inhibition of c-MYC Expression Through Disruption of an RNA*Protein interaction Using Antisense Oligonucleotides Christopher M. Coulis A thesis submitted in coafodty with the requirements for the degree of Master of Science Graduate Department of Pharmacology University of Toronto Q Copyright by Chnstopher M. Coulis 1999 National Libraiy Bibliothèque nationale l*l of,", du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Weilington OttawaON KlAW O(tawaON KlAûN4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive Licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distri'buer ou copies of this thesis in microform, vendre des copies de cetîe thèse sous paper or electronic formats. la fome de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts firom it Ni la thèse ni des extraits substantiels may be printed or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT The proto-oncogene c-myc encodes a protein that regdates cellular proliferation and differentiation. Conditions that alter the stability of c-myc mRNA can lead to overexpression of the gene resulting in uncontrolled cell growth. Synthetic therapeutic agents, known as Antisense Oligonucleotides (ODN), cm bind to target mRNA and inhibit its expression. A previously characterized protein, the coding region stability determinam-binding protein (CRD-BP),binds the coding region deteminant (CRD)of c-myc mRNA and is believed to protect it fmm endonucleolytic cleavage. Accordingly, we hypothesized that ODNs directed towards the CRD of c-m_vc mRNA could prevent CRD-BP*RNA interactions, thus decreasing c-myc expression. Using an in vitro gel shift assay we demonstrated that ODNs inhibit the CRD-BP*c-nryc mRNA interaction. The most effective ODN, CRD-ODN4, exhibited a sequence-specific and concentration-dependent inhibition of the RNA*CRD-BP interaction, with a maximal inhibition of 75% at 1 FM. K562 cells treated with a 2'-O-methyl derivative of CRD-0DN4 displayed a concentration4ependent decrease of c-myc expression. Up to 65% inhibition of protein expression and 45% inhibition of mRNA expression was observed with 200 nM of CRD-ODN4- Conversely, a 2'-O-methyl ODN derivative targeting the translational initiation codon (ODN- AUG) reduced c-myc protein but increased mRNA levels 2-fold. The effect of ODN-AUG was partially due to increased mRNA stability. ODN-AUG treatment increased the haii-life of c-myc mRNA from 30 min to 70 min whereas the half-life of c-myc mRNA after treatment with CRD- 0DN4 was not signif~wntlydifferent from the control. Additionally, CRD-0DN4 was more effective at inhibiting K562 ce11 growth, reducing ce11 number by - 7Wo after 48 h of exposure to 750 UMODN. The results observed both in vin0 and within cells support the hypothesis that CRD-ODN4 is capable of disrupting the interaction between CRD-BPand c-myc mRNA and this disruption decreases c-myc expression and functional effects in cells. 1 would like to than my supervisor, Dr. Rebecca Rokipcak for giving me the opportwity to do pduate work in her iaboratory, and for her invaluable support, encouragement and advice. 1 would like to offer special thanks to Leonardo Salmena and Vienthong Lam for their tutorials in Western Blot and MIT assays and for their helpful discussions and support. I also offer spetial thanks to Christine Albino for her support and discussions. Finally, 1 wish to thank my mother, whose continued support, understanding and patience made everything possible. 1 couidn't have doue it without any of these parties, and to al1 of you 1 dedicate this thesis. iii TABLE OF CONTENTS .. ABSTRACT 11 .-- ACKNOWLEDGEME:NTS 111 TABLE OF CONTENTS iv LIST OF TABLES vii LIST OF F'IGURES vü LIST OF ABBREVIATIONS ix 1. INTRODUCTION 1.1 Antisense Oiigonticleotides Bac kground OLigonucleotide Structural Modikations Oligonucleotide Afinity and Specifici ty Uptake and Distribution of Oligonucleotides Antisense OligonucIeotide Mechanisms of Action Antisense Oligonucteotide Toxicity Antisense Oligonucleotides in Cancer Chemotherapy 1.2 Proto-oncogenec-myc 1.2.1 Background 1.2.2 Structures of c-myc Gene and Protein 1.23 Mechanisms of Myc Regulation 1.2.4 Activation of Gene Expression by Mye 13 Myc as a Target of ODN Therapy 1.4 Ratiode for Ciiri~ntStrdy 1 -4.1 Preliminary Data 14.2 ffypothesis II. MATERIALS AND METHODS So-e of Materials Cell Line Oligonucleotide Preparation Probe Preparation for Northem Analysis Labeling DNA Probe Probe Pre paration for Ribonuclease Protection Assay Treatment of ceb with Antisense Oligonacleotides Transfection of ODN into Cells Expression of c-myc mRNA and Protein in K562 CeUs RNA Isolation RNA Eiectrophoresis and Northem Transfer Northern Hybridization Analysis Ri bonuclease Protection Assay Protein Isolation Protein Electrophoresis and Western Blot Analysis Estimation of c-myc mRNA =Life Aatisense Oligonncleotide Effect on CeU Gmwth MTT Growth Assay v 3.1 Assay Opthization Range of Detection for c-myc by Western Blot Analysis and RNase Protection Assay Carrier Molecule Selection K562 Exposure Time to ODN fffect of Superfect on K562 Cell Growth 3.2 Effeetg of CRD Airtisense Oiyonocieotides on c-nryc mRNA and Protein Levels in K562 Ceûs c-myc mRNA Levels c-myc Protein Levels Cornparison of 2'OM Versus PS Dose-Dependent Inhibition of c-myc Expression 33 Cornparison of the Effect of 0DN4 and ODN-AUG on c-myc mRNA Stability 3.4 Effect of 0DN4 and ODN-AUGon K562 Ceiï Proliferstion IV. DISCUSSION V. FUTURE DIRECTIONS VI. REFERENCES LIST OF TABLES Table 1. Examples of inhibition of Malignant Cell Growth in vino and in Animal Models by ODNs Table 2. ODN Sequences Used in Sudy vii LIST OF FIGURES Figum 1. Structure and Sites of Chemical Modifications Made to 5 Phosphdester Oligonucleotides Figure 2. Human c-myc Gene and Structural Domains of c-myc Protein 19 Fwre 3. Schematic Representation of Human c-myc mRNA 23 Figure 4. Pathways of Transcriptional Activation or Repression by c-myc 28 Fire5. Schematic Representation of Roposed ODN Mechanism of Action 3 1 Fiire 6. Gel- Shift Analysis of a Panel of ODNs Targeting the CRD of c-myc mRNA Figure 7. Optirnizatioa of RNase Protection and Western Blot Anal ysis Fii8. Comparison of Carrier MoIecules Figure 9. Optimization of ODN Treatment The Figure 10. Superfect Dose-response Curve Foire11. Effects of ODNs on c-myc mRNA Levels in Cells Fiire 12. Effects of ODNs on c-mye Rotein Levels in Cells Fire 13. Comparison of 0DN4 as Both a PS and 2'OM Derivative Figure 14. Concentration-dependent Effect of 0DN4 and ODN-AUG on c-myc mRNA Levels Figure 15. Concentration-dependent Effect of 0DN4 and ODN-AUG on c-myc Protein Levels Fignre 16. c-rnyc mRNA Decay Rate Following Treatment with ODN4 or ODN-AUG Figure 17. Effect of ODN4 and ODN-AUG on K562 Ce11 Growth viii 2'OM - 2'-O-methylribose Oiigonucleotide 3 '-UTR - 3'-untranslated region 4G - G-quartet 5'-UTR - 5'-untraasIated region A - Acidic region A, - absorbance at 260 nm ARE - Adenylateturidylate rich element AU - Adenylatehridylate AUG - initiation codon B - Basic regon bp - base pair cDNA - Complementary DNA reverse transcribed from RNA CML - Chronic myeloid leukemia CO, - Carbon dioxide CpG - Cytosine/Guanine residues flanked by two purines on 5'-end and two pnrnidines on 3'-end cpm - Counts per mitlion CRD - Coding region determinant CRDBP - Coding region stability determinant binding protein C-terminal - Carboxy terminal CUG - Initiation codon BSA - Bovine serum albumin dATP - Deoxyadenosine 5' triphosphate dCrP - Deoxycytidine 5' triphosphate ddH20 - Double distiiled water DEPC - Diethylpyrocarbonate DMSO - Dimethylsulfoxide DNA - DeoxyribonucIeic acid dNTPs - Deoxynucleotide triphosphates DRB - 56-dicloro-1-beta-D-~bofuranosylbenWmidazole d?TP - Deoxythymidine 5' triphosphate DTT - Dithiothreitol E-box - Enhancer box ECL - cherniluminescence detection system EDTA - Ethylenediamine tetracetic acid FBS - Fetal bovine serum GAPDH - Glyceraîdehyde-3-phosphate dehydrogenase GC - GiianinefCytosine HBV - Hepati tis B virus HCl - Hydrochloric acid Hepes-KOH - Hepesfpotassium hydroxide HIV - Human irnmunodeficiency Mnis HLH - Helix loop helix hrs - hours ix K562 - humau erythroleukemia cell line k, - Speed of association kb - Kilobase kDa - Kilodalton KH - Region of homology to RNA-binding domain of ribonucleoprotein K L - litre LZ - Leucine Ppper M - Molar mg - Miilignun grams) mins - minutes mg - Milli gram ( 1U3 grams) ml - Millilitre ( litres) mM - Millimolar ( 10'~ molar) MOPS - 3-fN-Morpholino] pmpanesulfonic acid mRNA - Messenger RNA MIT - 3-[4~dimeihylthiazol-2-yl]-2~diphenyltbromide NaCl - Sodium chloride NaH,PO, - Sodium phosphate ng - Nanogram (109grams) NLS - Nuclear localization signal nM - Nanomolar (l~-~molar) nt - Nucleotide N-terminal - Amino terminal ODN - Antisense Oligonucleotides OLB - Oligo labeling buffer PBS - Phosphate buffered saline PCR - Polymerase chah reaction PO - Phosphodiester Oligonucleotide PS - Phosphorothioate Oligonucleotide RGG - Arginine/guanine/guanine RNA - Ribonucleic acid RNase H - Ribonuclease H RNase L - Ribonuclease L RRM - RNA recognition