Abstract
In cancer and AIDS, overexpression of the MDR1 gene has important implications in the design of chemotherapy protocols because of the ability of its product, the ATPdependent drug efflux pump P-glycoprotein (Pgp), to confer selective advantage to tumor and HIV-infected cells in the form of multidrug resistance. To study Pgp expression and physiology, we designed a translational fusion between the MDR1 and enhanced green fluorescent protein (EGFP) genes. The chimeric protein, Pgp-EGFP, was concentrated mainly in the plasma membrane and in the Golgi when expressed in drug-sensitive KB- 3-1 cells. Doxorubicin, daunorubicin and rhodamine-123 efflux assays confirmed function of the chimeric pump. Also, at the single-cell level, an inverse relationship between Pgp-EGFP expression and nuclear doxorubicin accumulation was demonstrated. Polarized Pgp expression on the apical cell surface was confirmed by transfection of the MDR-EGFP fusion gene into MDCK cells. However, after colchicine selection, Pgp-EGFP was also detectable in the lateral domain of the transfected MDCK monolayers. These results indicate that drug selection affects not only expression, but cellular localization of Pgp. Furthermore, using a tet-based inducible expression system for Pgp-EGFP, we confirmed the stable nature of Pgp (t1 / 2 total Pgp-EGFP= 2.2 days), but revealed that surface-Pgp acquires extra stability as an active pump (t1 / 2 surface Pgp-EGFP= 3.7 days).
Keywords: p-glycoprotein, green fluorescent protein, drug selection, turnover, subcellular localization
Current Drug Delivery
Title: An MDR-EGFP Gene Fusion Allows for Direct Cellular Localization, Function and Stability Assessment of P-Glycoprotein
Volume: 1 Issue: 1
Author(s): Jordi Petriz, Michael M. Gottesman and Josep M. Aran
Affiliation:
Keywords: p-glycoprotein, green fluorescent protein, drug selection, turnover, subcellular localization
Abstract: In cancer and AIDS, overexpression of the MDR1 gene has important implications in the design of chemotherapy protocols because of the ability of its product, the ATPdependent drug efflux pump P-glycoprotein (Pgp), to confer selective advantage to tumor and HIV-infected cells in the form of multidrug resistance. To study Pgp expression and physiology, we designed a translational fusion between the MDR1 and enhanced green fluorescent protein (EGFP) genes. The chimeric protein, Pgp-EGFP, was concentrated mainly in the plasma membrane and in the Golgi when expressed in drug-sensitive KB- 3-1 cells. Doxorubicin, daunorubicin and rhodamine-123 efflux assays confirmed function of the chimeric pump. Also, at the single-cell level, an inverse relationship between Pgp-EGFP expression and nuclear doxorubicin accumulation was demonstrated. Polarized Pgp expression on the apical cell surface was confirmed by transfection of the MDR-EGFP fusion gene into MDCK cells. However, after colchicine selection, Pgp-EGFP was also detectable in the lateral domain of the transfected MDCK monolayers. These results indicate that drug selection affects not only expression, but cellular localization of Pgp. Furthermore, using a tet-based inducible expression system for Pgp-EGFP, we confirmed the stable nature of Pgp (t1 / 2 total Pgp-EGFP= 2.2 days), but revealed that surface-Pgp acquires extra stability as an active pump (t1 / 2 surface Pgp-EGFP= 3.7 days).
Export Options
About this article
Cite this article as:
Petriz Jordi, Gottesman M. Michael and Aran M. Josep, An MDR-EGFP Gene Fusion Allows for Direct Cellular Localization, Function and Stability Assessment of P-Glycoprotein, Current Drug Delivery 2004; 1 (1) . https://dx.doi.org/10.2174/1567201043480072
DOI https://dx.doi.org/10.2174/1567201043480072 |
Print ISSN 1567-2018 |
Publisher Name Bentham Science Publisher |
Online ISSN 1875-5704 |
Call for Papers in Thematic Issues
Advancements in Robotic and AI-based Drug Discovery and Delivery for Neurological Disorders
Neurological disorders, including Alzheimer's disease, Parkinson's disease, epilepsy, and brain tumors, pose significant challenges in drug delivery due to the blood-brain barrier's intricate structure and the complexity of neuronal networks. Traditional drug delivery methods often fall short in reaching therapeutic concentrations within the central nervous system, limiting treatment efficacy and ...read more
Advances of natural products, bio-actives and novel drug delivery system against emerging viral infections
Due to the increasing prevalence of viral infections and the ability of these human pathogens to develop resistance to current treatment strategies, there is a great need to find and develop new compounds to combat them. These molecules must have low toxicity, specific activity and high bioavailability. The most suitable ...read more
Electrospun Fibers as Drug Delivery Systems
In recent years, electrospun fibers have attracted considerable attention as potential platforms for drug delivery due to their distinctive properties and adaptability. These fibers feature a notable surface area-to-volume ratio and can be intentionally designed with high porosity, facilitating an increased capacity for drug loading and rendering them suitable for ...read more
Emerging Nanotherapeutics for Mitigation of Neurodegenerative Disorders
Conditions affecting the central nervous system (CNS) present a significant hurdle due to limited access of both treatments and diagnostic tools for the brain. The blood-brain barrier (BBB) acts as a barrier, restricting the passage of molecules from the bloodstream into the brain. The most formidable challenge facing scientists is ...read more
- Author Guidelines
- Graphical Abstracts
- Fabricating and Stating False Information
- Research Misconduct
- Post Publication Discussions and Corrections
- Publishing Ethics and Rectitude
- Increase Visibility of Your Article
- Archiving Policies
- Peer Review Workflow
- Order Your Article Before Print
- Promote Your Article
- Manuscript Transfer Facility
- Editorial Policies
- Allegations from Whistleblowers
Related Articles
-
Breast Cancer Stem Cells and Intrinsic Subtypes: Controversies Rage On
Current Stem Cell Research & Therapy Novel Colchicine Derivatives and their Anti-cancer Activity
Current Topics in Medicinal Chemistry The Redox Regulation of Thiol Dependent Signaling Pathways in Cancer
Current Pharmaceutical Design Update of Targeted Therapy-Induced Hypertension: Basics for Non-Oncology Providers
Current Hypertension Reviews Preclinical and Clinical Efficacy of the Bisphosphonate Ibandronate in Cancer Treatment
Current Clinical Pharmacology High Throughput Screening for Colorectal Cancer Specific Compounds
Combinatorial Chemistry & High Throughput Screening Gene Therapy in In Vivo Isolated Perfusion Models
Current Gene Therapy Dequalinium-Derived Nanoconstructs: A Promising Vehicle for Mitochondrial Targeting
Current Drug Delivery Synthesis, Computational Study, and Evaluation of In Vitro Antimicrobial, Antibiofilm, and Anticancer Activities of New Sulfanyl Aminonaphthoquinone Derivatives
Letters in Drug Design & Discovery Cellular Targets for Anticancer Strategies
Current Drug Targets Synthetic Approaches to Various Class of Topoisomerase II Inhibitors
Mini-Reviews in Organic Chemistry Anticancer Metallotherapeutics in Preclinical Development
Current Medicinal Chemistry Targeting RANK/RANKL in the Treatment of Solid Tumours and Myeloma
Current Pharmaceutical Design miRNA 21: Diagnostic Prognostic and Therapeutic Marker for Oral Cancer
MicroRNA Anti-GD2 Antibody Therapy for GD2-Expressing Tumors
Current Cancer Drug Targets Quantitative Structure-Activity Relationship Studies: Understanding the Mechanism of Tyrosine Kinase Inhibition
Current Enzyme Inhibition PET in Anti-Cancer Drug Development and Therapy
Recent Patents on Anti-Cancer Drug Discovery Attacking c-Myc: Targeted and Combined Therapies for Cancer
Current Pharmaceutical Design Polypharmacology of Approved Anticancer Drugs
Current Drug Targets Therapeutic Implications of Tocilizumab, A Humanized Anti-Interleukin-6 Receptor Antibody, for Various Immune-Mediated Diseases: An Update Review
Current Rheumatology Reviews