CONTENTS

Acknowledgement i
Abbreviations ii
Summary iii

Chapter 1: GENERAL INTRODUCTION................................................................... 1-44

1.1. LEUKEMIA................................................................................................................. 1
1.2. AEUTE MYELOID LEUKEMIA................................................................................ 2
1.2.1. Signs and symptoms of leukemia..............................................................................2
1.2.2. Risk factor for leukemia............................................................................................3
1.2.3. Diagnosis of AML...................................................................................................3
1.2.4. Classification............................................................................................................ 4
1.2.5. Treatment...................................................................................................................4
1.3. CHRONIC MYELOID LEUKEMIA...........................................................................5
1.3.1. Signs and symptoms................................................................................................. 5
1.3.2. Diagnosis...................................................................................................................5
1.3.3. Phases ofCML..................................... ................................................................... 6
1.3.3.1. Chronic phase........................................................................................................6
1.3.3.2. Accelerated phase..................................................................................................6
1.3.3.3. Blast crisis.............................................................................................................6
1.3.4. Treatment................................................................................................................. 6
1.3.4.1. Chronic phase...................................................................................................... 6
1.3.4.2. Blast crisis............................................................................................................7
1.4. THE CELL CYCLE......................................................................................................7
1.4.1. Cancer cell cycle...................................................................................................... 8
1.4.2. The Retinoblastoma Protein pRB in cancer...............................................................9
1.4.3. Cvclin dependent kinase inhibitors (CDKI)............................................................. 10
1.4.3.1. CIP family............................................................................................................. 10
1.4.3.2. INK4 family......................................................................................................... 11
1.4.4. The MDM2-p53 Network and cancer....................................................................... 11
1.4.5. Regulation of p53 stability by degradation through the proteasome......................... 12
1.4.6. MDM2........................................................................ :............................................ 14
1.4.7. INK4A/ARF...............................................................................................................14
1.4.8. BcI-2........................................................................................................................... 15
1.5. THE p53 FAMILY........................................................................................................16
1.5.1. p73.............................................................................................................................. 17
1.5.2. p63........................................................................................................... 19
1.6. ROLE OF EPIGENETICS IN GENE EXPRESSION.................................................. 20
1.6.1. DNA methylation at CpG islands.............................................................................. 21
1.6.2. DNA methylation regulation..................................................................................... 21
1.6.3. DNA methyltransferases family.................................................................................21
1.6.4. Chromatin Structure and DNA methylation..............................................................22
1.6.5. Methylation of genes in Cancer................................................................................. 24
1.6.6. Reversal of gene silencing to prevent or treatment of cancer.................................... 25
1.7. MOLECULAR GENETICS OF MYEOLIDLEUKEMIA............................................ 26
1.7.1. MOLECULAR PATHOGENESIS OFACUTE MYELOID LEUKEMIA..............26
 1.7.1.1. Mutations in AML that provide proliferative advantage to hematopoietic cells.. ..29
1.7.1.1.1. N-RAS and K-RAS Mutations............................................................................ 29
1.7.1.1.2. The roles of FLT3 in acute myeloid leukemia....................................................29
1.7.1.13. FLT3 mutation in human leukemias................................................................... 30
1.7.1.1.4. FLT3 Internal Tandem Duplications (ITDs).......................................................30
1.7.1.1.5. FLT3 “Activation Loop or Kinase Domain” Mutations in Human Leukemias.31
1.7.1.1.6. FLT3 as a target for AML therapy....................................................................... 31
1.7.2. MOLECULAR GENETICS OF CHRONIC MELOID LEUKEMIA........................32
1.7.2.1. Molecular structure of the BCR-ABL translocation............................................... 33
1.7.2.2. The physiologic function of the ABL and BCR.......................................................35
1.7.23. Molecular targets for therapy in CML......................................................................36
1.7.2.4. Signaling pathways and biologic properties of BCR-ABL positive cells............. 38
1.7.2.4.1. Altered adhesion properties................................................................................. 38
1.7.2.4.2. Mitogenic signaling pathways activation............................................................ 38
1.7.2.43. Inhibition of apoptosis.......................................................................................... 39
I.7.2.4.4. Degradation of inhibitory proteins........................................................................40
1.8. GENES WHICH ARE INVOLVED IN BIOACTIVATION OF PROCARCINOGEN AND
DETOXIFICATION OF CARCINOGEN...............................................................................40
1.8.1. GST classification and human polymorphisms............................................................ 41
1.8.1.1. Alpha family................................................................................................................ 41
1.8.1.2. Pi family.......................................................................................................................42
1.8.13. Zeta family................................................................................................................ 42
1.8.1.4. Omega family.............................................................................................................. 42
1.8.1.5. Mu family.....................................................................................................................43
1.8.1.6. Theta family................................................................................................................ 43
1.8.1.2. GST null genotype and cancer prognosis.................................................................. 44

Aims of the study.............................................................................................45

Chapter 2: General Materials and Methods.............................................46-58

2.1. Collection of bone marrow or blood samples................................................................... 46
2.2. Profile of patients and controls........................................................................................ 46
2.3. Cell culture.........................................................................................................................46
2.4. Genomic DNA isolation from peripheral whole blood or bone marrow........................46
2.5. Genomic DNA isolated using Qiagen Kit....................................................................... 47
2.6. Total RNA isolation...........................................................................................................47
2.7. cDNA synthesis.................................................................................................................47
2.8. Agarose gel electrophoresis................................................................................................48
2.9. Nondenaturing polyacrylamide gel electrophoresis........................................................48
2.10. Plasmid DNA Isolation...................................................................................................48
2.10.1. Kamal-Chowdhury Method...................................................................................... 48
2.10.2. Sequencing grade Plasmid Isolation...........................................................................49
2.10.3. Qiagen Miniprep kit plasmid isolation..................................................................... 49
2.11. Isolation of DNA fragments from polyacrylamide gels................................................ 49
2.12. Cloning target fragment of interest............................................................................... 50
2.13. Colony PCR.....................................................................................................................51
2.14. 5’- end labeling of the sequencing primer with y32P-ATP............................................ 51
2.15. DNA Sequencing............................................................................................................ 52
2.16. DNA sequencing by automated sequencer....................................................................... 52
2.17. Restriction digestion.................................................................................................................53
2.18. In vitro DNA methylation........................................................................................................53
2.1. TABLE OF PRIMERS USED.................................................................................................53
2.19. Resuspension of Oligos........................................................................................................... 56
2.20. Common reagents..................................................................................................................... 56
2.21. Statistical Analysis.................................................................................................................... 56
2.22. Composition of different mediaand solutions...................................................................... 56

PART-1
Chapter 3.1: Expression of cell cycle regulatory genes in acute myeloid leukemia
59-65
3.1.1. Introduction............................................................................................................................59
3.1. 2. Materials and Methods................................................................................................... 59
3.1.2.1. Patients.............................................................................................................................. 59
3.1.2.2. Analysis ofgene expression by RT-PCR...................................................................... 59
3.1.2.3. Statistical analysis........................................................................................................... 60
3.1.3. Results.......................................................................................................................................61
3.1.3.1. Gene expression differences between AML patients and controls........................ 61
3.1.3.2. Cell cycle genes on prognostic rote for survival of AML patients.......................... 63
3.1.4. Discussion...............................................................................................................................63

Chapter 3.2: Role of p73 in acute myeloid leukemia.............................66-74

3.2.1. Introduction...........................................................................................................................66
3.2.2. Materials and Methods...................................................................................................... 67
3.2.2.1. Patients.............................................................................................................................. 67
3.2.2.2. RNA and DNA Extraction...............................................................................................67
3.2.2.3. p73 isoforms analysis by RT-PCR................................................................................ 67
3.2.2.4. Analysis of ANp73 isoform expression.........................................................................68
3.2.2.5. Allelic status ofp73 gene................................................................................................68
3.2.2.6. Statistical analysis............................................. 68
3.2.3. Results..................................................................................................................................... 70
3.2.3.1. Expression of C-terminalp73 isoforms....................................................................... 70
3.2.3.2. ANp73 isoform expression............................................................................................ 70
3.2.3.3. Polymorphisms and allelic expression ofp73 gene.................................................. 70
3.2.3.4. p73 gene and survival of the A ML patients................................................................70
3.2.4. Discussion...............................................................................................................................72

PART-II
Chapter 4: Promoter methylation status in acute myeloid leukemia.75-83
4.1. Introduction.............................................................................................................................. 75
4.2. Materials and Methods..................................................................................... .................... 76
4.2.1. Patients................................................................................................................................... 76
4.2.2. Genomic DNA isolation......................................................................................................76
4.2.3 Bisulfite Modification..........................................................................................................76
4.2.4. Methylation specific polymerase chain reaction............................................................76
4.2.5. Statistical analysis...............................................................................................................77
4.3. Results........................................................................................................................................77
4.3.1. Profile ofpromoter CpG island methylation pattern in AML.................................... 77
 4.3.2. Correlation of methylation data with clinical parameters.......................................80
4.3.3. Prognostic role of hypermethylated genes for survival ofAML patients................. 80
4.4. Discussion..........................................................................................................................80

PART-III
Chapter 5.1: Detection of FLT3 alteration and AML1-ETO transcript in AML
84-90
5.1.1. Introduction.....................................................................................................................84
5.1.2. Materials and Methods..................................................................................................85
5.1.2.1. Patients....................................................................................................................... 85
5.1.2.2. Genomic DNA and RNA isolation.............................................................................85
5.1.2.3. Detection of FLT3 Internal Tandem Duplication (1TD).........................................85
5.1.2.4. Detection of FLT3-D835 mutation in kinase domain.............................................86
5.1.2.5. P.T-PCRfor detection of AML1-ETO transcript.....................................................87
5.1.2.6. Statistical analysis.....................................................................................................87
5.1.3. Results............................................................................................................................. 87
5.1.3.1. FLT3 alteration........................................................................................................ 87
5.1.3.2. AML1-ETO transcript detection............................................................................. 88
5.1.4. Discussion.......................................................................................................................89

Chapter 5.2: Characterization of BCR-ABL transcript in chronic myeloid

leukemia................................................................................................... 91-100
5.2.1. Introduction.....................................................................................................................91
5.2.2. Materials and Methods..................................................................................................93
5.2.2.1. Patients........................................................................................................................93
5.2.2.2. Sample collection and isolation ofgenomic DNA and RNA.................................. 93
5.2.2.3. Characterisation ofjunctional breakpoint by RT-PCR......................................... 93
5.2.2.4. Genotyping of BCR exon 13 polymorphism............................................................. 94
5.2.2.5. Genotyping of BCR intron 13 polymorphism.......................................................... 94
5.2.2.6. Sequencing reaction.................................................................................................. 94
5.2.3. Results.............................................................................................................................. 97
5.2.4. Discussion........................................................................................................................ 99

PART-IV
Chapter 6: Glutathione S-transferase genotype frequency in CML.101-106
6.1. Introduction...................................................................................................................... 101
6.2. Materials and methods.................................................................................................... 101
6.2.1. Subject...........................................................................................................................101
6.2.2. Blood sampling and DNA extraction.......................................................................... 102
6.2.3. Genotyping of GST...................................................................................................... 102
6.2.4. Statistical Analysis...................................................................................................... 102
6.3. Results...............................................................................................................................103
6.4. Discussion......................................................................................................................... 103

References................................................................................................ 107-132
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