Molecular and Cellular Biochemistry 118: 191-195, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.

Inhibition of ornithine decarboxylase and S-adenosylmethionine decarboxylase synthesis by antisense oligodeoxynucleotides Rentala Madhubala and Anthony E. Pegg Departments of Cellular and Molecular Physiology and of Pharmacology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Centre, P.O. Box 850, Hershey, PA 17033, USA Received 18 July 1991; accepted 13 August 1992

Abstract Oligodeoxynucleotides 18 nucleotides in length having sequences complementary to regions spanning the initiation codon regions of ornithine decarboyxlase or S-adenosylmethionine decarboxylase mRNAs were tested for their ability to inhibit translation of these mRNAs. In reticulocyte lysates, a strong and dose dependent reduction of ornithine decarboyxlase synthesis in response to mRNA from D-R L1210 cells was brought about by 5'-AAAGCT GCTCATGGTTCT-3' which is complementary to the sequence from - 6 to + 12 of the mRNA sequence but there was no inhibition by 5'-TGCAGCTTCCATCACCGT-3'. Conversely, the latter oligodeoxynucleotide which is complementary to the sequence from - 6 to + 12 of the mRNA of S-adenosyl methionine decarboxylase was a strong inhibitor of the synthesis of this enzyme in response to rat prostate mRNA and the antisense sequence from ornithine decarboxylase had no effect. The translation of ornithine decarboxylase mRNA in a wheat germ system was inhibited by the antisense oligodeoxynucleotide at much lower concentration than those needed in the reticulocyte lysate suggesting that degradation of the hybrid by ribonuclease H may be an important factor in this inhibition. These results indicate that such oligonucleotides may be useful to regulate cellular polyamine levels and as probes to study control of mRNA translation. (Mol Cell Biochem 118: 191-195, 1992)

Key words: polyamines, ornithine decarboxylase, S-adenosylmethionine decarboxylase, antisense oligodeoxynucleotides

Abbreviations: ODC - ornithine decarboxylase, AdoMetDC - S-adenosylmethionine decarboxylase, DFMO difluoromethylornithine

Introduction Polyamine levels in mammalian cells are regulated by the activities of two enzymes, ODC and AdoMetDC. These proteins are very highly inducible in response to growth promoting stimuli and have very rapid rates of

turnover [1, 2]. The importance of polyamines in cell growth has been demonstrated by the use of specific inhibitors of these enzymes and such inhibitors have considerable potential for use as therapeutic agents [3-5]. However, the rapid turnover of these enzymes and the increases in their synthesis rates which occur in response to depletion of cellular polyamines tend to

Address for offprints: R. Madhubala, School of Life Sciences, Jawaharlal Nehru University, New Delhi - 110 067, India

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mRNA from D-10 ceils [11] was translated in a reticulocyte lysate system [14] in the presence of [35S]methionineand the concentrations of 5'-AAAGCTGCTCATGGTTCT-3' (11; Anti-ODC) or 5'TGCAGCTI'CCATCACCGT-3' (0; Anti-AdoMetDC) indicated. The synthesis of ODC was then determined using immunoprecipitation and densitometric scanning of the band at 51,000 M.W. which corresponds to this enzyme [14]. Results are expressed as % of the activity in samples without added oligodeoxynucleotide.

MetDC. The mRNA from rat protate cells [11] was translated in a reticulocytelysate system [14]in the presence of [35S]methionineand the concentrations of 5'-AAAGCTGCTCATGGTI'CT-3' (11; AntiODC) or 5'-TGCAGCTI'CCATCACCGT-3'(0; Anti-AdoMetDC) indicated. The synthesis of AdoMetDC was then determined using immunoprecipitation and densitometric scanning of the sum of the proenzyme band at 38,000 M.W. and the enzyme subunit (M.W. 32,000) [13, 16]. Results are expressed as % of the activityin samples without added oligodeoxynucleotide.

minimize the effects of these inhibitors. Agents which selectively shut off the synthesis of these enzymes therefore have potential advantages for use to deplete cellular polyamines. At present the only tools available for this purpose arepolyamine analogs such as N 1, N12-bis (ethyl)spermine [5-7] and it is not clear to what extent these derivatives perturb cellular metabolism directly as well as by virtue of reduction of the content of natural polyamines. Recent studies have indicated that, in many cases, antisense D N A or R N A fragments can be used to block the translation of specific m R N A species [8-10]. In the present work, we have investigated the effects of oligodeoxynucleotides on cell-free translation of O D C m R N A from L1210 cells and A d o M e t D C m R N A from D F M O treated ventral prostate in rabbit reticulocyte lysate and in wheat germ extracts. The specificity of the oligonucleotide in inhibiting translation has been examined. Our results indicate that sequence-specific oligonucleotides corresponding to the region spanning the initiation codon of these m R N A are useful probes for inhibition of their synthesis and for exploring mechanisms of m R N A interaction with the translation apparatus in cell-free system.

Materials and methods Rabbit reticulocyte lysate was purchased from Bethesda Research Laboratories, Inc. Wheat germ extract was purchased from Promega Biotec. [35S] Methionine (1000 Ci/mmol) was purchased from New England Nuclear, Inc. Ribonuclease H was obtained from Pharmacia. All reagents used were either reagent grade or electrophoresis grade. Oligdeoxynucleotides were synthesized using the standard phosphotriester chemistry and purified by ion exchange, high pressure liquid chromatography. Total R N A and poly (A) m R N A were prepared by the standard protocols from L1210 D-10 cells which overproduce O D C [11] and from prostates of rats treated with D F M O to increase the content of A d o M e t D C m R N A [12]. Translation of m R N A from these sources was carried out in a reticulocyte or wheat germ extract using p S ] m e t h i o n i n e as labeled amino acid. Immediately prior to assay of translation, aliquots of the m R N A preparations were heated at 65°C for 10 min and the oligonucleotides indicated in the tables were annealed at 30°C for another 10 min. The proteins corresponding to O D C and A d o M e t D C were then immunoprecipitated, separated by polyacrylamide gel electrophoresis and quantitated by fluorography and

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Fig. 3. Effect of oligodeoxynucleotides on the total protein synthesis. The mRNA from D-f0 cells [11] was translated in a reticulocyte lysate system in the presence of [35S]methionine and the concentrations of AAAGCTGCTCATGGTTCT-3' (ll; Anti-ODC) or 5'-TGCAGC TTCCATCACCGT-3' (Q; Anti-AdoMetDC) as indicated. The effect on total protein synthesis was determined as previously described [14]. Results are expressed as % of the activity in samples without added oligodeoxynucleotide.

scanning of the protein bands with a laser densitometer [13, 14]. Conditions for translation in the reticulocyte lysate were as previously described [13]. The assay medium with the wheat germ extract contained 102 mM potassium acetate, 4.4 mM magnesium acetate, 5.1 mM [3-mercaptoethanol and 20 mM HEPES, pH 7.5. Based upon prior studies which suggest that complementary or antisense oligonucleotides directed at the 5' end of the gene or 5' leader sequence upstream from the target gene initiation codon are most inhibitory to translation, we synthesized 18 base oligonucleotides complementary to the initiation codon region of ODC and AdoMetDC (nucleotide s - 6 to + 12) [15-16].

Results The effect of these oligodeoxynucleotides on cell-free translation of D-10 m R N A in rabbit reticulocyte lysate is shown in Fig. 1. A strong and dose dependent reduction of ornithine decarboxylase synthesis in response to mRNA from D-RL1210 cells was brought about by 5'-AAAGCTGCTCATGGTTCT-3' which is complementary to the O D C m R N A but there was no inhibition by 5'-TGCAGCTTCCATCACCGT-3', which is complentary to AdoMetDC mRNA. Conversely, when these oligodeoxynucleotides were tested for their effects on the translation of m R N A from rat prostate, the sequence corresponding to the initiation codon region

Fig. 4. Effect of oligodeoxynucleotides on the synthesis of ODC in wheat germ extracts. The mRNA from D-10 cells [11] was translated in a wheat germ (0) or reticulocyte lysate (©) system in the presence of [35S]methionine and the concentrations of 5'-AAAGCTGCTCATGGTTCT-3' indicated. The synthesis of ODC was then determined as described in Fig. 1.

of AdoMetDC was strongly inhibitory towards the synthesis of this protein but the oligodeoxynucleotide complementary to ODC had no effect (Fig. 2). Total protein synthesis was not affected by these oligodeoxynucleotides at the concentrations used (Fig. 3). The effect of 5'-AAAGCTGCTCATGGTTCT-3' on the translation of ODC mRNA contained in the total mRNA from D-10 cells was also tested in a wheat germ translation system. In this system, inhibition of ODC synthesis was obtained with much lower concentrations than needed in the reticulocyte lysate with 50% inhibition produced by about 0.3 uM concentrations compared to 40 uM (Fig. 4). This additional inhibition may be the result of the greater content of ribonuclease H in the wheat germ system. Addition of one unit of ribonuclease H to the reticulocyte lysates greatly increased the ability of the oligodeoxynucleotide to inhibit the synthesis of ODC with 100% inhibition being produced by 10 uM concentrations (results not shown).

Discussion Previous work has indicated that double stranded R N A serves as a poor template for protein synthesis and the addition of complementary RNA or D N A blocks the translation of a number of mRNA species in cell free extracts ]8-10, 17]. The oligonucleotides used in the present study were designed to bind to the distinct regions spanning the initiation codon region of ODC or

194 AdoMetDC and the results presented here show these can selectively prevent translation of these mRNAs. The block to translation is the result of the duplex formation between the oligonucleotide and m R N A and is consequently very specific. Our studies indicate that oligonucleotides which bind to the initiation codon region of m R N A are very effective and selective inhibitors of translation. The mechanism of inhibition may involve blocking of the ribosome binding site and thus preventing attachment or movement of the ribosome on the mRNA. Alternately, oligomer binding could cause a change in the conformation of the mRNA. These conformation changes could be sufficient to prevent proper or productive binding of the ribosome with the message. These results suggest that by suitable choice of oligomer sequence and binding site, it will be possible to control translation or specific mRNAs. Therefore, this technique should provide a valuable tool for studying and/or controlling gene expression in mammalian cells. These results are in agreement with previous findings that oligonucleotides complementary to the initiation codon region of globin m R N A were able to inhibit translation in a rabbit reticulocyte lysate [17, 18] and were more potent in a wheat germ system [17]. The difference between results in the wheat germ extract and reticulocyte lysate system may be explained by the presence in the former of ribonuclease H which could act to cleave the m R N A strand of the duplex [19]. Our results indicate that the appropriate antisense oligonucleotides should prove to be useful tools for the study of polyamine function in the cell and may be useful as therapeutic agents. The major problem with such approaches relates to the uptake and biological stability of these agents. For investigations of polyamine function, it should prove possible to insert the cDNAs for ODC and AdoMetDC into mammalian expression vectors in the correct orientation to produce antisense R N A which could shut off the synthesis of these enzymes [9, 10]. Pharmacological approaches will require the external application of appropriate oligonucleotides but it is now well established that molecules of comparable size to those used in these studies are taken up in mammalian cells [8, 20], that conjugates with better uptake properties or increased activity can be synthesized [8, 9] and that more stable analogs such as methylphosphonates, phosphorothioates and a-oligodeoxynucleotides can be used for this purpose [8-10, 21, 22].

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195 translation by antisense oligonucleotides. Proc Natl Acad Sci USA 85: 5011-5015, 1988 20. Loke SL, Stein CA, Zhang XH, Mori K, Nakanishi M, Subasinghe C, Cohen JS, Neckers LM: Characterization of oligonucleotide transport into living cells. Proc Natl Acad Sci USA 86: 3474-3478, 1989

21. Maher LJ, Dolnick B J: Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxy-ribonucleoside methylphosphonates in a cell-free system. Nude Acids Res 16: 3341-3358, 1988 22. Eckstein F, Gish G: Phosphorothioates in molecular biology. TIBS 14: 97-100, 1989