James M. Croop

Mammalian multidrug resistance gene: Complete cDNA sequence indicates strong homology to bacterial transport proteins

The complete nucleotide and primary structure (1276 amino acids) of a full length mdr cDNA capable of conferring a complete multidrug-resistant phenotype is presented. The deduced amino acid sequence suggests that mdr is a membrane glycoprotein which includes six pairs of transmembrane domains and a cluster of potentially N-linked glycosylation sites near the amino terminus. A striking feature of the protein is an internal duplication that includes approximately 500 amino acids. Each duplicated segment includes a consensus ATP-binding site. Amino acid homology is observed between the mdr gene and a series of bacterial transport genes. This strong homology suggests that a highly conserved functional unit involved in membrane transport is present in the mdr polypeptide. We propose that an energy-dependent transport mechanism is responsible for the multidrug-resistant phenotype.

The three mouse multidrug resistance (mdr) genes are expressed in a tissue-specific manner in normal mouse tissues.

The gene responsible for multidrug resistance (mdr), which encodes the P-glycoprotein, is a member of a multigene family. We have identified distinct mdr gene transcripts encoded by three separate mdr genes in the mouse. Expression levels of each mdr gene are dramatically different in various mouse tissues. Specific mdr RNA transcripts of approximately 4.5, 5, and 6 kilobases have been detected. Each of the mdr genes has a specific RNA transcript pattern. These results should be considered in relation to understanding the normal physiological function of the mdr multigene family.

Isolation and characterization of Drosophila multidrug resistance gene homologs.

Mammalian multidrug-resistant cell lines, selected for resistance to a single cytotoxic agent, display cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. These cell lines overproduce a membrane protein, the P-glycoprotein, which is encoded by a member(s) of a multigene family, termed mdr or pgp. The amino acid sequence of the P-glycoprotein predicts an energy-dependent transport protein with homology to a large superfamily of proteins which transport a wide variety of substances. This report describes the isolation and characterization of two Drosophila homologs of the mammalian mdr gene. These homologs, located in chromosomal sections 49EF and 65A, encode proteins that share over 40% amino acid identity to the human and murine mdr P-glycoproteins. Fly strains bearing disruptions in the homolog in section 49EF have been constructed and implicate this gene in conferring colchicine resistance to the organism. This work sets the foundation for the molecular and genetic analysis of mdr homologs in Drosophila melanogaster.

Isolation and characterization of a mammalian homolog of the Drosophila white gene.

The Drosophila melanogaster white gene is a member of the ABC transporter superfamily of ATPase transmembrane proteins and is involved in the cellular uptake of guanine and tryptophan. We have cloned and sequenced human and mouse homologs of white which share 55-58% amino acid similarity with the Drosophila protein. Northern analysis reveals that the mammalian homolog is highly expressed in several tissues, including brain, spleen, lung and placenta. We have localized the gene to human chromosome 21q22.3 by means of fluorescence in situ hybridization and linkage analysis using a (CA)n polymorphism. The human homolog maps to the interval between D21S212 and D21S171, a region which includes loci for bipolar affective disorder and a recessive form of deafness. Since tryptophan is a precursor for the neurotransmitter serotonin and neurotoxic metabolites of the kynurenine pathway, we propose that the human homolog of white is a suitable candidate gene for these neurological disorders in humans.

Genetics of multidrug resistance.

Despite major advances in the treatment of cancer, resistance to multiple chemotherapeutic agents remains a major cause of treatment failure. Some tumors are initially resistant to many ofthe most active antineoplastic agents. Other tumors that are initially sensitive to cytotoxic agents often recur and frequently develop resistance to a broad range ofchemotherapeutic agents including those used during the initial therapy. Both the intrinsic and acquired resistance to chemotherapeutic agents have been inherently difficult to study in the clinical setting. However, drug-resistant cell lines have provided a tool for studying the mechanisms underlying resistance to the drugs used in chemotherapy. Although drug-resistant cell lines are usually derived by selection for resistance to a single cytotoxic agent, they often develop cross-resistance to a broad spectrum of structurally and functionally unrelated compounds. This phenomenon has been termed multidrug resistance. Recently, an understanding of the biological basis of multidrug resistance has begun to emerge through the application of molecular genetics. This approach has led to the identification, isolation, and characterization of a gene family whose members include genes with the capacity to confer multidrug resistance on otherwise drug-sensitive cells. Molecular probes and antibodies developed as a consequence of genetic analysis promise a new and precise level of understanding of the role of this gene family in human physiology and tumor biology. This knowledge will in turn be crucial for the development of strategies for circumventing multidrug resistance in the clinical setting.

Role of P-glycoprotein in dolastatin 10 resistance

Dolastatin 10, a cytotoxic pentapeptide isolated from the mollusk Dolabella auricularia, exhibits potent antitumor activity. The present studies demonstrated that sublines of murine PC4 and human U-937 leukemia cells expressing a multidrug resistance (MDR) phenotype are cross-resistant to this agent. We also demonstrated that such resistance was reversed by verapamil. While these findings suggested the involvement of the P-glycoprotein (P-gp) in dolastatin 10 resistance, we performed similar studies in a CHO cell line transfected with the human mdr1 cDNA. Expression of P-gp in the transfected cells was associated with resistance to dolastatin 10 by a verapamil-sensitive mechanism. The demonstration that photoaffinity labeling of P-gp was decreased in the presence of dolastatin 10 further supports the interaction of this cytotoxic peptide with P-gp. Taken together, these findings suggest that resistance to dolastatin 10 is conferred, at least in part, by P-gp and that this cytotoxic peptide is a novel member of the MDR phenotype.

P-glycoproteins encoded by mdr 1b in murine gravid uterus and multidrug resistant tumor cell lines are differentially glycosylated.

There are 3 members of the multidrug‐resitance gene family expressed in mouse. Only one of these, mdr lb, and its gene product P‐glycoprotein are induced to high levels in the mouse endometrium during pregnancy. It is shown here that P‐glycoprotein in the gravid uterus is significantly larger (M r 155000) compared to P‐glycoprotein encoded by mdr lb in a murine multidrug‐resistant cell line (M r 140000). However, both species co‐migrate after enzymatic removal of N‐linked sugars (M r 125000). These results demonstrate that differential glycosylation of the mdr lb gene product contributes to molecular heterogeneity found in P‐glycoprotein from normal and multidrug‐resistant cells.