A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants.

The plasma membranes of hamster, mouse, and human tumor cell lines that display multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. In every case, increased expression of a 170,000-dalton surface antigen was found to be correlated with multidrug resistance. This membrane component is of identical molecular size and shares some immunogenic homology with the previously characterized P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have application to cancer therapy.

https://science.sciencemag.org/content/sci/221/4617/1285.full.pdf

Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines.

One reason for the failure of chemotherapy in the treatment of advanced cancers may be the outgrowth of multidrug-resistant tumour cells1–5. Multidrug resistance has been modelled in numerous mammalian cell lines in which the phenotype is characterized by a pleiotropic cross-resistance to unrelated drugs1–7. In the study reported here, we have produced monoclonal antibodies whose binding to plasma membranes of different multidrug-resistant mammalian cells correlates with the degree of drug resistance. All these antibodies are specific for P-glycoprotein, a cell surface component of relative molecular mass (Mr) 170,000 (170K) that has been described previously8–15, and are directed against three spatially distinct epitopes which define a conserved cytoplasmic domain in the C-terminal region of the P-glycoprotein polypeptide. The conserved nature of P-glycoprotein and its low-level expression in drug-sensitive cells suggest that it has an important function at the cell surface. The monoclonal antibodies against P-glycoprotein described here might serve as diagnostic reagents for clinically unresponsive tumours16.

Detection of P-glycoprotein in multidrug-resistant cell lines by monoclonal antibodies

The ATP binding cassette (ABC) superfamily of membrane transporters is one of the largest protein classes known, and counts numerous proteins involved in the trafficking of biological molecules across cell membranes. The first known human ABC transporter was P-glycoprotein (P-gp), which confers multidrug resistance (MDR) to anticancer drugs. In recent years, we have obtained an increased understanding of the mechanism of action of P-gp as its ATPase activity, substrate specificity and pharmacokinetic interactions have been investigated. This review focuses on the functional characterization of P-gp, as well as other ABC transporters involved in MDR: the family of multidrug-resistance-associated proteins (MRP1-7), and the recently discovered ABC half-transporter MXR (also known as BCRP, ABCP and ABCG2). We describe recent progress in the analysis of protein structure-function relationships, and consider the conceptual problem of defining and identifying substrates and inhibitors of MDR. An in-depth discussion follows of how coupling of nucleotide hydrolysis to substrate transport takes place, and we propose a scheme for the mechanism of P-gp function. Finally, the clinical correlations, both for reversal of MDR in cancer and for drug delivery, are discussed.

From MDR to MXR: new understanding of multidrug resistance systems, their properties and clinical significance.

3.2. Other Potential in vivo Functions of Pgp 2992 3.3. Physiological Role of ABCG2 2992 3.4. Physiological Role of MRP1 2992 4. Structure of ABC Superfamily Drug Efflux Pumps 2992 4.1. Domain Structure of ABC Proteins 2992 4.2. Structure of Entire Bacterial ABC Proteins 2993 4.3. Structure of Pgp, MRP1, and ABCG2 2994 5. Substrate Specificity of ABC Drug Efflux Pumps 2995 5.1. MDR Spectrum Substrates 2995 5.2. Binding and Transport of Drugs 2998 5.3. Multidrug-Binding Pockets 2998 6. Catalytic Cycle of ABC Drug Efflux Pumps 3000 6.1. ATP Binding and Hydrolysis 3000 6.2. Occluded Nucleotide Conformation of Pgp 3000 6.3. Role of NBD Dimerization and the Occluded Conformation in the Catalytic Cycle of Pgp 3000

ABC efflux pump-based resistance to chemotherapy drugs.

Independent colchicine-resistant (CHR) mutants of Chinese hamster ovary cells displaying reduced permeability to colchicine have been isolated. A distinguishing feature of these membrane-altered mutants is their pleiotropic cross-resistance to a variety of unrelated compounds. Genetic characterization of the CHR lines indicate that colchicine resistance and cross-resistance to other drugs are of a dominant nature in somatic cell hybrids. Revertants of CHR have been isolated which display decreased resistance to colchicine and a corresponding decrease in resistance to other drugs. These results strongly suggest that colchicine resistance and the pleiotropic cross-resistance are the result of the same mutation(s). Biochemical studies indicate that although colchicine is transported into our cells by passive diffusion, no major alterations in the membrane lipids could be detected in mutant cells. However, there appears to be an energy-dependent process in these cells which actively maintains a permeability barrier against colchicine and other drugs. The CHR cells might be altered in this process. A new glycoprotein has been identified in mutant cell membranes which is not present in parental cells, and is greatly reduced in revertant cells. A model for colchicine-resistance is proposed which suggests that certain membrane proteins such as the new glycoprotein of CHR cells, are modulators of membrane fluidity (mmf proteins) whose molecular conformation regulates membrane permeability to a variety of compounds and that the CHR mutants are altered in their mmf proteins. The possible importance of the CHR cells as models for investigating aspects of chemotherapy related to drug resistance is discussed.

Victor Ling

Papers

Drug resistance and membrane alteration in mutants of mammalian cells.