Neuropeptides (1997) 31 (5), 409-413 © Harcourt Brace and Company Ltd 1997

Comparison between vasoactive intestinal polypeptide and pituitary adenylate cyclase activating polypept!de levels in neuroblastoma t u m o u r t i s s u e s

P. Vertongen 1, P. De Clerck 2, J.-C. Fournet s, H. Martelli s, P, H61ardot 5, C. Devalck 4, T, P e e t e r s 2, E, Sar iban 4, R R o b b e r e c h t ~

1Laboratoire de Chimie Biologique et de la Nutrition, Faculte de M6decine, Universit~ Libre de Bruxelles, Belgium 2Gut Hormone Laboratory, Department of Medical Research, Katholieke Universiteit Leuven (KUL), Belgium 3Groupe Hospitalier Necker- Enfants Malades, Paris, France 4D6partement d'H6mato-oncologie, H6pital Universitaire des Enfants Reine Fabiola (HUDERF), Universit6 Libre de Bruxelles, Belgium 5H6pital St. Vincent de Paul, Paris, France

Summary Vasoactive intestinal polypeptide (VIP) is reported to exert an autocrine control on neuroblastoma cell tumours: VIP is produced by the tumour and stimulates cell differentiation. This study tested the hypothesis that the parent peptide, the pituitary adenylate cyclase activating polypeptide (PACAP) may have a similar role. It was found that PACAP mRNA and PACAP were expressed in 12/12 tumours; it was also observed that PACAP receptor mRNA and functional PACAP receptors were expressed in 12/12 and 5/9 tumours, respectively. VIP mRNA and VIP were detected in 9/12 tumours. VIP receptor mRNA was expressed in 5/12 tumours and functional VIP receptors were never demonstrated. The tumours having the highest VIP levels also had the highest PACAP contents and were associated with a watery diarrhoea syndrome due to activation of intestinal VIP receptors. As PACAP recognizes the PACAP receptors and the VIP receptors with the same high affinity it may contribute to the syndrome and is a likely candidate for an autocrine control of neuroblastoma cell growth and differentiation.

INTRODUCTION

Pituitary adenylate cyclase activating polypeptide (P.4CAP) and vasoactive intestinal polypepfide WIP) have 68% sequence homology, ~ and are related to the glucagon/secretin family of peptides. PACAP occurs as two biologically active forms: the longest form of 38 amino acids representing only 90% of the total PACAP immunoreactivity, 2,3 and a shorter form corresponding to the N-terminal PACAP-38 sequence and synthesized by alternative processing.

Received 1 April 1997 Accepted28 July 1997

Correspondence to: P. Robberecht, Laboratoire de Chimie Biologique et de la Nutrition, Facult6 de Medecine, Universitb Libre de Bruxelles, B~.t. G/E, CP 611,808 Route de Lennik, B-1070 Brussels, Belgium. Tel: + 32-2-555-62-15, Fax + 32-2-555-62-30. E-mail: probbe@ulb.ac.be

PACAP and VIP interact with seven transmembrane G protein coupled receptors: the PACAP type I receptors, with a high affinity for both PACAPs and a low affinity for VIP, and the PACAP type II with a high affinity for both PACAPs and for VIP. The PACAP type II receptors are subdivided into two subclasses: the VIP~ and the VIP 2 receptors that have been cloned and identified in different tissues. 4,5

The two types of PACAP receptors are positively coupled to adenylate cyclase, but the type I receptor may also be coupled to the IPJcalcium cascade. 4 Recently, a PACAP type I receptor variant was reported to directly activate an L-type calcium channel. 6

A recent study showed that PACAP type I receptors were expressed in a vast majority of neuroblastoma cell lines and surgical specimens of human neuroblastoma. 7 The VIP t and VIP 2 receptors were only rarely detected. It

409

410 Vertongen et al

has also been shown by O'Dorisio et al, s that VIP is pro- duced by low grade neuroblastoma, could be responsible for tumour differentiation and that high levels of VIP decrease the expression of functional VIP receptors. It was suggested that an autocrine loop involving VIP synthesis and VIP receptor expression could modulate neuroblastoma growth and differentiation.

The aim of the present study was to test the hypothesis that PACAP may also constitute an autocrine growth reg- ulator for neuroblastomas by measuring the expression of PACAP in turnout samples.

M A T E R I A L A N D M E T H O D S

Collection of the samples

Two types of samples were processed:

1. Some surgical samples were rinsed in ice-cold saline in the operating room and sent immediately to the laboratory. Three tumour fragments were frozen in liquid nitrogen and stored at - 80°C until peptide extraction, RNA extraction and membrane preparation.

2. Other surgical samples were sent frozen to the laboratory for membrane preparation, RNA extraction and peptides extraction.

Quantitative determination of PACAP and VIP preparation of tissue extracts

The frozen surgical samples were immersed in boiling water for 5 rain, homogenized with a Polytron, cooled at 4°C, acidified with 1.0 M acetic acid and centrifuged at 50 000 x g for 30 min at 4°C. The supemate was absorbed on a C18 Cartridge (Sep-Pak, Waters, Milford, MA) equilibrated in 0.1% trifluoroacetic acid (TFA), 10% CH3CN. The peptides were eluted with 0.1% TFA, 50% CH3CN. After lyophylization the dry residue was dis- solved in 10mM phosphate buffer, pH 7.4, containing 150 mM NaC1, 1 mM EDTA, 1% BSA, 1 g/L sodium azide and 0.05% Tween 20.

Quantitative determination of PACAP-27 and PACAP-38

PACAP amounts were estimated by radioimmunoassays with two antisera, CH1/5 and CH10/5, obtained by immunization of rabbits against, respectively, PACAP-27 and PACAP-38 (coupled to bovine serum albumin by carbodiimide) as described previnusly?

Assays were performed in a final volume of 550 ~1. The antigen and antibody were pre-incubated overnight at 4°C. The tracer was then added and after a further incu- bation for 24 h, free peptide was separated from the

bound fraction by the addition of sheep antirabbit 7-globulin and I h later, 1 ml of 3% polyethylene glycol 20 000 before centrifugation. The antigen was diluted with 10 mM sodium phosphate buffer, pH 7.4, containing 0.15 M NaC1, 0.05% Tween 20, 0.1% sodium azide and I mM EDTA. The antisera were diluted in the same buffer containing normal rabbit serum (1 in 500). The CH1/5 antiserum was used at a final concentration of 1 in 250 000 with ~25I-acetyl-His~-PACAP-27 as tracer. The CH10/5 antiserum was used at a final concentration of 1 in 25 000 with ]25I-PACAP-29 as tracer?

Quantitative determination of VIP

The radioimmunoassay for VIP was performed according to the method described by Long and Bryant/° with syn- thetic human VIP as standard, antibodies to human VIP (Biogenesis, Poole, UK) and ~25I-VIP from NEN. The free and bound ligands were separated by the charcoal/ dextran method.

RNA extraction and reverse transcriptase polymerase chain reaction

Total RNA was extracted by homogenization of the frozen samples in guanidium thiocyanate and centrifugation on caesium chloride gradient. Total RNA was submitted to a DNAase treatment before reverse transcription as described previously. 7

The primers used for:

1. PACAP cDNA amplification were: sense: 5' G A T C T T C A C G G A C A G C T A C A G 3' and antisense: 5' G T T T G G A T A G A A C A C A C G A G C 3 ' corresponding, respectively, to the 11664-11684 bp and 11868-11899 of the human PACAP gene.1 ]

2. VIP cDNA amplification were: sense 5' T C A C T G A C A A C T A T C C C G C C 3' and antisense: 5' A C A G C A T A T G A A A T T G C A G G C 3 " corresponding, respectively, to the 419-439 bp and 789-809 bp of the human VIP cDNA? 2

3. PACAP type I receptor cDNA amplification were: sense: 5 ' T T A A C T T T G T G C T T T T A T T G G C 3' and antisense: 5' T C C C T T T T G C T G A C A T T C T C 3' corresponding, respectively, to the 1200-1222 bp and 1364-1382 bp of the human PACAP receptor. 13

4. VIP 1 receptor cDNA amplification: sense: 5' G A A G G C T G GA C G C A C C T G G A 3' and antisense: 5' T T A C A G C C A C G G A C C T G G A 3 ' corresponding, respectively, to the 416-436 bp and 722-742 bp of the human WIl~ 1 receptor cDNA. 14

5. VIP 2 receptor cDNA amplification were: sense: 5' C C AC C T G A A C C T G T T C C T G T 3 " a n d

Neuropeptides (1997) 31(5), 409-413 © Harcourt Brace and Company Ltd 1997

VIP and PACAP levels in neuroblastomas 411

Table Patients' characterist ics

Tumour Sex Age Stage PACAP PACAP VIP VIP mRNA mRNA

Adenylate PACAP VIP~ VIP 2 cyclase receptor receptor receptor activity mRNA mRNA mRNA

(pmol cAMP produced min/

mg protein)

ng/g t issue ng/g t issue B PACAP VIP N HOP

243 F 11 months I 9000 +++ 24 000 +++ 128 130 127 + +++ + + 340 M 2 years I 10 000 ++ 8000 +++ 145 142 146 + ++ - - 310 F 4 years II 10 000 +++ 4000 ++ 22 24 23 + + - - 326 F 3 years I 2000 +++ 900 + ND ND ND + +++ - + 223 M 1 month II 100 ++ 3 - 43 100 51 + + - - 104 F 3 days II 400 + ND - 26 91 29 + + - - 335 F 6 months II 200 + 115 ++ 103 528 247 ++ - + ++ 333 F 9 months II 400 ++ 450 +++ 155 220 154 + + - - 295 M 6 years II 750 ++ 10 + 23 25 22 + - - - 334 F 7 months III 2500 +++ 1500 +++ N D N D N D + + + - 336 F 4 months IVs 20 + 30 + N D N D N D - ++ + + 184 F 7 years IV 600 ++ 7 - 122 354 190 + +++ - -

*Tumour stage, levels of PACAP and VIP, identif ication of PACAP, VIP, PACAP I receptors, VIP 1 receptor and VIP~ receptor mRNA and identif ication of PACAP receptor by PACAP- and VIP-st imulated adenylate cyclase. Evaluat ion of adenylate cyclase activity was performed in triplicate.

antisense: 5" T A C T G C A G G A A G A A G A C C A G G C T 3' corresponding, respectively, to the 576-595 bp and 706-725 bp of the human VIP 2 receptor cDNA. ~5

Thirty PCR cycles (1 rain at 95°C, 1 rain at annealing temperatures and 1 rain at 72°C) were performed. The annealing temperatures were 62°C for PACAP cDNA amplification, 60°C for VIP, VIPa receptor and VIP 2 recep- tor cDNA amplification and 58°C for PACAP type I recep- tor amplification. [3-Actin cDNA was used as control. 5 ~tl of each PCR product were submitted to electrophoresis on a 1.2% agarose gel, stained with ethidium bromide and visualized under UV light.

Receptor identification

Adenylate cyclase activity was determined by the proce- dure of Salomon et al.~ Membrane protein (5-10 ~tg) was incubated in a total volume of 60 ~tl containing 0.5 mM [~-32p]ATP, 5raM MgCI2, 0.5 mM EGTA, 1 mM cyclic AMP, 1 mM theophylline, 10raM phospho(enol) pyru- vate, 30 ~tg/ml pyruvate kinase and 30 mM Tris-HCl at a final pH of 7.5. The reaction was initiated by adding the membranes and was terminated after incubation for 15-20 rain at 37°C by adding 0.5 mM ATP, 0.5 mM cyclic AMP, and 20 000 cpm [8-3HI-cAMP to determine cAMP recovery, cAMP was separated from ATP by two succes- sive chromatographies on Dowex 50-WX8 mad neutral alumina. PACAP-27, PACAP-38 and VIP were tested in the presence of 10 pM GTP.

R E S U L T S

Patient's characteristics

Some characteristics are given in the Table. The first three patients (nos. 243, 340, 310) were reported to have neuroblastoma tumour associated with severe diarrhoea attributed to hyperproduction of VIP ('Vemer Morrisson syndrome'). These three patients had a low grade turnout.

Antiserum characterization

PACAP-27 tumour levels were estimated by a radio- immunoassay using the CH1/5 antiserum and 125I-acetyl- Hisl-PACAP-27 as tracer. The antiserum characterization was as described previously. 3 The epitope involved in anti- body recognition was the C-terminal amide of PACAP-2Z PACAP-38 was poorly recognized by this antiserum, which did not recognize VIP and the other structurally related peptides.

Total PACAP irnmunoreactivity was estimated by RIA using the CH 10/5 antiserum with 125I-PACAP-29 as tracer and PACAP-38 as standard. PACAP-27 and PACAP-38 were equally well recognized whereas VIP, PHM and PHV were not recognized. The epitope involved in anti- body recognition was located in the middle part of PACAP: indeed, different analogues of PACAP-27 modi- fied in the 10-16 position ([R16]PACAP-27, [Leu~0]pACAP - 27, [Leu~3]PACAP-27) were poorly recognized (Figure).

It was verified that the antiserum ~° used to estimate VIP levels did not crossreact with PACAP-27 or PACAP-38.

© Harcourt Brace and Company Ltd 1997 Neuropeptides (1997) 31(5), 409-413

VIP and PACAP levels in neuroblastomas 413

reported to increase n e u r o n a l survival in the absence of

adenylate cyclase st imulation. 24 Finally, the parallel evolu t ion of VIP and PACAP levels

in the t u m o u r s might be explainable by an identical t ran-

scriptional regulat ion of bo th mRNA syntheses t h r ough cyclic AMP responsive elements. 13,25,26

In conclusion, neu rob las toma cell tu rnou ts were able to synthesize bo th VIP and PACAP. Both peptides may contr ibute to the symptomato logy w h e n large a moun t s of peptides are p roduced and PACAP might be involved ~an autocr ine regulat ion of cell growth and differentiation.

ACKNOWLEDGEMENTS

Aided by a grant TELEVIE and by a 'Pole d'Attraction

Interuniversi ta ire ' from the Federal Governmen t of

Belgium.

REFERENCES

1. Arimura A. Pituitary adenylate cyclase activating polypeptide (PACAP): discovery and current status of research. Regul Pept 1992; 37: 287-303.

2. Arimura A, Somogyv~ri-Vigh A, Miyata, A, Mizuno K, Coy DH, Kitada C. Tissue distribution of PACAP as determined by RIA: highly abundant in the rat brain and testes. Endocrinology 1991; 129: 2787-2789.

3. Vertongen P, Woussen-Colle MC, Cauvin A, Robberecht P, Christophe J. Distribution of PACAP-38 and PACAP-27 in rat brain. Biomed Res 1992; 13: 377-382.

4. Rawlings SR, Hezareh M. Pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP/vasoactive intestinal polypeptide receptors: actions on the anterior pituitary gland. Endocr Rev 1996; 17: 4-29.

5. Usdin TB, Bonner T I, Mezey E. Two receptors for vasoactive intestinal polypeptide with similar specificity and complementary distributions. Endocrinology 1994; 135: 2662-2680.

6. Chatterjee TK, Sharma RV, Fisher RA. Molecular cloning of a novel variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) receptor that stimulates calcium influx by activation of L-type calcium channels. J Biol Chem 1996; 271: 32226-32232.

Z Vertongen P, Devalck C, Sariban E et al. Pituitary adenylate cyclase activating peptide and its receptors are expressed in human neuroblastomas. J Cell Physiol 1996; 167: 36-46.

8. O'Dorisio MS, Fleshman DJ, Qualman SJ, O'Dorisio TM. Vasoactive intestinal peptide: autocrine growth factor in neuroblastoma. Regul Pept 1992; 37:213-226.

9. Van Rampelbergh J, Gourlet P, De Neef P, Robberecht P, Waelbroeck M. Properties of the PACAP I, PACAP II VIP~ and chimeric N-terminal PACAP/VIP 1 receptors: evidences for multiple receptor states. Mol Pharmacol 1996; 50: 1596-1605.

10. Long RG, Bryant MG. Vasoactive intestinal polypeptide. In: Bloom SR, Long RG, eds. Radioimmunoassay of Gut Regulatory Peptides. London: WB Saunders, 1982: 120-130.

11. Hosoya M, Kimura, C, Ogi K et al. Structure of the human pituitary adenylate cyclase activating polypeptide (PACAP) gene. Biochim Biophys Acta 1992; 1129: 199-206.

12. Itoh N, Obata K, Yanaihara N, Okamoto H. Human preprovasoactive intestinal polypeptide contains a novel PHI- 27-like peptide, PHM-27. Nature 1983; 304: 547-549.

13. Ogi K, Miyamoto Y, Masuda Yet al. Molecular cloning and functional expression of a cDNA encoding a human pituitary adenylate cyclase activating polypeptide receptor. Biochem Biophys Res Commun 1993; 196:1511-1521.

14. Sreedharan SP, Patel DR, Huang J-X, Goetzl EJ. Cloning and functional expression of a human neuroendocrine vasoactive intestinal peptide receptor. Biochem Biophys Res Commun 1993; 193: 546-553.

15. Svoboda M, Tastenoy M, Van Rampelbergh Jet al. Molecular cloning and functional characterization of a human VIP receptor from SUP-T 1 lymphoblasts. Biochem Biophys Res Commun 1994; 205: 1617-1624.

16. Salomon Y, Londos C, Rodbell M. A highly sensitive adenylate cyclase assay./kllal Biochem 1974; 58: 541-548.

17. Modlin IM, Bloom SR. VIPomas and the watery diarrhoea syndrome. S Mr MedJ 1978; 54: 53-56.

18. Laburthe MC, Dupont CM, Besson JD, Rousset M, Rosselln GE. A new bioassay of VIP: resuks in watery diarrhoea syndrome. Gut 1980; 21: 619-623.

19. Tiedemann K, Pritehard J, Long R, Bloom S R. Intractable diarrhoea in a patient with vasoactive intestinal peptide- secreting neuroblastoma. Eur J. Pediatr 1981; 137:217-219.

20. Mendelsohn G, Eggleston JC, Olson JL, Said SI, Baylin SB. Vasoactive intestinal peptide and its relationship to ganglion cell differentiation in neuroblasfic tumors. Lab invest 1979; 41: 144-149.

21. Couvineau A, Rouyer-Fessard C, Fournier A, St-Pierre S, Pipkorn R, Laburthe M. Structural requirements for VIP interaction with specific receptors in human and rat intestinal membranes: effect of nine partial sequences. Biochem Biophys Res Commun 1984; 121: 493-498.

22. Wollman Y, Lilllng G, Goldstein MN, Fridkin M, Gozes I. Vasoactive intestinal peptide: a growth promotor in neuroblastoma cells. Brain Res 1993; 624: 339-341.

23. Pence JF, Shorter NA. In vitro differentiation of human neuroblastoma cells caused by vasoactive intestinal peptide. Cancer Res 1990; 50: 5177-5183.

24. Gressens P, Hill JM, Paindaveine B, Gozes I, Fridkin M, Breimeman D E. Severe microcephaly induced by blockade of vasoactive intestinal peptide function in the primitive neuroepithellum of the mouse. J Clin Invest 1994; 94: 2020-2027.

25. Yamagami T, Ohsawa K, Nishizawa Met al. Complete nucleotide sequence of human vasoactive intestinal peptide/PHM-27 gene and its inducible promoter. In: Said. SI and Mutt V, eds. Vasoactive intestinal peptide and related peptides. Ann NY Acad Sci 1988; 527: 87-102.

26. Ohsawa K, Hayakawa Y, Nishisawa Met al. Synergistic stimulation of VIP/PHM-27 gene expression by cyclic AMP and phorbol esters in human neuroblastoma cells. Biochem Biophys Res Commun 1985; 132: 885-891.

© Harcourt Brace and Company Ltd 1997 Neuropeptides (1997) 31(5), 409--413