structural studies of multi-drug resistance p-glycoprotein by electron microscopy

Dunn, Co-Chairpersons Multi-drug resistance P-glycoprotein Pgp, a member of the ATP-binding cassette transporter superfamily, is associated with multi-drug resistance in cancer chemother

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ACKNOWLEDGEMENTS

With everybody’s wishes, | came to University of California, Riverside in

Fall 2000 and entered the graduate program in biochemistry and molecular biology The graduate school life began, and for nearly five years and half, | have reached another milestone in my life It has been sweet and bitter, but | have enjoyed everything including research, cultures and friendship

My parents are whom | want to thank first Without their support and

encouragement, | would not be able to pursue my scientific career and would not reach this far Secondly, | want to appreciate and dedicate my respect to my

Ph.D mentor, Dr Stephan Wilkens Like a friend and a teacher, he has guided

me through my research project, and helped me tackle many problems during my

dissertation study even although we have had disagreement each other in some aspects His supportiveness and encouragement have made me dedicate at my

project, and thus | have learned problem solving and extensive thinking regarding

research In addition, | would thank both Dr Alan E Senior from University of

Rochester Medical Center and Dr Ina L Urbatsch from Texas Tech University Health Science Center Not just collaborators for this project, they have also

given me advices for my research as well Moreover, without the help from Dr Vinzenz M Unger in Yale University School of Medicine, | would not be able to

properly set up the system for 3-D image reconstruction and to obtain a 3-D

model of P-glycoprotein

Next, | want to dedicate my appreciation to Norton Kitagawa and Erik

Kish-Trier who have helped me a lot in experiments, and have given me suggestions and feedbacks for the dissertation writing | also want to thank Bob Richards, Rod Nakayama, Tarek Hawasly, John Peloquin and Fernando Minauro From these fellows, | have learned a lot about lab skills, attitudes for research and prospects of life In addition, | cannot forget how much Dr Krassimir Bozhilov from the EM facilities at UCR has assisted me in the use of the electron microscopes Finally, | want to thank both Drs Michael E Adams and Michael F Dunn They have served as my committee members from the

very beginning of my graduate school, and advised me for my research progress every year During the lab move to Syracuse, both of them were very supportive

to my decision and helped me to finish my dissertation Not only the committee members, they are my research advisors and friends as well

The text of this dissertation, in part or in full, is a reprint of the material as

is appeared in Lee et al, 2002 The co-author, Stephan Wilkens, listed in that publication directed and supervised the research which forms the basis for this dissertation The other co-authors, Alan Senior and Ina Urbatsch, purified P- glycoprotein and provided biochemical data for that publication.

To Dad, Mom, and two younger brothers,

Who have supported me all the time;

To beloved great-grandma, grandpa and grandma,

Who passed away in 2004 and 2005 and will never celebrate with me;

and

To University of California, Riverside, Which offers me the opportunity to accomplish my Ph.D degree

ABSTRACT OF THE DISSERTATION

Structural Studies of Multi-Drug Resistance P-glycoprotein

by Electron Microscopy

by

Jyh-Yeuan Lee

Doctor of Philosophy, Graduate Program in Biochemistry and Molecular Biology

University of California, Riverside, March 2006 Drs Stephan Wilkens & Michael F Dunn, Co-Chairpersons

Multi-drug resistance P-glycoprotein (Pgp), a member of the ATP-binding cassette transporter superfamily, is associated with multi-drug resistance in

cancer chemotherapy In addition to cancer cells, Pgp can be found in organs or

normal tissues, such as blood-tissue barriers, gastrointestinal tract or kidney, where it provides protection against toxic or xenobiotic materials This indicates that Pgp plays a dual role: one in drug resistance in cancer and one in detoxification in normal ceils, and it is important to investigate the mechanism of Pgp for understanding multi-drug resistance in cancer and for elucidating its physiological functions

P-glycoprotein is a single polypeptide consisting of ~1280 amino acids The protein contains two cytoplamic nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs) It is believed that Pgp undergoes significant

to gain insight into the conformational changes, the structure of Pgp was studied

by electron microscopy and image analysis First, two-dimensional (2-D) crystals

of Pgp were developed by the lipid-monolayer technique A projection structure

of Pgp in the absence of nucleotide or drug substrates was solved to 22-A

resolution The projection shows two closely interacting domains, thus providing evidence of a closed conformation of the NBDs Second, a subtle rearrangement

of the two domains has been observed in projection structures under various nucleotide conditions, showing the two domains shifted away to each other This indicates that Pgp undergoes conformational changes during the ATP hydrolytic cycle Yet, the conformational changes are subtle and may not induce a dramatic domain movement as proposed from crystal structures of bacterial lipid-A flippase (MsbA; Chang & Roth, 2001) Third, three-dimensional (3-D) structures

of membrane-bound Pgp in the nucleotide-free and ATPyS-bound states were obtained by 3-D reconstruction from tilted images of negatively stained specimen Based on the projections and 3-D models, although at a modest 22-A

resolution, a refined mechanism is presented that describes the domain-domain

interaction during the catalytic cycle This study illustrate the first detailed structural information of lipid-bilayer-bound Pgp showing a_ conformational

change in the functional domains due to ATP binding to the NBDs

1.2 ATP-binding Cassette Transporters

1.3 Human ABC Transporters in MDR

1.4 P-Glycoprotein

1.4.1 Background

1.4.2 Mechanism of Drug Resistance by Pgp

1.4.2.1 Alternating ATP Hydrolysis Mechanism

1.4.2.2 Drug Transport Mechanism

1.4.2.3 ATP-coupled Drug Transport

1.4.3 Structures of P-glycoprotein and other ABC transporters

1.4.3.1 Progress in the Studies of Pgp Structure

1.4.3.2 Structures of other ABC Transporters

1.4.3.3 Summary

1.5 Studies in this Dissertation Research

Chapter 2: Projection Structure of P-glycoprotein by Electron

Microscopy: Evidence for a Closed Conformation of the

Nucleotide Binding Domains

2.1 Abstract

2.2 Introduction

2.2.1 Interaction between the N- and C-terminal Halves of Pgp

2.2.2 Need for High-resolution Structural Information for Pgp

2.2.3 Two-dimensional (2-D) Electron Crystallography

2.2.4 Projection Structure of Pgp in the Lipid Bilayer

2.3 Results

2.3.1 Characterization of Pgp Used for 2-D Crystallization

2.3.2 Two-Dimensional Crystallization of Pgp

2.3.3 Image Analysis by Correlation Averaging

2.3.4 Comparison of the Projection Structure of Pgp with the

crystal structures of E coli MsbA and BtuCD

2.4 Discussion and Conclusion

2.4.2 Comparison of Projection Structures

2.5 Materials and Methods

2.5.1 Materials and Reagents

2.5.2 Native Agarose Gel Electrophoresis

2.5.3 Two-dimensional Crystallization of Pgp

2.5.4 Transmission Electron Microscopy

2.5.5 Image Analysis of the Pgp 2-D Crystals

Endnotes

Chapter 3: Projection Structures of Nucleotide-bound P-glycoprotein by

Electron Microscopy: Evidence of Conformational Change in

the Nucleotide-binding Domains of P-glycoprotein

3.1 Abstract

3.2 Introduction

3.2.1 Close Interaction of the Nucleotide-binding Domains in

ABC Transporters 3.2.2 Alternating ATP Hydrolysis

3.2.3 Conformational Change of the Nucleotide-binding Domains

3.2.4 Projection Structures of Substrate-bound Pgp in the Lipid

3.3.2 Two-dimensional Crystallization of Cysteine-less Pgp

3.3.3 Image Analysis by Correlation Averaging

3.3.4 Image Analysis of Frozen-hydrated 2-D Crystals

3.4 Discussion and Conclusion

3.4.1 Domain-domain Interaction in P-glycoprotein

3.4.2 Conformational Change of Pgp in the ATP Hydrolysis

Cycle 3.5 Materials and Methods

3.5.1 Materials and Reagents

3.5.2 Source of Cysteine-less Pgp for 2-D Crystallization

3.5.3 Fragmentation of Anti-Pgp Antibody C219

4.2.4 Three-dimensional Structures of P-glycoprotein

4.3 Results

4.3.1 Pgp 2-D Crystals Prepared in the Absence of Nucleotide or

Drug Substrates 4.3.2 Pgp 2-D Crystals Prepared in the presence of Mg-ATPyS

4.4 Discussion and Conclusion

4.4.1 Conformational Change in the Nucleotide-binding Domains

and in the Transmembrane domains 4.4.2 Three Dimensional Model of P-glycoprotein

4.5 Materials and Methods

4.5.1 2-D Crystallization and Electron Microscopy

4.5.2 3-D Image Analysis

Endnotes

Chapter 5: Purification of P-glycoprotein by Fast Performance Liquid

Chromatography And Two-dimensional Crystallization

5.4 Discussion and Conclusion

5.4.1 Expression and Purification of Cysteine-iess Human P-

glycoprotein

5.4.2 Two-dimensional Crystallization by Dialysis Technique

5.5 Materials and Methods

5.5.1 Chemicals and Reagents

5.5.2 Growth of Pichia pastoris Yeasts and Detection of Pgp

Expression 5.5.3 Purification of P-glycoprotein by FPLC

5.5.4 2-D Crystallization of Pgp by Dialysis and Transmission

Electron Microscopy Chapter 6: Conclusions

LIST OF TABLES

Table 3.1 Substrate Conditions for 2-D Crystals of P-glycoprotein

Table 3.2 Unit Cell Parameters and the Dimension of Single Pgp

Molecule

Table 4.1 Results of ALLSPACE for +20° Tilted Series

Table 4.2 Image Statistics (untilted projection structure of +20°

tilted series)

Table 4.3 Summary of 3-D Data Analysis for +20° Tilted Series

Table 4.4 Results of ALLSPACE for +40° Tilted Series

Table 4.5 Image Statistics (untilted projection structure of +40°

tilted series)

Table 4.6 Summary of 3-D Data Analysis for 40° Tilted Series

Table 5.1 Purification of Human Cysteine-less Pgp

LIST OF FIGURES

Figure 1.1 Proposed Mechanisms of Drug Resistance for

Chemotherapy

Figure 1.2 Arrangement of the Functional Domains of Multi-drug

Resistance Associated ABC Transporters in Human

Figure 1.3 Modes of Drug Resistance in Tumor Cells

Figure 1.4 Model of the Alternating ATP Hydrolysis Cycle in Pgp

Nucleotide-binding Domains (NBDs)

Figure 1.5 Drug Transport by P-glycoprotein

Figure 1.6 Scheme of ATP Hydrolysis by Pgp Coupled to Drug

Transport

Figure 1.7 The ATP Switch Model for the Transport Cycle of the

ABC Transporters

Figure 1.8 Mechanism of the Intercellular Transfer of Pgp

Figure 2.1 2-D Crystallization by the Lipid-monolayer Technique

Figure 2.2 Analysis of Pgp by Native Gel Electrophoresis and

Electron Microscopy

Figure 2.3 Aggregates of P-glycoprotein during 2-D Crystallization

Figure 2.4 Two Dimensional Crystals of Pgp

Figure 2.5 Examination of Protein Binding on the Lipid Monolayer

Figure 2.6 Correlation Averaging of Pgp 2-D Crystals

Figure 2.7 Comparison of the Pgp Projection Structure with the

Crystal Structures of MsbA and BtuCD

Figure 2.8 Comparison of the Projection Structures of Mouse Pgp

and Hamster Pgp

Figure 3.1 Schematic Diagram of Head-to-Tail Arrangement in the

Nucleotide-binding Domains of ABC Transporters

Figure 3.2 Schematic Diagram of Pepsin Digestion of IgG Antibody

Figure 3.3 Fragmentation of Anti-Pgp Antibody C219 IgG2a

Figure 3.4 Immunoblot Analysis of P-glycoprotein by Dot-blot

Technique

Figure 3.5 Giant 2-D Crystal Tubes of P-glycoprotein

Figure 3.6 Electron Micrograph of Frozen-hydrated 2-D Crystals of

Pgp

Figure 3.7 Observation of 2-D Crystals of Pgp by Fluorescence

microscopy and Transmission Electron Microscopy

Figure 3.8 Projection Structure of Nucleotide-free P-glycoprotein

Figure 3.9 Image Analysis of Pgp 2-D Crystals by Correlation

Figure 3.12 Projection Structures of ATPyS/TNP-ATP-bound P-

glycoprotein

Figure 3.13 Projection Structure of Antibody-labeled P-glycoprotein

Figure 3.14 Image Analysis of the Frozen-hydrated 2-D Crystals

Figure 3.15 Schematic Diagram of the Domain-domain Shifting in

P-glycoprotein

Figure 3.16 Projection Structures of Pgp v.s Current ATP

Hydrolysis Schemes

Figure 3.17 The Modified ATP Hydrolysis Scheme

Figure 3.18 Proposed Mechanism of Conformational Changes in

the NBDs of P-glycoprotein

Figure 4.1 Flow Chart of 3-D Image Reconstruction

Figure 4.2 P-glycoprotein 2-D Crystals Used for 3-D Image

Analysis

Figure 4.3 Analysis of the 2-D Crystals of Pgp in the Absence of

Nucleotide or Drug Substrates

Figure 4.4 Quality of the 3-D Data (+20° Series)

Figure 4.5 3-D Structure of Substrate-free Pgp

Figure 4.6 Analysis of the ATPyS-treated 2-D Crystals of Pgp

Figure 4.7 Analysis of the Effective Resolution Cutoffs for 3-D

Figure 4.9 3-D Structure of Pgp from ATPyS-treaed 2-D Crystals

Figure 4.10 Cross-sections of the 3-D Structure of Pgp

Figure 4.11 Comparison of the Nucleotide-binding Domains of Pgp

to the X-ray Crystal Structures of ATP-bound MJ0796

and Nucleotide-free MJ1267

Figure 4.12 Rearrangement of the Extracellular Loops (EC loops)

of Pgp

Figure 4.13 Proposed Mechanism of the Conformational Change of

Pgp upon ATP Binding

Figure 5.1 Analysis of Protein Purity

Figure 5.2 Analysis of Pgp Expression in Pichia pastoris

Figure 5.3 FPLC Profile of P-glycoprotein Purification (I)

Figure 5.4 FPLC Profile of P-glycoprotein Purification (11)

Figure 5.5 Comparison of Reconstituted Pgp proteoliposomes by

Dialysis and by Lipid-monolayer Technique

Figure 6.1 Illustration of Conformational Change in the NBDs of P-

CHAPTER 1

Introduction

Cancer has had a major impact on human health According to the World

Health Organization (WHO), about 11 million people are diagnosed with cancer

every year, and the disease has contributed to about 12.5% of deaths worldwide (http:/Awww.who.int/cancer/en/) Chemotherapy is currently the most practiced among the therapies for cancer During chemotherapy, drug resistance has been found to cause the failure of chemotherapy, especially multi-drug resistance (MDR) (Section 1.1) In both clinical and basic research, a large effort has been undertaken in order to understand the mechanism of MDR and to look for methods to overcome it A similar mechanism has also been found in normal tissues that function in detoxification or metabolite transport (Sections 1.2 & 1.3) This introduction will present a brief summary of current knowledge in MDR and

the molecules (or molecular complexes) involved in the process The last section

will outline the main body of the dissertation research that will be presented in Chapters 2, 3, 4 and 5

do not respond to chemotherapeutic agents, and only a minor group of patients

(~5-10%) can reach long-term survival One major factor for the failure of chemotherapy is drug resistance in which the cancer cells have found a way to

prevent the drugs from entering targets inside the cells

For decades, extensive studies about the mechanism of drug resistance

were performed on both cellular and molecular levels Michael Gottesman and

colleagues have reviewed the progress of this research since the early 1990s (Gottesman & Pastan, 1993; Ambudkar et al, 1999; Gottesman et al, 2002) Five possible mechanisms of drug resistance can be summarized as follows First, efflux of drugs from the cytoplasm to the extracellular surroundings can be enhanced by ATP-dependent drug-efflux transporters (Figure 1.1A) Drugs in this

case are usually amphipathic and contain a large hydrophobic portion Second, studies of the resistance to some water-soluble drugs suggested decreased

uptake of drugs from the outside of cells, by which cells fail to accumulate the drugs without evidence of efflux (Figure 1.1B) Third, a detoxification system,

such as cytochrome P450, can be activated and used to metabolize the drugs before reaching the targets (Figure 1.1C) Fourth, drug resistance can be

mediated by the expression of certain nuclear receptors, such as steroid and

xenobiotic receptor (SXR), which activates the mechanisms to repair drug-

induced DNA damage (Figure 1.1D) Fifth, malignant cancer cells can acquire

defective apoptosis with decease of ceramide levels by expression of mutant or non-functional p53 that normally suppresses tumor-associated genes (Figure 1.1E) In recent years, more and more studies at the transcription level have

suggested that genetic or epigenetic changes may be associated with the expression of drug-efflux transporter (Section 1.3) This adds more complexity to

the mechanism of drug resistance in chemotherapy of cancer Due to the genetic heterogeneity of cancer cells, the drug resistance phenomenon can be caused

by the combination of more than one mechanism as mentioned above

Therefore, this is also called “multi-factorial” multi-drug resistance

Drug resistance is usually broad-spectrum, i.e., many structurally unrelated organic compounds are removed from cancer cells This is especially

the case in the drug-efflux mechanism in which certain drug pumps are capable

of exporting a variety of drugs This broad-spectrum resistance to chemotherapy

is therefore called multi-drug resistance, or MDR for short Among the drugs relevant in multi-drug resistance are the vinka alkaloids (e.g., vinblastine and

vincristine), the anthracyclines (e.g., doxorubicin and daunorubicin), the

transcription inhibitors (e.g., actinomycin-D), microtubule-stabilizing drugs (e.g.,

colchicines and paclitaxel), and highly water-soluble drugs (e.g., antifolates or nucleotide analogs) Although the diversity of drugs shows differences in chemistry and structure, without consideration of water-soluble drugs, the majority are amphipathic compounds that usually contain a large hydrophobic

portion

In addition to cancer cells, phenomena similar to MDR can also be

observed in cells of normal tissues or in other organisms, such as bacteria,

nematodes and plants In human, for example, drug-efflux ATP-binding cassette transporters (ABC transporters, see section 1.2) have been found in the blood-

brain barrier (BBB) (L6scher and Potschka, 2005) Similar efflux system is also observed in other normal tissues such as liver, intestine, kidney, testis, and placenta (Bodo et al, 2003; Young et al, 2003) Expression of such kind of transporters facilitates removal of toxic materials from normal cells In bacteria,

such as Lactococcus lactis, LmrA is a multi-drug resistance protein (Poelarends

et al, 2002), whereas in Escherichia coli (E coli), ArsAB, an arsenic efflux pump, provides resistance to arsenite and antimonite In nematodes, such as Caenorhabditis elegans (C elegans), expression of human ABC transporter homologues has been shown to contribute to drug resistance and/or heavy metal

resistance (Broeks et al, 1995, 1996) In plants, such as Arabidopsis thaliana, homologues to human ABC transporter ABCC family were discovered to be

associated with resistance to xenobiotics (Theodoulou, 2000) The existence of

the efflux system in many types of cells and in many organisms suggests that the

mechanism derived from multi-drug resistance may be relevant to normal physiological functions Thus, understanding of the drug resistance would not only enable the future design of cancer therapies, but also be helpful in understanding the detoxification mechanism in normal tissues

1.2 ATP-binding Cassette Transporters

Among the proposed mechanisms of MDR, studies have been mostly focused on the stimulation of drug efflux system, and drug efflux has been shown

to be associated with the increased expression of the ATP-dependent transporters on the cell membrane (Gottesman & Pastan, 1993; Ambudkar et al, 1999; Gottesman et al, 2002; Litman et al, 2001) These transporters belong to

the family of the ATP-binding cassette transporters that were first introduced by

Christopher Higgins in a 1992 review (Higgins, 1992) ABC transporters are identified by the presence of a highly conserved ATP-binding cassette which lies

in the currently known nucleotide-binding domains (Holland & Blight, 1999;

Gottesman & Ambudkar, 2001; Higgins, 2001; Borst & Elferink, 2002) The

consensus sequences in the ATP-binding cassette include the P-loop motif (also called homology A or Walker A motif), the Q-loop motif (also called homology B

or Walker B motif) and the ABC signature motif LSGGQ In cancer multi-drug

resistance, three major types of ABC transporters are associated with the drug- efflux mechanism (Section 1.3) This includes the products of multi-drug resistance genes (MDR/PGY/Pgp/GP170), multi-drug-resistance-associated

protein genes (MRP), and mitoxantrone-resistance protein genes (MXR/BCRP)

(Bates et al, 2001; Kruh et al, 2001; Borst & Elferink, 2002) From topological prediction based on polypeptide sequences, all of the active proteins/complex contain the core domains consisting of two integral transmembrane domains (TMD1 & TMD2) and two cytoplasmic nucleotide-binding domains (NBD1 & NBD2) (Figure 1.2) In addition to the core domains, many MRP families have an extra transmembrane domain (TMD0O) and an extra cytoplamic loop (LO) (Figure 1.2B)

In human, cancer cells are not the only cell type that expresses ABC

transporters as drug-efflux pumps As mentioned earlier, MDR-related ABC

transporters have been observed in normal tissues for regular physiological purposes Moreover, other ABC transporters have been reported to serve as: 1) nutrient or metabolite transporters (e.g., ABCA1 for phospholipids/cholesterol transport on apical plasma membrane), 2) peptide transporters (e.g., TAP1/TAP2 complex for antigen presentation), 3) bile salt transporters (e.g., MRP3 in bile

ducts), 4) plant sterol transporters (e.g., ABCG5/G8 complex in intestine or liver), and so forth Up to date, there are 48 functional ABC transporters reported from

the human genome (Appendix A; Dean, 2002), and each member may also

contribute to certain human disease In 1999, a new universal nomenclature scheme was implemented for the human ABC and mouse Abc genes All 48 members from human are then further categorized into seven subfamilies: A, B,

C, D, E, F and G (http://www.gene.ucl.ac.uk/nomenclature/genefamily/abc.html) For example, MDR1, the first P-glycoprotein identified, is named ABCB1, and MXR is named ABCG2 In this dissertation, this new scheme will be mainly used

in all chapters ABCB1, however, will also be called P-glycoprotein (or Pgp in short) for convenience

The ABC transporters can also be found in other organisms In E coli, this family comprises almost 5% of the genome (Higgins, 2001) One function of

these transporters is to mediate the uptake of nutrients, e.g., maltose transporter

(MalFGK2), histidine permease (HisQMP) or Vitamin B12 importer (BtuCD) (Ehrmann et al, 1998; Késter, 2001) In addition, certain bacterial ABC

transporters can serve as drug-resistance pumps, such as LmrA from Lactococcus lactis that renders the bacterial resistance to cytotoxic compounds

and antibiotics Also, ABC transporters expressed in bacteria or nematodes play

a role in detoxification of xenobiotics For example, PGPA from Leishmania is involved in resistance to arsenite and antimonite while in C elegans, the nematodes can acquire resistance to heavy metals by expressing homologues to

human ABCB1 and ABCC1 (Broeks et al, 1996; Légaré et al, 2001) In recent

years, plant ABC transporters have been quickly determined after the

Arabidopsis genome was released The number (>120 members) far exceeds the comparable genomes from mammal (~50 members), and many are believed to

be involved in plant development (Lin & Wang, 2005; Geisler & Murphy, 2006)

The mechanisms of ABC transporters may vary depending on the physiological functions and the requirement of accessory components In

prokaryotes, the nutrient uptake usually requires the substrate-binding proteins to carry the nutrients to the transporters, e.g., maltose-binding protein (MBP) as to

maltose transporter (MalFGK2) in E coli In eukaryotes, taking ABCC1 as an example, glutathione is required to conjugate substrate for drug transport (Section 1.3) However, as for the cooperation between the core domains, it is generally believed that substrate transport through transmembrane domains is coupled with the energy provided by ATP binding and hydrolysis on the nucleotide-binding domains Studies in Pgp by Alan Senior and colleagues have led to a proposed alternating catalytic model (Senior et al, 1995a; Senior, 1998) Details will be discussed in Section 1.4 A modified model based on the alternating catalysis, the ATP switch model, further supports the idea of the cooperation between the core domains (Higgins & Linton, 2004)

The increasing number of ABC transporters from various organisms has broadened our view on this family of membrane protein, and accumulated

studies in the mechanism of ABC transporters have provided insights into how

molecules may be shuffled on both sides of membrane Therefore, discovery and characterization of different ABC transporters and their mechanisms will enrich our knowledge in the understanding of molecule transport and protection in

organisms

1.3 Human ABC Transporters in MDR

In human, over-expression of MDR-related ABC transporters is believed

one major cause for the failure of chemotherapy As briefly mentioned in Section 1.1, the main participants are ABCA2, ABCB1, ABCC1-3 and ABCG2 (Appendix A) The common features shared by these members are 1) that they are

expressed on the plasma membrane of the cells, where drugs or toxic materials are pumped out from the cytoplasmic side to extracellular side and 2) that they transport a broad range of structurally unrelated drugs

However, the difference between these transporters depends on tissue localization, types of drugs transported or the topological arrangement of their

polypeptide domains ABCB1 was first discovered through cancer cells, and it

has also been found in tissues such as tissue-blood barriers, renal and intestinal ducts, where ABCA2 has also been localized Drug transport via ABCB1 (and possibly ABCA2 as well) does not need conjugation with other reducing agents

This type of ABC transporter consists of a single polypeptide with two

transmembrane domains and two nucleotide-binding domains in the order of

TMD1-NBD1-TMD2-NBD2 (Figure 1.2A) This forms the core domains in ABC transporters Detailed information on ABCB1 (Pgp/MDR1) will be discussed in

Section 1.4

ABCC1 was first discovered to overlap the resistance phenotype of Pgp to

natural product drugs such as anthracyclines or vinca alkaloids, but such as taxol, ABCC1 did not confer resistance to such drug typically removed by Pgp Moreover, ABCC subfamily members require reducing agents such as glutathione to form conjugated compounds in order to perform drug efflux, which

is not the case in Pgp (Kruh et al, 2001; Kruh & Belinsky, 2003) Yet, similar to Pgp, each ABCC member is a single polypeptide with the core domains, and contains an extra transmembrane domain (TMDO) and an extra cytoplasmic loop (LO) at the N-terminal end, except for ABCC4, 5, 8 & 9 that do not have TMDO (Figure 1.2B) In addition to drug resistance, some members of ABCC subfamily have physiological substrates For example, ABCC1 was determined on the

basement membrane to transport leukotrienes ABCC1 is thus thought to be part

of the immune response ABCC4 has been reported to transport cyclic nucleotides, which suggests its association with signal transduction pathways

(Chen et al, 2001)

The last major player in human MDR is ABCG2 ABCG2 confers better resistance to drugs such as mitoxantrone than ABCB1 or ABCC1 (Bates et al, 2001) In normal tissues, its expression pattern is similar to Pgp, and it does not require reducing agents to transport drugs, either However, ABCG2 is a half transporter, i.e., a single polypeptide of ABCG2 is approximately half the size of Pgp, and there are one nucleotide-binding domain (NBD1) and one transmembrane domain (TMD1) in the order of NBD1-TMD1 (Figure 1.2C)

(Doyle & Ross, 2003) The active unit is the homodimer of ABCG2, namely

ABCG2/G2 In addition to drug resistance in chemotherapy, ABCG2 has been largely determined in placenta or liver Expression in such organs suggests a role for ABCG2 in detoxification and metabolite excretion Indeed, for instance,

studies from ABCC members have shown the cooperation between ABCC

subfamily and ABCG2 in bile acid excretion (Kruh & Belinsky, 2003)

It has been known that drug efflux is coupled with ATP binding and

hydrolysis at the nucleotide-binding domains, and that the drugs are transported

through the transmembrane domains (Sections 1.2 & 1.4) However, in recent

years, it has been reported that regulation at the transcriptional level also plays

an important role in multi-drug resistance The pioneering work by Kathleen Scotto and colleagues has enriched our knowledge in the transcriptional

regulation of ABC transporters in MDR (Scotto, 2003) Briefly, the mechanisms of

the transcriptional regulation on MDR-related ABC transporters include the

following 1) For constitutive (non-drug-induced) transcription, the TATA-less

promoters interact with endogenous transcription activators, such as NF-Y, Sp1

or AP-1 Also, malignant transformation occurs due to malfunction of tumor

suppressors, such as p53 or Ras 2) For constitutive overexpression (drug-

induced), it involves activation of promoter-enhancing factors, such as MEF1, and induction of epigenetic modification, such as gene rearrangement of ABCB1 (Baker & El-Osta, 2004) 3) For constitutive repression, histone deacetylation and

DNA methylation can suppress the expression of MDR-related genes in drug-

sensitive cells 4) The stress-induced drug-resistance responds to various environmental signals (e.g., carcinogens or hypoxia) that induce the transcription

of ABC transporters by activating a postulated transcriptional complex called

enhancesome To summarize the transcriptional regulations as described above,

they can be categorized into three modes of drug resistance in tumor cells: intrinsic mode, selected mode and inducible mode (Figure 1.3)

Above all, ABC transporters have been shown to play a very important

role in the failure of chemotherapy with drug-efflux mechanism It is thus intriguing to know the mechanism and the regulation of these transporters, by

which new strategies may be designed to target cancer more efficiently

as P-glycoprotein (Juliano & Ling, 1976) Pgp was the first ABC transporter found

to be related to the failure of chemotherapy at the time when the issue of MDR had arisen in cancer (Shapiro & Ling, 1995; Gottesman & Ling, 2006) It shows poor specificity to many structurally and mechanically unrelated drugs in chemotherapy although the majority of these compounds are cationic with a large hydrophobic portion (Seelig & Landwojtowicz, 2000; Zheleznova et al, 2000; Salerno et al, 2002) Drug resistance by Pgp is thus also called multi-specific drug resistance

Pgp in human is the product of MDR genes that contain two subclasses: MDR1 and MDR2 (also called MDR3) (Gottesman & Pastan, 1993; Zhou et al, 1999) According to the new ABC gene nomenclature scheme, MDR1 and MDR2

are now named as ABCB1 and ABCB4 respectively (Dean, 2002) In human, ABCB1 ¡is associated with multi-drug resistance while ABCB4

(phosphatidylcholine flippase) transfers phosphatidylcholine across lipid bilayer (Borst et al, 2000) For Pgp in mouse, the human ABCB1 homologue is Abcb1a (also called mouse MDR3) while Abcb1b (also called mouse MDR1‘1) shares similar function of Abcb1a, and Abcb4 (also called mouse MDR2) is the homologue of human ABCB4 Pgp contains three potential glycosylation sites on the extracellular region of the N-terminal half and is composed of ~1280 amino acids with molecular weight ~170KD It consists of the core domains typical for

ABC transporters, TMD1-NBD1-TMD2-NBD2, in which TMD1 and NBD1 form

the N-terminal half (or domain) and TMD2 and NBD2 form the C-terminal half (or domain) N- and C-terminal halves share ~40% sequence identity and ~60% sequence similarity by gapped BLAST alignment (Altschul et al, 1997) The nucleotide-binding domains use ATP to provide the power stroke to facilitate the transport of drugs across the membrane, and according to thiol cross-linking

studies, the postulated chamber embedded inside the transmembrane domains

is believed to hold various kinds or sizes of drugs (Loo & Clarke, 2001a/b)

In addition to cancer cells, Pgp has also been found in normal tissues for defense (Leslie et al, 2005) At blood-tissue barriers, such as blood-brain barrier

(BBB), blood-testis barrier and placenta, the excretory function of these barriers

has been speculated to be associated with the relatively high expression of Pgp (Fromm, 2004) For example, Pgp expressed on endothelial cells in BBB protects the central nerve system (CNS) by eliminating drugs or toxic materials from

reaching neural cells This has raised concerns on the pharmacoresistance for some CNS diseases (Léscher & Potschka, 2005) Similar to BBB, Pgp in testis or placenta removes toxins to protect testicular tissues or the fetus respectively ABCBI1 is localized to the apical membranes of hepatocytes (liver cells), where it excretes drugs into bile ABCB4 is connected to lipid transport (Elferink et al, 1997; Hooiveid et ail, 2001) In the gastrointestinal tracts, it extrudes toxins from mucosal cells In kidneys, Pgp can be found in proximal tubule epithelium, disposing of xenobiotics (or antiviral drugs) and metabolites into the urine

(Izzedine et al, 2005; Leslie et al, 2005) The knockout experiment showed that

mdr1a/1b double knockout mice were more sensitive to acute arsenic toxicity than wild-type mice (Liu et al, 2002) In addition, it has been reported in recent years that the drug resistance in autoimmune diseases (e.g., rheumatoid arthritis)

is, at least in part, because of the Pgp expression on the peripheral lymphocytes (Richaud-Patin et al, 2004) All of these functions suggest the normal physiological role of Pgp in detoxification or protection of the body

1.4.2 Mechanism of Drug Resistance by Pgp

Pgp confers drug efflux by coupling the energy from ATP binding and hydrolysis on the nucleotide-binding domains (Al-Shawi & Senior, 1993; Urbatsch

et al, 1994; Senior et al, 1995b; Stein, 1997; Shapiro & Ling, 1998) As discussed

in Section 1.2, this is the common mechanism proposed for ABC transporters to

transport substrates The minimal functional unit was reported to be the

monomer (Loo & Clarke, 1996; Taylor et al, 2001), and well-documented studies

in ATP hydrolytic cycle and drug binding have enabled us to propose a possible

mechanism of Pgp In recent years, the drug-resistance by Pgp has been shown

to be tightly regulated at the transcriptional level (Section 1.3), and at the cellular level, intercellular Pgp transfer has been reported to be associated with a novel pathway to spread drug resistance (Ambudkar et al, 2005) In the text that

follows, discussion will be focused on the ATP-coupled drug transport, followed

by a brief introduction to intercellular transfer

1.4.2.1 Alternating ATP Hydrolysis Mechanism

The alternating hydrolysis catalytic cycle has been proposed as the working model of ATP-coupled pumping mechanism (Figure 1.4) (Senior et al, 1995a, 1998) Currently, this model has the most support based on many

biochemical studies, including nucleotide-trapping with vanadate (Loo & Clarke,

2002b; Urbatsch et al, 2003), covalently labeling of the nucleotide-binding

domains by fluorescent maleimide, photoaffinity labeling with Mg-8-azido-

[a**P]ATP on intact or separate NBDs (Loo & Clarke, 1995b; Urbatsch et al, 1995; Hrycyna et al, 1998, 1999), covalently inhibition (Al-Shawi et al, 1994; Sankaran et al, 1997a/b; Senior & Bhagat, 1998) and mutation of the Walker A residues (Urbatsch et al, 2000a) This model suggests that the two nucleotide-

binding domains show negatively cooperativity, i.e., binding of ATP on one site reduces the affinity on the other site Once the hydrolysis occurs, ATP can bind

to the second site presumably due to conformational change upon the ATP hydrolysis Then, ADP from the first site is released Another ATP will bind to the first site after the hydrolysis occurs at the second site By means of this alternating hydrolysis, the enzyme completes a catalytic cycle In addition, the hydrolytic activity can be stimulated by a factor of 3-10 fold in the presence of drugs compared to the basal activity (Scarborough, 1995; Senior et al, 1998; Loo

& Clarke, 2000a) This suggests that there might be a link between drug binding and ATP hydrolysis

However, recent ATP-binding studies showed that the binding only could

be enough to provide the power stroke allowing drug transport (Martin et al, 2000b) Vanadate-trapping experiments showed the switch of the NBDs between open and closed modes (Urbatsch et al, 2003), and fluorescent resonance

energy transfer (FRET) analysis also showed close association between the two

NBDs (Qu & Sharom, 2001) Moreover, the X-ray structures of the NBDs from DNA repair ABC, Rad50, and from Methanococcus jannaschii’s ABC, MJ1267

and MJ0796, have shown that ATP is sandwiched between the P-loop of one

NBD and the LSGGQ motif of the other NBD (Hopfner et al, 2000; Karpowich et

al, 2001; Yuan et al, 2001; Smith et al, 2002) Based on this new information and

the original alternating hydrolysis model, a revised model was proposed recently

by Christopher Higgins and colleagues: the ATP switch model, in which the two NBDs switch back and forth between the closed dimer and open dimer during the alternating catalytic cycle (Section 1.3.2.3, Higgins & Linton, 2004)

1.4.2.2 Drug Transport Mechanism

For the drug transport, there have been five possible pathways proposed

so far (Figure 1.5) 1) Flippase model (Gottesman & Pastan, 1993; Stein, 1997): The drug substrates bind to Pgp on the side exposed to the inner leaflet of lipid bilayer, and Pgp flips over the drugs onto the outer leaflet of the membrane and

secretes the drugs ino the extracellular space 2) Vacuum cleanser model (Gottesman & Pastan, 1993; Stein, 1997): When the drugs enter the membrane,

they are partitioned to where Pgp molecules are located, and the proteins

transport the drug compounds presumably through a channel which opens to the

extracellular space and to the hydrophobic region of membrane 3) Affiliated

transport model (Gottesman & Pastan, 1993; Stein, 1997): Drugs bind to the

cytoplasmic site of Pgp molecules and are pumped out by the energy generated

by ATP hydrolysis 4) Protonation model (Raghunand et al, 1999): It has been

shown that low pH occurs on the cytoplasmic side of the plasma membrane in multi-drug resistance cells and that many drugs are cationic Therefore, the drugs are neutralized and become more hydrophobic at low pH, which increases the rate of partitioning the drugs to membrane environment 5) Drug-ATP cotransport