Levels and molecular forms of chromogranins in human childhood neuroblastomas and ganglioneuromas

Neuroscience Letters 253 (1998) 17–20

Levels and molecular forms of chromogranins in human childhood neuroblastomas and ganglioneuromas Ursula Eder a, Reiner Fischer-Colbrie a ,*, Per Kogner b , c, Bernd Leitner a, Per Bjellerup b, Hans Winkler a a

Department of Pharmacology, University of Innsbruck, Peter-Mayr-Str. 1a, A-6020 Innsbruck, Austria b Department of Clinical Chemistry, Stockholm, Sweden c Paedriatrics, Karolinska Hospital, Stockholm, Sweden Received 8 June 1998; received in revised form 13 July 1998; accepted 15 July 1998

Abstract The chromogranins are a class of acidic proteins found in large secretory granules of neuroendocrine tissues and tumors derived from them. We measured the relative amounts and characterized the molecular forms of two members of this family, i.e. chromogranin A and secretogranin II, in 14 neuroblastomas and five ganglioneuromas. In all the tumors investigated significant amounts of chromogranin A and secretogranin II were found. Neuroblastomas contained two times and ganglioneuromas 45 times more secretogranin II compared to chromogranin A. Both proteins were processed in these tumors to a great extent to smaller peptides, only limited amounts of intact chromogranin A or secretogranin II were present. In general, proteolytic processing of secretogranin II to the small neuropeptide secretoneurin was more complete than that of chromogranin A to the peptide GE-25. Proteolytic processing of both chromogranins as well as the total amounts of these proteins were unrelated to tumor staging.  1998 Elsevier Science Ireland Ltd. All rights reserved

Keywords: Chromogranin A; Secretogranin II; Tumor; Neuropeptide; Proteolytic processing

Chromogranin A and secretogranin II are two acidic proteins belonging to the class of chromogranins [6,23] which are stored in the content of secretory vesicles. The chromogranins are expressed in many tissues of the neuronal and endocrine system which has made them very useful markers to identify elements of the diffuse neuroendocrine system. Thus chromogranin A is one of the most specific histochemical markers for neoplasms containing neuroendocrine secretory vesicles [4,17]. The presence of chromogranin A in peripheral neuroblastic tumors like neuroblastoma, ganglioneuroma and pheochromocytoma is well established by immunochemical and immunohistochemical data [2,8,12, 16,18–20]. For chromogranin A elevated serum levels have been reported in patients suffering from pheochromocytoma and neuroblastoma [8,9,12]. During recent years it became clear that factors secreted from neuroendocrine * Corresponding author. Tel.: +43 512 5073720; fax: +43 512 5072868; e-mail: [email protected]

cells can stimulate proliferation of tumors [1,5]. It has been speculated that chromogranins or smaller peptides derived from them might contribute to this process [1,5,13]. We therefore asked the following questions: (i) what is the concentration of chromogranin A and secretogranin II in peripheral neuroblastic tumors? (ii) Are the relative concentrations of the chromogranins different in neuroblastomas with favorable versus advanced stage? (iii) Are chromogranins processed proteolytically to smaller peptides in these tumors and does this correlate with tumor stage? In total 21 patients were included in the study, 14 children with neuroblastoma, five children with ganglioneuroma and two adults with pheochromocytoma. All children were diagnosed and staged according to the International Neuroblastoma Staging System (INSS) criteria [3]. Fresh tumor tissue was obtained at surgery, quick frozen on dry ice or liquid nitrogen and stored at −70°C. Tumor tissue was cut in frozen state, boiled in 10 volumes of 1 M acetic acid homo-

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00588- 6

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Table 1 Levels of chromogranins in neuroblastomas, ganglioneuromas and pheochromocytomas

Neuroblastomas stage 1 + 2 stage 4 + 4s Ganglioneuromas Pheochromocytomas

GE-25-IR (pmol/g)

Secretoneurin-IR (pmol/g)

Ratio

50.2 ± 6.6 (6) 54.6 (33.1,67.2) 48.0 ± 16.6 (8) 23.9 (7.3,88.7) 1.0 ± 0.4 (3) 0.7 (0,2.9) 54,320 (2)

116.1 ± 28.2 (6) 106.1 (43.6,188.6) 94.2 ± 31.6 (7) 33.8 (16.9,171.5) 45.5 ± 27.9 (5) 19.8 (2,122.8) 6,620 (2)

0.43 0.51 0.022 8.2

The concentration of GE-25 and SN-IR in tumor extracts was measured with specific RIAs. The values in the upper line represent the mean ± SEM. The number of experiments is given in parenthesis. The lower line indicates the median, the lower and upper 95% confidential intervals are given in parenthesis.

genized, and centrifuged. The remaining pellet was extracted with the same volume boiling water. Acid and neutral supernatants were lyophilized and stored at −70°C until analysis. Tumor extracts (up to 300 fmol of GE-25immunoreactivity (GE-25-IR) or secretoneurin-immunoreactivity (SN-IR) were applied to a Superose 12 HR 10/30 gel-filtration column (Pharmacia LKB, Sweden) in a volume of 200 ml. The proteins were eluted at a flow rate of 0.4 ml/min using a 37.5 mmol/l sodium phosphate buffer pH 7.4 containing 37.5 mmol/l NaCl as solvent. The void volume of the column was 8 ml and the total volume 24 ml. One minute fractions were collected and analyzed by radioimmunoassay (RIA) as described previously [10,11]. The recovery of the proteins separated was 70% for this column. The levels of GE-25-IR and SN-IR in peripheral neuroblastic tumors as determined by RIA are given in Table 1. Both, neuroblastomas and ganglioneuromas contained significant amounts of chromogranin A (GE-25)-IR and SNIR. There was no difference in IR between favorable (stage 1 and 2) and more advanced (stage 4 and 4s) neuroblastomas (Table 1). The relative concentration of GE-25 versus SN-IR in these tumors was quite different. Neuroblastomas expressed 2.3, however, ganglioneuromas 45 times more SN-IR versus chromogranin A-IR (Table 1). In extracts from two pheochromocytoma, shown for comparison, 8 times more GE-25 IR compared to SN-IR was present. Furthermore, pheochromocytomas contained 1000 times more GE-25-IR and 50 times more SN-IR when compared with neuroblastomas. The value of 54 nmol GE-25-IR found in this study agrees quite well with that of 80nmol/g wet weight chromogranin A-IR reported previously [8]. It is interesting to note that differentiation of SY5Y neuroblastoma cells to a more neuronal phenotype, as induced by a phorbol ester, increased secretogranin II mRNA [22]. Also, differentiation of PC12 tumor cells by NGF yielded elevated secretogranin II mRNA levels [14]. In vivo, secretogranin II is relatively more enriched in the nervous system when compared to chromogranin A [7,10,21]. Thus, the increasing relative amounts of secretogranin II versus chromogranin A from pheochromocytoma to neuroblastoma and to ganglioneuroma are compatible with the more neuronal

phenotype of ganglioneuromas versus neuroblastomas and both of them versus pheochromocytomas. Therefore, secretogranin II or the peptide secretoneurin are more sensitive markers for neuroblastomas and especially ganglioneuromas than chromogranin A. The absolute amounts of chromogranins present in neuroblastoma varied significantly between the individual tumor specimens. These variations result probably from different extents of necrotic areas or altered gene transcription in the individual tumors. There was no correlation of chromogranin levels with tumor staging. In a previous study increased secretogranin II mRNA levels correlated with a

Fig. 1. Molecular forms of secretoneurin-IR (SN-IR) in tumor samples. Extracts from neuroblastoma (NB) and ganglioneuroma (GN) were subjected to gel-filtration HPLC. The fractions eluted were analyzed by a RIA specific for the secretoneurin sequence. The elution volumes of secretogranin II (SgII) and the free peptide secretoneurin (SN) derived from the secretogranin II precursor are indicated by arrows. Two representative tumor extracts demonstrating lower (NB-1) or more extensive (NB-2) proteolytic processing are presented.

U. Eder et al. / Neuroscience Letters 253 (1998) 17–20

Fig. 2. Molecular forms of GE-25-IR in tumor samples. Extracts from neuroblastomas (NB) were subjected to gel filtration HPLC. The fractions eluted were analyzed by a RIA specific for the GE-25 sequence. The elution volumes of chromogranin A (CgA) and the free peptide GE-25 derived from the chromogranin A precursor are indicated by arrows. Two representative tumor extracts demonstrating lower (NB-1) or more extensive (NB-2) proteolytic processing are presented.

better outcome [16]. In our study, however, tumors from the advanced stages 4 and 4s displayed similar amounts of chromogranin A and secretogranin II-IR as tumors from stages 1 and 2. The proteolytic processing of chromogranins in these tumors was investigated by gel-filtration chromatography followed by radioimmunoassay (RIA; Figs. 1 and 2). Processing of both chromogranins varied to some extent between tumor specimens. In general, proteolytic processing of secretogranin II in neuroblastomas was more pronounced than that of chromogranin A. In all tumors investigated the small peptide secretoneurin was formed (Fig. 1), two out of six tumors contained in addition also the precursor secretogranin II (Fig. 1, NB-2). In ganglioneuromas secretogranin II was completely processed to SN (Fig. 1). For chromogranin A proteolytic processing was less complete, the majority of IR in neuroblastomas corresponded to intermediate-sized proteins (Fig. 2). Only small amounts of intact chromogranin A and of the peptide GE-25 were detected. In two out of five tumors processing to the peptide GE-25 was more advanced (Fig. 2, NB-2). In vivo, proteolytic processing of chromogranins varies between tissues [10,11,15]. In the nervous system secretogranin II is completely processed to the small peptide secretoneurin, whereas in the adrenal medulla large amounts of intact secretogranin II as well as intermediate-sized forms are present [10]. For chromogranin A processing in the adrenal medulla is also limited, but also in brain processing is not complete with significant amounts of intermediate peptides being present [11]. In pheochromocytomas in previous studies very limited processing with large amounts of

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unprocessed chromogranin A and secretogranin II were noted [8,20]. Thus, the degree of processing of chromogranin A and secretogranin II in the tumors of neuronal origin is comparable to that found in normal tissue. Since six different tumors were analyzed here, it is likely that these results are quite representative. Differences in proteolytic processing of chromogranins were unrelated to different stages of neuroblastomas. In this context it is interesting to note that in a previous study serum levels of chromogranin A-IR increased with advanced tumor stages [9] whereas those of pancreastatin, a proteolytic fragment derived from chromogranin A, were decreased in more advanced tumor stages [12]. One likely explanation for this discrepancy might be reduced proteolytic processing of chromogranin A to pancreastatin in advanced neuroblastomas. Our results, however, provided no evidence for such an hypothesis. Future studies have to demonstrate whether any of these chromogranin-derived peptides generated in neuroblastic tumors might have a trophic activity altering tumor proliferation. This work was supported by the Fonds zur Fo¨rderung der wissenschaftlichen Forschung, Austria (SFB 002-F206A) and Dr. Legerlotz Stiftung. [1] Abrahamsson, P.A. and Di Sant’Agnese, P.A., Neuroendocrine cells in the human prostate gland, J. Androl., 14 (1993) 307– 309. [2] Boomsma, F., Bhaggoe, U.M., Man in ’t Veld, A.J. and Schalekamp, M.A.D.H., Sensitivity and specificity of a new ELISA method for determination of chromogranin A in the diagnosis of pheochromocytoma and neuroblastoma, Clin. Chim. Acta, 239 (1995) 57–63. [3] Brodeur, G.M., Pritchard, J., Berthold, F., Carlsen, N.L., Castel, V., Castelberry, R.P., De Bernardi, B., Evans, A.E., Favrot, M., Hedborg, F., Kaneko, M., Kemshead, J., Lampert, F., Lee, R.E.J., Look, A.T., Pearson, A.D.J., Philip, T., Roald, B., Sawada, T., Seeger, R.C., Tsuchida, Y. and Voute, P.A., Revisions of the international criteria for neuroblastoma diagnosis, staging and response to treatment, J. Clin. Oncol., 11 (1993) 1466–1477. [4] Deftos, L.J., Chromogranin A: its role in endocrine function and as an endocrine and neuroendocrine tumor marker, Endocrine Reviews, 12 (1991) 181–187. [5] Di Sant’Agnese, P.A., Neuroendocrine differentiation in carcinoma of the prostate. Diagnostic, prognostic, and therapeutic implications, Cancer, 70 (1992) 254–268. [6] Fischer-Colbrie, R., Laslop, A. and Kirchmair, R., Secretogranin II: molecular properties, regulation of biosynthesis and processing to the neuropeptide secretoneurin, Prog. Neurobiol., 46 (1995) 49–70. [7] Hagn, C., Klein, R.L., Fischer-Colbrie, R., Douglas II, B.H. and Winkler, H., An immunological characterization of five common antigens of chromaffin granules and of large dense-cored vesicles of sympathetic nerve, Neurosci. Lett., 67 (1986) 295– 300. [8] Hsiao, R.J., Parmer, R.J., Takiyyuddin, M.A. and O’Connor, D.T., Chromogranin A storage and secretion: sensitivity and specificity for the diagnosis of pheochromocytoma, Medicine, 70 (1991) 33–45. [9] Hsiao, R.J., Seeger, R.C., Yu, A.L. and O’Connor, D.T., Chro-

20

[10]

[11]

[12]

[13]

[14]

[15]

[16]

U. Eder et al. / Neuroscience Letters 253 (1998) 17–20 mogranin A in children with neuroblastoma. Serum concentration parallels disease stage and predicts survival, J. Clin. Invest., 85 (1990) 1555–1559. Kirchmair, R., Hogue-Angeletti, R., Gutierrez, J., FischerColbrie, R. and Winkler, H., Secretoneurin - a neuropeptide generated in brain, adrenal medulla and other endocrine tissues by proteolytic processing of secretogranin II (chromogranin C), Neuroscience, 53 (1993) 359–365. Kirchmair, R., Leitner, B., Fischer-Colbrie, R., Marksteiner, J., Hogue-Angeletti, R. and Winkler, H., Large variations in the proteolytic formation of a chromogranin A-derived peptide (GE-25) in neuroendocrine tissues, Biochem. J., 310 (1995) 331–336. Kogner, P., Bjellerup, P., Svensson, T. and Theodorsson, E., Pancreastatin immunoreactivity in favourable childhood neuroblastoma and ganglioneuroma, Eur. J. Cancer, 31A (1995) 557–560. Lachmund, A., Gehrke, D., Krieglstein, K. and Unsicker, K., Trophic factors from chromaffin granules promote survival of peripheral and central nervous system neurons, Neuroscience, 62 (1994) 361–370. Laslop, A. and Tschernitz, C., Effects of nerve growth factor on the biosynthesis of chromogranin A and B, secretogranin II and carboxypeptidase H in rat PC12 cells, Neuroscience, 49 (1992) 443–450. Leitner, B., Fischer-Colbrie, R., Scherzer, G. and Winkler, H., Secretogranin II: relative amounts and processing to secretoneurin in various rat tissues, J. Neurochem., 66 (1996) 1312– 1317. Pagani, A., Forni, M., Tonini, G.P., Papotti, M. and Bussolati, G., Expression of members of the chromogranin family in pri-

[17]

[18]

[19]

[20]

[21]

[22]

[23]

mary neuroblastomas, Diagn. Mol. Pathol., 1 (1992) 16– 24. Rosa, P. and Gerdes, H.H., The granin protein family: markers for neuroendocrine cells and tools for the diagnosis of neuroendocrine tumors, J. Endocrinol. Invest., 17 (1994) 207–225. Schmid, K.W., Dockhorn-Dworniczak, B., Fahrenkamp, A., Kirchmair, R., To¨tsch, M., Fischer-Colbrie, R., Bo¨cker, W. and Winkler, H., Chromogranin A, secretogranin II and vasoactive intestinal peptide in phaeochromocytomas and ganglioneuromas, Histopathology, 22 (1993) 527–533. Schmid, K.W., Schro¨der, S., Dockhorn-Dworniczak, B., Kirchmair, R., To¨tsch, M., Bo¨cker, W. and Fischer-Colbrie, R., Immunohistochemical demonstration of chromogranin A, chromogranin B, and secretogranin II in extra-adrenal paragangliomas, Modern Pathol., 7 (1994) 347–353. Schober, M., Fischer-Colbrie, R., Schmid, K.W., Bussolati, G., O’Connor, D.T. and Winkler, H., Comparison of chromogranins A, B, and secretogranin II in human adrenal medulla and pheochromocytoma, Lab. Invest., 57 (1987) 385–391. Weiler, R., Marksteiner, J., Bellmann, R., Wohlfarter, T., Schober, M., Fischer-Colbrie, R., Sperk, G. and Winkler, H., Chromogranins in rat brain: characterization, topographical distribution and regulation of synthesis, Brain Res., 532 (1990) 87–94. Weiler, R., Meyerson, G., Fischer-Colbrie, R., Laslop, A., Pa˚hlman, S., Floor, E. and Winkler, H., Divergent changes of chromogranin A/secretogranin II levels in differentiating human neuroblastoma cells, FEBS Lett., 265 (1990) 27–29. Winkler, H. and Fischer-Colbrie, R., The chromogranins A and B: the first 25 years and future perspectives, Neuroscience, 49 (1992) 497–528.