile salts by rat multidrug resistance-associated protein 3 (Mrp3).
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We have previously shown that cloned rat multidrug resistance-associated protein 3 (Mrp3) has the ability to transport organic anions such as 17beta-estradiol 17-beta-D-glucuronide (E(2)17betaG) and has a different substrate specificity from MRP1 and MRP2 in that glutathione conjugates are poor substrates for Mrp3 (Hirohashi, T., Suzuki, H., and Sugiyama, Y. (1999) J. Biol. Chem. 274, 15181-15185). In the present study, the involvement of Mrp3 in the transport of endogenous bile salts was investigated using membrane vesicles from LLC-PK1 cells transfected with rat Mrp3 cDNA. The ATP-dependent uptake of [(3)H]taurocholate (TC), [(14)C]glycocholate (GC), [(3)H]taurochenodeoxycholate-3-sulfate (TCDC-S), and [(3)H]taurolithocholate-3-sulfate (TLC-S) was markedly stimulated by Mrp3 transfection in LLC-PK1 cells. The extent of Mrp3-mediated transport of bile salts was in the order, TLC-S > TCDC-S > TC > GC. The K(m) and V(max) values for the uptake of TC and TLC-S were K(m) = 15.9 +/- 4.9 microM and V(max) = 50.1 +/- 9.3 pmol/min/mg of protein and K(m) = 3.06 +/- 0.57 microM and V(max) = 161.9 +/- 21.7 pmol/min/mg of protein, respectively. At 55 nM [(3)H]E(2)17betaG and 1.2 microM [(3)H]TC, the apparent K(m) values for ATP were 1.36 and 0.66 mM, respectively. TC, GC, and TCDC-S inhibited the transport of [(3)H]E(2)17betaG and [(3)H]TC to the same extent with an apparent IC(50) of approximately 10 microM. TLC-S inhibited the uptake of [(3)H]E(2)17betaG and [(3)H]TC most potently (IC(50) of approximately 1 microM) among the bile salts examined, whereas cholate weakly inhibited the uptake (IC(50) approximately 75 microM). Although TC and GC are transported by bile salt export pump/sister of P-glycoprotein, but not by MRP2, and TCDC-S and TLC-S are transported by MRP2, but not by bile salt export pump/sister of P-glycoprotein, it was found that Mrp3 accepts all these bile salts as substrates. This information, together with the finding that MRP3 is extensively expressed on the basolateral membrane of human cholangiocytes, suggests that MRP3/Mrp3 plays a significant role in the cholehepatic circulation of bile salts. (+info)
Arsenic induces apoptosis of multidrug-resistant human myeloid leukemia cells that express Bcr-Abl or overexpress MDR, MRP, Bcl-2, or Bcl-x(L).
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We investigated the in vitro growth inhibitory and apoptotic effects of clinically achievable concentrations of As(2)O(3) (0.5 to 2.0 micromol/L) against human myeloid leukemia cells known to be resistant to a number of apoptotic stimuli. These included chronic myelocytic leukemia (CML) blast crisis K562 and HL-60/Bcr-Abl cells, which contain p210 and p185 Bcr-Abl, respectively, and HL-60 cell types that overexpress Bcl-2 (HL-60/Bcl-2), Bcl-x(L) (HL-60/Bcl-x(L)), MDR (HL-60/VCR), or MRP (HL-60/AR) protein. The growth-inhibitory IC(50) values for As(2)O(3) treatment for 7 days against all these cell types ranged from 0.8 to 1.5 micromol/L. Exposure to 2 micromol/L As(2)O(3) for 7 days induced apoptosis of all cell types, including HL-60/Bcr-Abl and K562 cells. This was associated with the cytosolic accumulation of cyt c and preapoptotic mitochondrial events, such as the loss of inner membrane potential (DeltaPsim) and the increase in reactive oxygen species (ROS). Treatment with As(2)O(3) (2 micromol/L) generated the activities of caspases, which produced the cleavage of the BH3 domain containing proapoptotic Bid protein and poly (ADP-ribose) polymerase. Significantly, As(2)O(3)-induced apoptosis of HL-60/Bcr-Abl and K562 cells was associated with a decline in Bcr-Abl protein levels, without any significant alterations in the levels of Bcl-x(L), Bax, Apaf-1, Fas, and FasL. Although As(2)O(3 )treatment caused a marked increase in the expression of the myeloid differentiation marker CD11b, it did not affect Hb levels in HL-60/Bcr-Abl, K562, or HL-60/neo cells. However, in these cells, As(2)O(3 )potently induced hyper-acetylation of the histones H3 and H4. These findings characterize As(2)O(3) as a growth inhibiting and apoptosis-inducing agent against a variety of myeloid leukemia cells resistant to multiple apoptotic stimuli. (+info)
Somatic hypermutation in MutS homologue (MSH)3-, MSH6-, and MSH3/MSH6-deficient mice reveals a role for the MSH2-MSH6 heterodimer in modulating the base substitution pattern.
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Although the primary function of the DNA mismatch repair (MMR) system is to identify and correct base mismatches that have been erroneously introduced during DNA replication, recent studies have further implicated several MMR components in somatic hypermutation of immunoglobulin (Ig) genes. We studied the immune response in mice deficient in MutS homologue (MSH)3 and MSH6, two mutually exclusive partners of MSH2 that have not been examined previously for their role in Ig hypermutation. In Msh6(-)/- and Msh3(-)/-/Msh6(-)/- mice, base substitutions are preferentially targeted to G and C nucleotides and to an RGYW hot spot, as has been shown previously in Msh2(-)/- mice. In contrast, Msh3(-)/- mice show no differences from their littermate controls. These findings indicate that the MSH2-MSH6 heterodimer, but not the MSH2-MSH3 complex, is responsible for modulating Ig hypermutation. (+info)
Mutations of the E2F4 gene in hematological malignancies having microsatellite instability.
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Mutations of coding repeats within the E2F4, TGF-betaRII, BAX, IGFIIR, and hMSH3 are critical targets of microsatellite instability (MSI) in many kinds of cancers. We analyzed 9 childhood acute lymphoblastic leukemia (ALL) samples, 5 acute myelocytic leukemia (AML) samples, and 10 adult T-cell leukemia (ATL) samples having MSI to determine whether they had mutations of the E2F4, TGF-betaRII, BAX, IGFIIR, and hMSH3 genes. Frameshift mutations were found at trinucleotide repeats within a coding exon of the E2F4 gene in 2 of 10 (20%) ATL samples and 1 of 9 (11%) childhood ALL samples. No mutations were found in the TGF-betaRII, BAX, IGFIIR, and hMSH3 genes. E2F4 is a transcription factor that influences the cell-cycle progression. These results suggest that mutations of the E2F4 gene, presumably caused by an abnormality of one of the DNA repair genes, may play an important role in development of ATL and childhood ALL. (Blood. 2000;95:1509-1510) (+info)
Synergy in a medicinal plant: antimicrobial action of berberine potentiated by 5'-methoxyhydnocarpin, a multidrug pump inhibitor.
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Multidrug resistance pumps (MDRs) protect microbial cells from both synthetic and natural antimicrobials. Amphipathic cations are preferred substrates of MDRs. Berberine alkaloids, which are cationic antimicrobials produced by a variety of plants, are readily extruded by MDRs. Several Berberis medicinal plants producing berberine were found also to synthesize an inhibitor of the NorA MDR pump of a human pathogen Staphylococcus aureus. The inhibitor was identified as 5'-methoxyhydnocarpin (5'-MHC), previously reported as a minor component of chaulmoogra oil, a traditional therapy for leprosy. 5'-MHC is an amphipathic weak acid and is distinctly different from the cationic substrates of NorA. 5'-MHC had no antimicrobial activity alone but strongly potentiated the action of berberine and other NorA substrates against S. aureus. MDR-dependent efflux of ethidium bromide and berberine from S. aureus cells was completely inhibited by 5'-MHC. The level of accumulation of berberine in the cells was increased strongly in the presence of 5'-MHC, indicating that this plant compound effectively disabled the bacterial resistance mechanism against the berberine antimicrobial. (+info)
Interactions of HIV protease inhibitors with a human organic cation transporter in a mammalian expression system.
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Recently, we cloned a human organic cation transporter, hOCT1, which is expressed primarily in the liver. hOCT1 plays an important role in the cellular uptake and elimination of various xenobiotics including therapeutically important drugs. HIV protease inhibitors are a new class of therapeutic agents. The purpose of this study was to elucidate the interactions of HIV protease inhibitors with hOCT1 and to determine whether hOCT1 is involved in the elimination of these compounds. We studied the interactions of HIV protease inhibitors with hOCT1 in a transiently transfected human cell line, HeLa. Uptake studies were carried out 40 h post-transfection using the radiolabeled model organic cation, [(14)C]tetraethylammonium (TEA), under different experimental conditions. In cis-inhibition studies, all of the HIV protease inhibitors tested, i.e., indinavir (IC(50) of 62 microM), nelfinavir (IC(50) of 22 microM), ritonavir (IC(50) of 5.2 microM), and saquinavir (IC(50) of 8.3 microM) inhibited TEA uptake in HeLa cells expressing hOCT1. However, none of the HIV protease inhibitors trans-stimulated [(14)C]TEA uptake, suggesting that they are poorly translocated by hOCT1. Nelfinavir, ritonavir, and saquinavir demonstrated an apparent "trans-inhibition" effect. No enhanced uptake of [(14)C]saquinavir was observed in hOCT1 DNA-transfected cells versus empty vector-transfected cells. These data suggest that HIV protease inhibitors are potent inhibitors, but poor substrates, of hOCT1. Some HIV protease inhibitors may potently inhibit the uptake and elimination of cationic drugs that are substrates for hOCT1, leading to potential drug-drug interactions. Other transporters, e.g., MDR1 and MRP1, in HIV-targeted cells may control the intracellular concentrations of HIV protease inhibitors. (+info)
Expression of deoxyribonucleic acid repair enzymes during spermatogenesis in mice.
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Meiotic recombination during gametogenesis is critical for proper chromosome segregation. However, the participating proteins and mechanics of recombination are not well understood in mammals. DNA repair enzymes play an essential role in both mitosis and meiosis in yeast. The mammalian mismatch repair system consists of homologues of the bacterial MutH, MutL, and MutS proteins. As part of our goal of understanding the function of enzymes that mediate meiotic recombination, we used a reverse transcription-polymerase chain reaction approach to identify germ cell transcripts for the MutL homologue, Pms2, and two members of the MutS family, Msh2 and Msh3. Both the Pms2 and the Msh2 genes were highly expressed in mitotically proliferating spermatogonia, and early in meiotic prophase in the leptotene and zygotene spermatocytes. Thereafter, expression declined in early and mid pachytene spermatocytes, and was negligible in postmeiotic spermatids. In contrast, expression of Msh3 was at its highest level in pachytene spermatocytes. Protein levels were similar to gene expression patterns, and both PMS2 and MSH2 were localized in spermatogonia and spermatocytes. These patterns of expression for genes encoding mismatch repair enzymes are consistent with the proposed roles of the gene products in mismatch repair during both DNA replication and recombination. (+info)
Methionine depletion enhances the antitumoral efficacy of cytotoxic agents in drug-resistant human tumor xenografts.
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Efficacy of chemotherapy is limited in numerous tumors by specific cellular mechanisms that inactivate cytotoxic antitumoral drugs, such as ATP-dependent drug efflux and/or drug detoxification by glutathione. In reducing ATP pools and/or glutathione synthesis, it might be possible to enhance the efficacy of drugs affected by such resistance mechanisms. Reduction of the ATP pool and glutathione content is achievable in cancer cells by depleting the exogenous methionine (Met) supply and ethionine. Thus, the rationale for the present study was to use Met depletion to decrease the ATP and glutathione pools so as to sensitize tumors refractory to cytotoxic anticancer drugs. Met depletion was achieved by feeding mice a methionine-free diet supplemented with homocysteine. The effects of Met depletion combined with ethionine and/or chemotherapeutic agents were studied using human solid cancers xenografted into nude mice. TC71-MA (a colon cancer) SCLC6 (a small cell lung cancer), and SNB19 (a glioma) were found to be refractory to cisplatin, doxorubicin, and carmustine, respectively. These three drugs are used to treat such tumors and are dependent for their activity on the lack of cellular ATP- or glutathione-dependent mechanisms of resistance. TC71-MA, SCLC6, and SNB19 were Met dependent because their proliferation in vitro and growth in vivo were reduced by Met depletion. Cisplatin was inactive in the treatment of TC71-MA colon cancer, whereas a methionine-free diet, alone or in combination with ethionine, prolonged the survival of mice by 2-fold and 2.8-fold, respectively. When all three approaches were combined, survival was prolonged by 3.3-fold. Doxorubicin did not affect the growth of SCLC6, a MDR1-MRP-expressing tumor. A Met-deprived diet and ethionine slightly decreased SCLC6 growth and, in combination with doxorubicin, an inhibition of 51% was obtained, with survival prolonged by 1.7-fold. Combined treatment produced greater tumor growth inhibition (74%) in SCLC6-Dox, a SCLC6 tumor pretreated with doxorubicin. Growth of SNB19 glioma was not inhibited by carmustine, but when it was combined with Met depletion, survival duration was prolonged by 2-fold, with a growth inhibition of 80%. These results indicate the potential of Met depletion to enhance the antitumoral effects of chemotherapeutic agents on drug-refractory tumors. (+info)