(CANCER RESEARCH 49. 7015-7019. Dci-cmhcr 15. 19S'>| Expression and Precursor Processing of Y in Human and Murine Neuroblastoma and Pheochromocytoma Cell Lines1

Mairead M. T. O'Hare2 and Thue VV.Schwartz laboratory of Molecular , L'nirersity Department of Clinical Chemistry, Rigshospitalet 6321, Blfgtlanisvej 9, DK-2IIH) Copenhagen. Denmark

ABSTRACT turally related peptide. pancreatic polypeptide (13). Since the activation of NPY was found to be very efficient in all the The synthesis and processing of the precursor for tumors studied, we have in the present investigation studied the (NPV) were studied in 16 human and murine neuroendocrine cell lines. expression and activation of NPY in a series of neuroendocrine Eight of the cell lines, NS-20Y, PCI 2, LA-N-5, CHP-234, SMS-KCNR, SH-SY5Y, SMS-KCN, and BE(2)-MI7, produced sufficient quantities cell lines, mainly human neuroblastoma cells. Such cell lines to permit Chromatographie characterization of the NPY immunoreactiv- could possibly serve as tools in studies on neuropeptide activa ity. Although the cell lines varied in the amount of NPY they produced, tion and on the possible autocrine function of NPY in cells both within and between cell lines, they displayed a relatively constant which produce both the peptide and the (9, 11). There pattern of posttranslational modifications. In contrast to observations in fore the cells were characterized by immunological and Chro tumor extracts (M. M. T. O'Hare and T. \V. Schwartz. Cancer Res., 49: matographie techniques for their capacity to perform the correct 7010-7014, 1989), all cell lines studied contained a substantial amount posttranslational modifications in the activation of NPY. of the intraccllular NPY in the form of the unprocessed propeptide, 57% (range, 33-72%) as characterized by both gel nitrations (32 experiments in 8 cell lines) and "in vitro conversion" with cndoprotcinasc Lys-C. In MATERIALS AND METHODS the majority, 4 of 6 cell lines studied, almost all of the NPY, which by size corresponded to the mature 36-amino acid form, was amidated as Cell Cultures. CHP-234, SMS-KCNR, SH-SY5Y, SMS-KCN, assessed by isoelectric focusing and by a radioimmunoassay specific for BE(2)-M17, SMS-MSN, CHP-212, SK-N-MC. MC-IXC, SMS-KAN, the COOII-terminal amide group of the pcptide. Both the propeptide LAI-5s, and SH-EP cells (14-16), derived from human neuroblasto and smaller molecular forms of NPY were secreted from the cell cultures; mas, were obtained from Dr. June L. Biedler and Dr. Barbara A. however, protcolytic degradation in the tissue culture medium prevented Spengler, Sloan Kettering Institute, New York, NY. LA-N-5 cells, also a detailed, meaningful characterization of these peptides. It is concluded human neuroblastoma cells, were provided by Dr. Robert C. Seegcr, that many neuroendocrine cell lines, especially those derived from human Department of Pediatrics, UCLA School of Medicine, Los Angeles, neuroblastomas, express the NPY gene; the cells display a partly im CA (17). NS-20Y and N18TG-2 cells, derived from the murine neuro paired dibasic processing capacity but they generally amidate the prod blastoma, C-1300, (18, 19), were provided by Dr. Marshall Nirenberg, ucts efficiently. Laboratory of Biochemical Genetics, N1H, Bethesda, MD. A subclone of PC12 cells (11-250), originally derived from a transplantable rat pheochromocytoma (20) was obtained from Dr. Hans Thoencn, Max INTRODUCTION Planck Institute for Psychiatry, Martinsried, Federal Republic of Ger NPY,1 a highly conserved 36-amino acid pcptide (1), was many. Tissue culture media, sera, and supplements were from G1BCO Laboratories (Grand Island, NY). initially isolated from porcine using a chemical assay for PCI 2 cells were maintained in Dulbecco's modified Eagle's mini detecting COOH-terminal amide functions in peptides (2). mum essential medium containing 10% (v/v) A/ycop/asma-screened NPY has since been shown to be widely distributed throughout horse serum and 5% (v/v) virus and Mycoplasma-screened fetal calf the central and peripheral nervous systems (3, 4). In the central serum. LA-N-5 cells were grown in RPMI 1640 containing 10% (v/v) fetal calf serum. All other cell lines were grown in Ham's nutrient , NPY appears to be involved in the regulation mixture F-10 containing Dulbecco's modified Eagle's medium 1885 of food intake, in circadian rhythm, and possibly also in mem ory (5). Peripherally, it functions as a , inter (1:1, v/v) with 15% (v/v) fetal calf serum, and 1% (v/v) nonesscntial amino acids. Penicillin (100 lU/ml), streptomycin (100 ^g/ml), and 2 acting with norepinephrine in the regulation of vascular tone miML-glutamine were added to all culture media. Cells were grown in (6, 7). Receptors for NPY have been localized in rat and porcine Nunclon culture flasks, 260 or 800 ml (Nunc, Roskilde, Denmark), and brain and are particularly abundant in the hippocampus, hy maintained in a humidified incubator at 37°Cwith 5% CO; in air. The pothalamus, and cortex (8-10). Two types of binding sites for production and secretion of NPY immunoreactivity was studied in cells NPY have also been described on pheochromocytoma and at the time when they were about to be passed. These cells, which tend neuroblastoma cell lines. These have been designated Y, and to grow in colonies, are generally not grown to complete confluency. Y; receptors and may explain the different presynaptic and At the time of passage or extraction, there were 6-20 x IO4cells/cm2. postsynaptic effects of the peptide (11). Extraction of Cells and Media. Cells (approximately IO7)were har Previously, we found NPY expression in all 16 pheochrom- vested by treatment for 3-5 min with 0.25% (w/v) trypsin (Gibco) in ocytomas studied and in 3 neuroblastoma tumors (12). The saline containing 0.5 M EDTA, washed twice in the appropriate culture activation of the NPY molecule requires a series of posttrans medium, and centrifuged prior to extraction. The cell pellet was boiled lational modifications similar to those described for the struc- for 3 min in 2 ml 50% (v/v) acetic acid (Merck. Darmstadt, Federal Republic of Germany) and sonicated for 15 min in a bath sonicator Received 5/25/89; revised 9/11/89; accepted 9/21/89. prior to centrifugation at 3000 x g for 15 min. The supernatant was The costs of publication of this article were defrayed in part by the payment dried in a vacuum centrifuge and reconstituted in assay buffer (see of page charges. This article must therefore be hereby marked advertisement in later). The protein content of the acetic acid extracts was determined accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This investigation was supported in part by the Danish Cancer Association, using the Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, and the Danish Medical Research Council. T. W. S. was the recipient of a CA). The tissue culture media were either assayed directly or aliquots Research Professorship in Molecular Endocrinology from the Danish Medical (usually 10 ml) were precipitated with acetone, 4 volumes (Merck) for Research Council and the Weimann Foundation. 1To whom requests for reprints should be addressed. Chromatographie characterization. The precipitate was dried under ' The abbreviations used are: NPV. neuropeptide Y; pro-NPY, proneuropep- nitrogen and reconstituted in 2 ml of 5% (v/v) formic acid (Merck) tide Y; HPLC. high performance liquid chromatography; NEPHGE. nonequilib- containing 10 mg/litcr of bovine serum albumin (BSA). rium pH gradient electrophoresis; BSA, bovine serum albumin. Radioimmunoassay Methods. The NPY radioimmunoassays were 7015 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1989 American Association for Cancer Research. NEUROPEPTIDE V IN NEUROENDOCRINE CELL LINES Table 1 Storage and intracellular processing ofpro-NPY in neuroendocrine cell lines Cellular NPY protein)Cell (fmol/mg efficiency"Median5955723347594162Range52-7042-5665-7520-4747-6732-7445-79n4353385Amidationefficiency*Median876584589492Range lineNS-20YPC! n83-98 360-87 2(11-250)NI8TG-2LA-N-5CUP

.3901.16057019010040<5<5<5<5<5Range1,970-15.70020-270<5-140100-64,000110-11.000160-5.91040-3.22010-160<5-15<5-7n3531254131143425243Processing756-60 234SMS-KCNRSH 287-100 SY5YSMS-KCNBE(2)-M17SMS-MSNCHP2I2SK-N-MCMC-IXCSMS-KANLAI 4

-5sSH-EPMedian3,6001701

' Percentage of total gel-filtered immunoreactivity in the NPY'"" peak. * Percentage of total NPY1'" migrating as the amidated species in NEPHGE.

performed as described previously ( 12). Two antisera were used; 337 NPY was raised to synthetic porcine NPY and 8999 to synthetic Cys-NPY31' (pill IpM) ". ['"I-7>T']- and ['2iI-7>r"']monoiodo-A7>)/ were used as tracers and MO were prepared as described previously (9). 600 Gel Filtration. Cell or media extracts were applied to Sephadex G- 400' 50 superfine columns (1.6 x 95 cm) eluted with 5% (v/v) formic acid 20 containing 10 mg/liter BSA. Fractions corresponding in size to pro neuropeptide Y (pro-NPY) or NPY' " were pooled and dried under vacuum for further characterization (12). LA-N-5ASMS-KCNRA High Performance Liquid Chromatography. Samples for HPLC analysis were reconstituted in 0.2 ml acetic acid (3 M) and analyzed 200-100-200 •¿300 using a Nucleosil C,8 column (0.4 x 25 cm) eluted with 0.1% (v/v) trifiuoroacetic acid in water and a 20-min linear gradient of 20-50% -500 acetonitrile (12). - 100450 Isoelectric Focusing. Gels containing Ampholyte pH 8-9.5 and Am- pholyte pH 7-9 (LKB, Bromma, Sweden) were prepared as described previously (12). The gels were cut into 2-mm slices and extracted for 12 h at 4°Cin 80% (v/v) formic acid-containing 10 mg/liter BSA. -

Endoproteinase Lys-C Digestion of Peptides. Gel-filtered pro-NPY ISO- was dried and reconstituted in 0.1 ml of 0.1 M A/-ethylmorpholine 300 adjusted to pH 8.65 with glacial acetic acid (Merck). Endoproteinase 100- •¿150 Lys-C (Boehringer Mannheim, Mannheim, Federal Republic of Ger 90-SMS-KCNAÂL..1 many) 30 milliunits in 10 /.il.was added prior to incubation for 60 min JlJLv,.SHSY-5YAA/1,,400 at 37°Casdescribed previously (12). 0.2 0.4 0.6 0.8 1.0 0 0.2 0.4 06 08 Calculations. The degree of dibasic processing was calculated as the Gelfiltration (G-50). Coefficient of distribution. Kd percentage of the total gel-filtered immunoreactivity eluting in the Fig. 1. Sephadex G-50 gel filtration of NPY immunoreactivity extracted from NPY1"3*peak compared to the total NPY immunoreactivity, excluding six human neuroblastoma cell lines. The columns were calibrated with BSA, NPY standard, and ['Hjleucine and eluted with 5% formic acid containing 10 mg/liter the material eluting in the void volume region. The degree of amidation is expressed as the percentage of the total NPY1'6 migrating as the BSA. Arrows, elution positions ofpro-NPY and synthetic NPY1 " standard. In some cases [SMS-KCN, SMS-KCNR, and BE(2)-M17] where up to 1 ml was amidated species in the NEPHGE gels. dried for radioimmunoassay, void volume interference was observed in the assay.

RESULTS corresponding in size to the mature NPY1 16 (Table 1). As shown in Fig. 1, the majority of the remaining NPY immuno NPY immunoreactivity was detected in 8 of the 13 human reactivity coeluted on gel filtration with pro-NPY. Although neuroblastoma cell lines, in both of the murine neuroblastoma pro-NPY and NPY1 16 are easily separated by gel filtration, cell lines, and in the rat pheochromocytoma cell line PCI2 they were not efficiently separated by HPLC (Fig. 2), which (Table 1). The individual cell lines displayed considerable vari may explain why pro-NPY was not described in PC 12 cells ation in the amount of NPY stored. Cells were at all times when only this technique was used (21). The pro-NPY material studied in early stationary phase of growth, and the variation was characterized further by endoproteinase Lys-C digestion. in NPY content was not directly correlated with the passage As reported previously for pro-NPY extracted from pheo- number, although a decline in production with increasing pas chromocytomas (12), the "in vitro conversion" with endopro sage number was often observed. Eight cell lines produced sufficient NPY immunoreactivity teinase Lys-C further substantiated that the large molecular for Chromatographie characterization. In contrast to the varia weight form of NPY immunoreactivity was pro-NPY (data not tion observed in the NPY production, the processing pattern shown). was relatively constant, both within cell lines and between cell The NPY-immunoreactive material which corresponded in size to NPY1"16was further characterized by HPLC analysis lines (Table 1). Compared to tumor extracts (12), only between 33 and 72% of the NPY immunoreactivity was found in a form and isoelectric focusing. Oxidation of NPY created heterogeneity 7016 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1989 American Association for Cancer Research. NEUROPEPTIDE Y IN NEUROENDOCRINE CELL LINES

800 ""I

600

400

JLJ/ 10 20 30 40 50 10 20 30 40 50

0 O.2 O.4 0.6 0.8 1.O C'i«:! ritieni »I disii ihulnin. k .,

600

50 400 S 400 40 * 30 I 20 S 10 IO 20 30 40 50 IO 20 30 40 50 0 5 10 15 20 25 Retention Tim«(min) 2,000

1.500

IO 20 30 40 50 IO 20 30 40 50

NEPHGE gel slice number 5 10 IS 20 25 Retention Time (min) Fig. 3. NEPHGE characterization of gel filtered NPY1 "-like immunoreactiv- ity extracted from six neuroblastoma cell lines. The gel slices were extracted with formic acid, dried, and assayed using both the 337 (•)and COOH-terminal Fig. 2. Characterization ofpro-NPY and NPY"6 from PCI2 cells by HPLC. specific 8999 (O) antisera. Closed and open arrows, positions taken by the Top, Sephadex G-50 elution profile of a PCI 2 cell extract. Fractions correspond nonoxidized and oxidized forms of synthetic human NPY1 '*, respectively. Double ing in size to pro-NPY (D) and NPY (•)were further characterized by HPLC on arrow, position taken by the free acid form of NPY, *, unidentified peak, which a Nucleosil Cig column (middle and bottom). Arrows, elution positions of oxidized may represent a fragment of NPY. (open arrow) and nonoxidized (closed arrow) synthetic human NPY; , acetonitrile gradient used. In all three experiments, antiserum 337 was used. in both systems as discussed previously (12). The amount of oxidized peptide extracted from the cell lines varied and occa sionally only nonoxidized peptide was detected (data not 300 shown). The final activation of NPY depends on the formation of the COOH-terminal amide function (22). We investigated CELL EXTRACT the amidation status of the cellular NPY by both Chromato 200 graphie and immunological techniques, as shown in Fig. 3 for NPY'"36-like immunoreactivity extracted from six of the cell lines. In all cell lines studied, the majority of the NPY immu 100 noreactivity migrated to a position corresponding to amidated NPY1"36,oxidized or nonoxidized (Fig. 3). Furthermore, this material was detected by both the general NPY antiserum and the antiserum specific for the COOH-terminal tyrosine amide,

8999. In two of the cell lines, SMS-KCNR and LA-N-5, a 300 significant amount of the immunoreactivity comigrated with the free acid form of NPY1 16.This material did not react with MEDIUM the amide-specific assay and could correspond to the glycine- 200 extended biosynthetic intermediate form or a degradation prod uct lacking the amide function (Fig. 3). All cell lines containing NPY also secreted the peptide in 100 proportional amounts to the cellular content. Chromatographie analysis of the NPY-immunoreactive material in media from the cell lines indicated that both pro-NPY-like and NPY-like material were present, as shown in Fig. 4 for NS-20Y cells. 0.2 O.4 O.6 0.8 1.O However, the smaller molecular weight form of NPY immu of dis K j noreactivity eluted slightly earlier than standard, synthetic Fig. 4. Sephadex G-50 gel filtration of NPY immunoreactivity secreted from murine neuroblastoma cells, NS-20Y (lop), detected by radioimmunoassay using NPY, and subsequent HPLC and NEPHG gel analysis of this the 337 antiserum. Bottom, gel filtration profile of 24-h medium collected from material collected from several cell lines revealed a complex NS-20Y cells and precipitated with acetone prior to analysis. 7017 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1989 American Association for Cancer Research. NEUROPEPTIDE Y IN NEUROENDOCRINE CELL LINES

100-, In the present investigation, the NPY produced by four of six neuroblastoma cell lines was almost totally of the amidated form as characterized by both Chromatographie and immuno- logical techniques. Thus, compared to the dibasic cleavage mechanism, the amidation of NPY was more efficient in the cell lines investigated and similar to that observed in the tumors (Fig. 5). It is surprising that the amidation activity, which is known to require certain cofactors (24) and which rapidly becomes impaired in primary cell cultures (25, 26). is well preserved in the cell lines. Conversely, the dibasic processing, Dibasic which is stable in primary cell cultures (13), is impaired in these processing Amidation cell lines (Fig. 5). Fig. 5. Summary of precursor processing and amidation of NPY in pheo- The neuroblastoma cells are potential tools in the character chromocylomas (•).neuroblastomas (O). and neuroendocrine cell lines. Bar. medians. ization of the biochemical activation mechanisms for regulatory peptides, as they can be manipulated to change their differen pattern indicating degradation of the released material (data tiation status (15, 20, 27). Since several of the cell lines both not shown). Thus the NPY found in the tissue culture media express NPY receptors (9, 11) and NPY itself, the possible was not further characterized in the present investigation. autocrine function of NPY in these cells lines and tumors should also be studied.

DISCUSSION ACKNOWLEDGMENTS In the present study a number of neuroendocrine cell lines have been screened for NPY production. The rat pheochro- Henny Jensen is thanked for excellent secretarial assistance, and Lone Bredo Pedersen, Lone Therkelsen. Margit Trelborg S0rensen, mocytoma cell line PC 12. the murine neuroblastoma cell line and Tina Jakobsen are thanked for tissue culture assistance. NS-20Y, and a number of human neuroblastoma cell lines synthesized the precursor for NPY. As shown in Fig. 5, proc REFERENCES essing at the dibasic cleavage site of the precursor was partly impaired in the cell lines as compared to pheochromocytomas 1. O'Hare, M. M. T., Tenmoku, S., Aakerlund. L.. Hilsted. L.. Johnsen, A., and neuroblastomas, whereas the cell lines amidated the NPY and Schwartz. T. VV.Neuropeplidc Y in guinea pig. rabbit, rat and man. Identical amino acid sequence and oxidation of methionine-17. Regul. Pep- as efficiently as the tumors. Some of the cell lines investigated tides. 20:293-304. 1988. produced NPY only sporadically, if at all. These included 2. Tatemólo. K.. Carlquist. M., and Mutt, V. Neuropeptide Y—anovel brain N18TG-2, SMS-MSN, CHP-212, SMS-KAN, Lal-5S, SH-EP peptide with structural similarities to peptide YY and pancreatic polypeplide. Nature (Lond.). 296: 659-660, 1982. cell lines and the cholinergic type human neuroblastoma cell 3. Adrian. T. E.. Allen. J. M., Bloom. S. R., Ghatei. M. A., Rossor, M. N., lines SK-N-MC and its subclone MC-IXC (14). Roberts. G. W., Crow. T. J., Tatemólo. K., and Polak. J. M. Neuropeptide Y distribution in . Nature (Lond.), 306: 584-586. 1983. NPY comprises approximately the amino-terminal half of its 4. Polak, J. M.. and Bloom, S. R. Regulatory peptides—the distribution of two propeptide and it is excised from this by processing at a com newly discovered peptides: PHI and NPY. Peptides. 5: 79-89. 1984. bined dibasic cleavage and amidation site (23). In normal tissue 5. Mutt. V'..HOkfelt. T.. Fuxe. K. and Lundberg, J. M. (eds.). Nobel Symposium XIV: Neuropeptide Y. New York: Raven Press. 1989. (10) and in tumor cells ( 12), only small quantities of the proform 6. \Vahlestcdt, C., Edvinsson, L.. Ekblad. E., and Hakanson, R. Neuropeptide are present, whereas in the PC 12 cells and the neuroblastoma Y potentiates noradrenaline-evoked vasoconstriction: mode of action. J. cell lines, the unprocessed pro-NPY accounted for 33 to 72% Pharmacol. Exp. Ther.. 234: 735-741. 1985. 7. Ekblad. E.. Edvinsson. L., Wahlestedt. C. Uddman. R.. Hakanson. R.. and of the total NPY immunoreactivity extracted. In one cell line, Sundler, F. Neuropeptide Y'co-exists and co-operates with noradrenaline in NS-20Y, intermediate molecular weight forms between pro- perivascular fibers. Regul. Peptides, 8: 225-235, 1984. 8. Unden, A., Tatemóte. K., Mutt. V.. and Bartfai. T. Neuropeptide Y'receptor NPY and NPY were reproducibly detected (Fig. 4), indicating in the rat brain. Eur. J. 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7019 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1989 American Association for Cancer Research. Expression and Precursor Processing of Neuropeptide Y in Human and Murine Neuroblastoma and Pheochromocytoma Cell Lines

Mairead M. T. O'Hare and Thue W. Schwartz

Cancer Res 1989;49:7015-7019.

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