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DEVELOPMENT OF DENATURING HIGH PERFORMANCE LIQUID

CHROMATOGRAPHY (dHPLC) IN DETECTION OF COMMON

POLYMORPHISMS IN THE MULTI-DRUG RESISTANCE (MDR1) GENE IN

MALAY PATIENTS WITH LEUKEMIA

BADRUL HISHAM YAHAYA

UNIVERSITI SAINS MALAYSIA

2006

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DEDICATION

Special thanks are dedicated to my loving parents, Mr. Yahaya Said and Mrs. Som Lazim

for their moral support and prayer, I thank Allah giving me such great parents who are

always encouraging me in whatever endeavor I take. For my sisters and brothers, thank

you for your love, encouragement, support and having confidence in me to finish my

studies, I love you all. Last but not least, to my wife, Azila Abd Aziz, thank you for your

support throughout my study.

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ACKNOWLEDGEMENTS

My deepest appreciation is dedicated to my main supervisor Dr Narazah Mohd Yusoff

and Dr Rosline Hassan for their supervision, advice, support and trust throughout my

study. You are such wonderful supervisors that I ever met in giving me an opportunity to

grow up by experiencing every single thing surround me during my study period. Your

encouragement and ideas during my study is greatly appreciated.

I also would like to express my deepest appreciation to Dr Zafarina Zainuddin (lecturer of

PPSK), Dr T.P Kannan (lecturer of PPSG), Dr Norsa’adah Bachok (Unit of Biostatistic,

PPSP) for reviewing this thesis and Mr Mohd Ros Sidek for the technical support. To

Kak Yah and all postgraduate students in Human Genome Center such as Ms Azlina

Ahmad, Farizan, Farini, Wati@Hayati, Surini, Thanaseelan and Khairul, thank you for

your support, help and always be there for me. Thank you for being such wonderful

fiends.

This acknowledgement also goes to staffs in Hematology Department i.e. Kak Sal and

Kak Sha for helping me in sample collection. Not forgetting those who had contributed to

this study either direct or indirectly, many thanks and may Allah bless you all. Last but

not least, I would like to thank Universiti Sains Malaysia for awarding me the Academic

Staff Training Scholarship (ASTS) and Institute of Graduate Studies or IPS for the

research grant Dana Penyelidikan Siswazah (Tabung Siswazah 308/AIPS/415401).

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LIST OF PUBLICATIONS & SEMINARS

Publication :

1) Y. Badrul Hisham, H. Rosline, W. M. Wan Maziah, S. Mohammad Ros, B. Abdul Aziz, B. Norsa’adah, M. Y. Narazah Rapid Screening for C3435T Polymorphism in Exon 26 of the Multi-drug Resistance (MDR1) Gene in Malay Patients with Acute Leukemia. The Malaysian Journal of Medical Sciences, Vol. 12, Suppl. 1, 2005, pg 61.

Oral Presentations :

1) Title : Analysis of Exon 21 of the acute lymphoblastic leukemia (ALL) and the chronic myeloid leukemia (CML) using dHPLC.

Authors : Badrul Hisham Y., Rosline H, Wan Maziah W.M, Abdul

Aziz B, Nor Sa’adah B. Narazah M.Y. Venue : 9th National Conference on Medical Sciences. School of

Medical Sciences, Health Campus, Universiti Sains Malaysia. Malaysia.

Date : 22nd -23rd May, 2004.

2) Title : Analysis of Nucleotide Changes in the Exons of Multi

Drug Resistance (MDR1) Gene Using dHPLC in Leukemia Patients.

Authors : Badrul Hisham Y., Rosline H, Wan Maziah W.M, Abdul

Aziz B, Nor Sa’adah B. Narazah M.Y.

Venue : 1st Postgraduate Research Colloquium. School of Health Sciences, Health Campus, Universiti Sains Malaysia. Malaysia.

Date : 14th August, 2004.

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3) Title : Rapid Screening for C3435T Polymorphism in Exon 26 of the Multi-drug ) Gene in Malay Patients with Acute Leukemia.

Authors : Y. Badrul Hisham, H. Rosline, W. M. Wan Maziah, S.

Mohammad Ros, B. Abdul Aziz, B. Norsa’adah, M. Y. Narazah

Venue : 10th National Conference on Medical Sciences. School of

Medical Sciences, Health Campus, Universiti Sains Malaysia, Malaysia.

Date : 21st -22nd of May, 2005.

Poster Presentation :

1) Title : Screening of Nucleotide in Exon 21 of the ABCB1 Gene Using Denaturing High Performance Liquid Chromatography (dHPLC) in Malay Patients with Acute Leukemia.

Authors : Badrul HishamY., Rosline H., Norsa’adah B.

Narazah M.Y. Venue : 5th Human Genetics Organization (HUGO) Pacific

Meeting & 6th Asia-Pacific Conference on Human Genetics, Biopolis Singapore.

Date : 17th-20th November 2004

International Course:

1) Title : 4th HUGO Mutation Detection Training Course

Venue : International Center for Life, Newcastle, UK

Date : 2nd – 6th September 2004

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LIST OF CONTENTS Contents Page

TITLE i

DEDICATION ii

ACKNOWLEDGEMENTS iii

LIST OF PUBLICATIONS & SEMINARS iv

LIST OF CONTENTS vi

LIST OF TABLES xi

LIST OF FIGURES xiv

ABBREVIATIONS xviii

ABSTRACT xx

ABSTRAK xxii

CHAPTER I: LITERATURE REVIEW

1.1 Research Background 1 1.1.1 Epidemiology and Etiology of Leukemia 1 1.1.1.1 Acute leukemia 3 1.1.1.2 Chronic Leukemia 12

1.2 Chemotherapy Resistance 13

1.2.1 MDR1 Gene also Known as Drug Transporter Gene 17

1.2.2 Mechanisms of Multi-drug Resistance 18

1.2.2.1 ABCB1 Gene Family 19 1.2.3 Expression of P-gp in Normal Cells 24

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1.2.4 Expression of P-gp in Hematological Malignancies 25

1.3 Genetic Variations and Methods to Detect Them 27

1.3.1 DHPLC as a Screening Method 30 1.3.1.1 Basic Principles of dHPLC 30 1.3.1.2 Detection of Heteroduplexes by dHPLC 36 1.3.1.3 Selection of Optimum Temperature 37 1.3.1.4 Application of dHPLC 38 1.3.1.4 (A) Mutation screening 38 1.3.1.4 (B) Detection of SNPs 39 1.3.2 Screening for Polymorphisms in the MDR1 gene 41 1.3.3 Study on the Effect of the SNPs to the P-gp

Expression and Function 49 1.4 Aim of the study 53 1.5 Flow chart of the study 54

CHAPTER II: MATERIALS AND METHODS 2.1 Materials 56 2.1.1 Blood Sampling From Patients 56 2.1.2 Reagent Preparation 56 2.1.2.1 DNA Extraction Reagents 56 2.1.2.1 (A) Ethanol (96 %) 56 2.1.2.1 (B) DNA Extraction kit (QIAamp DNA Mini kit) 57 2.1.2.1 (C) Proteinase K Stock Solution (100mg/ml) 57 2.1.2.1 (D) Buffer AL (Lysis buffer) 57

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2.1.2.1 (E) Buffer AW 1 (wash buffer 1) 57 2.1.2.1 (F) Buffer AW 2 (wash buffer 2) 57 2.1.2.2 Gel Electrophoresis 58 2.1.2.2 (A) Agarose Gel, 1.7 % 58 2.1.2.2 (B) SYBR Green 58 2.1.3 Mutational Analysis 59 2.1.3.1 DHPLC analysis 59 2.1.3.2 Buffer A 59 2.1.3.3 Buffer B 59 2.1.3.4 Acetonitrile 59 2.1.3.5 Isopropanol 59 2.1.3.6 pUC18 HaeIII dHPLC Standard 60 2.1.3.7 DYS271 dHPLC Standard 60 2.1.4 PCR Purification kit 60 2.1.5 Sequencing kit 60 2.1.6 PCR-Restriction Fragment Length Polymorphism

(RFLP) assays 61 2.2 Methods 63 2.2.1 DNA extraction 63 2.2.2 DNA Quantification 64 2.2.3 PCR Amplification 64 2.2.4 Agarose gel electrophoresis 68 2.2.5 Mutation Analysis 68 2.2.5.1 Predictions of melting temperature 68

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2.2.5.2 Standard Method for Mutational Analysis Using dHPLC 73

2.2.5.2 (A) Initiation Procedures 73 2.2.5.2 (B) Analysis Procedures 73 2.2.5.2 (C) Temperature Mapping 74 2.2.5.2 (D) Screening for Heteroduplex and

Homoduplex Samples 75 2.2.5.3 Confirmation of Known 1236C>T (exon 12), 2677G>T &

2677G>A (exon 21) and 3435C>T (exon 26) Polymorphisms 76

2.2.5.4 DNA sequencing 83 2.2.5.4 (A) PCR Purification 83 2.2.5.4 (B) Cycle Sequencing 84 2.2.5.5 (C) Ethanol/EDTA Precipitation 85 2.2.6 Statistical Analysis 88 CHAPTER III: RESULTS 3.1 Overview 89 3.2 Amplification of Exon 12, Exon 21 and Exon 26 of the MDR1

Gene using PCR Technique 89 3.3 Mutational Screening using dHPLC 94 3.3.1 Interpretation of dHPLC results 94 3.4 Confirmation of Results of dHPLC by RFLP 98 3.5 Confirmation of the Detected Polymorphisms

By DNA Sequencing 125 CHAPTER IV: DISCUSSION 4.1 Development of dHPLC for Screening of SNPs in the MDR1 Gene

In Patients with Leukemia in HUSM 152

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4.2 DHPLC as a Rapid and Cost-effective Method for the Screening of SNPs in the MDR1 Gene in Patients with Leukemia in HUSM 162

4.3 Combination of dHPLC and RFLP Assay 168 4.3.1 Combination of PCR-RFLP Assays and dHPLC Technique

for Detection of Known Polymorphisms in the MDR1 Gene 168 4.3.2 Distribution of Common SNPs in the MDR1 Gene among Malay

Patients with Leukemia 173 4.4 The 2677G>T/A and 3435C>T Polymorphisms: Significance to the

Diagnostic and Treatment of Malay Patients with Leukemia 174 4.5 Future and Further Investigation 180 4.5.1 DNA Sequencing for Homozygous Mutation in Exon 21 180 4.5.2 Screening for All 28 Exons of the MDR1 Gene 181 4.5.3 Association of Various Peak Patterns with Distribution of Mutations 182 4.5.4 The Need to Study the Effects on mRNA Levels in Patients with

Leukemia in HUSM-for the Future 183 CHAPTER V: SUMMARY 186 REFERENCES 189 APPENDIXES Appendix A 199 Appendix B 204

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LIST OF TABLES

Tables Page

1.1 The FAB Classification for ALL 5

1.2 The FAB and WHO classifications for Myeloid Malignancies 8 1.3 FAB classification for AML 9 1.4 Differences in Classification of AML by WHO and FAB 10 1.5 Modes of operations of nucleic acids analysis by dHPLC 35

1.6 Summary of MDR1 Genetic Variations in Different Ethnic Group 43 2.1 List of enzymes used for PCR-RFLP based assays 62 2.2 List of primer sequences used for PCR amplification for exons

12, 21 and 26 of the MDR1 gene 66 2.3 The reaction mixture (master mix) for PCR amplification 67 2.4 The gradient features for universal methods during

analyzing samples using denaturing HPLC. 77

2.5 Reaction mixtures for cycle sequencing (master mix) 87

3.1 Results for dHPLC Screening for Exons 12, 21 and 26 97

3.2 (a) The Association Between Exon 12 and Exon 21 in Distribution

of Polymorphisms among Malay Patients with ALL 105

3.2 (b) The Association Between Exon 12 and Exon 26 in Distribution

of Polymorphisms in ALL patients 106

3.2 (c) The Association Between Exon 21 and Exon 26 in Distribution

of Polymorphisms in ALL patients 107

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3.2 (d) The Association Between Sex and each exon in Distribution


3.2 (e) The Association Between Age and each exon in Distribution



of Polymorphisms among Malay patients with AML 112


of Polymorphisms among Malay Patients with AML 113



3.3(d) The Association Between Age and Each Exon in Distribution


3.3 (e) The Association Between Sex and Each Exon in Distribution



of Polymorphisms among Malay Patients with CML 119





3.4 (d) The Association Between Sex and Each Exon in Distribution


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3.5 The Distribution of Common Polymorphisms In Exons 12, 21 and 26

Of the MDR1 Gene According to the Types of Leukemia and Sex

Of the Patients 124

4.1 List of Reagents that Required and Should Be Avoided for the Purpose

Of using dHPLC for Mutation Screening 167

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LIST OF FIGURES

Figures Page

1.1 The phosphorylation process of P-gp 21 1.2 Schematic representation of P-gp 22

1.3 The ideogram for location of the MDR1 gene on chromosome 7 23

1.4 Basics principle of mutation detection by dHPLC 33

1.5 The Flow Chart of Methods Involved in Mutation Screening 55

2.1 DHPLC system by Helix System (VarianInc, USA) 70

2.2 Results for dHPLC Melt Program Interface for exon 26 71

2.3 An example for dHPLC Melt Program Report for exon 26 72

2.4 A pUC18 HaeIII restriction digests at non-denaturing temperature 78

2.5 Dys71 mutation standard with an A/G SNP 79

2.6 Results for temperature mapping analysis for exon 26 80

2.7 A typical wild type chromatogram 81

2.8 Flow chart for analyzing the homoduplex & heteroduplex samples

Using dHPLC 82

3.1 Results for PCR Amplification of Exon 12 91



3.4 Various Peak Patterns Obtained from dHPLC Analysis 96

3.5 Electrophoresis Pattern for 1236C>T Polymorphism Evaluated by

PCR-RFLP Based Assays 102

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3.6 Electrophoresis Pattern for 2677G>T/A Polymorphisms Evaluated by


3.7 Electrophoresis Pattern for 3435C>T Polymorphism Evaluated by


3.8 (a) Electropherogram Results for Homozygous Wild Type CC in Exon 12

At Position 1236 126

3.8 (b) The BLASTn Results obtained from NCBI Program for Homo Wild Type 127

3.8 (c) Electropherogram Results for Heterozygous Mutation CT at position 1236

Using Forward Primer 128

3.8 (d) The BLASTn Results Obtained from NCBI Program for Heterozygous

Mutation CT at Position 1236 129

3.8 (e) Electropherogram Results for Heterozygous Mutation CT at Position

1236 using Reverse Primer 130

3.8 (f) The BLASTn Results Obtained from NCBI Program for Heterozygous

Mutation CT at Position 1236 that amplified using Reverse Primer 131

3.8 (g) The BLAST Two Sequence Analysis Result for Both Primers for

Heterozygous Mutation CT at Position 1236 132

3.8 (h) Electropherogram Results for Homozygous Mutation TT at Position

1236 using Forward Primer 133

3.8 (i) The BLASTn Results Obtained from NCBI Program for Homozygous

Mutation TT at Position 1236 that amplified using Forward Primer 134

3.8 (j) Electropherogram Results for Homozygous Mutation TT at Position 1236

Using Reverse Primer 135

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3.8 (k) The BLASTn Results Obtained from NCBI Program for Homozygous

Mutation TT at position 1236 that amplified using Reverse Primer 136

3.8 (l) The BLAST Two Sequence results for Both Primers for Homozygous

Mutation TT at Position 1236 137

3.9 (a) Electropherogram Results for Homozygous Wild Type GG in Exon 21


3.9 (b) The BLASTn Results Obtained from NCBI Program for Homozygous

Wild Type GG at Position 2677 that amplified using forward Primer 139

3.10 (a) Electropherogram Results for Homozygous Wild Type CC in Exon 26


3.10 (b) The BLASTn Results Obtained from NCBI Program for Homozygous

Wild Type CC that amplified using Forward Primer 141

3.10 (c) Electropherogram Results for Homozygous Mutation TT in Exon 26

at Position 3435 that amplified using Forward Primer 142

3.10 (d) The BLASTn Results Obtained from NCBI Program for Homozygous

Mutation TT at Position 3435 that amplified using Forward Primer 143

3.10 (e) Electropherogram Results for Homozygous Mutation TT at Position

3435 that amplified using Reverse Primer 144

3.10 (f) The BLASTn Results Obtained from NCBI Program for Homozygous

Mutation TT at Position 3435 that amplified using Reverse Primer 145

3.10 (g) The BLAST Two Sequence Results for Both Primers for Homozygous

Mutation TT at Position 3435 146

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3.10 (h) Electropherogram Results for Heterozygous Mutation CT in Exon 26

at Position 3435 that amplified using Forward Primer 147

3.10 (i) The BLASTn Results Obtained from NCBI Program for Heterozygous

Mutation at Position 3435 that amplified using Forward Primer 148

3.10 (j) Electropherogram Results for Heterozygous Mutation CT at Position 3435

That amplified using Reverse Primer 149

3.10 (k) The BLASTn Results Obtained from NCBI Program for Heterozygous

Mutation CT at Position 3435 that amplified using Reverse Primer 150

3.10 (l) The BLAST Two Sequence Results for Both Primers for Heterozygous

Mutation CT at Position 3435 151

4.1 The Elution Peak Resulted from Analysis using dHPLC for Clean PCR

Product and Contaminated PCR Product 156

4.2 A Comparison Between Universal Method and Short Method using

dHPLC for Screening Homozygous Wild Type and Heterozygous

Mutation in Exon 12 164

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ABBREVIATIONS

MDR : Multi-drug resistance

P-gp : P-glycoprotein

DNA : Deoxyribonucleic acid

RNA : Ribonucleic acid

dsDNA : Double-stranded deoxyribonucleic acid

ssDNA : Single-stranded DNA

mRNA : Messenger ribonucleic acid

cDNA : Complementary deoxyribonucleic acid

PCR : Polymerase chain reaction

RFLP : Restriction fragment length polymorphism

SNP : Single nucleotide polymorphism

AML : Acute myeloid leukemia

ALL : Acute lymphoblastic leukemia

CML : chronic myeloid leukemia

CLL : Chronic lymphoblastic leukemia

FAB : French-American-British

WHO : World Health Organization

dHPLC : Denaturing high performance liquid chromatography

MDS : Myelodysplastic syndrome

ABCB1 : ATP-binding cassette sub-family B1

UV : Ultra violet

ATP : Adenine Tri-phosphate

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ASA : Allele specific amplification

ARMS : Amplification refractory mutation system

SSCP : Single strand conformation polymorphism

DGGE : Denaturing gradient gel electrophoresis

QRT-PCR : Quantitative reverse transcriptase polymerase chain reaction

STR : Short tandem repeat

TEAA : Triethylammonium acetate

FBN1 : Fibrillin 1

CFTR : Cystic fibrosis transmembrane conductance regulator

TSC : Tuberous sclerosis

BRCA : Breast cancer

APC : Adenomatous polyposis

Asn : Asparagine

Asp : Aspartate

Phe : Phenylalanine

Leu : Leucine

Arg : Arginine

Cys : Cystine

Gly : Glycine

Ser : Serine

Ala : Alanine

Thr : Threonine

Ile : Isoleucine

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ABSTRACT

Although the use of chemotherapy has evolved remarkably during the past 30 years for

treatment in leukemia patients, it remains a difficult disease to treat where the main

problem is drug resistance. This is due to over expression of multi-drug resistance gene

(MDR1) and it’s over expression has been described in many tumors resistant to

cytotoxic drugs including leukemias. This gene is polymorphic and studies in different

ethnic groups have indicated that the three most frequent single nucleotide

polymorphisms (SNPs) are located in exons 12, 21 and 26 of the MDR1 gene. Denaturing

high performance liquid chromatography (dHPLC) has been proven to be a feasible

method in detecting target DNA for presence of mutations and polymorphisms including

in the MDR1 gene. Studies on polymorphisms of the MDR1 gene, however has not been

carried out in patients in this institution. Thus, the main objectives of this study were to

develop and establish dHPLC technique for screening of these three most frequent

polymorphisms located in exons 12, 21 and 26 of the MDR1 gene, to investigate the

distribution of these polymorphisms of the MDR1 gene and to compare and combine the

results from dHPLC technique with restriction fragment length polymorphism (RFLP) in

detection of these polymorphisms of the MDR1 gene in Malay patients with leukemia in

this institution. A total of hundred and one DNA samples from Malay patients diagnosed

as leukemia were studied and subjected to dHPLC analysis and RFLP analysis. Statistical

analysis was done using chi-square (χ2) and Fisher Exact’s test. Out of these, there were

35 (34.7%) patients diagnosed as acute myeloid leukemia (AML), 48 (47.5%) as acute

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lymphoblastic leukemia (ALL) and 18 (17.8%) as chronic myeloid leukemia (CML).

There were 58 (57.4%) males and 43 (42.6%) females. From the results, if all of the three

exons were combined, 25 (25.7%) of the patients showed heterozygous mutation, 7

(6.7%) of them had wild type, while 2 (1.9%) showed homozygous mutation. In all of

these patients, there was a significant difference in the distribution of polymorphisms

between exons 21 and 26 with a p<0.05. PCR-RFLP assays had been performed to

confirm the results of dHPLC technique and there were 93.2%, 96.1% and 100% of

common polymorphisms detected in exons 12, 21 and 26, respectively. In this study

dHPLC was successfully developed for detection of common polymorphisms in the

MDR1 gene in Malay patients with leukemia in this institution. The clinical implications

of the distribution of these polymorphisms especially in the response of treatment in these

patients should be studied for the future.

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PEMBANGUNAN TEKNIK KROMATOGRAFI CECAIR DENATURASI

BERPRESTASI TINGGI (dHPLC) DALAM MENGESAN POLIMORFISME

YANG LAZIM PADA GEN KERINTANGAN PELBAGAI DADAH (MDR1) DI

KALANGAN PESAKIT MELAYU YANG MENGHIDAP LEUKIMIA

ABSTRAK

Walaupun keberkesanan penggunaan kemoterapi dalam rawatan terhadap pesakit

leukimia adalah jelas terbukti, namun leukimia tetap merupakan penyakit yang sukar

dirawat terutamanya dalam masalah kerintangan pesakit terhadap ubat-ubatan (dadah).

Ini disebabkan oleh pengekspresan gen kerintangan pelbagai dadah (multidrug resistance

gene, MDR1) yang berlebihan dan ianya telah dilaporkan dalam kebanyakan pesakit

tumor yang rintang terhadap dadah sitotoksik termasuk leukimia. Gen ini adalah gen

yang polimorfik dan kajian di dalam kumpulan etnik yang berlainan menunjukkan

bahawa tiga polimorfisme nukleotida tunggal (SNPs) yang paling kerap berlaku adalah

terletak pada ekson-ekson 12, 21 dan 26 pada gen MDR1. Teknik denaturasi cecair

berprestasi tinggi (dHPLC) telah dibuktikan sebagai suatu teknik yang berkesan dalam

pengesanan mutasi dan polimorfisme, begitu juga dalam gen MDR1. Kajian pengesanan

polimorfisme pada gen MDR1 masih belum pernah dijalankan dikalangan pesakit

leukimia di institusi ini. Oleh itu, objektif utama kajian ini adalah untuk membangunkan

teknik dHPLC bagi menyaring ketiga-tiga ekson tersebut; ekson 12, 21 dan 26 pada gen

MDR1, mengkaji taburan polimorfisme gen MDR1 dan seterusnya membanding serta

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menggabungkan hasil di antara teknik dHPLC dan restriction fragment length

polymorphism (RFLP) dalam mengesan polimorfisme ini dikalangan pesakit Melayu

yang menghidap leukemia. Sebanyak 101 sampel DNA pesakit-pesakit Melayu yang

menghidap leukimia telah digunakan dalam kajian ini. Analisa statistik telah dilakukan

menggunakan ujian-ujian chi-square (χ2) dan Fisher Exact. Daripada keseluruhan

sampel, sebanyak 35 (34.7%) adalah merupakan leukimia myeloid jenis akut (AML), 48

(47.5%) merupakan leukemia lymphoblastic jenis akut (ALL) and 18 (17.8%) merupakan

leukemia myeloid jenis kronik (CML). Sebanyak 58 (57.4%) sampel adalah merupakan

pesakit lelaki dan 43 (42.6%) sampel adalah perempuan terlibat di dalam kajian ini.

Sekiranya hasil daripada ketiga-tiga ekson digabungkan, didapati 25 (25.7%) sampel

daripada keseluruhan pesakit menunjukkan mutasi heterozigus, 7 (6.7%) sampel

menunjukkan profil jenis liar sementara 2 (1.9%) sampel menunjukkan mutasi

homozigus. Di kalangan pesakit-pesakit ini, terdapat perbezaan yang bererti bagi

penyebaran polimorfisme di antara ekson 21 dan ekson 26 dengan nilai p<0.05. Teknik

PCR-RFLP telah dijalankan bagi mengesahkan profil yang telah ditemui oleh teknik

dHPLC dan sebanyak 93.2%, 96.1% and 100% polimorfisme yang lazim telah

dikenalpasti hadir dalam setiap ekson-ekson 12, 21 dan 26 yang dikaji. Dalam kajian ini,

teknik dHPLC telah berjaya dibangunkan untuk mengesan polimorfisme yang kerap

berlaku pada gen MDR1 dikalangan pesakit Melayu yang menghidap leukemia di

institusi ini. Implikasi klinikal bagi penyebaran polimorfisme ini terutamanya dalam

respon pesakit Melayu yang menghidap leukemia terhadap rawatan haruslah dikaji

sebagai kajian lanjutan.

1

CHAPTER I

LITERATURE REVIEW

1.1 Research Background

Leukemia is defined as a neoplasm of the white blood cells (WBC) and its precursor

which leads to clonal proliferation and accumulation of WBC in bone marrow. Leukemia

is divided into two categories i.e. myeloid and lymphoid each representing the types of

cells involved (Denham and Chanarin, 1985). Leukemia can be acute or chronic

depending on presentation and progression of the clinical manifestations. The incidence

of leukemia varies with morphological type and is also dependent on age, sex, race and

locality as will be explained below.

1.1.1 Epidemiology and Etiology of Leukemia

The incidence of leukemia varies with the morphological type. The frequency of any

given type of leukemia is also dependent on age, sex, race and locality. In general, acute

lymphoblastic leukemia (ALL) is a disease of childhood whereas in acute myelogenous

leukemia (AML) there is a higher incidence with increasing of age (Munker et al., 2000).

In Malaysia, there are 26,089 patients diagnosed with cancers according to the first report

of the National Cancer Registry (NCR) from 1st January 2002 to 31st December 2002.

Out of these, there are 11,815 males and 14,274 females. Unregistered cases are

estimated to be 10,656. In terms of risk, 1 in 6 Malaysians is expected to get cancer in

his/her lifetime and if taking into account unregistered cases, the risk would be higher i.e.

2

1 in 4 Malaysians. The crude rate for males is 118.9 per 100,000 population and 148.4

per 100,000 populations in females. Leukemia ranked fourth among cancers in males and

fifth among cancers in females (Lim et al., 2002).

Etiology of leukemia remains unknown in the majority of patients. However, several risk

factors have been identified. The association of ionizing radiation in leukemia has been

firmly established where radiation exposure has been documented by the Atomic Bomb

Casualty Commission in Hiroshima and Nagasaki (Rowe and Liesveld, 1995). However,

the role of low-level irradiation and of diagnostic x-rays in the etiology of leukemia

remains doubtful. Benzene exposure has been strongly associated with development of

leukemia. Other chemicals such as phenylbutazone, arsenic and chloramphenicol

exposure have also been related to the development of leukemia (Rowe and Liesveld,

1995).

Environment has also been recognized as one of the factors contributing to the

development of leukemia. These include maternal irradiation, in utero irradiation, early

childhood viral diseases and maternal history of fetal wasting. These factors are often

difficult to measure and in many instances it is likely that the disease progression are by

multiple factors rather than a single factor (Rowe and Liesveld, 1995). It is also observed

that frequency of leukemia is increased in the identical twins of leukemia patient, also in

those with congenital disorders such as Down syndrome, Bloom syndrome, Fanconi

syndrome, ataxia-telangiectasia, Kleinfelter syndrome and congenital aneuploidy. Most

of these congenital disorders are associated with non-random chromosomal aberrations

3

where they might play a role in leukemeogenesis. Relatives of patients with cystic

fibrosis also may have an increased risk of leukemia (Rowe and Liesveld, 1995).

1.1.1.1 Acute leukemia

Acute leukemia is a heterogeneous group of malignant disorders characterized by

uncontrolled clonal proliferation and accumulation of blast cells in the bone marrow,

blood and other tissues. Acute leukemia is usually rapidly fatal if untreated while chronic

leukemia is associated with slower progression (Pallister, 1999). Acute leukemia affects

all age groups from neonates to old age (Provan and Henson, 1998). The accumulation of

cells in the bone marrow replaces haemopoietic precursor cells and results in bone

marrow failure. At presentation at least 30% and usually more than 80% of marrow cells

are blasts.

The disease is divided into two main subgroups, acute myeloid (myeloblastic) leukemia

(AML) and acute lymphoblastic leukemia (ALL), and further subdivided into various

subcategories. The French-American-British (FAB) scheme divides AML and ALL into

various subtypes. There are additional unusual types [see also World Health Organisation

(WHO)] classification in later part of this dissertation. ALL is the most common

malignancy of childhood where it accounts for 85% of childhood leukemia. The FAB

system classifies ALL (Table 1.1) into three subtypes L1, L2 and L3.

ALL is commonest in the age group of 2-10 years with a peak of incidence at 3-4 years.

The incidence then decreases with increasing age, though there is a secondary rise after

40 years. ALL affecting B lymphocyte precursors with CD10+ subtype is the most

4

common subtype in children and has an equal sex incidence. On the other hand, there is a

male preponderance for ALL of T lymphocyte precursor subtype (Provan and Henson,

1998).

Hematological investigations may reveal a normochromic, normocytic anemia with

thrombocytopenia in most cases of acute leukemia. The total white cell count may be

decreased, normal or increased up to 200x109/l or more. Blood film examination typically

shows variable number of blast cells. The bone marrow is hypercellular with more than

30% of leukaemic blasts (Munker et al., 2000).

The signs and symptoms of acute leukemia result from the infiltration of the malignant

blasts into bone marrow and other organs. Signs of anemia are pale skin and mucous

membranes and easy fatigability. Thrombocytopenia may be a feature as manifested by

gum bleeding, petechiae, retinal bleeding and easy bruising. Affected children may also

have bone and joint pains and sometimes a painful enlargement of the spleen.

5

Table 1.1: Table shows the French –American British (FAB) classification of acute lymphoblastic leukemia (ALL) into three subtypes. Adapted from (Munker et al., 2000)

FAB type Morphological features

L1 Small homogeneous with scanty cytoplasm, moderate basophilia,

inconspicuous nuclei.

L2 Larger, heterogeneous cells, variable cytoplasm, basophilia,

prominent nucleoli.

L3 Larger, homogeneous cells with dark basophilic cytoplasm,

prominent vacuoles, prominent nucleoli.

6

On the other hand, AML is the commonest leukemia (80%) of adulthood where the

median age at presentation is 60 years old. The incidence of AML is about two to four

new cases per 100,000 people. AML is rare in children and young adults (Munker et al.,

2000) and forms only a minor fraction of the leukemias in childhood. The sex

preponderance for AML is the same for both males and females (Provan and Henson,

1998).

However, this is not the case in older patients where males are more susceptible. This is

most likely due to the fact that myelodysplastic syndromes (MDS) are more common in

men, and advanced MDS frequently evolves into AML rather than ALL (Provan and

Henson, 1998). AML is an aggressive hematologic neoplasm that requires urgent

treatment. The myeloid stem cell is transformed into blasts or myeloblasts which then

proliferate in the blood and bone marrow and suppresses normal hematopoiesis.

Myeloblasts proliferate under the influence of myeloid growth factors, some of which are

produced by the leukemic cells themselves. Consistent with the clonal transformation of

myeloid stem cells, myeloblasts express cytogenetic abnormalities in 50-70% of cases.

Cytogenetic studies performed on bone marrow also provide important prognostic

information.

According to the FAB classification there are three groups of myeloid malignancies

whilst WHO classification system groups diseases that show similar characteristics of

both myeloproliferative and myelodysplastic conditions into a new separate group i.e.

myeloproliferative/myelodysplastic diseases. This difference in classification is compared

in Table 1.2.

7

The FAB classification, divides AML into eight sub-types (Table 1.3). The FAB

subtypes are based on characteristic patterns of cytochemical stains and

immunophenotype while WHO classification is based on chromosomal aberration

involved. Table 1.4 compares the different sub-types of AML classification by FAB and

WHO.

8

Table 1.2 Classification of myeloid malignancies based on French-American-British (FAB) and World Health Organization (WHO) (http://xenia.sote.hu/depts/pathophysiology/hematology/e/who-classification.html)

FAB WHO

Chronic myeloproliferative diseases Chronic myeloproliferative diseases

Myelodysplastic/myeloproliferative

diseases

Myelodysplastic syndromes Myelodysplastic syndromes

Acute myeloid leukemia Acute myeloid leukemia

9

Table 1.3: French-American-British (FAB) classification of acute myeloid leukemia (AML) into eight subtypes. Adapted from (Munker et al., 2000).

FAB types Morphological features

M0 Undifferentiated -lymphoid markers negative, reactive with myeloid markers (CD33, CD13), ultrastructural peroxidase positive

M1 Myeloid, no maturation -poorly differentiated blasts with rare azurophilic granules

M2 Myeloid with maturation -myeloblasts, promyelocytes, occasionally eosinophils and basophils, often Auer rods.

M3 Promyelocytic -Two variants: hypergranular (90%) and atypical microgranular (<10%)

M4 Myelomonocytic -leukemic myeloblasts and monoblasts, variant: M4Eo with abnormal eosinophils.

M5 Monoblastic -leukemia monoblasts

M6 Erythroleukemia -more than 50% abnormal erythropoietic cells, >30% myeloblasts of all nonerythroid cells

M7 Megakaryoblastic -usually associated with myelofibrosis, platelet markers present

10

Table 1.4: Shows difference in classification systems of acute myeloid leukemia (AML) by French American British (FAB) compared with World Health Organization (WHO) and classification systems. (http://xenia.sote.hu/depts/pathophysiology/hematology/e/who-classification.html

AML classification

FAB WHO

M0: minimally differentiated AML with recurrent cytogenetic

translocations

-AML with t(8;21)(q22;q22)

AML1/CBFalpha/ETO

-Acute promyelocytic leukemia:

AML with t(15;17)(q22;q12) and variants

PML/RARalpha

-AML with abnormal bone marrow

eosinophils inv(16)(p13;q22) vagy

t(16;16)(p13;q22) CBFbeta/MYH1

-AML with 11q23 MLL abnormalities

M1: myeloblastic leukemia without

maturation

M2: myeloblastic leukemia with

maturation

M3: hypergranular promyelocytic leukemia

M4: myelomonocytic leukemia

M4Eo: variant, increase in marrow

eosinophils

AML with multilineage dysplasia

With prior MDS

Without prior MDS

M5: monocytic leukemia

M6: erythroleukemia (DiGuglielmo's

disease)

11

AML Classification

FAB WHO

M7: megakaryoblastic leukemia

AML with myelodysplastic syndrome,

therapy related

Alkylating agent related

Epipodophyllotoxin related

Other types

AML not otherwise categorized

AML minimally differentiated

AML without maturation

AML with maturation

Acute myelomonocytic leukemia

Acute monocytic leukemia

Acute erythroid leukemia

Acute megakaryocytic leukemia

Acute basophilic leukemia

Acute panmyelosis with myelofibrosis

Table 1.4, continued

12

1.1.1.2 Chronic Leukemia

Chronic leukemia has a slower progression in terms of clinical manifestation as compared

to acute leukemia. There are two types of chronic leukemia, chronic myeloid leukemia

(CML) and chronic lymphocytic leukemia (CLL). CML is a clonal disorder of the

pluripotent stem cell and is classified as one of the myeloproliferative disorders. The

disease accounts for around 15% of all leukemias and may occur at any age.

This disease occurs in both sexes with a male: female ratio of 1.4:1 and most frequently

between the ages of 40 and 60 years. However, it may occur in children and neonates and

the very old (Pallister, 1999, Provan and Henson, 1998). The diagnosis of CML is rarely

difficult and is assisted by the characteristic presence of the Philadelphia (Ph)

chromosome. This results from the reciprocal translocation of chromosomes 9 and 22 as a

result of which part of the Abelson proto-oncogene (ABL) on chromosome 9 is moved to

the BCR gene on chromosome 22 and part of BCR gene on chromosome 22 moves to

ABL gene on chromosome 9. The abnormal chromosome 22 is the Ph chromosome or t

(9; 22) (q34; q11).

On the other hand, CLL has a peak incidence between 60 and 80 years of age and rarely

before the age of 40. The male to female ratio is 2:1 (Pallister, 1999, Provan and Henson,

1998). It is characterized by a chronic persistent lymphocytosis and subtypes can be

distinguished by morphology, immuno phenotype and cytogenetics. DNA studies may be

useful in showing a monoclonal rearrangement of either immunoglobulin or T-cell

receptor genes.

13

1.2 Chemotherapy Resistance

The use of chemotherapy in the treatment of leukemia has evolved remarkably during the

past 30 years, particularly in childhood ALL. However, leukemia remains a difficult

disease to treat despite recent progress in chemotherapy. The biggest problem limiting the

effective use of cytotoxic chemotherapy is drug resistance. Tumors differ in their initial

chemo sensitivity where some tumors, are very sensitive, resulting in a relatively high

percentage of responses. Others, such as small-cell lung cancer, are initially sensitive but

then recur; whereas in others, such as pancreatic cancer, cytotoxic drugs have little

activity. These differences reflect differences in the biology of the tumor, relating to

factors such as the growth fraction, the activity of specific drug resistance mechanisms,

and changes in proteins mediating apoptosis.

A tumor is commonly referred to as resistant if the activity of standard chemotherapy in

that patient is less than that to be expected in a typical population. Resistance can be

intrinsic, such that the tumor is resistant to a particular drug or drugs at presentation, or it

can be acquired, such that resistance develops during treatment. The cellular mechanisms

mediating drug sensitivity and resistance include target-related changes, changes in drug

efflux mechanism, drug deactivation, changes in DNA repair and changes in apoptosis. In

drug efflux process, a number of mechanisms for effluxing xenobiotics exist, their normal

function being to protect the cell from potentially damaging compounds. Not

surprisingly, many cytotoxic drugs are effluxed by this mechanism, resulting in decreased

intracellular drug concentrations.

14

The first of these pumps to be described was P-glycoprotein (P-gp), a member of the

adenosine triphosphate (ATP)-binding cassette (ABC) super family of transmembrane

transport proteins. Increased expression of this protein and activity of the pump have

been described in tumors that are resistant to specific cytotoxic drugs. Moreover, the

selection of drug-resistant cells by prolonged exposure to specific cytotoxic drugs that are

P-gp substitutes results in the accumulation of cells with increased P-gp activity that are

also resistant to a number of other structurally, unrelated compounds that are P-gp

substrates. This phenomenon has been termed multi-drug resistance (MDR) because

resistance to one P-gp-effluxed drug is associated with resistance to multiple other

agents.

P-gp is commonly detected in the leukemic cells of patients with AML in who increased

P-gp, or MDR1 expression, is correlated with increase risk for treatment failure. The

influence of P-gp and MDR1 on the outcome of patients with ALL is less clear. P-gp

consists of two homologous halves of six hundred and ten amino acids joined by a

flexible linker consisting of sixty amino acids (Shen et al., 1986). It is an ATP-dependent

efflux pump that confers protection to the body from environmental toxins. It transports a

huge variety of structurally diverse compounds and it was first identified by because it

was over expressed in human tumor cells.

Studies have helped to explain the mechanism of action of human P-gp which indicated

that mutations in membrane-spanning domains or nucleotide binding domains of P-gp are

involved. According to (Fojo et al., 1985), drug resistance is due to decrease

15

accumulation of these drugs because of an energy-dependent increase in drug efflux

which results from increased expression of a membrane glycoprotein.

Recent studies indicate that over expression of P-gp plays a more general anti-apoptotic

role that extends beyond resistance to chemotherapy. The cells over express P-gp are

resistant to a wide range of apoptotic inducers. These include serum starvation, fas ligand

ligation, UV-irradiation and also exposure to tumor necrosis factor (TNF) as well as

complement-mediated cytotoxic action (Scotto et al., 2003).

P-gp is embedded in lipid membranes, facilitate the import of nutrients into cells or

releasing the toxic products into the surrounding medium (Higgins, 1992). It is expressed

in very high levels of the secretory cell type such as adrenal gland and kidney. An

intermediate level has also been reported in the lung, liver, lower jejunum, colon and

rectum while low levels are reported in many other tissues where the normal function is

thought to involve the excretion of toxic metabolites (Schinkel, 1997, Fojo et al., 1986).

This protein is also highly expressed in hematopoietic stem cells, where it is thought to

protect these cells from toxins. It also has been shown to play a role in the migration of

dendritic cells (Schinkel et al., 1997). P-gp binds to ATP (Figure 1.1) and use energy to

drive the transport of various molecules across all cell membranes (Higgins, 1992, Dean

et al., 2001). This phosphorylation process will cause conformational changes that are

transmitted to the membrane-spanning domains where finally it transports substrates from

one side of the membrane to the other.

16

In general, P-gp transports a wide range of substances including ions, sugars, amino

acids, glycans, peptides, proteins, phospholipids, toxins and drugs. In eukaryotes, most P-

gp move compounds from the cytoplasm to the outside of the cell or into an intracellular

compartment such as endoplasmic reticulum (ER), mitochondria and peroxisome (Dean

et al., 2001).

The P-gp consists of two structurally homologous halves, each with six transmembrane

domains, one ATP binding site and highly conserved ‘Walker A’ and ‘Walker B’ motif

(Figure 1.2). These two halves are probably derived from internal gene duplication. It

was also reported that phosphorylation of P-gp might be essential for drug transport

(Hrycyna et al., 1996). However, mutation in the major phosphorylation sites within P-gp

does not affect its transport function. The glycosylated at extracellular sites of P-gp are

not involved in routing and stability of the protein but it also serve as an antigens for

monoclonal antibodies recognizing P-gp (Sonneveld, 2000, Schinkel et al., 1993). P-gp

not only removing an anticancer drug from the inside to the outside of the cell membrane

using ATP energy, but also prevents the influx of drugs through the cell membrane

(Juliano and Ling, 1976).

The classical phenomenon of MDR is seen where levels of P-gp expression was found in

a large range of cell-lines that were selected through exposure to different cytotoxic

agents (Kartner et al., 1983). There are two different genes encoding for P-gp in humans,

MDR1 gene and MDR3 gene, both located on chromosome 7. Despite their homology,

only MDR1 gene is a drug efflux protein which is related to MDR (Hrycyna et al., 1998).

Several studies suggested that phosphorylation of P-gp might be essential for drug

transport (Germann et al., 1996). The glycosylated sites of P-gp at the extracellular site

17

are probably involved in routing and stability of the protein as well as serve also as an

antigen for monoclonal antibodies in recognizing P-gp (Schinkel et al., 1993).

Several studies have reported the association between genotype variations with the

reduction of P-gp expression levels. A single nucleotide change in the coding region of

MDR1 gene has been found to be associated with the expression level of P-gp. These

single nucleotide changes are also known as single nucleotide polymorphisms (SNPs).

MDR represents a form of pleotropic drug resistance that has an adverse prognostic value

in AML, refractory multiple myeloma and non-Hodgkin’s lymphoma. It may affect the

outcome of current chemotherapy protocols (Sonneveld, 2000). Numerous studies have

investigated the expression of the MDR1 gene products in leukemia, lymphoma and

myeloma (Hegewisch-Becker and Hossfeld, 1996). Studies in myelogenous leukemia and

myeloma have so far provided the best evidence for a significant correlation between P-

gp expression and response to chemotherapy (Sonneveld, 2000).

1.2.1 MDR1 Gene also known as Drug Transporter Gene

Drug transporter gene is the gene that allows drugs to enter cells and in some cases, acts

to keep them out from membrane cell. Transporter genes may account for discrepancies

in the way drugs such as antidepressants, anticonvulsants, and chemotherapy agents work

in different people. The drug-resistant phenotype is due to a membrane alteration which

reduces the rate of drug permeation (Juliano and Ling, 1976). Surface labeling studies

reveal that in Chinese hamster, the ovary cell membranes possess a carbohydrate-

containing drug-resistant component. This component is shown to be a cell surface

18

glycoprotein and has a weight of 170 000 Dalton (170 kD) in size. Since this glycoprotein

is unique to mutant cells displaying altered drug permeability, it has been designated as

the P-gp. It has been reported to be an important factor in understanding a natural

resistance of tumors to cytotoxic drugs (Juliano and Ling, 1976).

1.2.2 Mechanisms of Multi-drug Resistance

There are two mechanisms of resistance to anticancer drugs, firstly, those that damage

delivery of anticancer drugs to tumor cells and secondly, those that arise in the cancer cell

it self due to genetic and epigenetic alterations which can affect drug sensitivity (Jain,

2001). Cancer cells can become resistant to a single anticancer drug or a class of drugs

with a similar mechanism of action (Gottesman et al., 2002). It arises either by altering

the drug’s cellular target or by increasing repair of drug-induced damage, commonly by

DNA. After selection for resistance to a single drug, cells might also show cross-

resistance to other structurally and mechanistically unrelated drugs.

Cancer cells are genetically heterogeneous because of the mutated phenotype. Tumor

cells that are exposed to chemotherapeutic agents will be selected for their ability to

survive and grow in the presence of cytotoxic drugs. Thus, in any population of cancer

cells that is exposed to chemotherapy, more than one mechanism of multi drug resistance

can be present. This phenomenon has been called as multi factorial MDR (Gottesman et

al., 2002).

MDR can also result from activation of coordinately regulated detoxifying systems, such

as DNA repair and the cytochrome P450 mixed function oxidases. It can be induced after

19

exposure to any drug (Schuetz et al., 1996). Recent evidence indicates that certain orphan

nuclear receptors might be involved in mediating global response to environmental stress

(Synold et al., 2001). MDR can also result from defective apoptotic pathways which

might occur as a result of malignant transformation as in those cancers with mutated or

non-functional p53 gene (Lowe et al., 1993). Alternatively, cells might acquire changes

in apoptotic pathways during exposure to chemotherapy, such as changes in cell-cycle

machinery, leading to an activated checkpoints which prevents initiation of apoptosis

(Lowe et al., 1993).

1.2.2.1 ABCB1 Gene Family

Classical MDR results from expression of ATP-dependent efflux pumps with broad drug

specificity (Gottesman et al., 2002). These pumps belong to a family of ATP-binding

cassette (ABC) transporters which share sequence and structural homology. So far,

fourty-eight human ABC genes have been identified and divided into seven distinct

subfamilies (ABCA, ABCB, ABCC, ABCD,… and ABCG) on the basis of their

sequence homology and domain organization (Dean et al., 2001). Drugs that are affected

by classical MDR include vinca alkaloids (vinblastine, vincristine), the anthracyclines

(doxorubicin, daunorubicin), the RNA transcription inhibitor actinomycin-D and the

microtubule-stabilizing drug paclitaxel (Ambudkar et al., 1999).

The ABCB1 gene maps to chromosome 7q21.1 and is the best characterized ABC drug

pump (Figure 1.3). Formerly known as MDR1 gene or P-gp, ABCB1 gene was the first

human ABC transporter cloned and characterized through its ability to confer a MDR

phenotype to cancer cells that had developed resistance to chemotherapy drugs (Roninson

20

et al., 1986, Juliano and Ling, 1976). The gene is thought to play an important role in

removing toxic metabolites from cells. It also expressed in cells at the blood brain barrier

and may play a role in transporting compounds into the brain that cannot be delivered by

diffusion (Hoffmeyer et al., 2000).

P-gp belongs to the ABC transporter subfamily B1 and is encoded by the ABCB1 gene

(Ueda et al., 1987). ABCB1 gene is found to be over expressed in all MDR human cell

lines. It consists of 28 exons and 26 introns and encodes a transporter P-gp.

21

Figure 1.1: The figure shows the process of phosphorylation of P-gp. This process is essential for transport activity of the drugs into the membrane cell. This figure cited from http://images.search.yahoo.com/search/images?p=+p-glycoprotein&ei=UTF 8&fl=0&imgsz=all&fr=FP-tab-img-t&b=41.

Extracellular site

Intracellular site

22

Figure 1.2: Schematic representation of P-gp. It contains two ATP-binding sites, phosphorylation sites and glycosylation sites as well as comprises of 1280 amino acid sequences (Sonneveld, 2000).

23

Figure 1.3: The MDR1 gene is located at the q-arm of chromosome 7 at position 21.1. The ideogram shows the location of MDR1 gene on the chromosome. (http://www.ncbi.nlm.nih.gov/mapview/maps.) The OMIM reference code: # 171650.

MDR1 gene located at 7q21.1 on chromosome 7.

24

1.2.3 Expression of P-gp in Normal Cells.

P-gp is differentially expressed in normal tissues as a consequence of differentiation

triggers and response to environmental challenges. High levels of P-gp expression are

found in the kidney and adrenal glands. Expression of P-gp is also high in the colon and

in the endothelial cells of the blood-brain and blood-testes barrier, where it plays a role in

the pharmacokinetic and bio-distribution of xenobiotics. Furthermore, environmental

factors such as heat shock, cytokines, hormones, differentiation agents,

chemotherapeutics, dietary products, UV and X-irradiation, receptor agonists, oncogenes

and tumor suppressor genes can influence the levels of specific P-gp isoforms in different

systems. Therefore, P-gp expression is controlled by the highly complex interplay of a

variety of factors that are involved in multiple regulatory pathways (Schwab et al., 2003).

P-gp is involved in limiting absorption of xenobiotics from the gut lumen, in protection

of sensitive tissues (brain, fetus, testes) and in biliary and urinary excretion of its

substrates (Schwab et al., 2003). P-pg has an important role in regulating central nervous

system permeability. The brain is protected against blood-borne toxins by the blood-brain

barrier (BBB) and the blood-cerebrospinal-fluid (CSF) barrier. P-gp is located on the

luminal surface, preventing the penetration of cytotoxins across the endothelium (Rao et

al., 1999).

P-gp plays an important role to protect the testicular tissues and transports toxins into the

capillary lumen in the testes (Cordon-Cardo et al., 1990). In the placenta, P-gp is

localized on the apical syncytiotrophoblast and involved in developing of fetus by

protecting from toxic cationic xenobiotic. In the liver, gastrointestinal tract and kidney, P-

gp is responsible to excrete toxin thus protecting the entire organisms (Schinkel, 1997).

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