Multidrug resistance and cystic fibrosis genes: complementarity of function?
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Biomedical subjects
V Ling
Publications and source records attributed to V Ling.
Multidrug resistance in cancers of childhood: clinical relevance and circumvention.
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Animals↗
The core promoter region of the P-glycoprotein gene is sufficient to confer differential responsiveness to wild-type and mutant p53.
The overexpression of P-glycoprotein is thought to be responsible for resistance to chemotherapy in some non-responsive cancers. The mechanism by which P-glycoprotein is overexpressed in human tumors is poorly understood. However, several lines of evidence suggest that the major regulatory mechanism of P-glycoprotein overexpression in human tumors is at the transcriptional level. During tumor progression one of the most commonly observed alterations is mutation of the p53 tumor-suppressor gene. It has been shown that the p53 protein plays a role in transcriptional regulation. To gain insight into the effect p53 protein may have on P-glycoprotein promoter activity, we transiently co-transfected plasmids containing the hamster pgp1 or human mdr1 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene with plasmids encoding either wild-type or mutant p53 protein into Chinese hamster ovary (CHO) cells. In this report, we show that wild-type p53 protein represses P-glycoprotein promoter activity, while mutant forms of p53 protein enhance P-glycoprotein promoter activity. Furthermore, we present data which indicate that the transcriptional regulatory effects of p53 are mediated through interactions with pgp1/mdr1 core promoter sequences. These findings have implications for our understanding of the molecular mechanism(s) by which p53 protein functions as a transcriptional regulator of gene expression. In addition, our results suggest a mechanism by which P-glycoprotein may be overexpressed in human cancers that also express mutant forms of p53 protein.
Animals↗
P-glycoprotein genes in the winter flounder, Pleuronectes americanus: isolation of two types of genomic clones carrying 3' terminal exons.
In mammals, P-glycoprotein (P-gp) is encoded by two or more highly conserved genes that differ in their abilities to transport drugs. One isoform class (class I) is consistently associated with the multidrug resistance phenotype, while the other (class III) is not. This study was designed to enumerate the P-gp genes in fish and determine how they are related to the two functional classes already defined in mammals. Southern blot analysis using a conserved single exon from the 3' terminal region of hamster P-gp cDNA (pEX1-172) as a probe indicated that there were two P-gp genes in right-eye flounders. Subsequently, two sets of clones were isolated from a winter flounder genomic library that correspond to the 3' ends of the two flounder P-gp genes. Sequence analysis was done on two key areas: the 3' ATP binding site and the 3' terminal exon, both of which were found to be homologous with their mammalian counterparts. Despite high levels of sequence identity in the predicted coding regions of the gene fragments it has not been possible to use these sequences to relate the homologs to particular mammalian classes of P-gp genes, perhaps because of gene conversion between mammalian P-gp genes. These cloned sequences are the first set of P-gp genes reported in lower vertebrates and will be useful for delineating the expression of P-gp genes in fish and understanding the role of P-gp in fish physiology.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
P-glycoprotein expression during tumor progression in the rat liver.
P-Glycoprotein (Pgp) has been shown to mediate multidrug resistance in tumor cell lines. Overexpression of Pgp has been detected in clinical cancer samples of many histological types. The basis and biological significance of such increases in Pgp expression are not well understood. In this study, the expression of Pgp during stepwise progression to rat liver cancer was examined to investigate the possible role of Pgp in carcinogenesis. An immunohistochemical technique was used to detect Pgp at the single-cell level, in a large number of liver nodules, hepatocellular carcinoma, and in distant metastases of the carcinomas. The results showed that distinct changes in Pgp expression occurred during stepwise liver carcinogenesis and that these changes were closely associated with the microscopic anatomy of the lesions. In contrast to gamma-glutamyl transpeptidase and glutathione S-transferase-7.7, whose expression appeared to correlate with the early steps of liver carcinogenesis, Pgp expression was higher in the large hyperplastic nodules and in hepatocellular carcinomas than in the early microscopic lesions. A particularly striking finding was the consistent expression of Pgp in the lung metastases. These findings suggested that Pgp was associated with a more progressed malignant phenotype in liver carcinogenesis.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Charles F. Kettering Prize. P-glycoprotein and resistance to anticancer drugs.
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ATP Binding Cassette Transporter, Subfamily B, Mem↗
Amino acid substitutions in the sixth transmembrane domain of P-glycoprotein alter multidrug resistance.
Eukaryotic cells can display resistance to a wide range of natural-product chemotheraputic agents by the expression of P-glycoprotein (pgp), a putative plasma membrane transporter that is thought to mediate the efflux of these agents from cells. We have identified, in cells selected for multidrug resistance with actinomycin D, a mutant form of pgp that contains two amino acid substitutions within the putative sixth transmembrane domain. In transfection experiments, this altered pgp confers a cross-resistance phenotype that is altered significantly from that conferred by the normal protein, displaying maximal resistance to actinomycin D. These results strongly implicate the sixth transmembrane domain in the mechanism of pgp drug recognition and efflux. Moreover, they indicate a close functional homology between pgp and the cystic fibrosis transmembrane regulator in which the sixth transmembrane domain has also been shown to influence substrate specificity.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Identification of distinct P-glycoprotein gene sequences in rat.
In higher vertebrates, P-glycoprotein is usually encoded by a small family of genes. We have determined that the rat contains three P-glycoprotein genes and have cloned distinct genomic fragments containing the putative 3' untranslated regions of these P-glycoprotein genes. Sequence analysis indicates that the rat P-glycoprotein genes belong to the three P-glycoprotein classes identified in mammals. These cloned sequences will be useful for delineating the expression of P-glycoprotein genes in the rat. We have also isolated a fourth clone which contains only a short, but highly conserved P-glycoprotein domain. This clone appears not be a member of the P-glycoprotein gene family, and its relationship to P-glycoprotein is unknown.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Characterization of the hemolysin transporter, HlyB, using an epitope insertion.
The prokaryotic hlyB gene product is a member of a superfamily of ATP-binding transport proteins that include the eukaryotic multidrug-resistance P-glycoprotein, the yeast STE6, and the cystic fibrosis CFTR gene products (Juranka, P. F., Zastawny, R. L., and Ling, V. (1989) FASEB J. 3, 2583-2592). Previous genetic studies have indicated that HlyB is involved in the transport of the 107-kDa HlyA protein from Escherichia coli; however, the HlyB protein has not been purified for biochemical studies due to its low abundance. In this study, we have engineered a monoclonal antibody epitope into the C-terminal end of HlyB that did not destroy its function. This has allowed us to use immunological methods to identify and localize various molecular forms of the HlyB protein present in vivo.
Amino Acid Sequence↗
Cellular distribution of calmodulin and calmodulin-binding proteins in Vicia faba L.
The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. 125I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with Mr 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (Mr 39,000, 88,000), stems (Mr 45,000, 60,000, 64,000), and roots (Mr 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.
Calmodulin↗
Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry.
A detailed tryptic map is presented for recombinant human tissue plasminogen activator (rt-PA). Electrospray ionization mass spectrometry is utilized as an on-line HPLC detector for tryptic mapping of this glycoprotein. The additional dimension provided by mass spectrometry gives considerably more detail about the complex tryptic map and significantly enhances the high-resolution chromatographic separation by distinguishing by mass any coeluting components. Through this improvement, the proline isomers of a tryptic peptide were observed eluting over a broad range of retention times. The glycopeptides of rt-PA are observed as well as any corresponding nonglycosylated peptides. In addition, the carbohydrate heterogeneity is readily observed, allowing analysis of the carbohydrate composition. The characteristic diagonal patterns formed by glycopeptides in a contour plot of the data allow rapid recognition of the glycopeptides.
Amino Acid Sequence↗
P-glycoprotein expression as a predictor of the outcome of therapy for neuroblastoma.
BACKGROUND AND METHODS: Multidrug resistance in chemotherapy for cancer is characterized by increased genetic expression of P-glycoprotein, which acts as an ATP-dependent drug-efflux pump. To determine whether P-glycoprotein levels are of prognostic value in such cases, we measured these levels immunohistochemically in a retrospective study of sequential tumor samples from 67 children with neuroblastoma. RESULTS: P-glycoprotein was not detected in pretreatment samples from either of the 2 patients with Stage I disease, any of the 21 with Stage II disease, or any of the 8 with Stage IVS disease, but it was detected in the samples from 1 of the 17 patients with Stage III disease (6 percent) and 12 of the 19 with Stage IV disease (63 percent). Of the 44 patients with nonlocalized neuroblastoma (Stage III, IVS, or IV), 26 of the 31 who were negative for P-glycoprotein had a complete response to primary treatment, as compared with 6 of the 13 who were positive for P-glycoprotein (84 percent vs. 46 percent, P = 0.0232 by Fisher's exact test). Log-rank analysis of outcome, with simultaneous stratification according to tumor stage and age, showed that the group that was negative for P-glycoprotein had significantly longer relapse-free survival (P = 0.0011) and overall survival (P = 0.0373) than the group that was positive. CONCLUSIONS: Expression of P-glycoprotein before treatment may predict the success or failure of therapy for nonlocalized neuroblastoma. Neuroblastoma may be a promising tumor to treat with anticancer drug therapy combined with a chemosensitizing agent capable of reversing P-glycoprotein-mediated multidrug resistance.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Study of membrane orientation and glycosylated extracellular loops of mouse P-glycoprotein by in vitro translation.
Increased expression of P-glycoprotein (Pgp) has been demonstrated to cause multidrug resistance (MDR) in vitro, and it may be responsible for chemotherapy failure in a number of human cancers. Pgp is a plasma membrane protein thought to function as an energy-dependent drug transporter. From its deduced protein sequence the topology of Pgp was proposed to contain 12 transmembrane domains with six extracellular loops and two cytoplasmic ATP-binding sites. To investigate further the membrane orientation of Pgp, we have expressed a full length cDNA of mouse mdr1, as well as its truncated forms, in a cell-free system supplemented with dog pancreatic microsomal membranes (RM). We determined which domains of the in vitro-synthesized Pgp had transversed the RM membranes by analyzing their resistance to protease digestion and their glycosylation state. To our surprise, this system revealed that a significant portion of in vitro-synthesized Pgp molecules has an additional glycosylated domain in the C-terminal half. Previously, only the first predicted extracellular loop near the N terminus had been thought to be glycosylated. Furthermore, we discovered that Pgp has at least two functional signal recognition particle/docking protein dependent signal sequences, one at the N-terminal half and the other at the C-terminal half. These findings suggest a new topological model for in vitro synthesized P-glycoprotein which may be relevant to its in vivo topology.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Isolation and characterization of a succinimide variant of methionyl human growth hormone.
Deamidation of asparagine and glutamine residues, isomerization of aspartic acid side chains, and racemization of the L- to the D-form of the amino acids are common spontaneous chemical reactions known to occur in proteins. Previous studies have implicated succinimides as intermediates in these reactions; however, the evidence has been indirect. Our results demonstrate, for the first time, the presence of a succinimide intermediate in an intact protein. The succinimide (cyclic imide) variant was isolated from thermally stressed recombinant methionyl human growth hormone (hGH) by high performance anion-exchange chromatography, further purified by reversed-phase high performance liquid chromatography, and analyzed by tryptic mapping. A later eluting tryptic peptide, compared with the native T12 peptide (residues 128-134, Leu-Glu-Asp-Gly-Ser-Pro-Arg), was analyzed by mass spectrometry (MS). This variant had a protonated molecular mass of 755.3 atomic mass units (u), as compared with 773.3 u for the native T12 peptide. A difference of 18 u, a loss of water, is consistent with the formation of a succinimide intermediate at Asp-130 of methionyl hGH. MS/MS analysis of the cyclic imide-containing peptide verified that the modification occurred at Asp-130. A difference of 18 u was also observed for the intact cyclic imide methionyl hGH variant (22,238 u), as measured by electrospray mass spectrometry, compared with native methionyl hGH (22,256 u).
Amino Acid Sequence↗
Deamidation of soluble CD4 at asparagine-52 results in reduced binding capacity for the HIV-1 envelope glycoprotein gp120.
High-performance cation-exchange chromatography of recombinant soluble CD4 (rCD4) allowed the resolution of four charge variants. This charge heterogeneity could be eliminated by neuraminidase treatment of rCD4 and therefore can be attributed to different degrees of sialylation of the carbohydrate portion of this glycoprotein. A single acidic variant was observed upon cation-exchange chromatography of neuraminidase-treated rCD4 that had been stored in liquid solution, pH 7.2, at 25 degrees C for 6 months. This acidic variant was isolated by semipreparative cation-exchange chromatography and subjected to tryptic mapping analysis. Tryptic peptides were characterized by fast atom bombardment mass spectrometry (FABMS). The results of this analysis demonstrated that the acidic variant of neuraminidase-treated rCD4 is generated from deamidation at Asn-52. Digestion of the deamidated rCD4 with endoproteinase Asp-N confirmed Asn-52 as the primary site of deamidation. The ability of the deamidated rCD4 variant to bind gp120 was assessed by use of an ELISA-based binding assay. The binding capacity of the deamidated variant was 24% of the binding capacity of unmodified rCD4. The overall structure of the V1 domain in the deamidated variant was not markedly different from that of the native protein as probed with eight conformationally dependent anti-V1 monoclonal antibodies. Therefore, it appears that Asn-52 is directly involved in binding to gp120.
Amino Acid Sequence↗
Modulation of ATP and drug binding by monoclonal antibodies against P-glycoprotein.
The role of P-glycoprotein in mediating the drug-resistance phenotype in multidrug resistant cells is now well documented. It is thought to function as an energy-dependent drug-efflux pump of broad specificity. Structurally, P-glycoprotein is an internally duplicated molecule containing two large multi-spanning transmembrane domains and two cytoplasmic ATP binding domains. In this report we demonstrate that monoclonal antibodies C219, C494, and C32 directed against short linear regions of the P-glycoprotein molecule inhibit ATP binding to P-glycoprotein in vitro. We also provide direct evidence that both predicted ATP-binding domains bind ATP and that there is co-operativity between the two sites. In addition, the capacity of P-glycoprotein to bind the calcium channel blocker, azidopine, is inhibited differentially by the antibodies. These observations are the first evidence linking specific perturbations of the P-glycoprotein molecule with ATP and drug binding.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Primary structures of Arabidopsis calmodulin isoforms deduced from the sequences of cDNA clones.
Complementary DNA (cDNA) clones encoding calmodulin isoforms were isolated from an Arabidopsis leaf lambdagt10 library by screening with cloned barley calmodulin cDNA probes. Two cDNAs, one a 626-base pair partial-length clone (ACaM-1) and one a 1400-base pair full-length clone (ACaM-2), encode calmodulin polypeptides that differ by four conservative amino acid substitutions. None of the amino acid sequence differences occur within the four Ca(2+)-binding domains of the proteins. Whereas the deduced amino acid sequences of the two Arabidopsis calmodulin isoforms share 97% identity, the nucleotide sequences encoding the two isoforms share 87% sequence identity. Most of these nucleotide sequence differences (80%) occur in codon wobble positions. ACaM-1 and ACaM-2 both hybridize with a distinct set of restriction fragments of Arabidopsis total DNA, indicating that they were derived from transcripts of separate genes; these genes are single- or very low-copy in the Arabidopsis genome. Both cDNAs hybridize to messenger RNA (mRNA) species of 0.8 kilobases that are expressed to a greater extent in developing siliques compared with leaves, flowers, and stems. Northern blot and polymerase chain reaction assays both indicate that ACaM-1 mRNA is more highly expressed than ACaM-2 mRNA in developing siliques. The steady-state levels of both isoform mRNAs increase as a result of touch stimulation; the kinetics and extent of increase are comparable for the two mRNAs.
Journal Article↗
Complete cDNA sequences encoding the Chinese hamster P-glycoprotein gene family.
The only function of the transport protein P-glycoprotein (Pgp) that has been identified to date in mammals is its ability to mediate multidrug resistance (MDR) in tumour cell lines. Rodents have three P-glycoprotein (pgp) genes (termed pgp or mdr 1, 2 and 3), and humans have two (mdr1 and mdr3/mdr2). Pgp isoforms differ in their drug transport capabilities: Pgp1 and Pgp2 can mediate MDR, while Pgp3 apparently cannot. The expression of the gene family members is tissue-specific, suggesting that they have unique physiological roles. We report in this paper the complete cDNA sequences for each of the three pgp genes in Chinese hamster. A comparison of the Chinese hamster cDNA sequences with those isolated from human and mouse confirms the identification of the gene family member homologues across these species. An analysis of mammalian Pgp sequences identifies conserved sequences which, it may be speculated, are important for Pgp activity. Previously, three different mdr3 (pgp3 homologous) transcripts, products of alternative splicing, have been reported in humans. Unexpectedly, we find no evidence for a similar alternative splicing event in Chinese hamster: it appears that the expression of pgp3 (mdr3) is different between rodents and humans.
ATP Binding Cassette Transporter, Subfamily B, Mem↗