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Interleukin-6 and corticotrophin-releasing hormone mRNA are modulated during differentiation of human neuroblastoma cells
Neuropeptides (1992) 23,45+9 0 Longman Group UK Ltd 1992
Interleukin-6 and Corticotrophin-releasing Hormone mRNA are Modulated During Differentiation of Human Neuroblastoma Cells A. STEPHANOU”, G. MELINOt, R. A. KNIGHT*, M. ANNICCHIARICO-PETRUZZELLlt, N. J. SARLIS*, A. FINAZZI-AGRO’t and S. L. LIGHTMAN” *Department of Neuroendocrinology, Charing Cross and Westminster Medical School, Charing Cross Hospital, London, W6 8RF, UK, tDepartment of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Rome, 00173, Italy, #Department of Cystic Fibrosis, Brompton Hospital, National Heart and Lung Institute, London, SW3 SLY, UK (Reprint requests to AS) Abstract-Two cell clones [BE(2)-C and BE(2)-Ml71 derived from the human neuroblastoma cell line SK-N-BE(2) express corticotrophin-releasing hormone as well as interleukind mRNA. Both genes are overexpressed, although with a different time course, following exposure to 5 PM retinoic acid, in parallel to the induction of neuroblastic differentiation. On the contrary, we are unable to detect interleukin-1P mRNA in these cell lines. Both cytokines are known to increase hypothalamic CRH mRNA. The production of cytokines and neuropeptides by neuroblastoma cells indicate a complex dialogue between tumour cells and anti-tumour immunity.
Introduction
retinoic acid (RA) (1, 2). The N-myc oncogene, which is amplified and overexpressed in undifferNeuroblastomas (NB) are catecholamine-producing entiated NB cells, shows reduced expression in RAtumours of neuroectodermal (neural-crest) origin, treated cells (4). presenting mainly in childhood. Several NB cell NB cell lines are known to express various neulines have been described, some of which can be ropeptides. Some of these may act as tumour growth induced to differentiate towards a neuroblastic pheregulators in an autocrine/paracrine fashion. Thus notype (l-3). For example, the NB line, SK-Nthe NB cell line, SK-N-AS, both express InsulinBE(2) and 2 clones derived from it, BE(2)C and like Growth Factor-II (IGF-II) and Type I IGF BE(2)-M17, show growth cone development and receptors, and its autonomous growth in vitro is neurite outgrowth, with increased expression of ‘yblocked by antibodies to the Type I IGF receptor (5, aminobutyric acid (GABA) after treatment with 6). Pro-opiomelanocortin (POMC) gene expression has also been described inNB cell lines (7), and since NB cells both express receptors for endogenous opiDate received 27 February 1992 ates, such as j%endorphin produced from the POMC Date accepted 5 May 1992 45
46 gene, and show altered growth characteristics both in vivo (8) and in vitro (9) in the presence of opiates antagonists, POMC products have also been proposed as NB autocrine/paracrine growth factors. Several neuropeptides produced by NB cells, including Corticotropin-Releasing Hormone (CRH) (lo), GrowthHormone-Releasing Hormone (GHRH) (11) and Neuropeptide Y (NPY) (12), as well as POMC gene products (7), have immunoregulatory properties in vitro. Since NB is frequently associated with an inflammatory infiltrate in vivo, and the rare cases of spontaneous regression of NB are thought to be immunologically mediated (13), it is possible that neuropeptides released from the tumour cells may exert paracrine effects on tumour infiltrating inflammatory cells. In contrast with the reports on neuropeptide production by tumour cells, there is little data on the more conventional cytokines by neural crest-derived turnours. It has recently been shown, that melanoma cells, which are also of neural crest-origin, produce Interleukin-1 (IL-l), which in turn induces the expression of the metastasis-associated marker, Intercellular Adhesion Molecule- 1 (ICAM- I), and thereby increases the metastatic potential of the cells (14). Futhermore, a number of glioblastoma cell lines have been shown to express IL-6, and IL-6 expression can be further enhanced by treatment with IL- 1 (15). Recently, a human phaeochromocytoma, another neural crest-derived tumour, has been shown to contain immunohistochemically detectable IL-6 peptide, which was associated with a paraneoplastic syndrome (16). Other early work, in the classical neuroendocrine and the immune system, is beginning to show a relationship between interleukins and neuropeptides. Both IL-l and IL-6 stimulate CRH release from hypothalamic neurons (17, 18) and IL1 and IL-2 increase POMC expression in pituitary and in lymphocytes (19). In the present study, we show that RA treatment ofNB cells rapidly enhances the expression of IL-6 mRNA and we attempt to relate this temporally to the expression of CRH mRNA, that also accompanies differentiation.
Materials and Methods All reagents for cell culture and differentiation, i.e. plastics, tissue culture media, trypsin, EDTA, 4-(2-
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hydroxyethyl)- 1-piperazine ethanesulfonic acid (Hepes), L-glutamine, sodium bicarbonate, phosphate buffered saline (PBS), foetal calf serum (FCS) and non-essential aminoacids were purchased from Flow Laboratories Ltd. (Her& UK). All-trans retinoic acid, glyoxal thiocyanate, agarose and RNAase were obtained from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). GeneScreen Plus membranes were from DuPont. (Richmond, CA, USA). XARX-ray film was purchased from Kodak Co. Ltd. (USA) and 32Pwas from Amersham (Berks, UK). Cell culture SK-N-BE(2) cells and their clones, BE(2)-C and BE(2)-M17, were generously provided by Dr June Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). All experiments were performed using cells after 60-66, 29-32 and 21-25 passages respectively. Cells were grown in monolayer cultures in a 1: 1 mixture of MEM and Hams F-12 media supplemented with 15% heat-inactivated FCS, sodium bicarbonate (1.2 mgml), Hepes buffer (15 n&I), L-glutamine (2 mM) and nonessential aminoacids (1% v/v). No antibiotics were used in the media. Cell lines were routinely screened for Mycoplasma infection. Cells were fed every 3-4 d, detached using trypsin/EDTA (0.025% : 0.02%) and were diluted between Vsand Vi0every week. Cells were routinely fed 24 h before harvest for experiments. In the differentiation experiments, 5000 to 10 000 cells per cm-2 were treated with medium containing 5 pM retinoic acid (5 mM stock solution in 70% absolute ethanol). 0.07% ethanol was added to control cultures. Fresh RA-containing or control medium was replaced daily. Cells were also grown for 24 h in the absence of serum towards confluency. This treatment leads to growth arrest that excludes any ambiguity of a possible modulation of RNA production due to changes in the cell cycle phase. Northern blot analysis For Northern analysis, total mRNA was prepared using the glyoxalthiocyanate method and Northern blotting was performed, loading 20 pg of total RNA per lane, as described previously (20), using GeneScreen Plus membranes. Blots were hybridised with nick-translated 32P-labelled cDNA probes for
CRH AND INTERLEUKIN-6
mRNA IN NEUROBLASTOMA
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47
BE
c
cf
4h
d#
e
Fig. 1 Northern blot analysis oftotal RNA extracted from BE(Z)-Ml7 (a), BE(Z)-C @) clones hybridised with IL-6 and CRH probes are shown under the headings M 17 and 2C respectively. Northern blot analysis of total RNA extracted from the parental line SK-NBE(2) hybridised with the IL-6 probe is designated as BE (c). Cells were treated with 5 pM RA and RNA was extracted at the time points shown (h: hours and d: days). The 18s rRNA intensity by ethidium bromide staining was used to evaluate the amount of RNA loaded on the gel. The cf lane indicates conthtent cells grown for 24 h in the absence of serum. The sizes of the CRH mRNA corresponds to approximately 1.4 Kb and the IL-6 mRNA 1.3 Kb.
48 human IL- 1p, IL-6 (both provided by the American Tissue Type Culture Collection, Rockville, MD, USA) and also with a oligonucleotide probe directed towards the exonic regions of the human CRH gene.
Results Very little expression of CRH mRNA was detected in the absence of R4 in all three NB cell lines. Following RA treatment, there was a dramatic increase in CRH mRNA accumulation in the clone BE(2)-Ml7 by day 3 of the culture, which was further increased on day 5 (Fig. la). However, in the clone BE(2)-C only a very slight increase in CRH mRNA accumulation was observed on days 3 and 5 post-R4 (Fig. lb). In the parent cell line, we were unable to detect CRH mRNA expression in the presence or absence of RA. (Fig. lc). No IL- 1p mRNA was detected in either parental or cloned cells either in the presence or absence of RA. In contrast, IL-6 mRNA was present in untreated cultures of both parental and cloned cells, with a stronger signal from the uncloned parental line (Fig. 1c). Following addition of RA to the cloned cells, the amount of IL-6 mRNA increased by 4 h and 8 h, declined towards control levels by day 1 and thereafter increased again by days 3 and 5 (Fig. la & Fig. lb). These changes were more marked with clone BE(2)-Ml7 than with clone BE(2)-C. Accumulation of IL-6 mRNA was slightly increased after day 1 to day 5 post&A treatment in parental cells.
Discussion The major finding of this study is that human neuroblastoma cell lines express IL-6 mRNA and that IL-6 mRNA accumulation is influenced by the induction of differentiation with R4. No IL-l p mRNA was detected in parental or cloned cells, irrespective of the presence or absence of RA. This is consistent with data presented by others showing that IL-l is rarely produced by tumours other than melanomas (21). On the other hand, CRH mRNA is undetectable in the absence of R4, while after R4-induced differentiation CRH mRNA became detectable by day
NEUROPEPTIDES
3 and reached a maximum on day 5, though only in the cells of clone BE(2)-M17. Clone BE(2)-C cells showed very little induction of CRH mRNA following RA treatment, despite marked morphological differentiation. Some of the interleukins are already known to be synthesized in the neuroendocrine system, as well as in leukocytes, and to influence expression of hypothalamic and pituitary peptides (22, 23). For example, both IL-l and IL-6 are present in the pituitary (24-26) and can increase hypothalamic expression of CRH mRNA (27). In the periphery, IL-l is a potent inducer of IL-6, although it is not known whether this also occurs in the pituitary (28). In the present study, although CRH mRNA accumulation follows that of IL-6, particularly in clone BE(2)M17, more evidence is needed before concluding that there is a direct effect of IL-6 on CRH in NB. In particular, it would be of interest to know whether inflammatory-derived IL- 1 enhances IL-6 expression in NB, as has been shown in glioblastoma (15). Using the same NB cell lines we have recently demonstrated the modulation of another hypothalamic releasing hormone, growth hormone-releasing hormone (GHRH). During RA-induced differentiation we again observed an increase in GHRH mRNA expression; we have also demonstrated that IGF-II mRNA levels are increased post&A (3). This is consistent with another finding in the human NB cell line SK-N-AS, which constituently expresses IGFII mRNA and is secreting IGF-II peptide that functions as paracrine/autocrine growth factor for this cell line (5,6). The data reported here and elsewhere show that NB cells are capable of expressing both cytokines and neuropeptides. The production of similar peptides by tumours and by the inflammatory cells that may infiltrate them suggests that a complex molecular dialogue exists between tumour cells and antitumour immunity. Thus, interleukins and neuropqtides elaborated by NB cells may not only influence the growth and differentiation of the tumour but also instruct the local immunological response. Correspondingly, products of the immune reaction may affect growth and differentiation of the NB in addition to their effects on the immune response itself. A complete understanding of this dialogue, and its therapeutic manipulation, is one of the most important tasks in neuroimmunology.
CRH AND INTERLEUKIN-6 mRNA IN NEUROBLASTOMA
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Acknowledgements Thiswork was partially supported by the CNR grant ‘Progetto Finalizzato Applicazioni Cliniche della Ricerca Gncologica’. We are also grateful for financial support from the Joint Research Committee of the Westminster Hospital Group. Dr N. J. Sarlis is supported by the Scholarship Committee of the Rotary Club of Athens (Rotary Foundation).
References 1. Melino, G., Farrace, M. G., Ceru’, M. P. and Piacentini, M. (1988). Correlation between transglutaminase activity and polyamine levels in retinoic acid and a-difluoromethylornithine induced differentiation of human neuroblastoma cells. Exp. Cell Res. 179: 429-445. 2. Melino, G., Piacentini, M., Pate], K., AnnicchiaricoPetruzzelli, M., Piredda, L. and Kemshead, J. T. (1991). Retinoic acid and a-difluoromethylomithine induce different expression of neural-specific cell adhesion molecules in differentiating neuroblastoma cells. Progr. Clin. Biol. Res. 366: 283-292. 3. Melino, G., Stephanou, A., Amiicchiarico-Petruzzelli, M., Knight, R. A., Biedler, J., Finazzi-Agro’, A. and Lightman, S. L. (1991). Modulation of GHRH and IGF-II expression in growth and differentiation of human neuroblastoma. Exp. Cell. Res. Submitted. 4. Ghibelli, L., Farrance, M. G., Guerrieri, P., Melino, G. and Piacentini, M. (1990). DMFO and retinoic acid induce two different types of morphological changes in a human neuroblastoma cell line. Clin. Chem. Enzym. Comms. 2: 250-256. 5. El-Badry, O., Helman, L. J., Chatten, J., Steinberg, S. M., Evans, A. E. and Israel, M. A. (1991). Insulin-like growth factor II-mediated proliferation of human neuroblastoma. J. Clin. Invest. 87: 648-657. 6 El-Badry, O., Romanus, J. A., Helman, L. J., Cooper, M. J., Rechler, M. and Israel, M. A. (1989). Autonomous growth of a human neuroblastoma cell line is mediated by insulinlike growth factor-II. J. Chn. Invest. 84: 829-839. 7 Stephanou, A., Knight, R. A., De Laurenzi, V., Melino, G. and Lightman, S. L. (1991). Expression of proopiomelanocortin mRNA in undifferentiated and in vitro differentiated human neuroblastoma cell lines. Adv. Neuroblastoma Res., Volume 3. Wiley-Liss Inc., New York, pp. 173-180. 8. McLaughlin, P. J. and Zargon, I. S. (1987). Modulation of human neuroblastoma transplanted into nude mice by endogenous opioid systems. Life Sci. 41: 1465-1472. 9. Yu, V. C., Richards, M. L. and Sadee, W. (1986). A human neuroblastoma cell line expresses u and 8 opioid receptor sites. J. Biol. Chem. 261: 1065-1070. 10. Usui, T., Nakai, Y., Tsukada, T., Jingami, H., Takahashi, H. and Imura, H. (1989). Expression of adrenocorticotropinreleasing hormone precursor gene in placenta and nonhypothalamic tissues in man. Mol. Endocrinol. 2: 871-875. I1 Christofides, N. D., Stephanou, A., Susuki, H., Yiangou, Y. and Bloom, S. R. (1984). Distibution of immunoreactive growth hormone-releasing hormone in the human brain and its production by tumours. J. Clin. Endocr. Metab. 123: 747-752.
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12. Cohen, P. S., Cooper, M. J., Helman, L. J., Thiele, C. J., Seeger, R. C. and Israel, M. A. (1990). Neuropeptide Y expression in the developing adrenal gland and in childhood neuroblastoma tumours. Cancer Res. 50: 6055-606 1. 13. Evans, A. E., Gerson, J. and Schnaufer, L. (1976). Spontaneous regression of neuroblastoma. Natl. Cancer Inst. Monogr. 44: 49-54. 14. Burrows, F. J., Haskard, D. O., Hart, I. R., Marshal, J. F., Selkirk, S., Poole, S. and Thorpe, P. E. (1991). Influence of tumour-derived interleukin 1 on melanoma-endothelial cell interaction in vitro. Cancer Res. 5 1: 4768-4777. 15. Van Meir, E., Sawamura, Y., Diserens, A.-C., Hamou, M.F. and De Tribolet, N. (1990). Human glioblastoma cells release interleukin-6 in vivo and in vitro. Cancer Res. 50: 6683-6688. 16. Fukumoto. S., Matsumoto, T., Harada, S.-I., Fuji&i, J., Kawano, M., Ogata, E. (1991). Pheochromocytoma with pyrexia and marked intlammatory signs: a paraneoplastic syndrome with possible relation to interleukin-6 production. J. Clin. Endocr. Metab. 73: 877-881. 17. Lyson, K., Milenkovic, L. and McCann, S. M. (1992). The stimulatory effect of interleukin-6 on corticotropin releasing factor (CRF) and thyrotropin-releasing factor (TRF) secretion in vitro. Progr. Neuroendocrinimmunol. Submitted. 18. Uehara, A., Gottschall, E., Dahl, R. R. and Arimura, A. (1987). Interleukin-1 stimulates ACTH release by an indirect action which requires endogenous corticotropin releasing factor. Endocrinology 121: 1580-1582. 19. Brown, S. L., Smith, L. R. and Blalock, J. E. (1987). Interleukin- 1 and interleukin-2 enhance POMC gene expression in pituitary cells. J. Immunol. 139: 3181-3183. 20. Thomas, P. (1980). Hybridisation of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77: 5201-5205. 21. Kock, A., Schwartz, A., Urbanski, Z., Peng, U., Vetterlein, M., Ansel, J. C., Kung, H. F. and Lunger, T. A. (1989). Expression and release of interleukin- 1 by different human melanoma cell lines. J. Natl. Cancer Inst. 81: 36-42. 22. Bemton, E. W., Beach, J. E., Holaday, J. W., Smallridge, R. C. and Fein. H. G. (1987). Release of multinle hormones bv direct action of interleukin 1 on pituitary ceils. Science 238: 519-521. 23. Besodovsky, H. and Del Rey, A. (1987). Neuroendocrine and metabolic responses induced by interleukin- 1. J. Neurosci. Res. 18: 172-178. 24. Koenig, J. I., Snow, K., Clark, B. D. et al. (1990). Intrinsic pituitary interleukin- 18 is induced by bacterial lipopolysaccharide. Endocrinology 126: 3053-3058. 25. Spangelo, B. L., MacLeod, R. M. and Isackson, P. C. (1990). Production of interleukind from anterior pituitary cells in vitro. Endocrinology 126: 582-586. 26. Vankelecom, H., C&meliet, P., Van Damme, J., Billiau, A. andDenef. C. (1989). Productionofinterleukin-6 bvfolliculostellate cells of the anterior pituitary gland in a his~otypic cell aggregate culture system. Neuroendocrinology49: 102-106. Suda, T., Tozawa, F., Ushiyama, Z., Sumitomo, T., Yamada, M. and Demura, H. (1990). Interleukin-1 stimulates corticotropin-releasing factor gene in the rat hypothalamus. Endocrinology 162: 1223-1228. 28. Kishimoto, T. (1987). B-cell stimulatory factor (BSFs): Molecular structure, biological structure, and regulation of expression. J. Clin. Immunol. 7: 343-355. L,.
Interleukin-6 and Corticotrophin-releasing Hormone mRNA are Modulated During Differentiation of Human Neuroblastoma Cells A. STEPHANOU”, G. MELINOt, R. A. KNIGHT*, M. ANNICCHIARICO-PETRUZZELLlt, N. J. SARLIS*, A. FINAZZI-AGRO’t and S. L. LIGHTMAN” *Department of Neuroendocrinology, Charing Cross and Westminster Medical School, Charing Cross Hospital, London, W6 8RF, UK, tDepartment of Experimental Medicine and Biochemical Sciences, University of Rome ‘Tor Vergata’, Rome, 00173, Italy, #Department of Cystic Fibrosis, Brompton Hospital, National Heart and Lung Institute, London, SW3 SLY, UK (Reprint requests to AS) Abstract-Two cell clones [BE(2)-C and BE(2)-Ml71 derived from the human neuroblastoma cell line SK-N-BE(2) express corticotrophin-releasing hormone as well as interleukind mRNA. Both genes are overexpressed, although with a different time course, following exposure to 5 PM retinoic acid, in parallel to the induction of neuroblastic differentiation. On the contrary, we are unable to detect interleukin-1P mRNA in these cell lines. Both cytokines are known to increase hypothalamic CRH mRNA. The production of cytokines and neuropeptides by neuroblastoma cells indicate a complex dialogue between tumour cells and anti-tumour immunity.
Introduction
retinoic acid (RA) (1, 2). The N-myc oncogene, which is amplified and overexpressed in undifferNeuroblastomas (NB) are catecholamine-producing entiated NB cells, shows reduced expression in RAtumours of neuroectodermal (neural-crest) origin, treated cells (4). presenting mainly in childhood. Several NB cell NB cell lines are known to express various neulines have been described, some of which can be ropeptides. Some of these may act as tumour growth induced to differentiate towards a neuroblastic pheregulators in an autocrine/paracrine fashion. Thus notype (l-3). For example, the NB line, SK-Nthe NB cell line, SK-N-AS, both express InsulinBE(2) and 2 clones derived from it, BE(2)C and like Growth Factor-II (IGF-II) and Type I IGF BE(2)-M17, show growth cone development and receptors, and its autonomous growth in vitro is neurite outgrowth, with increased expression of ‘yblocked by antibodies to the Type I IGF receptor (5, aminobutyric acid (GABA) after treatment with 6). Pro-opiomelanocortin (POMC) gene expression has also been described inNB cell lines (7), and since NB cells both express receptors for endogenous opiDate received 27 February 1992 ates, such as j%endorphin produced from the POMC Date accepted 5 May 1992 45
46 gene, and show altered growth characteristics both in vivo (8) and in vitro (9) in the presence of opiates antagonists, POMC products have also been proposed as NB autocrine/paracrine growth factors. Several neuropeptides produced by NB cells, including Corticotropin-Releasing Hormone (CRH) (lo), GrowthHormone-Releasing Hormone (GHRH) (11) and Neuropeptide Y (NPY) (12), as well as POMC gene products (7), have immunoregulatory properties in vitro. Since NB is frequently associated with an inflammatory infiltrate in vivo, and the rare cases of spontaneous regression of NB are thought to be immunologically mediated (13), it is possible that neuropeptides released from the tumour cells may exert paracrine effects on tumour infiltrating inflammatory cells. In contrast with the reports on neuropeptide production by tumour cells, there is little data on the more conventional cytokines by neural crest-derived turnours. It has recently been shown, that melanoma cells, which are also of neural crest-origin, produce Interleukin-1 (IL-l), which in turn induces the expression of the metastasis-associated marker, Intercellular Adhesion Molecule- 1 (ICAM- I), and thereby increases the metastatic potential of the cells (14). Futhermore, a number of glioblastoma cell lines have been shown to express IL-6, and IL-6 expression can be further enhanced by treatment with IL- 1 (15). Recently, a human phaeochromocytoma, another neural crest-derived tumour, has been shown to contain immunohistochemically detectable IL-6 peptide, which was associated with a paraneoplastic syndrome (16). Other early work, in the classical neuroendocrine and the immune system, is beginning to show a relationship between interleukins and neuropeptides. Both IL-l and IL-6 stimulate CRH release from hypothalamic neurons (17, 18) and IL1 and IL-2 increase POMC expression in pituitary and in lymphocytes (19). In the present study, we show that RA treatment ofNB cells rapidly enhances the expression of IL-6 mRNA and we attempt to relate this temporally to the expression of CRH mRNA, that also accompanies differentiation.
Materials and Methods All reagents for cell culture and differentiation, i.e. plastics, tissue culture media, trypsin, EDTA, 4-(2-
NEUROPEPTIDES
hydroxyethyl)- 1-piperazine ethanesulfonic acid (Hepes), L-glutamine, sodium bicarbonate, phosphate buffered saline (PBS), foetal calf serum (FCS) and non-essential aminoacids were purchased from Flow Laboratories Ltd. (Her& UK). All-trans retinoic acid, glyoxal thiocyanate, agarose and RNAase were obtained from Sigma Chemical Co. Ltd. (St. Louis, MO, USA). GeneScreen Plus membranes were from DuPont. (Richmond, CA, USA). XARX-ray film was purchased from Kodak Co. Ltd. (USA) and 32Pwas from Amersham (Berks, UK). Cell culture SK-N-BE(2) cells and their clones, BE(2)-C and BE(2)-M17, were generously provided by Dr June Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY, USA). All experiments were performed using cells after 60-66, 29-32 and 21-25 passages respectively. Cells were grown in monolayer cultures in a 1: 1 mixture of MEM and Hams F-12 media supplemented with 15% heat-inactivated FCS, sodium bicarbonate (1.2 mgml), Hepes buffer (15 n&I), L-glutamine (2 mM) and nonessential aminoacids (1% v/v). No antibiotics were used in the media. Cell lines were routinely screened for Mycoplasma infection. Cells were fed every 3-4 d, detached using trypsin/EDTA (0.025% : 0.02%) and were diluted between Vsand Vi0every week. Cells were routinely fed 24 h before harvest for experiments. In the differentiation experiments, 5000 to 10 000 cells per cm-2 were treated with medium containing 5 pM retinoic acid (5 mM stock solution in 70% absolute ethanol). 0.07% ethanol was added to control cultures. Fresh RA-containing or control medium was replaced daily. Cells were also grown for 24 h in the absence of serum towards confluency. This treatment leads to growth arrest that excludes any ambiguity of a possible modulation of RNA production due to changes in the cell cycle phase. Northern blot analysis For Northern analysis, total mRNA was prepared using the glyoxalthiocyanate method and Northern blotting was performed, loading 20 pg of total RNA per lane, as described previously (20), using GeneScreen Plus membranes. Blots were hybridised with nick-translated 32P-labelled cDNA probes for
CRH AND INTERLEUKIN-6
mRNA IN NEUROBLASTOMA
CELL LINES
47
BE
c
cf
4h
d#
e
Fig. 1 Northern blot analysis oftotal RNA extracted from BE(Z)-Ml7 (a), BE(Z)-C @) clones hybridised with IL-6 and CRH probes are shown under the headings M 17 and 2C respectively. Northern blot analysis of total RNA extracted from the parental line SK-NBE(2) hybridised with the IL-6 probe is designated as BE (c). Cells were treated with 5 pM RA and RNA was extracted at the time points shown (h: hours and d: days). The 18s rRNA intensity by ethidium bromide staining was used to evaluate the amount of RNA loaded on the gel. The cf lane indicates conthtent cells grown for 24 h in the absence of serum. The sizes of the CRH mRNA corresponds to approximately 1.4 Kb and the IL-6 mRNA 1.3 Kb.
48 human IL- 1p, IL-6 (both provided by the American Tissue Type Culture Collection, Rockville, MD, USA) and also with a oligonucleotide probe directed towards the exonic regions of the human CRH gene.
Results Very little expression of CRH mRNA was detected in the absence of R4 in all three NB cell lines. Following RA treatment, there was a dramatic increase in CRH mRNA accumulation in the clone BE(2)-Ml7 by day 3 of the culture, which was further increased on day 5 (Fig. la). However, in the clone BE(2)-C only a very slight increase in CRH mRNA accumulation was observed on days 3 and 5 post-R4 (Fig. lb). In the parent cell line, we were unable to detect CRH mRNA expression in the presence or absence of RA. (Fig. lc). No IL- 1p mRNA was detected in either parental or cloned cells either in the presence or absence of RA. In contrast, IL-6 mRNA was present in untreated cultures of both parental and cloned cells, with a stronger signal from the uncloned parental line (Fig. 1c). Following addition of RA to the cloned cells, the amount of IL-6 mRNA increased by 4 h and 8 h, declined towards control levels by day 1 and thereafter increased again by days 3 and 5 (Fig. la & Fig. lb). These changes were more marked with clone BE(2)-Ml7 than with clone BE(2)-C. Accumulation of IL-6 mRNA was slightly increased after day 1 to day 5 post&A treatment in parental cells.
Discussion The major finding of this study is that human neuroblastoma cell lines express IL-6 mRNA and that IL-6 mRNA accumulation is influenced by the induction of differentiation with R4. No IL-l p mRNA was detected in parental or cloned cells, irrespective of the presence or absence of RA. This is consistent with data presented by others showing that IL-l is rarely produced by tumours other than melanomas (21). On the other hand, CRH mRNA is undetectable in the absence of R4, while after R4-induced differentiation CRH mRNA became detectable by day
NEUROPEPTIDES
3 and reached a maximum on day 5, though only in the cells of clone BE(2)-M17. Clone BE(2)-C cells showed very little induction of CRH mRNA following RA treatment, despite marked morphological differentiation. Some of the interleukins are already known to be synthesized in the neuroendocrine system, as well as in leukocytes, and to influence expression of hypothalamic and pituitary peptides (22, 23). For example, both IL-l and IL-6 are present in the pituitary (24-26) and can increase hypothalamic expression of CRH mRNA (27). In the periphery, IL-l is a potent inducer of IL-6, although it is not known whether this also occurs in the pituitary (28). In the present study, although CRH mRNA accumulation follows that of IL-6, particularly in clone BE(2)M17, more evidence is needed before concluding that there is a direct effect of IL-6 on CRH in NB. In particular, it would be of interest to know whether inflammatory-derived IL- 1 enhances IL-6 expression in NB, as has been shown in glioblastoma (15). Using the same NB cell lines we have recently demonstrated the modulation of another hypothalamic releasing hormone, growth hormone-releasing hormone (GHRH). During RA-induced differentiation we again observed an increase in GHRH mRNA expression; we have also demonstrated that IGF-II mRNA levels are increased post&A (3). This is consistent with another finding in the human NB cell line SK-N-AS, which constituently expresses IGFII mRNA and is secreting IGF-II peptide that functions as paracrine/autocrine growth factor for this cell line (5,6). The data reported here and elsewhere show that NB cells are capable of expressing both cytokines and neuropeptides. The production of similar peptides by tumours and by the inflammatory cells that may infiltrate them suggests that a complex molecular dialogue exists between tumour cells and antitumour immunity. Thus, interleukins and neuropqtides elaborated by NB cells may not only influence the growth and differentiation of the tumour but also instruct the local immunological response. Correspondingly, products of the immune reaction may affect growth and differentiation of the NB in addition to their effects on the immune response itself. A complete understanding of this dialogue, and its therapeutic manipulation, is one of the most important tasks in neuroimmunology.
CRH AND INTERLEUKIN-6 mRNA IN NEUROBLASTOMA
CELL LINES
Acknowledgements Thiswork was partially supported by the CNR grant ‘Progetto Finalizzato Applicazioni Cliniche della Ricerca Gncologica’. We are also grateful for financial support from the Joint Research Committee of the Westminster Hospital Group. Dr N. J. Sarlis is supported by the Scholarship Committee of the Rotary Club of Athens (Rotary Foundation).
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