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Identification of 5-HT1A recognition sites in human ganglioneuroblastoma
European Journal of Pharmacology, 170 (1989) 127-128
127
Elsevier EJP 0223R
Rapid communication
Identification of 5-HT~A recognition sites in human ganglioneuroblastoma M i c h a e l R. P r a n z a t e l l i a n d J o a n n e Balletti Departments of Neurology and Pediatrics, Columbia University, 630 West 168th Street, New York, N Y 10032, U.S.A.
Received 28 September 1989, accepted 29 September 1989
5-HydroxytryptaminelA (5-HT1A) receptors have been characterized extensively in the central nervous system in m a n y species, including humans. They exhibit a distinct neuroanatomic distribution, linkage to an adenylate cyclase, and several physiologic and behavioral functions. Neurotransmitter receptors are often also sought on tumors derived from neural crest cells, the embryologic origin of central nervous tissue. However, the use of hybrid tumor-brain cell lines raises the possibility of alteration of receptor properties in tissue culture. Therefore, we used direct surgical specimens from pediatric patients to survey 5-HT 1 receptor subtypes in h u m a n neural crest-derived tumors. We characterized the binding of [3H]8O H - D P A T ([3H]DPAT), a selective 5-HTIA radioligand, in three different tumor types: neuroblastoma, ganglioneuroblastoma and ganglioneuroma. Radioligand binding assays were performed as described by Gozlan et al. (1983). Briefly, frozen tumor specimens were homogenized in 50 volumes of 50 m M Tris-HC1 buffer ( p H 7.4 at 37°C), preincubated for 15 rain at 37 o C, and centrifuged at 49000 × g for 20 min. The pellet was resuspended in 50 volumes of assay buffer (Tris-HC1, p H 7.4 at 37°C), 0.1% ascorbic acid, 4 m M CaC12 and 10 btM pargyline. Binding assays consisted of 0.2 ml tissue homogenate, 0.025 ml [3H]DPAT
Correspondence to: M.R. Pranzatelli, Departments of Neurology and Pediatrics, Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A.
(Amersham, 171-240 C i / m m o l ) , and 0.025 ml displacing drug or blank. The final concentration of [3H]DPAT used was 1 n M for competition experiments and 10-0.3 nM for Scatchard experiments. A 10 min incubation at 3 7 ° C was terminated by filtration through a Brandel M48-R Harvester (Schleicher and Schuell No. 32 glass fiber filters) with two 5 ml washes of ice-cold Tris-HC1 buffer ( p H 7.7 at 25 o C). Radioactivity was measured by liquid scintillation spectroscopy in 8 ml of 3a70 scintillation cocktail (Research Products International) at 58% counting efficiency. Specific binding, which was a mean of 40-60% at highest to lowest radioligand concentration, was defined as radioligand binding in the presence of 10 /~M unlabeled 5-HT. [3H]DPAT binding sites were found on ganglioneuroblastomas (n = 8) but not neuroblastomas (n = 6) or ganglioneuromas (n = 5). The density of sites (Bmax) was 1.10 + 0.13 p m o l / g wet weight tissue. Protein concentration, as determined by the Biorad assay using BSA, was 182 _+ 21 /~g protein per assay. K o was 2.80 _+ 0.52 and the Hill coefficient (nil) was 0 . 9 4 + 0 . 0 3 . Drug competition studies demonstrated that D P A T and 5-HT display highest affinity for [3H]DPAT-labeled sites in ganglioneuroblastoma (table 1). The order of drug potency was consistent with drug affinities for the 5-HTIA receptor. Therefore, the pharmacologic properties of [3H]DPAT-labeled membrane recognition sites in ganglioneuroblastoma agree well with those in rat brain (Gozlan et al., 1983). The novel finding of this study was the identification of an apparent 5-HT~A binding site on
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
128 TABLE 1 Drug affinities for recognition sites labeled by [3H]DPAT in human ganglioneuroblastoma. Radioligand binding studies were performed as described in the text using 1 nM [3H]DPAT. ICs0 values were determined by EBDA. Data are the means of three to four separate experiments performed in duplicate. Drug
IC50 (nM)
5-HT 8-OH-DPAT RU 24969 Methysergide DOI Methiothepin, Ketanserin Mianserin
5.9 50.8 10 53 38 ___ 7 5388 5-1227 7540 5-2006 8160 5- 3960 11945 _+ 925 > 100 000
h u m a n g a n g l i o n e u r o b l a s t o m a s which was n o t f o u n d on two other types of n e u r a l crest-derived tumor. [3H]5-HT b i n d i n g sites have b e e n identified o n the clonal n e u r o b l a s t o m a × b r a i n e x p l a n t h y b r i d cell line N C B - 2 0 that was a b s e n t from the p a r e n t n e u r o b l a s t o m a (Berry-Kravis a n d D a w s o n , 1983). However, to our knowledge, n o studies of 5 - H T 1 receptor subtypes have b e e n reported in n e u r a l crest-derived tumors. Other studies will be necessary to d e t e r m i n e if this is a f u n c t i o n a l 5HT1A receptor. T h e use of n e u r o t r a n s m i t t e r receptors as a biologic t u m o r m a r k e r also has clinical implications. H u m a n n e u r a l crest-derived tumors, as examples of primitive n e u r o e c t o d e r m a l t u m o r s ( P N E T S ) , often pose diagnostic difficulties for n e u r o p a thologists due to small cell size a n d o v e r l a p p i n g pathologic features (Rorke, 1983). N e w biologic markers m a y aid the diagnosis of P N E T s . 5-HTIA sites appear to distinguish g a n g l i o n e u r o b l a s t o m a s from other n e u r a l crest-derived t u m o r s studied. R a d i o l i g a n d b i n d i n g studies allow for the r a p i d a n d accurate screening of surgical t u m o r specimens. N e u r o t r a n s m i t t e r receptors m a y also explain some of the remote neurologic c o m p l i c a t i o n s of
h u m a n n e u r a l crest-derived tumors, such as the o p s o c l o n u s - m y o c l o n u s - a t a x i a syndrome, a movem e n t disorder which is most often associated with g a n g l i o n e u r o b l a s t o m a w h e n it is tumor-associated (Bray et al., 1969). I n e x p e r i m e n t a l animals, 5HT1A receptor agonists, such as D P A T , i n d u c e m y o c l o n u s (Tricklebank, 1985). 5-HTIA receptors m a y be the focus of p r o p o s e d a n t i b o d y - m e d i a t e d i n j u r y to the central n e r v o u s system in this synd r o m e (Bray et al., 1969). F u r t h e r studies are in progress to explore this possibility.
Acknowledgements This work was supported by NIH Grant 1-KO8-NS01158, the United Cerebral Palsy Research and Education Foundation and William Randolph Hearst Foundation (R381-88), and the Myoclonus Research Fund. Tumor specimens were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. The study was approved by our Institutional Review Board (No. X0308). We would like to thank Ann Zaragoza for excellent word processing.
References Berry-Kravis, E. and G. Dawson, 1983, Characterization of an adenylate cyclase-linked serotonin (5-HT1) receptor in a neuroblastoma × brain explant hybrid cell line (NCB-20), J. Neurochem. 40, 977. Bray, P.F., F.A. Ziter, M.E. Lahey and C.G. Myers, 1969, The coincidence of neuroblastoma and acute cerebellar encephalopathy, J. Pediat. 75, 983. Gozlan, H., S. Mestikawy, L. Pichat, J. Glowinski and M. Hamon, 1983, Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT, Nature 305. Rorke, L.B., 1983, The cerebellar medulloblastoma and its relationship to primitive neuroectodermal tumors, J. Neuropathol. Exp. Neurol. 42, 1. Tricklebank, M.D., C. Forler and J.R. Fozard, 1985, The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioral response to 8-hydroxy-2-(di-N-propylamino)tetralin in the rat, European J. Pharmacol. 106, 271.
127
Elsevier EJP 0223R
Rapid communication
Identification of 5-HT~A recognition sites in human ganglioneuroblastoma M i c h a e l R. P r a n z a t e l l i a n d J o a n n e Balletti Departments of Neurology and Pediatrics, Columbia University, 630 West 168th Street, New York, N Y 10032, U.S.A.
Received 28 September 1989, accepted 29 September 1989
5-HydroxytryptaminelA (5-HT1A) receptors have been characterized extensively in the central nervous system in m a n y species, including humans. They exhibit a distinct neuroanatomic distribution, linkage to an adenylate cyclase, and several physiologic and behavioral functions. Neurotransmitter receptors are often also sought on tumors derived from neural crest cells, the embryologic origin of central nervous tissue. However, the use of hybrid tumor-brain cell lines raises the possibility of alteration of receptor properties in tissue culture. Therefore, we used direct surgical specimens from pediatric patients to survey 5-HT 1 receptor subtypes in h u m a n neural crest-derived tumors. We characterized the binding of [3H]8O H - D P A T ([3H]DPAT), a selective 5-HTIA radioligand, in three different tumor types: neuroblastoma, ganglioneuroblastoma and ganglioneuroma. Radioligand binding assays were performed as described by Gozlan et al. (1983). Briefly, frozen tumor specimens were homogenized in 50 volumes of 50 m M Tris-HC1 buffer ( p H 7.4 at 37°C), preincubated for 15 rain at 37 o C, and centrifuged at 49000 × g for 20 min. The pellet was resuspended in 50 volumes of assay buffer (Tris-HC1, p H 7.4 at 37°C), 0.1% ascorbic acid, 4 m M CaC12 and 10 btM pargyline. Binding assays consisted of 0.2 ml tissue homogenate, 0.025 ml [3H]DPAT
Correspondence to: M.R. Pranzatelli, Departments of Neurology and Pediatrics, Columbia University, 630 West 168th Street, New York, NY 10032, U.S.A.
(Amersham, 171-240 C i / m m o l ) , and 0.025 ml displacing drug or blank. The final concentration of [3H]DPAT used was 1 n M for competition experiments and 10-0.3 nM for Scatchard experiments. A 10 min incubation at 3 7 ° C was terminated by filtration through a Brandel M48-R Harvester (Schleicher and Schuell No. 32 glass fiber filters) with two 5 ml washes of ice-cold Tris-HC1 buffer ( p H 7.7 at 25 o C). Radioactivity was measured by liquid scintillation spectroscopy in 8 ml of 3a70 scintillation cocktail (Research Products International) at 58% counting efficiency. Specific binding, which was a mean of 40-60% at highest to lowest radioligand concentration, was defined as radioligand binding in the presence of 10 /~M unlabeled 5-HT. [3H]DPAT binding sites were found on ganglioneuroblastomas (n = 8) but not neuroblastomas (n = 6) or ganglioneuromas (n = 5). The density of sites (Bmax) was 1.10 + 0.13 p m o l / g wet weight tissue. Protein concentration, as determined by the Biorad assay using BSA, was 182 _+ 21 /~g protein per assay. K o was 2.80 _+ 0.52 and the Hill coefficient (nil) was 0 . 9 4 + 0 . 0 3 . Drug competition studies demonstrated that D P A T and 5-HT display highest affinity for [3H]DPAT-labeled sites in ganglioneuroblastoma (table 1). The order of drug potency was consistent with drug affinities for the 5-HTIA receptor. Therefore, the pharmacologic properties of [3H]DPAT-labeled membrane recognition sites in ganglioneuroblastoma agree well with those in rat brain (Gozlan et al., 1983). The novel finding of this study was the identification of an apparent 5-HT~A binding site on
0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
128 TABLE 1 Drug affinities for recognition sites labeled by [3H]DPAT in human ganglioneuroblastoma. Radioligand binding studies were performed as described in the text using 1 nM [3H]DPAT. ICs0 values were determined by EBDA. Data are the means of three to four separate experiments performed in duplicate. Drug
IC50 (nM)
5-HT 8-OH-DPAT RU 24969 Methysergide DOI Methiothepin, Ketanserin Mianserin
5.9 50.8 10 53 38 ___ 7 5388 5-1227 7540 5-2006 8160 5- 3960 11945 _+ 925 > 100 000
h u m a n g a n g l i o n e u r o b l a s t o m a s which was n o t f o u n d on two other types of n e u r a l crest-derived tumor. [3H]5-HT b i n d i n g sites have b e e n identified o n the clonal n e u r o b l a s t o m a × b r a i n e x p l a n t h y b r i d cell line N C B - 2 0 that was a b s e n t from the p a r e n t n e u r o b l a s t o m a (Berry-Kravis a n d D a w s o n , 1983). However, to our knowledge, n o studies of 5 - H T 1 receptor subtypes have b e e n reported in n e u r a l crest-derived tumors. Other studies will be necessary to d e t e r m i n e if this is a f u n c t i o n a l 5HT1A receptor. T h e use of n e u r o t r a n s m i t t e r receptors as a biologic t u m o r m a r k e r also has clinical implications. H u m a n n e u r a l crest-derived tumors, as examples of primitive n e u r o e c t o d e r m a l t u m o r s ( P N E T S ) , often pose diagnostic difficulties for n e u r o p a thologists due to small cell size a n d o v e r l a p p i n g pathologic features (Rorke, 1983). N e w biologic markers m a y aid the diagnosis of P N E T s . 5-HTIA sites appear to distinguish g a n g l i o n e u r o b l a s t o m a s from other n e u r a l crest-derived t u m o r s studied. R a d i o l i g a n d b i n d i n g studies allow for the r a p i d a n d accurate screening of surgical t u m o r specimens. N e u r o t r a n s m i t t e r receptors m a y also explain some of the remote neurologic c o m p l i c a t i o n s of
h u m a n n e u r a l crest-derived tumors, such as the o p s o c l o n u s - m y o c l o n u s - a t a x i a syndrome, a movem e n t disorder which is most often associated with g a n g l i o n e u r o b l a s t o m a w h e n it is tumor-associated (Bray et al., 1969). I n e x p e r i m e n t a l animals, 5HT1A receptor agonists, such as D P A T , i n d u c e m y o c l o n u s (Tricklebank, 1985). 5-HTIA receptors m a y be the focus of p r o p o s e d a n t i b o d y - m e d i a t e d i n j u r y to the central n e r v o u s system in this synd r o m e (Bray et al., 1969). F u r t h e r studies are in progress to explore this possibility.
Acknowledgements This work was supported by NIH Grant 1-KO8-NS01158, the United Cerebral Palsy Research and Education Foundation and William Randolph Hearst Foundation (R381-88), and the Myoclonus Research Fund. Tumor specimens were provided by the Cooperative Human Tissue Network, which is funded by the National Cancer Institute. The study was approved by our Institutional Review Board (No. X0308). We would like to thank Ann Zaragoza for excellent word processing.
References Berry-Kravis, E. and G. Dawson, 1983, Characterization of an adenylate cyclase-linked serotonin (5-HT1) receptor in a neuroblastoma × brain explant hybrid cell line (NCB-20), J. Neurochem. 40, 977. Bray, P.F., F.A. Ziter, M.E. Lahey and C.G. Myers, 1969, The coincidence of neuroblastoma and acute cerebellar encephalopathy, J. Pediat. 75, 983. Gozlan, H., S. Mestikawy, L. Pichat, J. Glowinski and M. Hamon, 1983, Identification of presynaptic serotonin autoreceptors using a new ligand: 3H-PAT, Nature 305. Rorke, L.B., 1983, The cerebellar medulloblastoma and its relationship to primitive neuroectodermal tumors, J. Neuropathol. Exp. Neurol. 42, 1. Tricklebank, M.D., C. Forler and J.R. Fozard, 1985, The involvement of subtypes of the 5-HT1 receptor and of catecholaminergic systems in the behavioral response to 8-hydroxy-2-(di-N-propylamino)tetralin in the rat, European J. Pharmacol. 106, 271.