Proc. Natl. Acad. Sci. USA Vol. 91, pp. 8072-8076, August 1994 Pharmacology Inhibition of proopiomelanocortin expression by an oligodeoxynucleotide complementary to fendorphin mRNA SANTI SPAMPINATO*t, MARCO CANOSSA*t, LUCIA CARBONI*, GABRIELE CAMPANA*, GIAMPIERO LEANZA§, AND SERGIO FERRI* *Department of Pharmacology, University of Bologna, Irnerio 48, 40126 Bologna, Italy; and 1Institute of Human Physiology, University of Catania, 95125 Catania, Italy Communicated by Avram Goldstein, May 10, 1994

ABSTRACT Gene expression in mammalian cells can be peptides by antisense oligonucleotides. POMC, the common suppressed by oligonucleotides complementary to the target precursor of adrenocorticotropin (ACTH), the melanocyte- mRNA. This strategy was explored as a means of arresting stimulating hormones, and a family of opioid peptides ((- translation of the probormone precursor proopiomelanocortin lipotropin and the endorphins) (8), is expressed either in the (POMC), used as a model system ofpeptide messengers that are pituitary gland or in a subset of neurons arising from the synthesized and released from endocrine and neuronal cells. arcuate nucleus in the hypothalamus and projecting to target The synthesis ofthe POMC-derived peptides adrenocorticotro- areas throughout the rest of the brain (9). This communica- pin (ACTH) and (endorphin (*-END) was markedly reduced tion reports that the synthesis of POMC-derived peptides is by an oligodeoxynucleotide (ODN) complementary to a region markedly reduced by antisense oligodeoxynucleotide (ODN) of -END mRNA in AtT-20 cells, which retain many of the treatment in vitro in the AtT-20 cell line, which retains many differentiated phenotypes of corticotrophs; this treatment did of the differentiated phenotypes of corticotrophs (10), and in not affect the steady-state levels of POMC mRNA. Antisense vivo after microinfusion of the antisense ODN in the hypo- ODN was stable in cell culture medium for 24 h, and cellular thalamic arcuate nucleus. uptake was low (-2.5% of the added ODN); however, the intracellular levels of the ODN were sufficient to form a MATERIALS AND METHODS ribonuclease-resistant duplex with complementary cellular mRNA. Addition of ODN to the cell culture did not affect the ODNs. Unmodified 30-mer ODNs were synthesized by cellular levels of chromogranin A-(264-314)/pancreastatin or phosphoramidite chemistry on an Applied Biosystems 380B cell viability and proliferation, as evidenced by bromodeoxy- DNA synthesizer and purified by reverse-phase high- uridine incorporation and ornithine decarboxylase activity. performance liquid chromatography (11). The antisense Microinfusion of the antisense ODN in the rat hypothalamic ODN used was complementary to POMC mRNA encoding arcuate nucleus, where the majority of POMC-positive brain the sequence for the first 10 aa of murine j3-endorphin perikarya are located, significantly reduced ACTH- and (jS-END; 5 '-TACGGTGGCTTCATGACCTCCGAGAA- (.END-immunoposltive neurons, and antisense ODN-treated GAGC-3'; in this region there is 100% amino acid sequence rats showed substantially less ofthe grooming behavior usually homology with rat (-END and only a single-base mismatch). observed in a novel environment. Two control ODNs were prepared for comparison, one corresponding to the sense sequence (5'-ATGCCAC- Significant advances have been made in understanding the CGAAGTACTGGAGGCTCTTCTGG-3') and the other with biological role of chemical messengers released from endo- base content identical to the antisense sequence but in a crine and neuronal cells by employing substances that limit or scrambled order (5'-IICGTCATCGIIATGCACGA- selectively block their synthesis. Chemical tools have been CAAACGGCG-iC-3'; nucleotides complementary to the in- developed that interfere, by diverse mechanisms, with the tended target sequence are underlined). A GenBank search production or metabolism of small transmitter molecules, indicated that these ODNs were not complementary to such as acetylcholine, amino acid neurotransmitters, and mRNA sequences in any murine or rat genes so far entered catecholamines (1). However, this strategy gives limited in the data base. Concentrations were measured by assuming results when investigating peptide transmitters originating, in 1 A260 unit = 33 pg/ml. Purity was evaluated by running multiple forms, from high molecular weight protein precur- 32P-labeled samples on a 20% polyacrylamide/7 M urea gel sors by endoproteolytic and other enzymatic modifications (ref. 12, pp. 11.21-11.28). ODNs were 5' end-labeled with (2, 3). [y.32P]ATP using polynucleotide kinase (ref. 12, pp. 10.59- The regulation of gene expression by nucleic acid se- 10.61). quences complementary to a target RNA (defined as an- Cell Culture. AtT-20 cells (American Type Culture Collec- tisense sequences) has been successfully employed to arrest tion, CCL89) were grown in suspension in Dulbecco's mod- the translation of mRNA in eukaryotic cells. Most studies ified Eagle's medium (GIBCO) supplemented with 10Wo (vol/ have focused on the effects of antisense RNA or oligonucle- vol) heat-inactivated fetal calf serum (650C, 30 min), glucose otides on the expression of proliferation-associated cell pro- (25 mM), ampicillin (100 pg/ml), and kanamycin (100 yg/ml) teins (for a review, see ref. 4) or receptors in vitro and in vivo and grown in an atmosphere of 5% C02/95% air. (5). The cells were seeded at a density of 5 x 10W cells per well We chose the well-characterized proopiomelanocortin (5 ml) in serum-free medium and treated with ODN dissolved (POMC) as a model system to see whether this approach in culture medium (100 ul); fetal calf serum was added 3 h worked for arresting the translation of prohormone precur- sors and thus the synthesis and release of the processed Abbreviations: ACTH, adrenocorticotropin; a-END, p-endorphin; ODN, oligodeoxynucleotide; ir, immunoreactive; ODC, ornithine decarboxylase; POMC, proopiomelanocortin; CgA, chromogranin. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tPresent address: Department ofNeurobiology, Stanford University in accordance with 18 U.S.C. §1734 solely to indicate this fact. School of Medicine, Stanford, CA 94305. 8072 Downloaded by guest on September 29, 2021 Pharmacology: Spampinato et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8073 later. ODNs were added to the culture plates again after 24 samples were subjected to RNase A digestion (24); after and 48 h without replacing the medium. After 24 or 60 h in the phenol/chloroform extraction, stable hybrids were ethanol- presence of the ODN, the cells were centrifuged (400 x g, 5 precipitated, resuspended in formamide sample buffer, de- min, 250C), washed twice with phosphate-buffered saline (pH naturated (95(C, 5 min), separated in a 20% polyacrylamide/7 7.4), and incubated for 3 h in fresh, complete culture medium M urea gel and exposed to x-ray film (ref. 12, pp. E.21-E.27). without ODN to evaluate hormone secretion. Animals and Surgery. Adult male Sprague-Dawley rats In an additional protocol, treated cells were allowed to (280-320 g) were anesthetized with chloral hydrate, and a recover from the effects of the antisense ODN by seeding 30-gauge stainless cannula was stereotaxically implanted them in culture medium without ODN for 60 h and then unilaterally into the hypothalamic arcuate nucleus [coordi- incubating them for 3 h in fresh medium. At the end of the nates: x, 0.2 mm right, y, -2.3 mm; z, -9.6 mm from the incubation, cells and media were collected, separated by bregma according to Paxinos and Watson (25)] and connected centrifugation, and processed. to an Alzet 1003D miniosmotic pump (Alza) with a flow rate Peptide Levels. Peptide levels were measured by RIA on of 1 1l/h, filled with ODN (0.625 pyg/,l) dissolved in sterile samples of the culture medium and of an acetic acid extract saline for a 60-h infusion. At the end ofthe infusion, rats were of the cell pellet. The latter was washed twice with phos- subjected to behavioral analysis, anesthetized again, and phate-buffered saline (pH 7.4) and homogenized by sonica- with tion (3 x 20 s) in 0.1 M acetic acid (900C). After removal of transcardially perfused cold phosphate-buffered saline an aliquot for protein determination (13), the cell homogenate and then with 4% paraformaldehyde in 0.1 M phosphate was centrifuged (12,000 x g, 20 min, 40C), and the superna- buffer (pH 7.4). tant was saved. Immunoreactive (ir) 3-END, ir-ACTH, and Behavioral Analysis. The total time of grooming episodes immunoreactive chromogranin A (ir-CgA) were measured by performed by the rats in 1 h was recorded by placing the double-antibody precipitation RIA using commercially avail- animals in a novel environment as described by Gandolfi et able antisera (Peninsula Laboratories) according to the man- al. (26). ufacturer's directions. The antibody employed in the ACTH Immunohistochenistry. Cryostat sections (40 um thick) RIA was specific for rat and mouse ACTH-(1-39), and there were cut through the hypothalamus by standard techniques was no cross-reactivity with other peptides derived from the (27). Alternate sections were immunostained with antisera to POMC precursor. The /3-END antibody reacted equally well rat ACTH-(1-39) or (3END (Peninsula). Reactivity was with (-END and 3-lipotropin and recognized other interme- visualized by the avidin:biotinylated horseradish peroxidase diate, high molecular weight products of the precursor pro- method (28). Reagents were obtained from Vector Labora- cessing (14). The antibody employed in the CgA RIA was tories. raised against rat CgA-(264-314)/pancreastatin (15), and A stereological method for obtaining estimates of the total there was no cross-reactivity with porcine pancreastatin-(33- number of ACTH- or ,B-END-immunopositive neurons in the 49) and rat amylin amide or with high molecular weight rat hypothalamic arcuate nucleus was used (29). The antero- products of the precursor CgA. posterior extent of the arcuate nucleus was from the caudal RNA Isolation and Quantitation. Total cellular RNA was part of the suprachiasmatic nucleus to the disappearance of isolated as described (16), and RNA concentrations were the mamillary recess of the third ventricle, whereas the estimated by absorbance at 260 nm (=5 ,g per 106 cells). To lateral edges were set at +0.9 mm from the midline (25). quantitate POMC mRNA levels, a solution hybridization/ Computer analysis was performed by GRID software (Inter- RNase protection assay was done (17) using 1 pg of total activision, Silkeborg, Denmark). RNA and a 32P-labeled 923-base Riboprobe (2 x 105 cpm) All data are presented as mean ± SEM. Significant differ- derived from the pMKSU16 plasmid containing the mouse ences were evaluated by analysis of variance and Duncan's POMC cDNA (18), subcloned into a pGEM3 plasmid multiple-range test. (Promega). A sense transcript was used to generate a stan- dard curve (19). Hybridization solutions were incubated at 550C for 16 hand then treated with RNase A (5 pg/ml, 15 min, RESULTS 40C); protected species were electrophoretically resolved on Stability and Cellular Uptake of Antisense ODN. The 32p a 5% nondenaturing polyacrylamide gel. Gels were exposed end-labeled antisense ODN was very stable when exposed to to x-ray film, and the specific hybrid bands were excised and cell culture medium containing 10%6 heat-inactivated fetal calf Cerenkov-counted. POMC mRNA levels were established by serum, since no degradation was detectable over a 24-h comparison with the standard curve run in parallel (19). period at 370C (Fig. 1A). However, the oligomer was de- Cell Viability and Proliferation Assay. Cell number and viability were determined by staining with fluorescein diac- 0. S 2 b6 24rti 05i1 b1- etate/propidium iodide (20). The rate of 5-bromodeoxyuri- dine (BrdUrd) incorporation (21) was measured by adding 100 nmol of BrdUrd per 106 cells (Boehringer Mannheim, no. 1444611) 16 h before harvesting (i.e., 44 h after the addition of ODN) and measuring the absorbance at 405 nm according to the manufacturer's directions. A B Ornithine decarboxylase (ODC; EC 4.1.1.17) activity was evaluated in AtT-20 cells as described (22). FIG. 1. Stability of 32P-labeled antisense ODN in AtT-20 cell Detection of ODN-RNA Duplexes. Briefly, the cells were culture (one of three experiments). (A) Cells were exposed to harvested after incubation at 37°C for 6 or 24 h with antisense antisense ODN (100 nM) plus trace amounts of the corresponding DNA (100 nM) plus trace amounts of the corresponding 32P-labeled ODN (2 x 106 cpm/ml), and at timed intervals samples 32P-labeled ODN (2 x 106 cpm/ml), washed three times with of the medium containing heat-inactivated serum were removed, warm (370C) 5 mM sodium phosphate buffer (pH 7.4)/100 Cerenkov-counted, and subjected to electrophoresis on a 20%o poly- mM NaCl and resuspended in ice-cold 150 mM Hepes buffer acrylamide/7 M urea gel. An autoradiogram ofthe dried gel is shown. St., unexposed 32P-labeled 30-mer ODN (control). (B) The experi- (pH 7.4). The cell suspension was lysed with 1/10th vol of 5% ment was carried out as in A, but normal fetal calf serum was added Triton X-100 and separated into cytoplasm and nuclei by to the cell culture medium. Moreover, 32P-labeled ODNs were centrifugation (15,000 x g, 2 min, 40C), and the nuclei were degraded when added to culture medium containing normal fetal calf dissolved in Hepes buffer containing 0.1% SDS (23). The serum but lacking cells (data not shown). Downloaded by guest on September 29, 2021 8074 Pharmacology: Spampinato et al. Proc. Natl. Acad. Sci. USA 91 (1994) graded in the presence of normal serum (Fig. 1B). Cellular Levels of ir-f-END, ir-ACTH, and ir-CgA in Cell Extracts uptake ofthe radiolabeled ODN was rapid but then increased and Culture Medium. A significant dose- and time-dependent only slightly over the exposure time. The maximal concen- decrease in the levels and secretion of ir-f3-END and ir- tration of cell-associated radioactivity was =2.5% in 24 h ACTH was observed in AtT-20 cells exposed to antisense (Fig. 2A). To examine whether the antisense ODN formed a ODN but not to sense or scrambled ODN. Approximately with POMC mRNA, AtT-20 cells 48-50% of the reduction was detected after a 24-h exposure stable intracellular duplex to 100 nM antisense ODN, and the effect appeared to peak were exposed for 6 or 24 h to 32P end-labeled ODN. After after 60 h of treatment with 10 and 100 nM ODN (Fig. 3). Cell RNA isolation, a ribonuclease-resistant ODN-RNA duplex of content and release of the peptides returned to control values the same size as the intact ODN was detected either in the in cells exposed to antisense ODN for 60 h and then cultured cytoplasm or in the nucleus. The double-stranded ODN-RNA in fresh medium for an additional 60 h (Fig. 3). Exposure of duplex was more abundant in the cells exposed to antisense AtT-20 cells to antisense ODN (100 nM, 60 h) had no effect ODN for 24 h (Fig. 2B). To ensure that the duplex was not on ir-CgA levels evaluated in cell extract (215 ± 20.1 vs. 263 due to any artifact of RNA isolation, a lysate of AtT-20 cells ± 16.8 fmol/mg of protein in controls; n = 7) and in cell not exposed to ODN was mixed with 32P-labeled and unla- culture medium (303 ± 38.4 vs. 303 ± 27.6 fmol per ml per mg beled ODN equivalent to the maximal amount of cell asso- of protein in controls; n = 7). CgA is the precursor of ciated radioactivity (-2.5% of the amount added to the cell bioactive peptides and occurs in a wide variety of endocrine culture; see Fig. 2A). No duplexes were observed in these cells (30). CgA-related peptides are localized within secretory samples (data not shown). In AtT-20 cells exposed to sense granules and released with the resident peptides; they may play a role in the assembly of the vesicles and/or regulate or scrambled ODN, no ribonuclease-resistant duplexes were processing and release (31). Therefore, present results sup- detected (data not shown). port the hypothesis that the effect of antisense ODN on POMC-derived peptides is not due to any alteration ofthe cell secretory pathway. POMC mRNA in AtT-20 Cells. As shown in Fig. 4, the steady-state levels of POMC mRNA were not modified by antisense ODN exposure (100 nM, 60 h). Moreover, an- tisense ODN (100 nM) did not affect the steady-state levels of POMC mRNA in AtT-20 cells exposed to the oligomer for 0 E, Q 24 h (288 ± 66 vs. 264 ± 89 pg/pg of total RNA in controls; n = 7) or 48 h (310 ± 60 vs. 332 ± 88 pg/pug of total RNA in controls; n = 7). Cell Viability and Proliferation. Exposure ofAtT-20 cells to antisense ODN (100 nM, 60 h) did not affect cell growth as evidenced by the total number of cells (5.47 ± 0.06 x 105 vs. 5.50 ± 0.09 x 105 in control cells; n = 7) and their viability (76% ± 0.8% vs. 75% ± 0.7% in control cells; n = 7). Time (h) Antisense ODN (100 nM, 60 h) also induced no morpholog- ical changes and did not alter cell proliferation [BrdUrd B 1 2 3 4 5 incorporation assay absorbance values were 1.88 ± 0.15 (n = 5) in vehicle-treated cells and 2.02 ± 0.12 (n = 5) in ODN- treated cells (=7 x 105 cells per well)]. Finally, ODC activity in AtT-20 cells exposed to 100 nM antisense ODN for 60 h was superimposable on that of control cells (Table 1). ODC activity is a marker of changes in polyamine metab- olism and plays a key role in protein synthesis (32). Thus, these results further confirm that the blockade of POMC biosynthesis by antisense ODN is not caused by cellular damage. Effect of in Vivo Infusion of Antisense ODN on POMC- Containing Neurons ofthe Arcuate Nucleus. In agreement with earlier studies (33), we observed numerous ACTH-positive FIG. 2. Cellular uptake and ODN-RNA duplex formation in perikarya in the arcuate nucleus and periarcuate regions of AtT-20 cells exposed to antisense ODN (100 nM) plus trace amounts the medial basal hypothalamus of rats infused with vehicle of the corresponding 32P-labeled ODN (2 x 106 cpm/ml). (A) (Fig. 5). Immunopositive neurons were significantly de- Cell-associated radioactivity detected in AtT-20 cells. At the given creased in rats treated with antisense ODN (Fig. 5), but there time points, the cells were pelleted and washed three times with was no change in rats infused with the same dose of sense or phosphate-buffered saline solution (pH 7.4), suspended in lysis buffer, separated into cytoplasm and nuclei (24), and Cerenkov- scrambled ODN (see legend to Fig. 5). counted; -35% of radioactivity was associated with the nuclei and Antisense ODN-treated rats presented substantially less was not substantially modified over 24 h. The total radioactivity grooming behavior when exposed to a novel environment 60 measured in the cytoplasm and in the nuclei is expressed as percent h after the start of infusion. The grooming time in 1 h was of the amount added to the cell culture. Each point represents the 650.66 ± 40.61 s in rats infused with vehicle (n = 7) but only mean ± SEM of three independent experiments. (B) Detection of 262.33 ± 52.65 s (n = 7; P < 0.01) in the antisense-treated intracellular ODN-RNA duplex. An autoradiograin of ribonuclease- group. Grooming time was not modified in rats microinfused resistant duplexes formed between antisense ODN and cellular RNA with sense (643.80 ± 54.63 s; n = 7) or scrambled (664.21 ± is shown. Lanes 1 and 2 show the hybrid detected, after a 24-h n = 7) ODN. incubation, in the cytoplasmic and nuclear fractions, respectively. 58.88 s; Lanes 3 and 4 show the hybrid detected, after a 6-h incubation, in the cytoplasmic and nuclear fractions, respectively. Lane 5 shows DISCUSSION 32P-labeled antisense ODN (standard). This is representative ofthree independent experiments performed as described in Materials and This study indicates that antisense oligonucleotides may Methods. serve as an invaluable tool to arrest the translation of Downloaded by guest on September 29, 2021 Pharmacology: Spampinato et al. Proc. Natl. Acad. Sci. USA 91 (1994) 8075 .- 3.0 0) *6 70- Cell extr;'act o 2.5 20-60- r-Wa- 2.0 m 15 1050- .U C 1.0 ~!24 h)30- FIG. 3. Time course of ODN effects O 0.5 E on ir-,¢END (Left) and ir-ACTH (Right) . 0.0 content and release in AtT-20 cells [bars, 60 h 120 h from left to right: control (no treatment), r- 10. antisense ODN (1 nM), antisense ODN *5 150 Medium 0=10* 14* Medium (10 nM), antisense ODN (100 nM), ° 125 212- scrambled ODN (100 nM), and sense ODN (100 nM)]. Cells were exposed to = 100 ODN for 24 or 60 h or allowed to recover U 0 from the effects of the antisense by seed- 75 ing them in culture medium without ODN 50 for another 60 h (120 h from the start of 'E the experiment). Results are expressed 25 <260- 24 h as mean ± SEM of seven dishes per O treatment. **, P < 0.05; *, P < 0.01 vs. t~~tI124 h 60 h 120 h control (Duncan's multiple-range test af- ter ANOVA).

prohormone precursors either in cell lines or in the central the use of antibodies recognizing the substances released nervous system, with the aim of better elucidating the func- (35). tional role of these gene products. This strategy has been Although several potential mechanisms could account for successfully employed in vitro and in vivo to inhibit the arrest of POMC translation, the most likely is that the expression of numerous viral or mammalian genes (for a antisense ODN specifically binds to the target complemen- review, see ref. 4). As regards neuronal genes, several tary sequences of POMC mRNA, leading to the decrease of reports have described the suppression of receptors (5) or precursor translation. This is based on four observations: (i) microtubule-associate proteins (34) after exposure to an- antisense ODN formed a stable intracellular duplex with tisense constructs. complementary mRNA in AtT-20 cells; (ii) a scrambled or a The present report focuses on the feasibility ofinterrupting sense ODN had no inhibitory effects on POMC translation; cell communication by arresting the synthesis of peptide (iii) ACTH synthesis was reduced by antisense treatment, messengers. Neurons are secretory cells par excellence; although the ODN was complementary only to a sequence of therefore, the possibility of arresting the translation of pep- ,(3END mRNA placed in the 3' terminal region of the gene, tide messengers by antisense ODNs may offer an alternative thus suggesting that the expression of the entire protein may to pharmacological blockade of the target receptors (1) or to be reduced; (iv) exposure to antisense ODN did not reduce steady-state levels of POMC mRNA in AtT-20 cells. There- A 400- fore, our data do no support any major participation by RNase H (36). z cy- In agreement with previous studies (37, 38), cellular uptake *i 300° of ODN by cultured cells was low. However, there is a 0 :=: significant correlation between the levels of ODN and POMC /- :1 mRNA in AtT-20 cells; in fact, an uptake of 1% of the higher <-200 dose of antisense ODN (100 nM, added to 5 x 105 cells per z ix 5 ml) corresponds to -3 x 107 molecules per cell while the E W :. POMC mRNA concentration is -2 x 103 molecules per cell. C-) . 100 In agreement with in vitro findings, microinfusion of an- 0o tisense ODN reduced the number of hypothalamic POMC- 0- positive neurons in rats, which exhibited significantly less grooming when exposed to a novel environment. This be- 0 havior is induced by ACTH injection in the brain lateral ventricle (39) and reduced in rats treated with an antibody B 1 2 3 4 raised against this peptide (40). The effectiveness of the antisense ODN in vivo may be a result of the penetration and stability of nucleic acids administered in discrete brain areas (6). It has been previously reported that unmodified ODNs Table 1. ODC response to ODN exposure in AtT-20 cells ODC activity FIG. 4. (A) Steady-state levels of POMC mRNA in AtT-20 cells exposed to ODN for 60 h [bars, from left to right: control (no Treatment Basal Stimulated treatment), antisense ODN (100 nM), scrambled ODN (100 nM), and Vehicle 0.95 ± 0.15 6.62 ± 0.39 sense ODN (100 nM)]. Values are the mean SEM (n = 7). (B) Representative autoradiogram indicating POMC mRNA expression Antisense ODN 0.94 ± 0.14 5.86 ± 0.54 in control cells (lanes 1 and 2) and in cells exposed to 100 nM ODC activity is given in nmol of CO2 per mg of protein per h at antisense ODN for 60 h (lanes 3 and 4). The arrow indicates the 370C. Each value is the mean ± SEM of three samples. ODC position of a 947-bp radiolabeled DNA marker. The overall pattern response was established in cells exposed to ODN (100 nM) for 60 h of POMC mRNA expression was reproducible in separate experi- and then harvested (Basal) or else further diluted (1:3) and suspended ments. in fresh medium for 4 h before harvesting (Stimulated). Downloaded by guest on September 29, 2021 8076 Pharmacology: Spampinato et al. Proc. Natl. Acad. Sci. USA 91 (1994) M.C., L.C., and G.C. contributed equally to this work. Stereo- logical analysis was performed at the Department of Medical Cell Research, University ofLund, and we thank Prof. A. Bjorklund. We are grateful to Prof. F. Flamigni for assistance in ODC determination and to Dr. R. Rimondini-Giorgini and Dr. R. Dall'Olio for helpful advice about in vivo experiments. We acknowledge the generous support of the Fondazione Marino Golinelli (Bologna, Italy) and of the Consiglio Nazionale delle Ricerche target project "Biotechnol- ogy and Bioinstrumentation." 1. Cooper, J. R., Bloom, F. E. & Roth, R. H. (1991) TheBiochemicalBasis of Neuropharmacology (Oxford Univ. Press, Oxford, U.K.), 5th Ed. 2. Mains, R. E. & Eipper, B. A. (1990) Trends Endocrinol. Metab. 1, 388-394. 3. Jung, L. J. & Scheller, R. H. (1991) Science 251, 1330-1335. 4. Baserga, R. & Denhardt, D. T., eds. (1992) Ann. N. Y. Acad. Sci. 660, 1-353. 5. Wahlestedt, C. (1994) Trends Pharmacol. Sci. 15, 42-46. 6. Maciejewski-Lenoir, D., Jirikowski, G. F., Sanna, P. P. & Bloom, F. E. (1993) Proc. Nati. Acad. Sci. USA 90, 1435-1439. 7. Woolf, T. M., Melton, D. A. & Jennings, C. G. B. (1992) Proc. NatI. Acad. Sci. USA 89, 7305-7309. 8. Peng Loh, Y. (1992) Biochem. Pharmacol. 44, 843-849. 9. Civelli, O., Birnberg, N. & Herbert, H. (1982) J. Biol. Chem. 257, 6783-6787. 10. Giagnoni, G., Sabol, S. L. & Niremberg, M. (1977) Proc. Natl. Acad. Sci. USA 74, 2259-2263. 11. Boiziau, C., Thuong, N. T. & Toulm6, J.-J. (1992) Proc. Natl. Acad. Sci. USA 89, 768-772. 12. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, Plainview, NY), 2nd Ed. 13. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254. 14. Young, E., Bronstein, D. & Akil, H. (1993) in Opioids, ed. Herz, A. (Springer, Berlin), Vol. 1, pp. 393-421. 15. Iacangelo, A., Okayama, H. & Eiden, L. E. (1988) FEES Lett. 227, 115-121. 16. Chomczynsky, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. 17. Krause, J. E., Cremins, J. D., Carter, M. S., Brown, E. R. & MacDon- ald, M. R. (1989) Methods Enzymol. 268, 634-652. 18. Uhler, M. & Herbert, E. (1983) J. Biol. Chem. 258, 257-261. FIG. 5. Typical frontal plane pattern of ir-ACTH perikarya and 19. Canossa, M., Ventura, C., Vaona, I., Carboni, L., Guarnieri, C. & fibers in the hypothalamic arcuate nucleus of the rat (x50). Animals Spampinato, S. (1993) Biochim. Biophys. Acta 1172, 247-250. were treated with vehicle (A), antisense ODN (B), or scrambled ODN 20. Jones, K. H. & Senft, J. A. (1985) J. Histochem. Cytochem. 33, 77-79. (C). In vehicle-treated rats, 2590 ± 90 (n = 7) immunopositive 21. Magaud, J. P., Sargent, I. & Mason, D. Y. (1988) J. Immunol. Methods neurons were counted in the entire arcuate nucleus compared with 106, 95-100. 1430 ± 87 (n = 7; P < 0.01) in antisense ODN-treated rats. In rats 22. Flamigni, F., Campana, G., Carboni, L., Guarnieri, C. & Spampinato, S. microinfused with scrambled ODN, 3190 + 180 (n = 7) immunopos- (1994) Biochem. J. 299, 515-519. 23. Citro, G., Perrotti, D., Cucco, C., D'Agnano, I., Sacchi, A., Zuppi, G. itive neurons were and 3380 ± 121 = counted, (n 7) immunopositive & Calabretta, B. (1992) Proc. Natd. Acad. Sci. USA 89, 7031-7035. neurons were counted in sense ODN-treated rats. Results were 24. Teichman-Weinberg, A., Littauer, U. Z. & Ginzburg, I. (1988) Gene 72, comparable in brain sections stained with an antibody raised against 297-307. rat P-END (data not shown) and agree with Khachaturian et al. (33) 25. Paxinos, G. & Watson, C. (1986) The Rat Brain in Stereotaxic Coordi- who demonstrated P-END, P-lipotropin, and ACTH immunoreac- nates (Academic, Sydney). tivities in the same arcuate neuronal perikarya. The morphology of 26. Gandolfi, O., Dall'Olio, R., Vaccheri, A., Roncada, P. & Montanaro, N. immunopositive neurons did not appear to be altered, and ODN- (1988) Pharmacol. Biochem. Behav. 30, 463-469. treated rats presented no gross neurological abnormality. 27. Hollands, B. (1962) in Progress in Medical Laboratory Technique, ed. Baker, F. J. (Butterworth, London), pp. 112-135. 28. Hsu, S. M., Raine, L. & Fanger, H. (1981)J. Histochem. Cytochem. 29, diffuse through brain tissue and enter neurons following 577-580. 29. West, M. J., Slomianka, L. & Gundersen, H. J. G. (1991) Anat. Rec. intracerebroventricular administration; are effective when 231, 482-497. injected in the striatum, nucleus accumbens, and hypothal- 30. Wand, G. S., Takiyyuddin, M., O'Connor, D. T. & Levine, M. A. (1991) amus; and are relatively stable in the cerebrospinal fluid (for Endocrinology 128, 1345-1351. 31. Fasciotto, B. H., Trauss, C. A., Greeley, G. H. & Cohn, D. V. (1993) a review, see ref. 5). Therefore, the bilateral reduction of Endocrinology 133, 461-466. POMC-related peptides observed in the hypothalamus, fol- 32. Pegg, A. (1986) Biochem. J. 234, 249-262. lowing unilateral infusion of antisense ODN, could be due to 33. Khachaturian, H., Lewis, M. E., Tsou, K. & Watson, S. J. (1985) in the Handbook ofChemical Neuroanatomy, eds. Bjorklund, A. & Hokfelt, T. difusion of the oligomer through the brain parenchyma (Elsevier, Amsterdam), Vol. 4, pp. 216-272. and across the third ventricle. 34. Caceres, A., Potrebic, S. & Kosik, K. S. (1991) J. Neurosci. 11, Although in vitro studies have established that the an- 1515-1523. tisense ODN can inhibit the gene gross 35. Tilders, F. J., van Oers, J. W., White, A., Menzaghi, F. & Burlet, A. target without toxic (1990) Adv. Exp. Med. Biol. 274, 135-146. effects on cultured cells, its specificity and possible side 36. Toulmd, J. J. (1992) in Antisense RNA and DNA, ed. Murray, J. A. H. effects have yet to be established. In fact, ODNs could block (Wiley-Liss, New York), pp. 175-194. the translation of different mRNA sequences by hybridizing 37. Yakubov, L. A., Deeva, E., Zarytova, V. F., Ivanova, E. M., Ryte, A. S., Yurchenko, L. V. & Vlassov, V. V. (1989) Proc. Natl. Acad. Sci. with them, with fortuitous partial matches arising with ODN USA 86, 6454-6458. degraded by cellular nucleases (7). Further studies are clearly 38. Loke, S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., necessary to clarify the direct correlations between activity Subasinghe, C., Cohen, J. S. & Neckers, L. M. (1989) Proc. Natl. Acad. Sci. USA 86, 3474-3478. and ODN base composition so as to increase the discrimi- 39. Gispen, W. H. & Isaacson, R. L. (1981) Pharmacol. Ther. 12, 209-246. nation between target and nontarget sequences. 40. Dunn, A. J., Green, E. J. & Isaacson, R. L. (1979) Science 203,283-284. Downloaded by guest on September 29, 2021