Tohru Komano

P-glycoprotein gene (MDR1) cDNA from human adrenal: normal P-glycoprotein carries Gly185 with an altered pattern of multidrug resistance

We isolated a full-length MDR1 cDNA from human adrenal where P-glycoprotein is expressed at high level. The deduced amino acid sequence shows two amino acid differences from the sequence of P-glycoprotein obtained from colchicine-selected multidrug resistant cultured cells. The amino acid substitution Gly----Val at codon 185 in P-glycoprotein from colchicine resistant cells occurred during selection of cells in colchicine. As previously reported, cells transfected with the MDR1 cDNA carrying Val185 acquire increased resistance to colchicine compared to other drugs. The other amino acid substitution Ser----Ala at codon 893 probably reflects genetic polymorphism. The MDR1 gene, the major member of the P-glycoprotein gene family expressed in human adrenal, is sufficient to confer multidrug-resistance on culture cells.

Anti-cancer drugs and glutathione stimulate vanadate-induced trapping of nucleotide in multidrug resistance-associated protein (MRP)

Multidrug resistance‐associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP‐dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate‐induced trapping of 8‐azido‐ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti‐cancer drugs VP‐16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8‐azido‐[α‐32P]ATP by the vanadate‐trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP·MgADP·Vi, an analog of the MRP·MgADP·Pi transition state complex, is formed in the presence of vanadate. Glutathione as well as anti‐cancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP.

P-glycoprotein-mediated transcellular transport of MDR-reversing agents

Understanding of the interactions between P‐glycoprotein and multidrug resistance (MDR) reversing agents is important in designing more effective MDR modulators. We examined transcellular transport of several MDR modulators by using a drug‐sensitive epithelial cell line, LLC‐PK1 and its transformant cell line, LLC‐GA5‐COL300, which expresses human P‐glycoprotein on the apical surface. Basal‐to‐apical transports of azidopine and diltiazem across the LLC‐GA5‐COL300 monolayer were increased and apical‐to‐basal transports were decreased compared to those across the LLC‐PK1 monolayer, indicating that P‐glycoprotein transports azidopine and diltiazem. Movements of nitrendipine and staurosporine across the epithelial monolayer were not affected by P‐glycoprotein. These results suggests that some MDR modulators exert their inhibitory effect not only by blocking the initial binding of anticancer drugs but throughout the course of the transport process.

Heat-shock responsive elements in the induction of the multidrug resistance gene (MDR1)

The MDR1 gene, considered to be involved in multidrug resistance of cancer cells, is expressed in liver, kidney, small intestine and the blood‐brain barrier. We investigated MDR1 gene expression in the well‐differentiated hepatoma cell line HepG2 after exposure to several stresses and found that sodium arsenite treatment increased MDR1 gene expression 2.6‐fold. Deletion analysis of the MDR1 promoter indicated that the transcriptional activation after exposure to arsenite depends on a 60‐bp region containing two heat‐shock responsive elements.

Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene (MDR1) expression caused by arsenite

Expression of the MDR1 gene, which encodes P‐glycoprotein, is increased under some stress conditions. We have reported that quercetin, a bioflavonoid, inhibits the expression of heat‐shock proteins. We have identified the effects of quercetin on the MDR1 gene expression in the human hepatocarcinoma cells line, HepG2. The increase of P‐glycoprotein synthesis and MDR1 mRNA accumulation caused by exposure to arsenite were inhibited by quercetin. The CAT assay suggested that quercetin suppressed the transcriptional activation of the MDR1 gene after exposure to arsenite. Although many drugs that prevent the P‐glycoprotein function have been reported, this is the first report to describe the inhibition of MDR1 expression by a reagent.

Structure and organization of Marchantia polymorpha chloroplast genome. IV: Inverted repeat and small single copy regions

We characterized the genes in the regions of large inverted repeats (IRA and IRB, 10,058 base-pairs each) and a small single copy (SSC 19,813 bp) of chloroplast DNA from Marchantia polymorpha. The inverted repeat (IR) regions contain genes for four ribosomal RNAs (16 S, 23 S, 4.5 S and 5 S rRNAs) and five transfer RNAs (valine tRNA(GAC), isoleucine tRNA(GAU), alanine tRNA(UGC), arginine tRNA(ACG) and asparagine tRNA(GUU)). The gene organization of the IR regions in the liverwort chloroplast genome is conserved, although the IR regions are smaller (10,058 base-pairs) than any reported in higher plant chloroplasts. The small single-copy region (19,813 base-pairs) encoded genes for 17 open reading frames, a leucine tRNA(UAG) and a proline tRNA(GGG)-like sequence. We identified 12 open reading frames by homology of their coding sequences to a 4Fe-4S-type ferredoxin protein, a bacterial nitrogenase reductase component (Fe-protein), five human mitochondrial components of NADH dehydrogenase (ND1, ND4, ND4L, ND5 and ND6), two Escherichia coli ribosomal proteins (S15 and L21), two putative proteins encoded in the kinetoplast maxicircle DNA of Leishmania tarentolae (LtORF 3 and LtORF 4), and a bacterial permease inner membrane component (encoded by malF in E. coli or hisQ in Salmonella typhimurium).

Functional analysis of the fic gene involved in regulation of cell division

We have shown that cAMP may be a regulation factor in cell division of Escherichia coli (Utsumi et al., 1982, 1989). The fic (filamentation induced by cAMP) gene of this system found previously (Utsumi, et al., 1982) was analysed in this study. The open reading frame of the fic gene coded for 200 amino acids. The pabA (p-aminobenzoate synthase) gene was found downstream from the fic gene. The distance between the end of fic and the start of pabA was 31 base pairs. To deduce the function of Fic protein, the fic gene was destroyed by the kanamycin-resistant (Kmr) gene and the fic gene was shown to be essential for growth of E. coli. Such mutants required PAB (p-aminobenzoate) or folate for growth. These data suggested that the Fic protein is involved in the synthesis of PAB or folate and the fic gene could be part of a pab operon. In cells starved of them, cell division was inhibited. Addition of folate also repressed the filamentation induced by cAMP at 43 degrees C in the fic-1 mutant. These results would indicate that Fic protein and cAMP are involved in a new regulatory mechanism of cell division via folate metabolism. Furthermore, it is also shown that cell division could be controlled by coordination of cAMP, Fic and Fts proteins.

Divergent mRNA transcription in the chloroplast psbB operon.

The genes psbB, psbH, petB and petD for the components in photosystem II and the cytochrome b6/f complex are clustered and co‐transcribed in liverwort Marchantia polymorpha chloroplasts. On the opposite DNA strand in the spacer region between the genes psbB and psbH, we deduced an open reading frame consisting of 43 sense codons, and designated it as the ORF43 gene. The ORF43 gene was actively transcribed in liverwort chloroplasts. The ORF43 transcripts were entirely complementary to a part of the primary transcripts of the psbB operon. Heterogeneous Northern hybridization showed that the mRNA transcripts for the ORF43 gene increased with greening in pea seedlings. This is the first demonstration of divergent overlapping transcription in chloroplasts.

Physical mappings of chloroplast DNA from liverwort Marchantia polymorpha L. cell suspension cultures

SummaryPhysical maps of liverwort Marchantia polymorpha chloroplast DNA were constructed with restriction endonucleases, BamHI, SmaI, KpnI, and XhoI. The molecular size, calculated from the sum of the restriction fragments, is 121.0±1.0 kilobase (kb) pairs. This value is in good agreement with that of 118.7±2.0 kb pairs obtained from contour length measurements of the chloroplast DNA electron micrographs. The physical map indicates that the chloroplast DNA contains two copies of inverted repeats. The length of the inverted repeat is at most 11.7 kb pairs. Southern hybridization analysis indicates that two sets of the chloroplast ribosomal RNA genes are located in the inverted repeat regions. The site of the ribulose bisphosphate (RuBP) carboxylase (large subunit) gene was also determined on a BamHI fragment, Ba5, by using 32P-labeled DNA fragments containing the tobacco chloroplast RuBP carboxylase gene as a probe.

Non-equivalent cooperation between the two nucleotide-binding folds of P-glycoprotein

To identify the roles of the two nucleotide-binding folds (NBFs) in the function of human P-glycoprotein, a multidrug transporter, we mutated the key lysine residues to methionines and the cysteine residues to alanines in the Walker A (WA) motifs (the core consensus sequence) in the NBFs. We examined the effects of these mutations on N-ethylmaleimide (NEM) and ATP binding, as well as on the vanadate-induced nucleotide trapping with 8-azido-[alpha-32P]ATP. Mutation of the WA lysine or NEM binding cysteine in either of the NBFs blocked vanadate-induced nucleotide trapping of P-glycoprotein. These results suggest that if one NBF is non-functional, there is no ATP hydrolysis even if the other functional NBF contains a bound nucleotide, further indicating the strong cooperation between the two NBFs of P-glycoprotein. However, we found that the effect of NEM modification at one NBF on ATP binding at the other NBF was not equivalent, suggesting a non-equivalency of the role of the two NBFs in P-glycoprotein function.