Inhibition of C-MYC Expression Through Disruption of an RNA*Protein Interaction Using Antisense Oligonucleotides

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

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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 motif SDS - Sodium dodecyl sulfate SDS-Page - Sodium dodecyl sulfate-polyacrylamide gel electrophoresis SE - standard error SSC - Sodium chloride/sodium sulfate TAD - Transactivation domain TBS-T- Tris-buffered saline-tween TNF-a - Tissue necrosis factor alpha TTP - Tristetrapolin UTP - Uridine 5'-triphospahte pg - Microgram (104grams) pl - Microlitre (IO4 litres) WM- Micromolar (IO4 molar) Genetic abnormalities can induce a variety of disorders. For example, alterations in DNA caused by mutations, deletions, and translocations can cause cancer, viral infection, and

inflammatory disease (Cannistra, 1990; Weyand et al., 1998; Zhang et al., 1997). Identifying a

method for gene manipulation could provide an extremely useful tool for elucidation of the de of a particular gene, or for therapeutic intervention of an overexpressed gene.

Gene expression may be disrupted or manipulated through a variety of strategies, which

vary widely and depend primarily on the target molecule. These strategies can be thought of as either "anti-gene" or "anti-rnRNA" (Branch, 1996; Gryaznov et al., 19%). Anti-gene strategies reiy primarily on homologous recombination, triple helix formation, as well as transcription factor decoys (Helene, 1991). These techniques disrupt expression of the gene of interest however, they tend to be inefficient, have difficulty with delivery as welI as intracellular localization, and are Iimited to a srnaIl number of ce11 types (Yu et al., 1997).

Anti-niRNA approaches are undertaken with both ribozymes and oligonucleotides.

Ribozymes are catalytic RNA molecules that cleave mRNA to which they hybridize, but they require incorporation into a vector for ce11 delivery (Welch et al., 1998). Oligonucleotides on the other hand are usually DNA molecules that hybridize to their complementq mRNA and alter gene expression through a variety of mechanisms. ODNs are more attractive as gene therapy tools because they only require a unique target sequence and rely on direct application for their mode of delivery (Crooke, 1992; Zon, 1995a) 1.1 Antisense Ollgonucleotides

1.1.1 Background

Antisense oligonucleotides (ODN) have attracted much interest as a novel class of therapeutic agents for the treatment of a number of genetic disorders. These agents were first proposed in 1978 by Zamencik and Stephenson, who reported inhibition of viral replication with a short (13 mer) DNA oligonucleotide, antisense to the Rous sarcoma virus (Zamecnik and

Stephenson, 1978). The term "antisense" is used to describe the interaction between oligonucleotides complementary to (sense) pre-mRNA and mRNA molecules (Crooke, 1992).

ODNs are short (12-25 bases in length) synthetic sequences of DNA or DNA-analogs that are designed to hybridize to pre-selected target mRNA following Watson-Crick base pairing rules

(Gewirtz, 1997; Zon, 1995a). They interact with nucleic acid targets and inhibit mRNA intermediary metabolism, essentially blocking the flow of information from gene to protein

(Wagner, 1994).

RNA intermediary metabolism begins with transcription and ends with the destruction of the mature mRNA. RNA polymerases recognize and transcnbe one strand of DNA (antisense) into a sense pre-mRNA molecule. This pre-mRNA is spliced, then stabilized by capping the 5' end with a methyl-guanosine and polyadenylating the 3' end. These modifications aid in the transport of the mature mRNA out of the nucleus and into the cytoplasm where they are then translated by ribosomes. Each of the steps involved in mRNA intermediary metabolism is a potentiaî site for antisense oligonucleotide intervention.

Antisense effects of oligonucleotides have been reported in numerous in vitro expenments (Crooke, 1992; Helene and Toulme, 1990; Veal et al., 1998; Yu et al., 1997), and several in vivo studies (Crooke, 1996; Stein and Cheng, 1993; Zon, 1995a). Clinical trials are now in progress to evaluate the therapeutic potential of antisense oligonucleotides in several human diseases, including human immunodeficiency virus-1, acute myologenous leukemia,

human papillomavirus and Herpes virus (Bayever et al., 1993; Gewirtz et al., 1996; Webb et al.,

1997; Zon, 1995b).

1.1.2 Oligonucleotide Structural Modifications

In order for an oligonucleotide to be biologically active it must posses certain charactenstics. It must have the ability to resist nuclease degradation, it must display affinity for target mRNA, and it must posses a mechanism for inhibiting the expression of the target gene

(McKay et al., 1999). Oligonucleotides may be degraded by either exonucleases (from the 5' or

3' end) or endonucleases (which cleave internally) (Crooke, 1992). If an oligonucleotide loses as few as two residues to nuclease cleavage, a metabolic product with dramatically reduced antisense ac ti vity wi Il be produced (Graham et al., 1998)- Addi tionally, the nucleotide degradation products can affect cellular proli feration and di f ferentiation (Wagner, 1994).

The original or "native" oligonucleotide is known as the phosphodiester (PO) (figure 1).

It is quite soluble in aqueous environments but has a limited stability in both semm and cells

(Matsukura et al., 1987). Its limited stability is the result of rapid degradation by nucleases that are present in both extra and intracellular compartments (Rowley et al., 1996). Phosphodiesters have a half-life of approximately 5 mins in senirn and 30 mins in living cells (Shaw et al., 1991;

Woolf et al., 1990). It was this instability that dismissed their further use as therapeutic agents

(Boulme et al., 1998).

To alleviate the problem of nucleolytic degradation, chemical modifications to the native phosphodiester backbone have been introduced into oligonucleotides (Cook, 1991) (figure 1).

By exchanging one of the non-bndging oxygen atoms of the phosphate backbone with a sulfur atom, a phosphorothioate oligonucleotide (PS) is produced (figure 1) (Agrawal et al., 1988; Furdon et al., 1989; Stein et al., 1988). This simple alteration improves the ODN's resistance to most nucleases, thereby increasing its half-life to over 24 hrs in serum and allowing it to remain active for 48-72 hrs (Campbell et al., 1990; Matsukura et al., 1987). Furthemore, in vivo over

50% of phosphorothioates remain intact after 24 hrs (Graham et al., 1998), whereas phophodiester oligonuc1eotides can begin to degrade as quickly as 5 mins (Soni et al., 1998).

A second modification to the diester compound can be made to the 2'- suga. position

(figure 1). This alteration results in the 2'-O-methyl (2'OM) derivative, which has increased duplex stability and increased nuclease resistance (Baker et al., 1997; Iribarren et al., 1990)- Phosphornthioate -Base Base

Phosphdiester 2'-O-methyl ribose

Figure 1. Structure and Sites of Chernical Modifications Made to Phosphodiester Oligonucleotides

Substitution of oxygen with sulfur produces a phosphorothioate oligonucleotide. Modifications at the ?'-position produce a 2'-0- methyl denvative. Each modification alters the oligonucleotides specificity, affinity, stability and mechanism of action (adapted from Wagner et al., 1994) 1.1.3 Oligonucleotide Affhity and Specincity

Three characteristics required for successful drug design are specificity, affinity and potency (efficacy). An ODN is deemed to have high specificity and affinity if it produces no gross loss of ce11 viability, a significant decrease in target mRNA, and lower target protein quantity than a control (Branch, 1998)-

Affinity and specificity are influenced by the length and sequence of the oligonucIeotide as well as the melting temperature of the duplex and secondary structure of the ODN (Agrawai et al., 1988; Breslauer et al., 1986; Matsukura et al., 1987). Statistically, a sequence of 15-17 nucleotides is likely to occur only once in the genome (Branch, 1996), therefore a longer ODN will possess a higher specificity for a given sequence (Bacon and Wickstrom, 1991; Crooke,

1996; Saijo et al., 1997).

A decrease in affinity is exhibited when mismatches between the oligonucleotide and target molecule are present. The relative decrease depends upon the specific mismatch, the position of the mismatch, and the sequence surrounding the mismatch (Maltese et al., 1995).

Single mismatches can result in changes of affinity of approximately 500-fold (Freier et al.,

1991). Additionally, the GC content of the ODN will influence the specificity. If the ODN sequence contains more than 60% GC residues, non-specific binding may arise due to increased rnelting temperature which stabilizes the RNA-DNA hybrid (Reed et al., 1990b). Moreover, if palindromic sequences or interna1 complementary regions exist within an ODN, intramolecular binding or homodimerization may result, thereby preventing an ODN's interaction with target

RNA (Catsicas et al., 1996).

Modifications to native phosphodiester ODNs alter their specificity and affinity.

Phosphodiesters have a relatively high binding affinity to RNA. Phosphorothioates on the other hand, exhibit a decreased binding afinity for single-stranded RNA (Gryaznov et al., 1996) as each thioate modification produces a chiral center that reduces the melting temperature of ODN-

RNA duplexes by 1- 0.5 OC(Rowley et al., 19%)-

2'0-methyl derivatives have a much higher k, (speed of assoc.) with mRNA than PO derivatives and are more specific than PS derivatives (Baker et al., 1997; Lamond and Sproat,

1993). This occurs because each 2'OM center incorporated into an oligonucleotide increases its affini ty by 1°C/base (McKay et al., 1999). This enables the 2'OM derivative to have a twofold increase in afinity over thioates in A549 cells (McKay et al., 1999) and tenfold over its parent compound, the diester, in a ce11 free systern (Boulme et al., 1998).

An important factor in determining the rate of ODN-RNA hybridization is sequence accessibility. Sequence accessibility may be influenced by the presence of a vanety of RNA three-dimensional structures, each of which is induced by intramolecular hybridizations. Two of the more common structures are the stem loop and the pseudoknot (Crooke, 1993; Puglisi et al.,

1990). These structures provide additional stability for RNA, act as recognition motifs for proteins and nucleic acids, and are responsible for movement of mature mRNA to the cytoplasm

(Gait and Karn, 1993; Le Tinevez et al., 1998). They have also been reported to decrease the affinity that unmodified ODNs (PO) exhibit for their target (Ecker et al., 1992) while having little or no effect on modified ODNs (2'OM) affinity (Lamond and Sproat, 1993). Furthexmore, following hybridization, RNA-ODN duplexes may be unwound by various repair and editing enzymes, in particular, helicase and RNA unwindase (Nellen and Lichtenstein, 1993). This limits the duration of interaction and effectiveness that each ODN would display on its mRNA targe t.

A second factor capable of effecting ODN specificity and affinity is known as the G- quartet (4G), which arises when there exits four contiguous guanosines within a sequence

(Cheng and van Dyke, 1997; Saijo et al., 1997). A 4G can interact with a variety of cellular components, in addition to its target, producing sequence non-specific effects (Heikkila et al.,

1987; Wickstrom et al., 1988). Saijo et al. (1997) demonstrated that a scrambled sequence containing the 4G inhibited growth of the A549 ceIl line to the same extent as the unscrambled antisense sequence. Secondly, 4G are capable of self-association. By increasing the frequency of G-clusters within an oligonucleotide, one will increase the probability of forming intramolecular associations, thus decreasing the chance of intermolecular interaction. 4G1s remain associated both inter and intrarnoIecularly for extended periods of time as their dissociation is governed by slow kinetics (Cheng and van Dyke, 1997).

Another structure that influences ODN specificity is the "CpG" motif, It is characterized by CG residues fianked by two purines on the S'end and two pyrimidines on the 3'-end (Krieg et al., 1995). The CpG motif possesses the capabiIity to alter the expression of genes other than the intended target. Oligonucleotides containing this motif induce 95% of al1 spleen B cells to enter the ce11 cycle (Chavany et al., 1995; Krieg et al., 1996). Additionally, the CpG motif can activate macrophages to release TNF-a,IFN--y, IL-6 and IL12 (Lipford et al., 1997) and promote immune desensitization to known allergens (Kiine et al., 1998).

1.1.4 Uptake and Distribution of Oiigonucleotides

Cetlular uptake of ODNs is an active concentration-dependent process that varies as a function of ODN length, sequence and modification in addition to the tissue and ce11 line used-

Loke et al. (1989) reported a decrease in uptake by HI.60, MOLT-4, and DA1 cells as the length of the ODN increased, Biessen et al. (1998) observed an impaired Iiver uptake of ODNs containing a high number of G residues within the sequence, and Dluzewski et al. (1992) reported that ODNs are incapable of penetrating adult erythrocytes. Funhermore, due to their slightly lipophilic nature and negative charge, PS ODNs have been reported to bind to a variety of proteins, including heparin, fibronectin and serum albumin thus reducing cell-association

(Stein, 1995). However, even though they exhibit non-specific binding, PS still remain more efficient at intemalization than PO ODNs and TOM (Boulme et al., 1998; Kanamaru et al.,

1998). PO ODNs have a limited uptake in most ceIl lines (Rowley et al., 19%) and 2'OM lack the affinity for cell-surface binding that PS ODNs experience (Zhao et al., 1993).

In addition to rapid degradation in a biological medium, a low cellular uptake and a temperature-dependent efflux mechanism may be Iimiting factors for the therapeutic use of

ODNs. Wickstrom et al. (1988) demonstrated that following a 24 hr treatment only 2% of a PO

ODN was internalized by HL-60,Rauscher erythroleukemia cells and Daudi cells. Moreover, sevenl groups have reported a loss of intracellular ODN concentration (as much as 80%) within three hrs of administration even though the half-life of the ODN (in medium) was much longer

(Crooke, 1991; Thierry and Dritshilo, 1992). Inefficient uptake and cellular efflux will require an increase in ODN dosage in order to obtain a significant inhibitory effect. This may result in cytotoxicity and sequence non-specific effects thereby Iimiting the therapeutic potential of

ODNs.

Uptake of ODNs can be enhanced by the addition of a delivery molecule or rnethod which may result in a decreased dosage required for a biological effect (nM vs. piid). Cationic lipids were first proposed as carriers by Felgner et al. (1987) and have since been applied to the delivery of a number of ODNs (Bennett et al., 1992; Colige et al,, 1993; Williams et al., 1996).

Addi tional deli very systems include liposomes (Juliano and Akhtar, 1992; Legendre and Szoka,

1992), dendrirners (Bielinska et al., 1996), polycations (Boussif et al., 1995), polymeric nanospheres (TondeIli et al., 1998), and membrane permeabilization by electroporation (Regnier and Preat, 1998) and streptolysin O (Giles et al., 1998). Each delivery system employs a distinct mechanism for ODN intemalization, which may be conducive for distinct cellular environments. Based on numerous in vitro studies, multiple cellular uptake mechanisms for ODNs have been proposed, Yabukov et al. (1989) and Stein and Cheng (1993) observed that in fibroblasts and HL-60 cells uptake depends on membrane binding of ODNs. Likewise, Pearson et al.

(i993) and Biessen et al. (1998) reported that ODN uptake was mediated by scavenger receptors in CHO and liver cells. These studies confinn that ODN uptake is mediated by a receptor or adsorptive endocytosis pathway. In contrast, others suggest ODN internalization is dependant on extracellular calcium levels and tempemure and not solely on membrane binding (Crooke, 1992;

Wu-Pong et al., 1994).

Upon in vivo administration. ODNs display heterogeneous distribution throughout the body. ODN uptake is observed mainly in the liver and kidney as well as the intestine, spleen, skin, and bone marrow (Zhao et al., 1998) and is not observed in the brain (Bennett, 1998;

Calabretta et al., 1996). Furthemore, oligonucleotides do not distribute uniformly within a specific tissue but accumulate within certain ce11 populations. They tend to be taken up preferentially by endothelial and Kuppfer cells in the liver and proximal tubule cells in the kidney (Biessen et al., 1998). Moreover, depending on the ce11 type and ODN concentration, intracellular distribution may differ. Graham et al. (1998) and Kanamaru et al. (1998) found a majority of ODN deposited in lysosomal (cytoplasm and membrane) compartments and less within the nucleus of liver nonparenchymal cells and human promonocytic leukemia U937 cells.

In contrast, Shoeman et al. (1997) and Li et al. (1997) found a more equal distribution of ODN within the nucleus and lysosomal compartrnents of epithelial PtK2 cells and adrenal cells respective1y.

When developing any type of therapeutic intervention, one must consider the relative uptake by diseased cells compared to their disease free counterparts. If no discrepancy exists, normal cells wiII be affected to the sarne extent as diseased cells thus lowenng the therapeutic efficacy of the agent It has been demonstrated that ODNs have an approximate four fold higher uptake by transformed cells over normal cells, thus justifj4ng their therapeutic potential in cancer therapy (Gray et al., 1997; Skorski et al., 1997; Zhao et al., 1996). Additionally, some oncogenes are activated upon mutation. Targeting these mutated genes will ensure ce11 specificity as normal cells will not contain these sequences. One example of this is the philadelphia chromosome (Saglio et al., 1999).

1.1.5 An tisense Oligonucleotide Mechanisms of Action

Antisense oligonuclectides can participate in numerous mechanisms that are dependant on both their composition (sugar, backbone, and base residues) and location of the mRNA binding site (5'-UTR, coding region, 3'-UTR) (Bennett, 1998; Crooke, 1996). One of the most widely used mechanisms is the degradation of RNA via ribonuclease H (RNase H) (Eder and

Walder, 1991; Minshull and Hunt, 1986; Veal et al., 1998). RNase H is a ubiquitous cellular enzyme that recognizes and cleaves the RNA component of RNA-DNA duplexes and is found in a variety of organisms as diverse as humans and viruses (HBV, HIV) (Branch, 1996; Le Tinevez et al., 1998). This enzyme is primarily detected in the nucleus with only trace amounts found in the cytoplasm, resulting from nuclear leakage (Crum et al., 1988). Levels of RNase H activity

Vary during development, differentiation and rate of ce11 division (Crooke, 1993). Besides

RNase H, additional enzymes located in the nucleus are capable of degrading target RNA. For example, RNaseL has ken irnplicated in selectively degrading bcr/abl mRNA in an mRNA-

ODN complex in CML cells (Maran et ai., 1998).

ODN modifications alter the molecule's ability to serve as a substrate for RNase H.

Sugar modifications that result in RNA-like oligonucleotides (for example TOM) inhibit the activity of this enzyme (Baker et al., 1997). In contrat, altentions to the backbone wil1 increase the ability of the ODN to activate RNase H: PS ODNs are excellent substrates for this enzyme

(Veal et al., 1998).

Despite the fact that a majoriq of ODNs employ RNase H for their mechanism of action, the mechanism for which most ODNs have ken designed is translational arrest. In this approach, ODNs are designed to bind to the translational initiation codon and block movement or assembly of the ribosomes (Baker et al., 1997). Target RNA species that have ken reported to be inhibited by this mechanism include L-myc (Dosaka-Akita et al., 1995), intercellular adhesion molecule 1 (Baker et al., 1997; Chiang et al., 1991), c-myc (Bacon and Wickstrom,

1991 ), and HTV (Agrawal er al., 1988) among others.

A third potential mechanism requires that the ODN disrupt RNA secondary structures.

As stated before, RNA adopts a variety of three-dimensional structures that are required for numerous activities; disniption of these structures will alter RNA function. Within Xenopus 28s rRNA a Ioop structure exists that is responsible for protein synthesis and ribosomal stability.

ODNs designed to hybridize to this loop structure inhibited protein synthesis when injected into a Xenopus oocyte (Saxena and Ackennan, 1990). Likewise, Petryshyn et al. (1997) synthesized a number of ODNs that bound to and disrupted the secondary structure of a cellular regulatory

RNA which activates an RNA dependent protein kinase, thereby preventing activation of the kinase.

Regulation of pre-mRNA maturation is yet another potential mechanism by which ODNs can in hi bit gene expression. Sierakowska et al. (1996) and Zirbes et al. (1997) demonstrated that oligonucleotides are able to alter splicing of $-globin mRNA in mammalian cells. The ODN rnasked the original spiice site forcing cleavage at an alternate site. Similarly, Hodges and

Crooke (1995) reported selective blocking of splice sites within an adenovims tmnscnpt.

Other noted mechanisms of action include: inhibition of 5'-capping (Bacon and Wickstrom, 1991; Williams et al., 1996) and 3' polyadenylation (Goodchild et al., 1988), transcriptional arrest (Helene et al., 19971, and oligonucleotide-induced cleavage (Manson et al.,

1990; Zamecni k and Stephenson, 1978).

1.1-6 Antisense Oligonucleotide Toxicity

The study of a number of potential therapeutic agents has been discontinued as a result of their high toxicity. PO ODNs are believed to be the least toxic ODNs in part because they are rapidly degraded. When 30 pM of a 15-mer PO ODN was incubated with human lymphocytes for 4 hrs, no toxicity was observed (Heikkila et al., 1987). Similarly, incubation of a transformed leukemic ceIl tine with 50 FM PO ODN demonstrated no decrease in cell viability

(Reed er al., i990a). In addition, 2'OM display low toxicities because they bind targets with the high specificity, thereby reducing sequence non-specific effects, and they do not sequester

RNase H.

PS ODNs on the other hand have been associated with slight toxicities. They exhibit non-specific binding and inhibition of DNA polymerases, reverse transcriptases, and nuclease when incubated in a cell-free system (Crooke et al., 1996; Crooke et al., 1995; Gao et al., 1989) as well as non-specific activation of the SPI transcription factor (Perez et al., 1994; Shoeman et al., 1997). Furthemore, high concentrations have been shown to interact and interfere with the activity of heparin-binding growth factors and the clotting cascade, and to induce immune stimulatory effects in rodents (Monia et al., 1996b). Additionally, when low doses are administered by fast infusion, thioates can cause hypotension, cardiovascular collapse and death in Rhesus monkeys (Calabretta et al., 1996; Galbraith et al., 1994).

Inadvertent hybridization of ODNs to non-target mRNA (resulting from the 4G, CpG or partially matched sequences, such as m.Asfor related proteins) can be potentially hazardous, particularily if the ODN employs RNase H. RNase H has been reported to be responsible for non-specific inhibition due to cleavage of imperfect hybrids resulting from ODNs binding to non-targeted mRNA (Le Tinevez et al., 1998; Giles et al., 1998). RNase H doesn't require sufficiently long recognition sequences to be highly specific (Branch, 1996); in vitro it recognizes four base pairs and in vivo ten (Donis-Keller, 1979; Woolf et al., 1992). Therefore, it is difficult to target single RNA targets without affecting other non-target sequences.

1.1.7 Antisense Oligonucleotides in Cancer Chemotherapy

Since oncogenes play a key role in the pathogenesis of several malignant diseases, they appear the most suitable targets for antisense-mediated treatment. Numerous reports have surfaced implicating ODNs as potential chernotherapeutic agents that inhibit a variety of oncogenes in tumors of different histotypes (Citro et al., 1992; Leonetti et al., 1996; Skorski et al., 1995). ODNs designed to target the Philadelphia chromosome (bcr-abl), c-myb, and c-myc have been shown to suppress leukemic cell proliferation while sparing the growth of normal marrow progenitors in ce11 lines denved from patients with chronic myelogenous leukemia

(CML) (Szczylik et al., 1991). Moreover, following transplantation of CML cells into severe combined immunodeficient rnice (SCID) and treatment with ODNs for 9 consecutive days there was a marked decrease in bcr-ab1 transcripts, leukemic ce11 number as well as fatality rate

(Ratajczak et al., 1992; Skorski et al., 1994).

ODNs have also been shown to inhibit expression of MLL-LTG19 (derived from a 1lq23 translocation), the anti-apoptotic members of the hi-2 family, and erbB-2 in addition to L-myc, c-raf kinase, p53 and Ha-ras (table 1). Inhibiting expression of these genes has led to decreased cellular proliferation and in certain cases increased sensitivity of malignant cells to cytotoxic chemotherapeutic agents (Citro et al., 1998; Skorski et al., 1993). It has ken clearly demonstrated that ODNs are effective inhibitors of gene expression and tumor formation both in vitro and within animal models. This has led to the investigation of their efficacy at treating diseases such as ovarian, prostate, breast, brain, colon, hg, and pancreas cancer in human subjects. Additionally, solid tumors, non-Hodgkin's Iymphoma, chronic myelogenous leukemia and acute myelogenous leukemia have been targeted for clinical trials (Agrawal, 1996; Bishop et al., 1996; de Fabritiis et al., 1998; Gewirtz et al., 1996; Webb et al., 1997). Preliminary results based on these trials suggest that ODNs are efficacious for the treatment of patients with malignant diseases. Table 1. Examples of Inhibition of Malignant Ce11 Growth In vhand in Animal Modeb by ODNs

Target Gene Target Cells Reference c-myc promyelocytic leukemia, (Larc her et al., 1992) Burkitt's lymphoma, melanoma, (Balaji et al., 1997) neuroblastoma, CML, prostate, (Broaddus et al., 1997) lung, breast and colon cancer (Cerutti et al., 1996)

L-mye small ce11 lung cancer (Dosaka-Akita et al., 1995) c-nty b CML, melanoma (Ratajczak et al., 1992) bcr-ab1 CML (Rowley et al., 1996)

Ha-ras bladder carcinoma, solid tumors (Saison-Behmoaras et al., 1991)

C-ruf kinase lung, bladder, breast carcinoma (Monia et al., 19%a) hl-2 follicular lymphoma, prostate (Kitada et al., 1994) cancer erbB-2 breast, pancreatic, ovarian, gastric (Slamon et al., 1987); non-small ce11 lung, and (Coussens et al., 1985); endometrial cancer (Berchuck et al,, 1991)

~53 AML (Bayever et al., 1994)

MLL-LTG 19 B-ce11 infantile acute leukemia (Akao et al., 1998)

1.2 Proto-oncogene c-myc

1.2.1 Background

The myc family of nuclear genes is comprised of five closely related members that include c-myc, L-myc, N-myc, B-myc and s-myc (Atchley and Fitch, 1995). These genes encode proteins that influence ce11 proliferation and differentiation (Cole, 1990; Spencer and Groudine,

1991). The c-myc gene was the first characterîzed member of the rnyc gene family and is the cellular homolog of the avian retroviral transforming gene, v-myc (Watson et al., 1983). c-mye is expressed at al1 stages of embryological development and dunng proliferation in a wide variety of adult tissues (Hesketh, 1994). It displays maximal expression in lymphoid organs and minimal in liver and brain (Morelle, et al., 1989; Meichle et al., 1992).

Cellular proliferation is controlled at least in part at the level of gene transcription, and activation of transcription factors is a necessary step in the transition from quiescence to proli feration. c-myc is an important transcriptional factor that regulates ce11 proliferation in addition to apoptosis and is important for normal ce11 function (Mihich, 1996). In resting cells, c-nzyc expression is undetectable, whereas entry of cells into the ce11 cycle produces a 50-fold upregulation of the gene (Blanchard et al,, 1985). Sirnilarly, inhibition of myc severely impairs growth-factor-induced proliferation of cells in culture (Roussel et al., 1996) and knockout mimals, in which c-myc has been disnrpted, die early in embryogenesis (Davis er al., 1993).

1.2.2 Structures of c-myc Gene and Protein

The c-myc gene is located on chromosome 8 and is encoded in a three-exon structure.

The major protein-coding domain is contained within the second and third exons and a majority of the first and third exons constitute the 5' and 3' untranslated regions respectively. The first exon can play an important role in goveming oncogenic potency (Kato et al., 1990).

Transcription of the c-myc gene is initiated at four distinct promoters (Ryan and Birnie,

1996) (figure 2a). The two major promoters, Pl and P2, are separated by 160 nucleotides and contribute to 10-25 and 75-90% of cellular transcnpts respectively (Marcu et al., 1992).

Approximately 1500 nucleotides downstream of P2 is the third promoter, P3 which accounts for only 5% of c-mye mRNA. Of similar activity and present only in human c-myc mRNAs, is a fourth promoter, PO. Differential usage of each promoter has ken observed in many ce11 lines and upon deregulated expression of the gene (Ryan and Bimie, 19%). The c-myc protein can exist as both a 64 and 67 Daspecies with a relatively short half-

life (DePinho et al,, 1991). Each arises from separate translation initiation sites, one is AUG and

the other is CUG (Amati et al., 1992). The protein product shares two regions of homology with

DN A-binding transcriptionai regdatory proteins (figure Sb). The first region, termed the leucine zipper (LZ), is shared with a group of proteins that include fos and jun proto-oncogenes

(Zimrnerman and Alt, 1990). It consists of a 20 to 30 nucleotide sequence with a leucine residue situated every second helical turn (Landschulz er al., 1988). Deietion analysis indicates the leucine zipper is involved in protein-protein interactions (Crouch et al., 1990). The second region covers a block of amino acids Iocated 30 residues before the C-terminal and is a composed of two amphipathic a-helices flanking a looped-out segment preceeded by a basic region (B). It is termed the Helix-Loop-Helix (HLH) domain. Deletion analysis indicates that the HLH domain is responsible for homo and hetero protein-protein interactions, DNA binding and transcriptional activation (Davis et al., 1990). Moreover, the type of dimer formed through the HLH interaction can determine the affinity and specificity of DNA binding (Benezn et al., l!WO).

These regions are important for many different functions of myc. Extensive studies have shown that the HLH and zipper domains are critical for direct transformation of the Rat 1A ce11 line and primary chick embryo fibroblasts (Stone et al., 1987; Crouch et al,, 1990). Furthemore, they are required for autoregulation (Crouch et al., 1990; Penn et al., 1990b) and inhibition of both preadipoc yte and murine erythroleukemia ce11 differentiation (Smith et al., 1990). NLS BHLHLZ *

Basic Region Helix 1 \"/ Helix II

Figure 2. Human c-myc Gene and Structurai Dom* of c-myc Protein

A. c-myc gene structure. The solid regions of exon 2 and exon 3 are the protein coding ngions, the gray/white (AUGKUG) sections represent the 5'-UTR and the striped section represents the 3'-UTR. Transcription is initiated froni the two major promoters: Pl and P2 in exon 1. Two c- myc proteins (p64/p67) with different Ktemiiai are translated from alternative translation initiation codons; AUG in exon 2 and CUG near the 3'-end of exon 1. B. Structural domains of the 64 kDa c-myc species. The domains are indicated at the top: a transcriptional activation domain mAD), an acidic region (A), a nuclear localization signai (NLS),a basic region (B), a helix-loophelix motif (HLH) and a leucine zipper domain 0.The location of the major in vivo phosphorylation sites are indicated by an asterisk (*). The acidic region and B-HLHregions are involved in interactions with other pmteins and DNA (adapted from Luscher and Esenman. 1990). 1.23 Mechanisms of Myc Regdation

Regulated expression of the c-mye gene plays a pivotal role in regulating ce11 growth and differentiation. Constitutive expression of c-myc can force cells to enter the S-phase of the ce11 cycle and inhibit induced differentiation of mouse erythroleukemia cells and human monocytic cells (Coppola and Cole, 1986), or in environments devoid of growth factors, cause cells to undergo apoptosis (Wagner et al., 1994). In contrast, deregulation of c-mye expression can alter the normally opposing processes of proliferation and differentiation resulting in neoplasia. For example, overexpression of c-myc has been observed in a variety of human leukernias and solid tumors (Blackwood and Eisenman, 199 1; Field and Spandidos, 1990; Hann et al., 1988; Klein and Klein, 1986).

During ceIl growth and differentiation, c-myc expression may be regulated transcriptionally (Bentiey and Groudine, 1986; Eick and Bomkamm, 1986; Mechti et al., 1986) or posttranscriptiondly (Herrick and Ross, 1994; Paulin et al., 1998; Yeilding et al., 1996). The contribution of each of these mechanisms to the determination of steady state levels of c-myc expression varies as a function of ce11 type (Swartwout and Kinniburgh, 1989) and in some cases may be the result of a combination of mechanisrns (Zimmerman and Alt, 1990).

Numerous studies have shown that steady-state levels of c-mye mRNA are more a function of the mRNA half-life then the rate of transcription (Levy et al., 1996a; Levy et al,,

1996b; Yielding et al., 1998; More110 et al, 1990; Sobczak et al, 1989). The control of messenger RNA turnover is an important means of regulating both the level and timing of gene expression. mRNAs whose protein products influence proliferation and differentiation are often relatively unstabie, with half-lives of 1 hr or less. As a result of this instability, modest changes in their turnover rates affect their steady-state levels over a relatively short time period (Brewer,

1998). Kelly et al. (1983) showed that an increased transcription rate observed during semm stimulation is not sufficient to account for the 20-40 fold increase in c-myc mRNA levels.

Similarly, increased c-myc expression in proliferating cells cm be attributed to increased rnRNA stability, as the transcription rate does not change. The half-life of c-myc is 15-30 mins in quiescent cells, whereas in proliferating cells it is up to 120 mins (Hemck and Ross. 1994). Its half-Iife can depend on extracellular signals, cytoplasmic regulatory factors (trans-acting), and inherent elements (cis-acting).

Two regions within c-mye mRNA (cis-acting) have been implicated as instability determinants (figure 3). The first is comprised of an AU-rich element (ARE) found within the

3'-untranslated region (UTR)(Shaw and Kamen, 1986; Aghib et al., 1990; Bonnieu et al., 1990) and the second is found in the carboxy-terminal portion of the coding region (Bonnieu et al.,

1990; Hemck and Ross, 1994; Prokipcak et al., 1994; Swartwout and Kinniburgh, 1989). In vitro experiments have suggested that each instability determinant segment functions independently, is recognized by different reguIatory factors, and specifies a unique decay pathway (Dani et al., 1984; Hemck and Ross, 1994).

The ARE consists of multiple copies of the pentamer AUUUA (Aghib et al., 1990) often in conjunction with one or more U-rich regions (Shaw and Kamen, 1986). As the frequency of this pentamer increases, the stability of the transcnpt decreases (Shaw and Karnen, 1986). ARES are present in unstable oncogene and cytokine transcripts (Lagnado et al., 1994; Zubiaga er al.,

1995) functioning as mEWA destabilizing elements (Bonnieu et al., 1990). The c-myc transcript contains two AUUUA motifs separated by 25 nucleotides (DeMaria and Brewer, 1996).

Additionally, the sequence flanking the ARE is 53% uridine residues (Alberta et al., 1994). This cis-acting domain has been shown to determine rnRNA turnover in proliferating but not differentiating C2C 12 cells fleilding et al., 1996).

The other stability determinant in c-myc rnRNA is found within the coding region of exon 3. More specifically, within the C-tenninus of the coding region, a purine nch 180-nucleotide sequence (coding region determinant), 1705-1886 nt, has been shown to function as an rnRNA destabilizer (Bonnieu et al., 1990; Herrick and Ross, 1994; Prokipcak et al., 1994; Swartwout and Kinniburgh, 1989). Depending on the ceIl type, this region may play a more prominent role in regulating the stability of the mRNA than the 3'-UTR (Laird-Offringa et al., 1991). The coding region determinant (CRD) is necessary for translational-dependent downregulation of c- mye during C2C12 myoblast differentiation (Yielding and Lee, 1997). Ribosomes must translocate through the CRD to destabilize the transcript (Wisdom and Lee, 1990; Wisdom and

Lee, 199 1). Moreover, Lavenu et al. (1995) suggested the exon 3 coding sequence is responsible for tissue specific expression patterns of c-myc and induction of myc rnRNA in a regenerating li ver.

A variety of models exist for the degradation of an mRNA species. One major

mechanism is initiated by the shortening of the poIy(A) tail (Decker and Parker, 1993), which is directed by specific elements or sequences within the coding region and 3WTR of the transcript

(Brewer, 1998; Ross, 1995; Wellington et al., 1993). This deadenylation can lead to a decapping reaction that is followed by 5'-3' exonuclease activity (Muhlrad et al., 1994) or it can promote interna1 cleavage or degradation of the mRNA via a 33' exonuclease pathway (Muhlrad er al.,

1995; Muhlrad and Parker, 1994). Decapping can also arise through deadenylation-independent pathways. Muhkad and Parker (1994) demonstrated that premature translational termination in yeast can trigger decapping of rnRNA, thus exposing the uanscnpt to 5'-3' degradation.

An alternative mode1 implies that endonucleases recognize and cleave specific sequences within the transcript followed by rapid degradation catalyzed by a 3'4' exonuclease (Chen and

Shyu, 1995; Shyu et al., 1989). This may be the case with the surrounding areas of ARES of hiphly unstabie rnRNAs (Stoeckle and Hanafusa, 1989), the 3' stem-loop of histone mRNAs

(Gick et al., 1986) and an element in the 3'-UTR of transfemn receptor mRNA (Roberts and

Griswold, 1990). Additionally, ribosome-associated factors may degrade the transcript within the open reading frame. When ribosomal translocation is inhibited, almost al1 mRNAs are stabilized (Hemck, 1990)-

c-myc transcripts lacking the CRD do not produce an endonuclease decay product, but instead experience 3'-5' exonuclease decay (Herrick and ROSS,1994) suggesting that this element is cleaved by a specific endonuclease. Additionally, single point mutations in each of the three AWAmotifs within c-fos ARE indicate that the ARE is involved in rapid removal of the poly(A) tail and degradation of the transcribed portion of the message. However, the rapid removal of the poly(A) tail does not require the intact pentarner, whereas the degradation of the transcript does (Shyu et al., 199 1). In both models, it is believed that specific trans-acting factors recognize and bind to the cis-acting elernents, altering mRNA metabolism (Rajagopalan and Malter, 1994)- These RNA- binding factors can influence mRNA transport, localization, translation, and degradation

(Frankel et al., 1991; McCarthy and Kollmus, 1995). One such ARE-binding factor is AUFl. It was reported that the binding affinity of AZTFl correlated with its ability to direct rnRNA degradation, suggesting that this protein has a dein ARE-regulated turnover of c-myc mRNA

(Brewer, 1991). AUFl is comprised of two RNA recognition Motifs (RRM) and a glutamate- rich region present within the C-terminal (Ehrenman et al., 1994).

A second binding factor is a member of the Elav-like protein farnily. This protein, Ha, contains three RRMs (Kenan et al., 199 1), exhibits a high binding affinity for c-mye (Ma et al.,

1996) and is expressed in a wide variety of human tissues (Gw~,1995). It has been reported that HuR stabilizes vascular endothelial growth factor mRNA 3-8 fold following hybridization to its ARE. This is accomplished by eithet inhibiting endonucleolytic cleavage at the ARE or preventing the deadenylation of the mRNA (Levy er al., 1998).

A third tram-acting factor that as of yet has not been implicated in c-myc regulation, is

Tristetrapolin (TTP). This zinc-finger protein has been implicated in posttranscriptionai regulation of the ARE-containing TNF-amRNA and could therefore possibly regulate c-myc

(Carballo et al., 1998)-

In addition to the ARE, several factors bind to the CRD and affect mRNA stability; the most notable of which is the CRD binding protein (CRD-BP). CRD-BP is a member of the family of RNA-binding proteins containing two RRM, an RGG box and four KH regions (Doyle et al., 1998). It is a 70 kDa polysomal associated protein that binds to the coding region determinant and is believed to prevent ribosome-associated endonucleolytic cleavage of c-myc mRNA (Prokipcak et al., 1994; Lee et al., 1998). Evidence for this theory comes from studies that report those transcripts bound by CRD-BP experience m.Adecay through the 3'-5' exonuclease pathway (Herrick and Ross, 1994; Leeds et al., 1997). When CRD-BP is titrated off by cornpetitor RNA,c-mye mRNA is destabilized 8-fold (Bernstein et al., 1992). CRD-BP is a developmentally regulated oncofetal protein expressed in fetal tissue and transformed cells

(Doyle et al., 1998) and its levels directly correlate with c-myc expression during rat liver development (Leeds et al., 1997).

A second trans-acting factor implicated in c-mye stability through the CRD is thymïdylate synthase. This protein binds to the CRD of c-mye in human colon cancer cells and prevents trandation, thus increasing stability of the transcript (Chu et al., 1995). When titrated off c-myc mRNA, translational inhibition is relieved and the transcript destabilized.

1.2.4 Activation of Gene Expression &y Myc

The proto-oncogene c-mye is a nuclear phosphoprotein t hat functions as a transcriptional factor for genes that regulate cellular proliferation and differentiation. An independently regulated protein, Max, acts as a partner protein and dimerizes with c-myc to form a specific

DNA-binding complex (Blackwood and Eisenman, 199 1) (figure 4). Max, like rnyc, has a helix- loop-helix zipper motif, and MycfMax complexes recognize and bind to enhancer box (E-box)

DNA elements with the core sequence CAC(G/A)TG (Blackwood et al., 1992; Kato et al.,

1992). This binding is highly dependent on the existence of both the HLH leucine zipper domains and the basic region (Blackwood and Eisenman, 1991; Prendergast et al., 1991).

Furthermore, transcriptional activation is dependent on the N-terminal activation domain in c- rnyc, which is non-existent in Max (Kato et al., 1990).

In contrast, Max cm also act as a suppressor of c-myc transcriptional activity (Amati et al.,

1993; Blackwood et al., 1992). Max can form complexes with proteins including Mad. Mxi-1 and Mnt. Homodimeric complexes of Max and heterodimeric complexes of Max and Mad Mxi-

1 or Mnt repress gene-specific transcription (figure 4). Mad proteins accumulate during differentiation in several ce11 types (Ayer and Eisenman, 1993). Thus, interactions between Myc and Max constitute a sensitive switch that controls entry and exit fkom the ce11 cycle (Amati et al., 1993).

Numerous target genes of Myc-Max have been isolated through a variety of approaches.

These genes are thought to encode regulators of ce11 cycle activation and apoptosis. These include ornithine decarboxylase (involved in polyamine synthesis) (Bello-Femandez and

Cleveland, 1992), Cdc25A (essential for transition from Gl to S-phase under growth stimulation conditions and in absence of growth conditions helps induce apoptosis activity) (Galaktionov et al., 1996), cyclin Dl (Philipp et al., 1994), a-prothymosin (transcriptional factor) (Eilers et al.,

1991), ECA39 (regulator of G1-S transition) (F3envenisty et al., 1992), eIF4E (translational- initiation factor) (Jones et al., 1996), MrDb (encodes an RNA-helicase) (Grandon et al., 1996) and cad (Miltenberger et al., 1995).

Although it has been obsewed that c-myc can activate transcription of specific genes, cells transfonned by c-myc also demonstrate loss of expression of certain genes (Judware and Culp,

1995; Penn et al., 199ûa) (figure 4). One such repressed gene encodes the transcription factor c/EB P-a, which induces ce11-cycle arrest and commi ts cells to adipocyte di fferen tiation (Freytag and Geddes, 1992) and another, p27 Cm, encodes an inhibitor of Cdk2 kinase activity Uone et al., 1997). This repression is achieved by interactions between c-myc and YY-1, TFII-1, and

Miz- 1. Both YY-1 and TFII- 1 activate transcription from initiator elements; binding of c-myc to these proteins inhibits their interaction with the TATA-binding protein (Shrivastava et al., 1996), whereas c-mye inhibits Miz-1 induced growth arrest (Peukert et al., 1997). Myc-induced suppression of gene expression may also play a role in tumorigenesis. Heterodimerization with other factors Heterodimerization: Max-Mad 1 Max-Mxi- l -CAG(G/A)TG- h) 00 Max-Mnt I

Activation of transcri ption Repression of transcription

Figure 4. Pathways of transcriptional Activation or Reprcssion by c-myc

MyclMax heterodimers are transcriptional activators that bind to the E-box (-CAC(G1A)TG-) motif, found in target genes. Max can ah form homodimers or heterodimers with Mad. These complexes are transcriptional repressors that remit histone deacetylases through the Sin3 protein. In addition to the formation of heterodimers with Max, Myc protein can interact with and inhibit tramactivation by YY-1, TFiI- 1 and Miz- 1 (Bouchard et al., 1998). 1.3 Myc as a Target of ODN therapy

Numerous studies have demonstrated that c-myc is a potential and effective target for

ODN action in cancer chemotherapy (Balaji et al., 1997; Broaddus et al., 1997; Cerutti et al.,

1996b; Gryaznov et al., 1996). The most widely used ODN targets the initiation codon and the next four codons of the transcript (named ODN-AUG). Its sequence is

AACG'ITGAGGGGCAT (Balaji et al., 1997; Bennett et al., 1994; Broaddus et al., 1997) and it is synthesized as a phosphorothioate derivative. The initiation codon is popular as a target site because it is devoid of secondary structures; therefore it is relatively open to hybridization by

ODNs (Konings et al., 1987). Targeting this sequence assumes that ODN inhibition results from hybridization arrest of mRNA translation by ribosomes andor cleavage by RNase H cleavage

(Zamecnik and Stephenson, 1978).

ODN-AUG has ken effective at inhibiting c-rnyc expression in malignant glioma cells,

CML, prostate cancer, melanoma and thyroid carcinoma cells among others (Bacon and

Wickstrom, 1991; Balaji et al., 1997; Broaddus et al., 1997; Cerutti et al., 1996a; Citro et al.,

1998). It has been proven to be specific as well as dose-dependent, exhibiting a maximal 70% inhibition of c-nzyc protein expression at 10 pM in HL-60cells (Bacon and Wickstrom, 1991), an

80% inhibition after treatment of 4 pM to the AR0 ce11 line (Cerutti et al., 1996a), and a 40% inhibition after a 4 day treatment of Ml4 cells with 200 nM (Citro et al-, 1998). Additionally, in the LNCaP ceIl line, 5 PM of ODN-AUG reduced ce11 growth by more than 80% (Balaji et al.,

1997) while in RT-2 cells a 10 pM treatment of ODN-AUG decreased ce11 growth by 75%

(Broaddus et al., 1997).

However, despite being employed in the majority of studies to date, ODN-AUG may not be the most ideal agent for inhibition of c-myc expression. The presence of both a 4G and CpG motif in addition to the PS backbone increases the probability of non-specific effects (Saijo et al., 1997; Chavany et al, 1995; Krieg et al., 1996). Furthemore, for some transcripts, sites within the 5'-UTR (Bacon and Wickstrom, 1991; Larrouy et al., 1995)- protein coding

(Gryaznov et al., 19%)- and 3'-UTR (Monia et al., 1992; Tu et al., 1998) have been shown to be more sensitive to ODN action than the initiation codon. Therefore, the initiation codon may not be the best suitor for c-myc inhibition.

1.4 Rationale for Current Study

The previously reported ODNs were designed to anneal to various target sites within the transcript and disrupt translation or induce cleavage via RNase H. Preceding studies have demonstrated that cleavage mediated by RNase H is not always specific (Le Tinevez et al., 1998;

GiIes et al., 1998), and translationai inhibition leads to increased stability of c-myc mRNA

(Yielding and Lee, 1997). Both mechanisms may produce undesired effects; the ODN may inhibit expression of non-target genes, or possibly induce a rebound reaction whereby the stabilization of mRNA will eventually lead to the overexpression of c-myc protein.

With the recent identification and characterization of the protein, CRD-BP (Bernstein et al., 1992; Prokipcak et al., 1994), a new strategy may be employed. By utilizing an ODN to occlude the CRD-BP €rom binding to the CRD, one might leave the c-myc rnRNA vulnerable to endonucleolytic attack, thus destabilizing it (figure 5). "Breathing" of RNA @protein corn plex

"Naked" mRNA Endonucleol ytic cleavage of mRNA

Figure 5. Schematic Representation of Pmposed ODN Mechanism of Action

The CRD-BP is believed to bind to and protect the c-myc mRNA from endonucleolytic cleavage. Fnquent "breathing" of RNA.protein complex may allow ODNs to hybridize the mRNA. Once CRD-BP has been displaced from c-mye mRNA,the transcript is vulnerable to cleavage and degradation by endonucleases. 1.41 Prelimiaary Data

It has ken previously determined that the interaction between the CRD-BP and c-mye mRNA is Iocalized to the last 180 nt of the coding region of exon 3 (Bernstein et al., 1992). To specifically map the site of protein binding and determine the effectiveness of ODNs at inhibiting RNAmprotein interactions, a panel of ODNs complementary to sections along the 180 nt CRD region was prepared (table 2). Gel-shift analysis were canied in the presence of 1 pmol/çll (1 FM) of ODN, Ribosomal Salt Wash (RSW) and 3'~-labelled RNA. ODN4, which covers nucleotides 1763-1777, was the most effective at preventing the RNAaprotein interaction foI1owed by ODN5, ODN3, and ODN8 (figure 6). These ODNs are believed to bind the c-myc mRNA at the CRD and block sites important for protein binding.

Table 2. ODN Sequences Used in Study Sequence Location ODN-AUG" AACGïTGAGGGGCAT 570-584 (Exon 2) AUG-Scram" AGCTGGGGTAGCAAT ODN 1 TGïTTTCCAACTCCG 1714-1728 (Exon 3) 0DN2 TTTCA'ITGTl-ITCCA 1720- 1734 (Exon 3) 0DN3 GGATAACTACCITGG 1741-1755 (Exon 3) 0DN4 ATGTATGCTGTGGCT 1763-1777 (Exon 3) ODN4-MM ATGCATACAGTGGCT ODNS ACGGACAGGATGTAT 1772-1788 (Exon 3) ODNS-MM ACGGAAAGCAGGTAT 0DN6 AGCTTITGCTCCTCT 1793-1807 (Exon 3) 0DN7 GTlTCCGCTTC'ITGT 1822-1836 (Exon 3) ODN8 TCCGTAGCTGlTCAA 1861-1875 (Exon 3) "contains 4G motif bcontains CpG motif underlined nucleotides represent mismatches as compared to parent sequence - Prottin-RNA Complex Free - RNA

Figure 6. GeCShift Anslysis of Panel of ODNs Tqeting the CRD of c-myc mRNA

A. Autoradiograph of a gel-shift experiment. Lane 1 contains 32~-labelledCRD RNA alone. Lane 2 contains RNA in the presence Ribosomal Sait Waoh (RSW). Lane 3 thiough 10 contains 1 pM of indicated ODN in addition to RNA and RSW. B. Data fmm gel-shift ex-ents was quantitated using a Phosphorlmager (Moleculsr Dynamics) and signal intensity was expresscd as percent of control signal flanc 2). Numbers indicate ODN used. To further characterize the effect that ODN4 exhibits on CRD-BP.RNA interaction, additional gel-shift experiments were carried out with increasing concentrations of 0DN4 and

ODN5. Inhibition of complex formation was observed with as little as 10 nM 0DN4 and was maximai at 1 (75% inhibition). ODNS, which was less effective than ODN4, required 100 nM to observe an effect. To assess the specificity of 0DN4 and ODN5, three mismatches were added to the sequences (ODN4-MM and ODNS-MM). Neither ODN4-MM nor ODNS-MM were able to inhibit the CRD-BP.RNA interaction suggesting that the observed effect was specific (Coulis et al.,manuscript submitted). 1.4.2 Hypo t hesis

We have proven that, in vitro, it is possible to obstmct the interaction between CRD-BP

and c-myc mRNA by antisense oligonucleotides. We hypothesize that these results will be replicated within a cellular environment. We will employ an ODN derivative that does not support Nase H activity or induce sequence non-specific effects (TOM)and compare its efficacy at inhibiting c-myc expression to an ODN targeting the initiation codon. If CRD-BP does indeed function by preventing endonucleolytic cleavage of c-mye mRNA, then by disrupting the interaction we may decrease c-mye expression within cells.

Because the CRD-BP is an oncofetal protein, this strategy may be useful in targeting transforrned tissues or ce11 lines that overexpress c-myc. It has been demonstrated that prevention of myc function and expression is sufficient to inhibit DNA synthesis and cellular proli feration (Barone and Courtneidge, 1995; Wickstrom et al., 1988). Therefore an ODN designed to inhi bit the CRD-BP.CRD interaction could speci fically inhibit the uncontrolled ce11 proliferation that is characteristic of cancer. 2.1 Source of Materials

Ce11 Line. The human erythroleukemia cell line, K562, was established from bone marrow

samples taken from a woman with chronic myelogenous leukemia (CML)(American Type

Culture Collection, Rockville, Maryland)- K562 cells were cultured in suspension at 37OC and

5% C01/95% room air in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) (Gibco

BRL, Burlington, ON).

Oligonucleoride Preparation. 2'-O-methyl derivatives, with a phosphodiester backbone, and

Phosphorothioate antisense oligonucleotides were synthesized by Dalton Chemicals (North

York, ON). The sequences were designed to overlap the initiation codon (AUG) (Balaji et al.,

1997; Bennett et al., 1994; Broaddus et al., 1997) and the coding region determinant (CRD) found within exon 3 of c-myc mRNA (table 2). Al1 ODNs were 15 nucleotides long, those targeting the CRD had a 47% G/C content and those targeting AUG had a 53% G/C content.

OIigonucleotides were resuspended in RPMI-1640 containing no serum to give a final concentration of 1 rnM. Final concentrations varied depending on experiment performed and are provided in figure legends.

Probe Preparafion for Northern Analysis. c-myc and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) probes were synthesized by Polymerase Chain Reaction (PCR).

Oligomers at each end of the coding region for c-myc and GAPDH were synthesized by General

Synthesis and Diagnostics (Toronto, ON) and were added to 1 ng of plasmid DNA containing the corresponding cDNA as a template for probe preparation. The primer sequences have been previously described (Andreou and Prokipcak, 1998). The c-myc probe spanned 1.3 kb of the coding region; the GAPDH probe approximately 0.69 kb corresponding to the GAPDH coding region. The PCR products of 10 reaction tubes were pooled and underwent phenoYchloroform extraction to isolate the DNA found in the aqueous phase. The DNA was then precipitated in

100% ethanol and resuspended into a final volume of 400 pl ddH1O. The probes were then separated from primers, excess nucleotides and primer-dimer side products using Amicon microcon-100 spin filters (Amicon Canada Ltd., Oakville, ON) following the manufacturer's instructions. 1-3 11of each probe and a LOO bp DNA ladder (Pharmacia) were then separated on a 1% agarose gel to verify the size and concentration of the DNA.

iubeling DNA Probe. 100 to 200 ng of DNA probe was placed in a mi-crofuge tube with enough ddH,O to give a final volume of 16 pl. The sample was then placed on a heat block (95- lOO0C) for 10 min, then placed immediately on ice. 5 pl of Oligo labeling buffer (OLB)(0.24/0.024 M

TrisMgCL, 2 mM Dm, 0.1 mM dATP O. 1 mM dGTP, 0.1 rnM dm,1 M Hepes (pH 6.6), 15 units random primers (Pharmacia)) 1 pl BSA (New England Biolabs), 1 pl Klenow fragment

(Pharmacia), and 2.5 11 "P dCTP were added and the solution was incubated at room temperature for 2-3 hrs. 225 pl stop solution (RNase InactivatiodPrecipitation mixture,

Ambion) was added and double stranded DNA was denatured by heating the sample at 95°C for

5 min followed by placing it irnmediately on ice. The sample was then added to 15 ml of hybridization solution and incubated with the membrane for 18 to 24 hrs.

Probe Preparation for Ribonuclease Protection Assay. c-myc and GAPDH probes were synthesized by an in vitro transcription reaction. 0.5 pg of pTRI-cmyc or pTRI-gapdh DNA template (Ambion) was incubated with 6 pl dcWZO, 1 pl RNA guard (Pharmacia), 1 x T7/T3 buffer (Gibco BRL), 5 rnM DIT,0.5 m.each of ATP, GTP and CTP and 50 pM UTP,50 uni&

T7 RNA polymerase (Gibco BRL), and 3 pl ''P UTP at 37OC for 1.5 to 2 hrs. 10 units of RNase- free DNase (Pharmacia) was added and incubated for a further 15 min. The RNA probe was cleaned up using an RNaid kit (BI0 10 1, Inc-). To each sample, 60 pl RNA binding salt and 5 pl

RNAmatrix was added and incubated at room temperature for S to 10 min followed by a quick spin for 1 min. The supernatant was discarded into radioactive waste and the pellet washed twice with 500 pl solution. After one final spin, the excess liquid was removed and the pellet air-dried for 5 min. The pellet was resuspended into 50 pl DEPC-treated water and incubated for

5-10 min at 50-5S°C followed by a 1 min spin. The RNA, in the supernatant, was transferred to a clean tube and re-spun to remove any matrix carryover. The supernatant was then carefully removed without disturbing the leftover matrix and stored at -20°C. 1 pl of probe was then diluted to 10 pi for determination of total cpm in a liquid scintillation counter, The size of the probe was also verified on a 5% acrylamide/7 M urea minigel with "P markers. After the size and cpm had been determined, the probe was diluted to 90,000 cprnfpl.

2.2 Treatment of cells with Antisense Oligonucleotides

Transfection of ODN into cells 5 ml of 4 x ld K562 celldml were piated on 60-mm ce11 culture dishes in RPMI- 1640 with 10% FBS. After 24 hr, cells were collected by centrifugation at 1000 rpm and the medium was removed. For treatment with oligonucleotides in the presence of Superfect (Qiagen), the cells were washed once with 1 ml of phosphate buffered saline (PBS), resuspended in 5 ml of fresh RPMI-1640 containing 10% FBS and plated on 60-mmcell culture dishes. ODNs were diluted in 150 pl serum-free RPMI, and Superfect was added to give a final concentration of 6 pl Superfect reagent per pg oligonucleotide, up to a maximum of 30 pl

Superfect per 5 ml culture medium. The solution was vortexed for 10 seconds then incubated at room temperature for 5-10 min, after which it was added to the ceils in a dropwise manner. Cells were incubated for 6, 24 or 48 hrs before RNA and protein isolation or evaluation of cell number. Concentrations of ODNs are expressed as the final concentrations in the ce11 culture medium, and ranged from 10 nM to 750 nM.

For treatment with ODNs in the presence of Lipofectin (Gibco BRL), 2 ml of 1 x 106

K562 celldml were plated in RPMI-1640 containing 10% FBS and incubated at 37OC in a CO2 incubator for 24 hrs. The cells were collected by centrifugation at 10rpm, washed once with

RPMI-1640 without serum and resuspsended in serum-free medium. Two solutions were then prepared in sterile microtubes; in one ODNs were diluted into 100 pl RPMI-1640 with no serum to give a final concentration of 5 pM in 5 ml and kept on ice for 30 min. In the second tube, 5 pl

Lipofectin was incubated with 100 pl RPMI-1640 with no serurn at rwm temperature for 30 min. The two solutions were then combined, mixed gently and left at room temperature for 10-

15 min. 0.8 ml serum-free medium was added to the complexes and then added to the cells.

Following a 5 hr incubation at 37OC in a CO, incubator, 2 ml of RPMi-1640 in the presence of

10% FBS was added to the cells and incubated for 19 hrs, giving a total ODN exposure time of

24 hrs. RNA and protein isolation and evaluation followed.

For treatment with ODNs in the presence of DME-C (Gibco BRL). 2 ml of 1 x 106

K562 cellsfml were plated in RPMi-1640 containing 10% FBS and incubated at 37OC in a CO, incubator for 24 hrs. The cells were collected by centrifugation, washed once with RPMI-1640 without serum and resuspended in 2 ml serum-free medium. Two solutions were prepared in sterile microtubes. In one tube, ODNs were diluted into 500 pl of medium without serum to give a final concentration of 5 pM in 5 ml. In the second tube, 5 pg of DMRIE-C was added to 500 pl of serum-free medium and mixed. The two solutions were then combined, mixed and incubated at room temperature for 30 min. ODN-DMRIEC complexes were added to the cells and incubated for 5 hrs at 37OC in a CO2incubator, followed by addition of 2 ml of RPMI-1640 in the presence of 10% FBS. The cells were incubated for the next 19 tus giving a total exposure time to the ODN of 24 hrs. RNA and protein were then isolated and evaluated.

23 Expression of c-myc mRNA and Protein in K562 cells

RNA Isolation. To examine c-mye gene expression in K562 cells following ODN treatment, total celIuIar RNA was extracted using TRIzol reagent (Gibco BRL, Burlington, ON). Following a 24 hrs treatment with ODNs, approximately 4 x 106 cells were collected by centrifugation at

1000 rpm and the supernatant was removed. Cells were lysed in I ml TRIzol reagent and lysates were incubated at room temperature for 5 mins. 2 ml of Chloroform was added and the mixture was shaken vigorously for 15 seconds and was incubated at room temperature for an additional 2 mins. Cellular debris was removed by centrifugation in an Eppendorf microfuge 5415 C at

12,000 x g for 15 mins at 4°C and the top aqueous phase was transferred to new microfuge tubes.

0.5 ml of isopropyl alcohol was added and the mixture was incubated at roorn temperature for 10 min to precipitate RNA. Samples were centrifuged at 12,000 x g for 10 min at 4OC and the supernatant was removed. The RNA pellet was washed with 1.5 ml of 75% ethano1 and centrifuged at 7,500 x g for 5 min at 4OC. Following removal of the ethanol with a pipette, the

RNA pellet was air dried, whiIe on ice, for ten mins. The dried pellet was then resuspended in

20 to 30 pl ddH1O followed by treatment with 2 pl oFDNase 1 (10U/1ûûpl;Pharmacia) at 37OC for 15 min and 55°C for ten mins. Concentrations of extracted RNA were determined by spectrophotometric analysis of absorbance of the sample at 260 nm (A,) and the punty of the sample was assessed by examining the A*, ratio. RNA Elecîrophoresis and Northem Trunsfer- Detection of c-myc and GAPDH mRNA was performed using Northern Blot analysis (Sambrook et al., 1989). 20 pg of total RNA was denatured in a sample buffer containing 50% deionized formamide (v/v), 2.2 M formaldehyde,

50 mM 3-IN-Morpholino] propanesulfonicacid (MOPS) and RNA loading dye (50% glycerol,

0.25% bromophenol blue, 0.25% xylene cyanol) for 10 mins at 70°C. The RNA was then electrophoresed through a 1% agarose, 6.6% fonnaldehyde denaturing gel (1% agarose (wlv), 50 rnM MOPS, 2.2 M formaldehyde) in a 50 mM MOPS running buffer. The electrophoresis was stopped when the gel dye front migrated to = 2 cm from the end of the gei (approx. 2 hrs at 150

V) and the gel was nnsed in distilled water followed by incubation for 20 mins in 10 x SSC (1.5

M NaCI, 0.15 M Na Citrate). The RNA was then transferred to a Zeta-probe blotting membrane

(Bio-Rad) by capillary transfer in 10 x SSC. Following an overnight transfer, the membrane was washed briefly in distilled water, and UV cross-Iinked to stabilize the RNA interaction with the membrane. To assess the effîciency of the transfer, the membrane was stained with methylene blue (0.04% methylene blue (w/v) in 0.5 M sodium acetate, pH 5.2) and intact 28s and 18s nbosomal bands were visualized.

Northem Hybriditation Analysis. Membranes were pre-hybridized in 10 ml hybridization buffer (50% deionized formamide (v/v), 2% SDS, 30 mM NaH,PO,, 125 mM NaCl, 0.5 rnM

EDTA) for 2-3 hrs at 42OC in a hybridization oven. The prehybndization solution was discarded and hybndizations were performed for 20 hrs in hybridization buffer containing 3ZP-labelled cDNA probes for human c-mye and mouse GAPDH. The membrane was then washed in 2 x

SSC/O.l% SDS for 15 min at room temperature followed by a wash in 0.1 x SSC/O. 1% SDS at room temperature for 15 mins and 0.1 x SSCIO. 1% SDS at 5S°C for 15 mins for c-myc- For

GAPDH, the membrane was washed in 2 x SSClO.195 SDS for 15 min at room temperature followed by 1 x SSUO.l% SDS at room temperature for 15 min and 1 x SSC/O. 1% SDS at 37°C

for 15 min. Blots were then exposed for 24-36 hr at -70°C to Kodak X-OMATScientific

Irnaging film using intensifying screens followed by quantitation with a Phosphorlmager

(Molecular Dynamics).

Ribonuclease Protection Assay- mase Protection was camed out using a commercial kit

(Ambion RPA II kit). To examine c-myc mRNA levels following ueatrnent with ODN-AUG in the presence of various carrier molecules, 15 pg of total RNA was incubated with 1 pl c-myc and

GAPDH radiolabelled probes (2-8 x 10' cpm per IO pg total RNA) and 20 pl hybridization buffer (80% deionized formamide, 100 mM sodium citrate pH 6.4, 300 rnM sodium acetate pH

6.4, 1 mM EDTA) at 42OC for 18 hrs. After incubation, 200 pl of digestion buffer (1: 100 RNase

AIRNase Tl) was added to each sample followed by another incubation at 37OC for 30 min. 300

~1 of precipitation solution (RNase Inactivatiodprecipitation mixture) was then added and the solutions were stored at -20°C for 1-2 hrs. The samples were then spun for 20 min at 12,000 rpm and the supernatant decanted. Excess supernatant was removed following a second quick spin.

The RNA pellets were resuspended in 7 ~l loading dye, heated at 95OC for 3-4 min and run on a

5% acrylamide/7M urea gel in 1 x TBE (80 mM Tris base, 80 mM boric acid, 2.5 rnM EDTA) for 1.5-2 hrs at 250 V. Gels were then dried for 1 hr and exposed for 24-36 hr at -70°C to Kodak

X-OMAT Scientific Imaging film using intensifying screens followed by quantitation with a

P hosp horImager (MoIecular Dynamics).

Protein Isourtion. To examine c-myc protein levels in K562 cells following ODN treatment, 4 x

106 cells were lysed directly in a lysis buffer (50 mM Hepes-KOH (pH 7.0), 1 mM EDTA, 420 mM NaCl, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyi fluoride, 10 pghl Aprotinin, and 10 pg/ml Leupeptin). After incubation on ice for 30 mins, cellular debris was removed by centrifugation in an Eppendorf microfuge 5415 C for 10 min at 12,000 x g at 4OC (Sambrook et al., 1989). The supernatant was stored at -70°C and protein concentration was measured by the

Bradford method (B ioRad) (Bradford, 1976).

Protein Elechophoresis and Western BIot Anaiysk. Total cellular protein (50 pg) was rnixed with an equal volume of 2xSDS-polyacrylarnide gel electrophoresis (PAGE) sample buffer (4%

SDS, 20% glycerol, 200 mM DTï, 120 rnM Tris (pH 6.8), and 0.01% bromophenol blue) and boiled for 5 min. The protein was then resolved by a 10% SDS-Page at 150V for 1.5 hrs and was transferred ont0 a pol yvin ylidene fluoride membrane (Immobilon PTMillipore, Mississauga,

ON) using a wet transfer system (Bio-Rad, Mississauga, ON). Membranes were blocked overnight to prevent non-specific protein binding with TBS-T (0.1% Tween 20, 20 rnM Tris-

HCl, 137 mM NaCl) containing 5% skim milk powder at 4°C. The membrane was incubated with anti c-nzyc monoclonal antibody (AB-1, Calbiochem) at a 1:1000 dilution in TBS-T for L hr at room temperature. Blots were washed 1x15 min followed by 2x5 min with TBS-T then incubated with a horseradish-peroxidase conjugated anti mouse IgG antibody (Amersham) at a

1:5W dilution for 1 hr, followed by washing with TBS-Tas before. A solution of 2 ml of each of the cherniluminescent reagents (reagent 1 and reagent 2) (ECL detection, Boehringer

Mannheim, Germany) was poured on the blot for 1 min and the membrane was exposed to

Kodak X-OMAT Scientific Imaging film for 5 to 15 min to visualize the c-myc protein.

To confirm equal loading of protein, biots were stripped with a stripping buffer (62 mM

Tris-HCI, 2% SDS, 100 mM 2p-mercaptoethanol) and reprobed with anti-glyceraldehyde-3- phosphate dehydrogenase monoclonal antibody (Chemicon International, Temecula, CA) diluted to 1500 in TBS-T. Washing, incubation with secondary antibody, chemiluminescence solution and exposure were identical to those described above for c-myc. Exposed films for both c-mye and GAPDH were scanned with a Super Vista S-12 scanner (UMAX Data Systems, Hsinchu,

Taiwan) and densitomatric analysis was perfonned with abGel H (Signal Anaiytics Corp.).

2.4 Estimation of c-myc mRNA HaELife

4 x 106K562 cells were treated with 200 nM of ODN for 6 or 24 hrs, after which time the cells were collected by centrifugation at 100rpm and the supernatant was removed. The medium was repiaced with 5 ml RPMI-1640 with 10% FBS containing 5,6-dicloro-1-beta-D- ribofuranosylbenzimidazole (Dm) at a final concentration of 100 @lin 0.1% DMSO for up to

3 hrs. Total RNA was isolated from cells using TRIzol reagent (as described above) at 0, 15, 30,

45, 60, 90, 120, and 240 min. The RNA was then subjected to Northem Blot analysis as descri bed above.

2.5 Antisense Oligonucleotide Effect on Ceii growth

MTT Growth Assay. Exponentially growing cells were plated at 3 x 10' cells/ml in 96-well microtitre plates and were incubated at 37°C with 5% CO2 for 24 hr. 100 pl of oligonucleotide compiexed with Superfect or Superfect alone at the same concentrations used for the ODN treatments was added to each well to give a final concentration of 10-750 nM. Two control plates were prepared. In the first, oligonucleotide and Superfect were added to plates in the absence of cells. In the second, untreated cells were plated, After an incubation of 45 hrs with treatment, 20 pl of 0.45 mg/ml MTT (3-[4,5-dimethy1thiazol-2-yI]-2,5-diphenylte~~olium bromide) (Sigma Chemical Co.) was added to each well, followed by an additional 3 hr incubation for a totai drug incubation of 48 hr. Plates were centrifuged at 2500 rpm for 10 min and 150 pl medium was removed from each well. MTT fonnazan crystals in each well were resolubilized by the addition of 150 pl DMSO and the plates were placed on a shaker for 15 min. The absorbance of eac h well was measured spectrophotometrïcally using a Titrete k Mu1 tiscan automated microplate reader (provided by Dr. D. S. Riddick) at a wavelength of 540 nm and data was collected using Deltasoft. Background absorbance from the control plates was subtracted from the absorbance of each well and data from peripheral wells of each plate was excluded in the final analysis since evaporation occurred in these wells. Absohance in the wells treated with

Superfect and ODN or Superfect alone was expressed as a percent of the absorbance fiom the untreated celIs within the same plate. III. RESULTS

3.1 Assay Optimization

Range of Detection for omyc by Western Blot AnaCysi's and RNase Protection Assay. Western blot analysis and the RNase Protection assay are cornmon methods used for the quantitation of protein and RNA. In order to use these methods, it was necessary to ensure that detection oçcurred in a linear relationship with the amount of protein and RNA loaded.

Total RNA was isoiated, as described in Materials and Methods, and increasing amounts of RNA (5-20 pg) were incubated with the 32~c-myc RNA probe, followed by RNase digestion and analysis by electrophoresis on a 5% acrylamide/7 M urea gel. As the amount of RNA in the incubation increased, the signal for c-myc increased until 17.5 pg, where it began to plateau

(figure 7a). Therefore, for subsequent analysis of c-myc expression by RNase Protection, 15 pg total RNA was used in the incubation.

Following protein isolation as described in Matenals and Methods, increasing amounts of protein were loaded ont0 a LO% SDS-Page gel and separated by electrophoresis. After transfer to the Immobilon P membrane, the samples were analyzed for c-myc as described in Materials and Methods. Similar to the results obtained with total RNA, increasing amounts of protein produced an increased c-mye signal intensity until60 pg, after which the signal began to plateau.

Therefore, for subsequent analysis of c-myc protein levels, 50 pg of total protein was used

(figure 7b).

Carrier Molecule Seleetion- Numerous studies have reported that carrier molecules are able to compensate for the low cellular uptake of naked ODNs. This increased uptake is beneficial because it can lead to a decrease in the dosage required to observe a biological response. Many forms of commercial carrier molecules exist, each claiming superiority over the others. To determine which product is rnost efficient at increasing cellular uptake of ODNs and to optirnize the transfection protocol, K526 cells were treated with (PS) ODN-AUG in the presence of

Lipofectin, Dm-Cor Superfect. This particular ODN (derivative and target sequence) was chosen because it has been previously characterized (Balaji et al., 1997; Bennett et ai., 1994;

Broaddus et al., 1997; Tu et al., 1998). Following a 24 hr treatment with 5 pM ODN-AUG and carrier, RNA and protein were isolated and c-myc expression levels were analyzed by RNase

Protection Assay and Western blot analysis. c-mye mRNA levels were 118% f 8% for

Lipofectin, 132% f 18% for DMRIE-C, and 148% + 14% for Superfect compared to a control that was exposed to ODN-AUG alone (n=3). Conversely, c-myc protein levels were 78.5% f

Io%, 61% + 10%- and 39% t 8% respectively (n=3)(figure 8). Superfect was significantly more effective at reducing c-niyc protein levels but not mRNA levels as compared to the other carrier molecules (p c 0.05, paired t-test). From these results, 1 concluded that Superfect was more effective at increasing intracellular concentrations of ODN-AUG.

K562 Exposure Time to ODN. ODN exposure time may also play a factor in determining the level of inhibition of c-myc expression. For determination of optimal treatment time, cells were transfected with (PS) ODN-AUG in the presence of Superfect for 15, 24, 38, 48, and 62 tus.

Following treatment with 5 pM ODN-AUG and 12 pl Superfect, c-myc mRNA levels were 85%

+ 6% at 15 hrs, 148% + 14% at 24 hrs, 137% + 5% at 8 hrs, 142.5% t 7% at 48 hrs, and 137.5% t 8% at 62 hrs compared to an untreated control (figure 9). GAPDH mRNA

c-mye mRNA

Cl Lipofectin DMWEC SupdM Liposomal Carrier

Figure 8. Cornparison of Catrier Moiecaies K562 cells were treated with 5w (PS) ODN-AUGin the presence of either Lipofectia @l), DMRlE-C (5 pl) or Superkt (12 pi). Following a 24 hour incubation, total RNA and protein was isolated and separated by gel electrophomis or SDS-Wge. Radioactive signals were detected by autoradiography (top) and quanîitated by densitorneûy and myc mRNA levels were quantita ed by using the PhosphoImager (Molecular Dynamics) and norrnalized to GAPDH. Protein levels were detected by cherniluminescence (middle) and quantitated by densitornetry. Levels were expresseci as a percent of cells exposed to ODN without carrier molecule (bottom). Data shown are a means + SE for three septeexpcriments. The asterisk (*) indicates sam ples that are signifi cantly different from the other samples (p < 0.05). Treatment Time (hr)

Figure 9. Optimization of ODN Treatment Time K562 Cells were treated for O to 62 hours with 5 pM (PS) ODN-AUG in the presence of 12 pl Superfect. Following treatment, totai RNA was isolated and analyzed by an RNase Protection Assay as described in Materiais and Methods. Radioactive signais were detected by autoradiopphy and c-myc mRNA levels were quantitated by using the STORM and normalized to GAPDH. LRvels were expressed as a percent of untreated cells. Data shown are a mean t SE for three separate experiments . These results suggest that cells treated with ODN and Superfect for 24 hrs will exhibit a similar effect as those incubated for 62 hrs. It has been previously reported that PS ODNs are most effective between 24 and 48 hrs because of increased degradation and efflux after this penod (Crooke, 1991). Therefore, for subsequent transfections, 1 treated cells for 24 hrs with

ODN and Superfect.

Effect of Superfect on K562 Cell Growth. For al1 initial transfections, a constant volume of

Superfect (12 pl) was used regardtess of the concentration of ODN. FolIowing optimization of the transfection protocol (exposure time and carrier molecule selection), the manufacturer of

Superfect published a report that suggested using a ratio of 6 pl of Superfect per pg of ODN.

Toxicity was observed at concentrations above 30 pi of Superfect (> 200 nM ODN) per 5 ml ce11 culture (figure 10). For subsequent experiments, a ratio of 6 pl Superfect: lpg ODN was used up to ODN concentrations of I 200 nM, after which constant levels of Superfect (30 pl) were used. Protein and mRNA were isolated from treated cells following a 24 hr ODN treatment and sarnples were analyzed for c-myc MAand protein levels. Superfect volume (p U5 ml medium)

Figure 10. Superfect Dose-response Came

K562 cells were exposed to increasing concentrations of Superkt for 48 hours and cytotoxicity was assessed by the MTï assay as described in Materials and Methods. Manufacnirer's instructions suggest using 6 pl Superfect per pg ODN. Therefore 107 M ODN would require 14.7 p1 Superfect per 5 ml media. Ce11 toxicity was quite substantial at 70 pi Superfect. For subsequent transfections, no more than 30 pl SuperfectlS ml medium will be used even if the concentration of ODN exceeds 200 nM(in 5 ml medium, 200 nM contains approx. 5 pg ODN which requires 30 pi Superfect). 3.2 Effects of CRD Antisense Oligonucleotides on cmyc mRNA and Protein Levels in

K562 Cells

ODNs directed towards the coding region determinant have ken shown to be effective at

inhibiting the interaction between c-mye mRNA and CRD-BP in vitro (figure 6). The most

effective ODN (ODN4) targeted nucleotides 1763-1777 and was followed by ODN5 and ODN3.

ODN7, targeting 186 1-1 875, was the least effective at inhibiting the RNA*protein interaction

suggesting the CRD-BP does not come in contact with or anchor the RNA at this site.

It is believed that the CRD-BPprotects c-myc mRNA fiom endonucleolytic attack (Lee et al., 1998; Prokipcak et al., 1994). If this hypothesis is true, then disruption of the complex will

increase the degradation of the mRNA and decrease c-myc expression in cells. Because PO

ODNs are rapidly broken down in biological systems and PS ODNs have exhibited non-specific

activity, 2'OM ODNs were used for this study. Moreover, 2'OM do not induce cleavage via

RNase H; therefore any effect observed will be directly due to disniption of the CRDCRD-BP

interaction.

To determine if inhibition of complex formation altered c-mye expression in cells, 100 nM of ODN3,ODN4 and ODN5 were added to K562 cells using Superfect (14.7 pl) as a carrier molecule. c-myc mRNA Levels. c-mye mRNA levels were determined by Northern analysis and were nonnalized to GMDH levels for each sample to ensure equal loading. Values are shown as mean ISE for three separate experiments. Treatment of K562 cells with 0DN4 resulted in the greatest inhibition of c-mye expression, producing a 45% 2 6% decrease in c-myc mRNA compared to cells treated wi th Superfect alone. This in hibition of expression was statisticall y significant (p < 0.05, paired t-test). In contrast, 0DN3 treatment reduced c-mye mRNA by 23% t 15% and ODN5 by 3 1% + 8%. Neither of these results were significant compared to the control (p > 0.05, paired t-test) (figure Il).

To ascertain the specificity of ODN4, alterations were made to its sequence (table 2).

ODN-MM is composed of the same nucleotides as ODN4, however three mismatches exist between it and ODN4. ODN-MM did not significantly reduce c-mye mRNA levels as compared to cells exposed to Superfect alone (15% k 15% reduction, p > 0.05, paired t-test) (figure 11).

c-myc Protein Levels. Decreased c-myc mRNA levels do not infer a simiiar decrease in c-myc protein levels because levels of protein expression rely on translational efficiency in addition to the rate of protein degradation. Therefore, variations in c-myc protein levels following ODN treatment were examined by Western blot analysis using the AB-1 monoclonal antibody, which recognizes the epitope EQKLZSEEDL and reacts with the 64 kDa c-mye protein and its cleavage products (Evan et al., 1985). As was the case with mRNA levels, 0DN4 had the greatest inhibitory effect on c-myc protein expression. Treatment with ODN4 resulted in a 55% -+ 5% reduction of c-myc protein levels compared to only 5% -t 19% for ODN-MM (n=3). ODNS reduced protein levels by 45% + 7%, whereas 0DN3 caused a 19% + 11% reduction

(n=3)(figure 12). Inhibition caused by 0DN4 and ODNS was statistically different from the control that was exposed to Superfect alone (p < 0.05. paired t-test) while the decrease in expression due to 0DN3 and ODNeMM was not (p > 0.05, paired t-test). Controi ODN3 0DN4 ODNS ODNbMM AnUsense oligonucleotide (0.1 pM)

Figure 11. Effeets of ODNs on c-myc mRNA Levels in Ceiis

K.562cells were treated with 100 nMof 2'-O'methyl derivatives of the indicated ODN in the presence of 14.7 @ of Superfect for 24 hours. RNA was isolated and 2û pg was scparated by gel eiectrophoresis as described in Materials and Methods. Radioactive signais were detected by autoradiography (top panel). c-myc mRNA levels were quantitated using the Phosph* rlmager (Molecuîar Dynamics) and nonnalized to GAPDH. Levels are expressed as a pet- cent of Supedect control (bottom panel). Daia shown are meam & SE for 3 septe experiments. The asterisk (*) indicates that samples are sigdicantly Meratfrom control (paired t-test, p < 0.05). Control 0DN3 ODN4 ODN5 ODN4-MM Antisense oligonucleotide (0.1 pM)

------Figure 12. Effects of ODN on c-mye Protein Leveis in Cells

KS62 ceiis were treated with 100 nMof 2'OM derivatives of the indicated ODN in the presence of 14.7 pl of Superfect for 24 hours. Total pmtein was isolated and 50 M was sep-- ed by SDS-Page as descnbed in Materials and Methods. c-myc pmtein levels were detected by the AB-1 monoclonal antibody (top panel). c-myc protein levels were quantitated by densitomatric analysis and normaiized to GAPDH protein signais. Levels are expnssed as a percent of Superfect control (bottom panel). Data shown are me- *SE for 3 separate experiments. Asterislcs indicates samples that are signüicantly different fmm the control (paired t-test, p < 0.05). The results suggest that the disniption of the CRD and CRD-BP cornplex can leave the c- rnyc mRNA vulnerable to degradation, thus decreasing mRNA levels and preventing protein synthesis. Therefore, it is possible to decrease c-myc expression in a cellular environment with

ODNs targeting the CRD.

Cornparison of 2'OM Versus PS. Our previous results are consistent with the belief that 2'OM-

0DN4 can cause occlusion of the CRD-BP from c-mye mRNA, resulting in decreased c-myc expression. We then tested the hypothesis that an ODN capable of mediating RNase H activity could inhibit c-myc expression to an even greater extent. The PS derivative of 0DN4 could potentially act through both the activation of the endogenous endonuclease to degrade the c-myc mRNATas well as targeting the mRNA for degradation by RNase H. K562 cells were txeated for

24 hrs with 100 nM of PS-0DN4 or TOM-0DN4 in the presence of 14.7 pl Superfect. mRNA and protein levels were nonnalized to GAPDH and values are-shown as a mean + SE for three separate trials. TOM-0DN4 was significantly more effficient than PS-0DN4 at decreasing c- rnyc protein levels (p4.05, t-test), but not at decreasing mRNA levels. Treatment with TOM-

ODN4 resulted in 40% 4 9% decrease in mRNA levels and 55% + 9% decrease in protein levels compared to a 25% & 12% (rnRNA) and 20% + 11% (protein) decrease by PS-0DN4 (figure 13).

This difference in efficacy may be attributed to a decrease in affinity for RNA by the PS-ODN.

Furtherrnore, the PS-ODN may interact with serum proteins or non-targeted RNA thereby reducing the amount available to hybridize c-myc MA. I

& RNA omyc expression

Figure 13. Cornparison of 0DN4 as Both a PS and 2'OM Derivative

K562 ceils were treated with 100 nM of 0DN4 for 24 hours foiiowed by isolation of total RNA and protein and separation by gel electrophoresis or SDS-Page as described in Material and Methods. c-myc expression levels werc determined by both Northem and Western analysis and normalized to GAPDH (top panel). Levels are expressed as a percent of the Super- fect coatrol (bottom panel). Data shown are means %SE for thra separate experiments. The asterisk indicates where a significant dBercllce between ODN derivatives exisîs (pairrd t- test, p < 0.05). Duse-dependent Inhibition of c-myc Expression. A dose-dependent study was undertaken to characterize 20M-0DN4 and compare its efficacy at inhibiting c-myc expression to a 2'OM derivative of the previously characterized ODN-AUG (Balaji et al., 1997; Bennett et al., 1994;

Broaddus et al., 1997). K562 cells were treated with a dose range [IO nM (0.25pg/Sml) to 200 nM(5pg/5ml)] of either 0DN4 or ODN-AUG in the presence of Superfect (6pYpg ODN) for 24 hrs. Northern blot analysis demonstrated that reduction of c-myc mRNA was dose-dependent with a maximum reduction of 50% by 0DN4 (figure 14). Similady, ODN-AUG demonstrated a dose-dependent effect on c-myc mRNA levels. However, it induced an increase in c-nzyc m'NA levels with a maximal level greater than 2-fold.

In contrast, western blot analysis demonstrated similar effects on c-myc protein expression by both ODN4 and ODN-AUG. Both ODNs exhibited a dose-dependent decrease in c-myc protein levels with a maximum reduction of 66% + 5% for 0DN4 and 38% + 6% for

ODN-AUG (figure 15).

3.3 Cornparison of the Effect of 0DN4 and ODN-AUG on c-myc mRNA Stability

The differing effects produced by 0DN4 and ODN-AUG on c-myc mRNA levels suggest that each ODN may be acting through a distinct mechanism. The disruption of CRDaCRD-BP by ODN4 may leave the mRNA vulnerable to endonucleolytic attack (Bernstein et al., 1992; Lee et al., 1998; Prokipcak et al., 1994) thus destabilizing the transcript. Conversely, ODN-AUG may be inhibiting translation of the transcript. Numerous studies have reported that stability of c-myc mRNA is translation-dependent, with stability king higher when translation is inhibited

(Brewer and Ross, 1989; Yielding and Lee, 1997). To test the hypothesis that the two ODNs are acting through different mechanisms, we analyzed the decay rate of c-myc mRNA following a 6 hr treatment with either 200 nM ODN4 or ODN-AUG in the presence of 30 pl Superfect and O! m m m. wwm~ w . w. ..ma w .J O 10-8 Ic7 ODN concentration (M)

Figure 14. Concentr&on-dependent Eff't of ODN4 and ODN-AUG on c mRNA LeveIs

K562 cells were treated for 24 hours with 0, 10,20,50, 100, or 200 nMof either 2'OM-ODN4 (squares) or 2'0M-ODN-AUG (triangles) in the presence of Superfect (6 pVyg ODN) followed by isolation of RNA as described in Materials and Methods. 20 pg of RNA was separated by gel electrophoresis, transferred to a Zetaprobe membrane and probed for c-mye and GAPDH mRNA. Radioactive signals were quantitated using the Phosphodmager (Molecular Dynamics) and detected by autoradiography (top). Levels were expressed as a percent of the Superfect control. Data shown are me- + SE for three experiments. - c-myc Ot)N AUG - GAPDH

- c-myc 0DN4 - GAPDH

0 ! w w . .W., w . w .W..., w w' O IW i0-7 ODN concentration (M)

Figure 15. Concentcation-dependentEfkt of 0DN4 and ODN-AUG on c-w Protein Leveb

K562 cells were treated with 0, 10,20,50, 100, or 200 nMof either 2'OM-ODN4 or TOM-ODN-AUGin the presence of Superfect (6 pVpg ODN)for 24 hours. Total protein was isolated,separated by SDS-Page and tranferred to Immobilon-P membrane as described in Materials and Methods. Bound antibody was detected by cherniluminescence (top) and c-yc protein leveis were quantitated by densitometry and normalized to GAPDH. Levels were expressed as a percent of Superfect contrd. Data shown are means 2 SE for three separate experiments. compared it to a control that was exposed to Superfect alone (30 pl). Afier exposure to the ODN or Superfect, the ODNs and Superfect were removed and the cells were treated with the transcriptional in hibi tor DRB . Following increasing exposure times of transcriptional inhi bition,

RNA was isolated and analyzed by Northern analysis (figure 16). Those cells treated with

Superfect alone exhibited a hdf-life of 0.57 t 0.03 hn for c-myc mRNA (n=3). Following treatment with 200 nM of 0DN4 or ODN-AUG, the half-life was 0.49 i 0.08 hrs (n=3) and 1-15

+ 0.1 hrs (n=3). ODN-AUG dtered the decay rate of c-myc mRNA significantly (p < 0.05, t- test), whereas ODN4 did not (p > 0.05) as determined by a t-test for the significance of difference between decay rates.

To detennine if an increased exposure time to ODN4, ODN-AUG or Superfect would aiter rnRNA stability more so than at 6 hrs we incubated K562 cells for 24 hr with the reagents.

The half-life of c-mye mRNA was 0.52 hrs (n=2) and 0.55 (n=2) hrs following treatment with

Superfect alone and Superfect with ODN4. These results were similar to the 6 hr treatment, suggesting that enough ODN had ken incorporated into the ce11 and had hybridized the mRNA by 6 hrs. However, following a 24 hr treatment with ODN-AUG, the half-life of c-myc mRNA decreased to 0.94 (n=2) hrs suggesting that this ODN may be most effective at 6 hrs. A ODN-AUG m ODN4

Figure 16. c-ngc mRNA Decay Rate Following Treatment with 0DN4 or ODN-AUG

K562 cells were treated with 200 nMof 2'OM-0DN4 (solid square) or 2'0M-ODN-AUG (solid triangle) in the presence of 30 pl Superfect or 30 pl Superfect alone (open squares) for 6 hrs. Media and ODNs were then aspirated off and replaced with RPM1-1640 media containing 100 uMDRB. RNA was isolated at the indicated times as described in Materials and Methods and separated by electrophoresis. RNA was then transferred to a Zetaprobe membrane and probed for c-nyc and GAPDH mRNA. Radioactive signals were detected by autoradiography and c-mye mRNA levels were quantitated using the PhosphorIrnager (Molecular Dynamics) and normalized to GAPDH. Data shown are representative of three separate experiments. 3.4 Effect of 0DN4 and ODN-AUG on H62Ceii Pmliferatioa

To determine if a reduction in c-mye expression would result in inhibition of cell growth,

K562 cells were treated with either 0DN4 or ODN-AUG in the presence of Superfect (at a constant ratio 6 püpg ODN up to 200 nM (Spg/SmL), after which it was used at a constant volume of 30 pl per 5 ml culture) for 48 tus. Effects on ce11 growth were assessed by the MTT growth assay. Scrambled versions of the ODNs were used as controls, in addition to Superfect treatment alone. Although the cytotoxicity of Superfect lirnited the uptake of ODNs greater than

200 nM, we still observed a concentration-dependent effect on cell growth (figure 17). Both

ODN4 and ODN-AUG were capable of reducing ce11 growth at concentrations ranging from 10 to 750 nM. Maximal inhibition (70% k 4%, n=3) was seen with 0DN4 which was significantly more effective (p c 0.05, paired t-test) than ODN-AUG (50% f 4% reduction in ce11 growth, n=3) and Superfect treatment alone (15% k'3% reduction in ce11 growth, n=3). ODN4 was also more effective than ODN4-Scram (3296,n=2) and ODN-MM (45%, n=2).

Additionally, the effects produced by ODN-AUG were greater than its scrambled counterpart, AUG-Scram, which had a minor effect on ce11 growth (38%, n=2).

It can be concluded that cell growth is dependent on c-myc expression levels and ODN4, which is the most effective at reducing levels of c-myc, is a more selective and potent inhibitor of ceIl growth than ODN-AUG. A ODN-AUG-Scram A ODN-AUG

01 m m m m .v 1 . 1 m m... 10-7 10.6 ODN concentration (M)

Figrire 17. Effect of 0DN4 and ODN-AUG on 8562 Cell Growth

K562 cells were treated with 10 to 750 nM of 2'OM-ODN4, TOM-ODN-AUG, or the corresponding scrambled controls in the presence of Superfect (6 pYpg ODN until200 nM ODN) for 48 hours. Analysis of surviving ceils was camied out as described in Materials and Methods. Absorbances were corrected for background and expressed as a percentage of untreated cells. Six replicate wells were analyzed for each ODN and the experiment was replicated twice (ODN4Sctam and AUG-Scram) or three times (ODN4, ODN-AUGand Supenect alone). Data shown are means I SE. ODN4 (solid squares). ODN-AUG(solid triangles), ODNQScnun (open squares), AUG-Scram (open triangles), Superfect alone (asterisk). IV. DISCUSSION

Numerous expenments have revealed an important link between mRNA-binding proteins and mRNA translocation, translation, processing and stability (Politz et al., 1995; Ross et al.,

1997). Targeting protein binding sites with antisense technoiogy has not received much attention in part because it is believed that proteins prevent or interfere with ODN binding (Wagner,

1994). However, several groups have reported that these regions are accessible to ODN attack due to frequent "breathing" of the RNA-protein complexes. For example, ODNs are able to interact with poly(A) regions and f3-actin mRNA in intact cells despite the presence of both poly(A) binding proteins and the $-actin Zipcode binding protein (ZBP-1)(Politz et al., 1995;

Ross et al., 1997). Therefore, this study set out to determine if ODNs could inhibit specific

RNAmprotein interactions and encourage efficient degradation of c-mye mRNA.

We previously determined that in vitro, ODNs directed towards the CRD of c-mye mRNA are able to inhibit RNA-protein binding (figure 6). This novel approach of using a Gel- shift assay allows rapid screening of potentially useful ODNs instead of the time consuming approach of testing ODNs within intact cells. Using sub-cellular extracts obtained from the human erythroleukemia ce11 line K562, we localized the site of protein binding to nucleotides

1763-1777 (ODN4). Inhibition of RNA-protein complex formation by 0DN4 was sequence- specific, for when mismatches were added to the sequence, no inhibition of complex formation was observed. Additionally, despite overlapping by 6 nucleotides, ODN4 was more effective at lower concentrations than ODN5 (Coulis et al. manuscnpt submitted).

To detennine if these results could be replicated within a cellular environment, we treated the human erythroleukemia cell line K562 with 2'-O-methyl derivatives of ODNs. This derivative of ODN was chosen because it displays a greater resistance to nuclease degradation than PO ODNs and a higher affinity for RNA than the PS ODNs (Baker et al., 1997; Iribarren et al., 1990; Lamond and Sproat, 1993; Larrouy et al., 1995; Monia er al., 1993). A higher affinity may be required to allow for efficient competition with CRD-BP for the mRNA and may explain why the PS-0DN4 was less effective at decreasing c-myc protein levels compared to the 2'OM-

ODN4 molecule (figure 13). Additionally, because 2'OM ODNs do not employ RNase H activity, any observed effect would be due to inhibition of the CRD-BPdWA interaction (Baker et al., 1997).

In the present study, it was detennined that the relative ability of the 2'OM ODNs to decrease c-myc expression levels and ce11 growth resembIed their activity in the gel shift assay.

ODN4 was the most effective at preventing the interaction between CRD-BP and RNA in addition to being the most effective at decreasing c-myc expression and cellular proliferation

(figure 15 & 17). This correlation between in vitro and cellular effects supports the hypothesis that the mechanism of action by 0DN4 is to disrupt an RNA-protein interaction resulting in altered c-myc expression within a cell,

Treatment of cells with 0DN4 resulted in a decrease in c-myc mRNA as well as protein.

The decrease in mRNA suggests that the effect of ODN4 is due to its sequence rather than its chemistry as TOM ODNs do not target mRNAs for degradation by RNase H (or any other enzyme for that matter) (Chiang et al., 1991; Monia et al., 1993). Based on our mode1 of CRD-

BP function, we would predict that removal of CRD-BP from c-myc rnRNA would promote degradation of the transcript (figure 5). However, following treatment with ODN4, we were unable to detect an alteration in its hdf-life. This rnay be explained by technical limitations.

Untreated c-myc mRNA has such a short half-life (= 30 mins) that detecting an increased decay rate is difficult. Additionally, the transcriptional inhibitor, DRB, has been shown to have little effect on complexes that have already initiated transcription (Sehgal and Tamm, 1978). The continued synthesis of these initiated transcripts, in addition to time lag required for cellular DRB uptake, may make it difficult to measure very short half-lives. Therefore, it may be

worthwhile, in future experiments, to attempt to detect c-myc degradation fragments following

treatment with 0DN4 to determine if it is in fact destabilized.

Altematively, ODN4 could disnipt other functions of CRD-BP. CRD-BP contains one

putative nuclear localization sequence and two putative nuclear export sequences in addition to

sharing high sequence homology with RNA binding proteins (RBP) of other systems (Doyle et

al., 1998). These RBPs include ZBP-1 (a protein that binds chicken $-actin rnRNA) (Ross et al.,

1997), Vg LBP (a protein that binds to the 3'-UTR of the Vg 1 mRNA from Xenopus oocytes)

(Deshler et al., 1998), and IMP-1 (an insulin-like growth factor binding protein) (Nielsen er al.,

1999). ZBP-1, and VglBP are believed to recognize specific mRNAs in the nucleus and

translocate with them to the cytoplasm. If CRD-BP functions in the same manner then

dismption of the CRD-BPoRNA complex may inhibit translocation of c-myc mRNA to the

cytoplasm. This would prevent the transcript from coming in contact with the translation

machinery and account for the decrease in c-myc protein levels (figure 15). Funherrnore, it

would also account for the unaltered mRNA half-life because the endonuclease that cleaves c- myc in the CRD is predominantly polysomal-associated (Lee et al., 1998) (figure 16).

Additionally, IMP-1 has been implicated in translational regulation. Disruption of îts activity

may alter the stability of the mRNA while preventing the formation of new protein.

Another protein that contains conserved RBP sequences, which are also present in CRD-

BP, is HuD. This RBP is believed to bind specifically to both exonic and intronic N-myc mRNA sequences and play a role in its nuclear processing (Lazarova et al., 1999). If CRD-BP also functions in this manner, then occluding it from c-myc mRNA would result in inhibition of the production of mature mRNA. This may help to explain why levels of c-myc mRNA decreased yet there was no change in its stability following treatment with 2'OM-ODN4. Unlike ODN4, 2'OM ODN-AUG treatment caused an increase in c-mye mRNA levels

that was associated with an increase in mRNA stability. This effect is most likely the result of

translational inhibition because c-mye mRNA stabifity is translationai-dependent (Yielding and

Lee, 1997); ribosomes must translocate through the entire coding region to destabilize the

transcript (Wisdom and Lee, 1990; 1991). ODNs targeting the AUG start codon can potentially

inhibit binding or sliding of the 40s ribosomal subunit andor the association of factors involved

in the translational initiation process (Baker et ai., 1997; Broaddus et al., 1997). Similarly,

inhibitors of translation such as cyctoheximide have been shown to stabilize severai mRNAs

including c-myc (Brewer and Ross, 1989). This increase in mRNA stability might prove to be hazardous if a rebound reaction occurs once the ODN has been degraded. The high levels of

rnRNA rnay be translated thus leading to an overabundance of c-mye protein.

Despite the capability to mediate RNase H decay, PS-ODN-AUG treatment also resulted in an increase in c-myc mRNA levels. This rnay be the result of ODN localization- If ODNs are deposited in the cytoplasm they rnay be unable to sequester RNase H as it is primarily found in the nucleus. Additionally, carrier molecules rnay prevent the ODNs from interacting with the

RNase. It rnay also be due to endogenous levels of RNase H. K562 cells rnay not have high enough levels of the enzyme to produce a noticeable effect. Another possibility rnay be that

RNase H degradation rnay exist, however inhibition of translation rnay be far more prominent thereby masking any signs of it occuring. When comparing the results of this study to previous reports that utilized PS-ODN-AUG, it was found that virtually al1 of the past studies neglected to report the effect the ODN had on c-mye mRNA expression (Bacon and Wickstrom, 1991; Balaji et al., 1997; Broaddus et al., 1997; Cerutti et al., 1996a). The reasoning for this could be twofold: either the investigators were only interested in c-mye protein levels and were not concerned with mRNA, or they observed increases in rnRNA levels and could not explain why they occurred. Therefore, an increase in c-myc mRNA following PS-ODN-AUG treatment may not be unexpected.

When comparing ODN-AUG to 0DN4 it was detennined that 0DN4 was more effective than ODN-AUG at decreasing c-myc protein levels. This may be explained by the fact that c- mye mRNA contains two translation initiation sites (figure 2). If translation is initiated at the

CUG start codon, ODN activity may be limited as ODN-AUG is not complementary to regions surrounding this area. Therefore, the necessary components may still be able to attach to the transcript and initiate translation.

This study has met the criteria for demonstrating sequence-specific antisense effects

(Wagner et al., 1994). A scrarnbled sequence in addition to an ODN with three mismatches

(ODN-MM) were employed and were much less effective compared to their parent ODN

(ODN4) (figure 12 & 17). Furthermore, when Genbank was screened for similar sequences to

ODN4, there were no matches with more than 75% homology. There were, however, several sequences that had a match of 11 of 15 nucleotides. These matches represent proteins that contain the HLH domain (for example the transcriptional factor AREB6) that is also present in c- myc.

Even though substantial effects were observed following treatment with ODN4 there is still the possibility for greater inhibition of c-myc expression and ce11 growth. Sensitivity to reduced c-mye expression varies as a function of ce11 type. Cemtti et ol(1996a) reported that an

80% decrease in c-myc protein levels correlated with an 80% decrease in ce11 growth in AR0 cells whereas Broaddus et al (1997) showed a 70% inhibition of c-myc protein resulted in only a

50% decrease in RT-2 cell growth. Therefore KS62 cells may not be the most responsive ce11 line to changes in c-myc expression levels.

Total inhibition of c-myc expression may require longer exposure times to an ODN (i.e. 24 hrs rnay not be sufficient enough) or higher concentrations of ODN. Because 2'OM ODNs have a half-life > 24 hrs (Crooke, 1992)' it might be beneficial to re-administer the ODN at set intervals (20 hrs) instead of leaving the cells exposed to the original ODN for an extended period of time. Similarly, if higher concentrations of ODN are use& the molecules rnay remain in cells for a longer pend time. Altering treatment in either of these manners could decrease mRNA levels more effectively as well as allow sufficient time for the depletion of cellular reservoirs of c-myc protein.

Re-administenng ODNs could also produce more significant results on ce11 growth. c- myc is one of many initiators of the ce11 growth process; it activates growth factors which in tum stimulate cellular proliferation. Therefore, it is highly unlikely that proliferation will occur imrnediately following c-myc overexpression. A greater decrease in proliferation rnay require a longer exposure time to an ODN. If it takes 24-48 hrs to decrease c-mye protein expression significantly, then it rnay take 74 hrs of ODN treatrnent before a substantial effect on growth is observed.

Furthemore, because c-myc is but one cornponent in the ce11 growth process, it rnay be beneficial to target a second transcription factor or the CRD-BP for ODN attack. Targeting multiple rnRNAs rnay inhibit ce11 proliferation to a greater extent as certain genes can be responsible for specific ce11 growth (table 1). Similarly, if levels of CRD-BP are downregulated in conjunction with inhibition of CRD-BP.RNA interactions then c-myc rnRNA levels rnay be decreased much more.

Data we obtained from the gel-shift analysis suggests that the CRD-BP rnay be able to cover an extended region of c-myc mRNA. The region of mRNA identified as king the most important for interaction with CRD-BP (ODN4 1764-1785 nt) is = 30 nt 3' to the endonucleolytic cleavage site (L,ee et al., 1998). Despite king 84 nucleotides 3' of ODN4, ODN8 was also effective at disrupting the interaction between CRD-BP and c-myc mRNA

(figure 6). Therefore, changes made to the sequence of the ODN molecule could enhance its activity- Increasing its length or shifting the sequence left or right dong the CRD rnight occlude the CRD-BP from the mRNA more efficient1y. Additionally, dtenng the melting temperature of the ODN may dso increase an ODNs activity, as those ODNs targeting the CRD had on1 y a 47%

G/Ccontent.

Modifications to the backbone of ODN4 may also improve its efficacy. New generation

ODNs (N3'-PS phosphoramidate, 2'-O-alkyl) are much more resistant to nucleases and display a higher aîfinity for target RNA (Boiziau et al., 1995; Gryaznov et al., 1996). Furthemore,

ODNs that mediate RNA decay through RNase H may also prove to be more effective. These molecules can utilize both the endogenous endonuclease as well as RNase H to degrade the transcript. Recent studies have also reported that uniform modification of oligonucleotides are not necessary to confer enhanced stability and activity (Shaw et al., 1991; Woolf et al., 1990). In certain cases, ODNs with chirnera backbones have ken shown to be more effective than those with homogenous backbones (Maran et al., 1998; McKay et al., 1999). Therefore an ODN with a nuclease-resistant backbone on its 5' and 3' outermost nucleotides and internai modifications that allow it to sequester RNase H may prove to be the most effective at decreasing c-mye expression levels.

The decrease in c-mye expression resulting from disruption of the CRD-BP*RNA interaction suggests that ODN4 can have a more prominent effect in cells expressing the protein.

Previous reports have shown that CRD-BP is developmen ta11 y expressed, king observed in fetal tissues but not in adult tissues. It is also detected in transfonned cells suggesting it is an oncofetal protein (Doyle et al.. L998; Leeds et al., 1997). This may enhance the selectivity of

0DN4 for cancer cells, as a minimal or absence of effect may be observed in cells without the protein.

In conclusion, the data supports a role for the CRD-BP in regulating expression of c-mye mRNA. The data also suggests that targeting RNA-protein interactions may be a useful strategy for antisense action. V. FUTURE DIRECTIONS

Having established that disruption of an RNAaprotein interaction by antisense oligonucleotides can decrease expression levels and inhibit ceIl growth, several experiments cm now be initiated to determine the mechanisms of ODN action on c-mye mRNA.

Targeting the coding region with ODNs can produce numerous effects on the structure, translation, localization and stability of -A. We determined that 0DN4 altered c-myc expression levels, however, we have not clearly addressed how this occurred- It will be necessary to perform an RNase Protection assay or RT-RCR following RNA ligation to detect any degradation fragments that result from occlusion of the CRD-BP from the mRNA (Veal et al., 1998). If the CRD-BP does indeed function by protecting the mRNA from endonuclease attack, then fragments may be detected.

Secondly, by binding to the coding region, ODNs may directly inhibit translation of the transcript or may interfere with CRD-BP's influence in translation. This may explain why the decrease in c-myc rnRNA levels were not as pronounced as the decrease in protein levels. To detect translational inhibition, a polysomal profile assay could be performed (Baker et al., 1997).

If translation is altered (more specifically ribosomal assembly) a change in the polysomal profile will result.

Thirdly, mRNA binding proteins detect and bind to recognizable sequences or structures within the mRNA. The coding region of c-mye niRNA is believed to be comprised of three stem-loop structures. 0DN4 treatrnent could possibly alter the secondary structure of the transcript thus masking it from its binding protein. To determine if 0DN4 treatment alters the secondary structure, an RNA fwtprint could be perforrned.

0DN4 may also interfere with nuclear-cytoplasmic transport of the mRNA. To test for this, we could observe the distribution of c-myc mRNA in nuclear and cytosolic fractions following treatment with ODN4.

It may also be beneficial to define the RNA sequences necessary for CRD-BP binding.

To determine this, Gel-shifi assays with purified CRD-BP and radiolabelled c-myc mRNA could be incubated with ODNs spanning the coding region. This expriment will differ from the previous one (figure 6) in that ODNs with shorter sequences will be used. Additionally, changes to the sequence of ODN4 will be made. By shifting it slightly to the left or nght we rnay be able to occlude the protein from the mRNA more effectively.

We have detennined that ODNs are capable of interfering with RNA.protein interactions and this interference can alter expression of a gene within a cell. The findings of this study provide the foundation necessary to determine the role RNA binding proteins play in mRNA expression and identify a novel target or approach for antisense oligonucleotides action. VI. REFERENCES

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