Histochemistry (1991) 95 : 555-559 0301556491000472
Histochemistry © Springer-Verlag 1991
In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors K. Funa 1, B. Eriksson 2, E. Wilander 3, and K. Oberg 1' 2 1 Ludwig Institute for Cancer Research, University Hospital, S-75185 Uppsala, Sweden 2 Department of Internal Medicine, University Hospital, Uppsala 3 Department of Pathology, University Hospital, Uppsala ReceivedAugust 6, 1990 / AcceptedNovember 14, 1990 Summary. The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with 3~S-labeled oligonucleotide probes in formalinfixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the m R N A for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of m R N A expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.
Introduction Chromogranins are acidic secretory proteins which are stored in chromaffin granules of neuroendocrine cells and tumors (Simon and Aunis 1989; Fisher-Colbrie and Frischenschlager 1985). The main component, chromoOffprint requests to: K. Funa
granin A, has been isolated and characterized (Smiths and Winkler 1967) and the amino acid sequence of bovine chromogranin A has been determined (Benedum et al. 1986; Icangelo et al. 1986), Chromogranin A is synthesized as a precursor molecule which is processed by endogenous proteases and gives rise to a family of chromogranin A proteins (Kilpatrick etal. 1983; Winkler et al. 1986). The cDNA cloning of human counterpart revealed that it contains a pancreastatin sequence flanked by sites for proteolytic processing (Konecki et al. 1987). Chromogranin B (secretogranin I) and chromogranin C (secretogranin II) are two other members of the acidic chromogranin protein family that are also found in chromaffin granules in neuroendocrine cells (Fisher-Colbrie and Frischenschlager 1985; Winkler et al. 1986). Chromogranin B cDNA was also cloned and the sequence was shown to have amino acid homology with chromogranin A at their amino acid and carboxyl termini but diverges in the intervening region (Benedum et al. 1987). The biological functions of chromogranins have not yet been established, but they have been proposed to play a regulatory role increasing the storage capacity of various hormones (Simon and Aunis 1989). Chromogranins seem to be differentially processed and regulated (Fischer-Colbrie et al. 1988), and some differences in the distribution of these three proteins have been demonstrated (Schmid et al. 1989). The chromogranins, especially chromogranin A, have been shown to be good markers of neuroendocrine cells and tumors (Eriksson et al. 1990; Linnoila et al. 1988; Lloyd et al. 1989). In this communication, we report the expression pattern of chromogranins A and B in various types of carcinoid tumors examined by in situ hybridization technique using sequence-specific oligonucleotide probes. The results were also compared with the expression of the corresponding proteins determined by immunohistochemistry, using a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranin A and B.
556
Materials and methods
Materials Formalin-fixed paraffin-embedded tumor tissues consisting of 18 bronchial carcinoid tumors, 15 mid-gut carcinoid tumors and 5 rectal carcinoid tumors were collected (Table 1). In several cases more than one tissue specimen from the same tumor were examined. Seven mid-gut carcinoid tissues (patients 19 25) were from liver metastases which were taken before initiation of interferon (IFN) therapy. The corresponding liver biopsies from 4 patients after 1FN therapy (more than 3 months) were examined (patients 19-22) with the same chromogranin A probe and developed at the same time. Most of the other tissues were derived from primary tumors or regional metastases. The tissues were sectioned at a thickness of about 5 gm and mounted on 3-gamma-propyltriethoxy silane-coated slides (Sigma St. Louis, Mo., USA) to assure the attachment of the tissue to the slides (Belfik et al. 1989).
Probes The sequences of oligonucleotides, used for the probes, were selected so that no cross-hybridization would occur between chromogranin A and B on the one hand and any other sequence on the other, as examined by the Genepro computer program. A 51 base probe complementary to mRNA of human chromogranin A coding for amino acids 104-120 and a 39 base probe to mRNA of human chromogranin B coding for amino acid 413-425 were synthesized by an DNA synthesizer (Applied Biosystems, Foster City, Calif., USA, Model 380B) at the Ludwig Institute in Stockholm and cleaned through Sephadex G25 column (Pharmacia, Uppsala, Sweden). The corresponding sense oligonucleotide probes were also prepared in the same manner. The oligonucleotides were labeled by the 3'-end labeling method. Briefly, ten picomoles of oligonucleotide were incubated with 50 gCi 35S dATP (1300 Ci/mmol), 2 mM COC12, 0.5 mM dithiothreitol, and 20 units of terminal deoxynucleotidyl transferase in 100 mM potassium cacodylate (pH 7.2) for 45 rain at 37° C. The reaction was stopped by adding 400 gl 0.1 M Tris-HC1, 10 mM triethylamine, I mM ethylene-diaminetetraacetic acid (EDTA, pH 7.5). The labeled probes were separated by running the reaction products through NENSORB cartridge (DuPont Scandinavia, Stockholm, Sweden) according to vendors' description.
In situ hybridization The paraffin-embedded sections were deparaffinized by immersing the sections in xylene, graded ethanols and digested with 0.1 mg/ml proteinase K (Sigma) at 37°C for 15 rain before hybridization. The sections were rinsed in 2 x SSC (1 x SSC; 0.15 M sodium chloride and 0.0125 M trisodium citrate) and incubated in 0.25 % acetic anhydride in 0.1 tool/1 triethanolamine for 10 rain. They were then incubated in 0.1 tool/1 Tris-HC1 and 0A tool/1 glycine for 30 rain. They were rinsed in 2 x SSC and prehydridized with 40 ~tl of hybridization mixture consisting of 2 x SSC, 10% dextrane sulphate, 1 x Denhardt's solution (0.20% Ficoll, 0.02% bovine serum aiburain and 0.02% polyvinylpyrrolidone), 50% formamide, 1 mg/ml sheared herring-sperm DNA, and 1 mg/ml Escherichia coli tRNA for 1 h at 40 ° C. The slides were rinsed once in 50% formamide-2 x SSC at 40 ° C and were coated with 10-30 gl of the same hybridization mixture but containing 10 mmoi/1 dithiothreitol and the labeled probe (3 x 105 cpm per section), covered with cover slips, sealed with rubber cement, and incubated at 40 ° C in a humidified chamber overnight. Slides were rinsed in 2 x SSC for 2 h and i x SSC for 1 h at 40 ° C and dehydrated in graded ethanol. Hybridized preparations were autoradiographed with NTB 2 nuclear track emulsion (Eastman Kodak) diluted 1 : 1 with distilled water. After exposure for a week at 4° C, slides were developed in Dektol (Kodak Rochester, NY, USA) developer at 15°C for
4 rain, rinsed in distilled water, fixed for 5 min, soaked in distilled water for 5 min and air-dried. Slides were counterstained with Mayer's hematoxylin and eosin. Control slides included the following. (1) A normal pancreas and a carcinoid tumor section which had previously been shown by immunohistochemistry to contain both chromogranin A and B. (2) The positive tissues were treated with RNase before hybridization in order to see that the probe hybridized with cellular RNA. (3) The hybridization was carried out with addition of 100 times excess cold oligonucleotides for chromogranin A or chromogranin B, respectively, to see that the signals were diminished by the competition for the target mRNA. (4) The tissues which were negative for any of the chromogranins were checked for the presence of cellular mRNA by hybridizing with the RNA probe for human fl-actin (Funa et al. 1987). (5) Finally, a few tissues which were positive for both chromogranin A and B were hybridized with labeled sense oligonucleotide probe for each mRNA. Semiquantitative evaluation of the results was performed independently by two of the authors.
Immunohistochemistry and Grimelius' argyrophil staining The tissue sections were deparaffinized, rehydrated and treated with peroxide in methanol to inactivate endogenous peroxidase. Immunoperoxidase staining was performed using Vectastain ABC kit (Vector Laboratories, Burlingame, Calif., USA) according to the vendor's description and aminoethylcarbazole as chromogen. A mouse monoclonal antibody against chromogranin A (Lloyd and Wilson 1983) and a rabbit polyclonal antiserum which recognizes both chromogranin A and B (Eriksson et al. 1990) were used. In each assay, normal goat serum used for blocking or normal rabbit serum were substituted for primary antibodies as a negative control. All the sections were stained for argyrophil reaction according to Grimelius (Grimelius and Wilander 1980).
Results T h e expression o f c h r o m o g r a n i n s A a n d B m R N A s a n d the c o r r e s p o n d i n g p r o t e i n expressions in the c a r c i n o i d t u m o r tissues are s u m m a r i z e d in Table 1. A l l m i d - g u t c a r c i n o i d t u m o r s (15 cases) expressed m R N A s for b o t h c h r o m o g r a n i n A a n d B (Fig. 1). In one case, I p r i m a r y intestinal t u m o r tissue, 5 r e g i o n a l m e t a s t a s e s as well as 1 o m e n t u m m e t a s t a s i s were e x a m i n e d ( p a t i e n t 27). A l l these tissues s h o w e d s t r o n g signals with m i n o r differences. T h e b i o p s y m a t e r i a l s f r o m liver m e t a s t a s e s (patients 19-25) t a k e n f r o m the p a t i e n t s b e f o r e s t a r t o f I F N t h e r a p y s h o w e d s t r o n g signals for b o t h m R N A species. T h e c h r o m o g r a n i n A m R N A e x p r e s s i o n was c o m p a r e d with the c o r r e s p o n d i n g b i o p s y specimens after I F N thera p y (patients 19-22). A l l f o u r p a t i e n t s s h o w e d r e d u c e d e x p r e s s i o n o f c h r o m o g r a n i n A m R N A o n the liver metastasis o b t a i n e d after I F N t h e r a p y (Fig. 2, Table 1). O n l y 12 o f 18 e x a m i n e d cases o f b r o n c h i a l c a r c i n o i d a n d 1 o f 5 rectal c a r c i n o i d t u m o r s s h o w e d b o t h types o f c h r o m o g r a n i n m R N A . Two b r o n c h i a l c a r c i n o i d (patients 7, 13) a n d one rectal c a r c i n o i d ( p a t i e n t s 3 5 ) s h o w e d o n l y c h r o m o g r a n i n B m R N A , b u t these b r o n chial c a r c i n o i d s (patients 7, 13) s h o w e d i m m u n o r e a c t i v i ty for c h r o m o g r a n i n A. In a n o t h e r two cases o f b r o n c h i al c a r c i n o i d s (patients 15, 17), c h r o m o g r a n i n m R N A (especially c h r o m o g r a n i n A ) was expressed in all t u m o r cells b u t o n l y a focal staining was o b t a i n e d b y i m m u n o h i s t o c h e m i s t r y with b o t h a n t i b o d i e s (Fig. 3).
557 1. Results of in situ hybridization, immunohistochemistry for chromogranin A and B mRNA, and protein in carcinoid tumors, compared with Grimelius' argyrophil reaction
Table
Patient In situ hybridization Immunohistochemistry Grimelius' stailling ChrA ChrB ChrA ChrA + B A. Bronchial carcinoid tumors 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 t6 17 18
-
. + + ++ ++ -+ + + + + ++ + + +
-
.
. + + ++ ++ + + + + ± ± + + + + ND
-
ND
+ + ++ + + + + + + ± + + ± + ±
+ + ++ + + + ± + + + + ++ + ± ±
ND + ++ ++ ND ++ ++ _+ ++ ++ ++ ND ++ + ++ ++ ++
+
+
+
ND ND ND ND ND ND ND ++ + + + + + + +
ND ND ND ND ND ND ND ++ + ++ + ++ + + +
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
ND
± ± ± ± ND
++ ++ ± ++
.
+
+
B. Mid-gut carcinoid tumors
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
++(+)"+ + + +(+) + + + +(+) + + + + ( + ) ND ++ ++ + + ++ ++ ++ ++ ++ ++ + + ++ ++ ++ ++ + + + + + ++
C. Rectal carcinoid tumors
34 35 36 37 38
++
+ -+
"Tissue materials taken after IFN treatment stated within parentheses. Comparison is semiquantitative and not aimed to he performed between different probes or antibodies + + Strong hybridization signals (immunoreaction) on all tumor cells + Positive hybridization signals (immunoreaction) on tumor cells +_ Weak and/or focal hybridization signals (immunoreaction) on tumor cells - No hybridization signals (immunoreaction) ND Not Determined For Grimelius' staining:
+ + More than half of the tumor cells positive + Less than half of the tumor cells positive -t- Rare positive cells No positive cells ND Not Determined
No tumor tissue expressed only chromogranin A m R N A . Normal stromal cells adjacent to tumor cells did not express any of these m R N A s , except neuronal cells in a ganglion which showed chromogranin B m R N A (Fig. 4) and also chromogranin A m R N A at low level (not shown). Normal endocrine ceils, but not exocrine cells in pancreas, expressed the two types of chromogranin m R N A s (not shown). No normal intestinal mucosal cell expressed clear distinct signals for chromogranin m R N A , whereas a few scattered cells expressed the corresponding proteins when determined by immunohistochemistry (not shown). Treatment of the tissue section with RNase before hybridization reduced the signals greatly, indicating that the probe hybridized with cellular R N A . Hybridization with addition of 100 times excess cold oligonucleotides for chromogranin A or B resulted in reduction of the signals by competition for the target m R N A (not shown). Two tissues which were positive for both chromogranin A and B m R N A (patients 15, 26) did not hybridize with sense oligonucleotide probes (not shown). All but one carcinoid tumor tissues were shown to be positive for Grimelius' argyrophil staining. Mid-gut carcinoid tumor were uniformly positive, while in 3 bronchial and 1 rectal carcinoid tumor, only less than half of the tumor cells were positive and 1 rectal carcinoid tumor was completely negative for the reaction (Table 1). This tumor was also negative for m R N A of chromogranin A and B.
Discussion
The carcinoid tumors examined showed an almost complete concordance between chromogranin A and B m R N A expression. The expression of m R N A and protein of chromogranin A also displayed almost complete concordance, except in three cases of bronchial carcinoid tumors where the protein had been detected by immunohistochemistry, but no m R N A having been shown. This might be due to difference in sensitivity, since a single normal mucosal endocrine cell was shown to contain chromogranin immunoreactivity but not distinct hybridization signals for m R N A . Length of half-life and stability of the molecules may also account for the differences. However, in two cases of bronchial carcinoid tumors, the m R N A expression was stronger as well as more homogeneously spread than the protein expression. Immunohistochemistry showed only focal staining in small areas. The reason of the discrepancy is unclear. Chromogranin A and B may function as precursors for biologically active substances and might undergo enzymatic cleavage into smaller molecules. Thus, antigen determinants recognized by these antibodies m a y not be present in the tissue, whereas m R N A s for the precursors was recognized by oligonucleotide probes. However, the high concordance of these two methods indicates that they complement each other quite well in localizing the gene products and proteins in the tissues. We have found that chromogranin A m R N A expression in the mid-gut carcinoids was weaker after initiation
558
Fig. 1 A-D, Microphotographs of mid-gut carcinoid tumor tissue (patient 30). (A) Hybridization with probe against chromogranin A mRNA. (B) A section hybridized with a probe against chromogranin B mRNA. (C) Chromogranin A immunoreactivity with a monoclonal antibody. (D) Immunoreactivity against chromogranin A and B with a polyclonal antiserum in the corresponding area (magnification x 270) Fig. 2 A, B. Microphotographs of liver biopsies from mid-gut carciholds hybridized with chromogranin A probe (patient 22). (A) Tissue section obtained before IFN therapy. (B) Tissue section obtained after IFN therapy (magnification x 270). A few groups of tumor cells are present in fibrotic tissue
Fig. 3 A-D, Microphotographs of bronchial carcinoids (patient 15). (A) Hybridization with probe against chromogranin A mRNA. (B) Immunoreactivity against chromogranin A and B with a polyclonal antiserum in the corresponding area (magnification x 270). (C) An adjacent group of tumor cells expressing the same level of chromogranin A mRNA. (D) The corresponding area showing very weak immunoreactivity with a polyclonal antiserum (magnification x 270) Fig. 4. Microphotograph of neuronaI cells in an intestinal wall ganglion hybridized with chromogranin B probe (magnification x 270)
o f I F N treatment, c o m p a r e d with that before therapy. This finding indicates that I F N might inhibit the hormone production at the transcription level in the t u m o r cells (De Maeyer et al. 1988). This might explain our previous observation of a rather rapid decrease of circulating h o r m o n e levels after IFN-c~ therapy (Oberg et al. 1983).
All mid-gut carcinoid tumors expressed both types of chromogranins, whereas 80% o f bronchial and only 40% of rectal carcinoid tumors expressed one or both m R N A s . Cases of mid-gut carcinoid tumors are also reported to present the highest circulating chromogranin A and B levels (Eriksson et al. 1990). The same tendency was seen with the argyrophil reaction, where most o f
559 the m i d - g u t t u m o r cells were u n i f o r m l y stained, while c o n s i d e r a b l e v a r i a t i o n o f r e a c t i v i t y was seen in s o m e o f the b r o n c h i a l a n d rectal c a r c i n o i d t u m o r cells. M i d - g u t c a r c i n o i d s are h i g h l y d i f f e r e n t i a t e d n e u r o e n docrine tumors with homogenous pattern of hormone p r o d u c t i o n , g r o w t h rate a n d clinical course. R e c t a l carcin o i d t u m o r s , h o w e v e r , r e p r e s e n t a m i x t u r e o f t u m o r subtypes w i t h v a r y i n g degrees o f n e u r o e n d o c r i n e differentiation, a n d they u s u a l l y store m u c h less s e r o t o n i n c o m p a r e d w i t h m i d - g u t c a r c i n o i d ( F i o c c a et al. 1980). It is k n o w n t h a t c h r o m o g r a n i n s f o r m the m a j o r m a t r i x substance o f m a n y different t y p e s o f dense core granules, w h i c h is a c r y t o p l a s m i c s t o r a g e site o f the specific p e p tides a n d b i o g e n i c a m i n e p r o d u c t s o f n e u r o e n d o c r i n e cells. It is also p o s s i b l e t h a t the m a i n p r o t e i n m a t r i x o f the s e c r e t o r y g r a n u l e s o f rectal c a r c i n o i d s consists o f p r o t e i n c o m p o n e n t s t h a t are still unidentified. I n c o n c l u s i o n , the m R N A for c h r o m o g r a n i n A a n d B are g o o d m a r k e r s for c a r c i n o i d t u m o r s , especially those o f m i d - g u t origin. F o r e - g u t , m i d - g u t a n d rectal c a r c i n o i d t u m o r s are different in their n e u r o e n d o c r i n e d i f f e r e n t i a t i o n a n d p r o p e r t i e s r e g a r d i n g the e x p r e s s i o n o f the different c h r o m o g r a n i n s .
Acknowledgements. We thank Mrs. A.-C. Mel6n and Mrs. A. Nagy for skilful technical assistance and Mr. S. Kvist at the Ludwig Institute for Cancer Research in Stockholm for synthesis of the oligonucleotides. This work was partly supported by Swedish Medical Council and Swedish Cancer Society. References Bel/tk K, Funa K, Kelly R, Belfik S (1989) Rapid diagnosis of Aujeszky's disease in pigs by improved in situ hybridization using biotinylated probes on paraffin-embedded tissue sections, J Vet Med B36:10-20 Benedum UM, Baeuele PA, Konecki DS, Frank R, Powell J, Mallet J, Huttner WB (1986) The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells. EMBO J 5:1495-1502 Benedum UM, Lamouroux A, Konecki DS, Rosa P, Hille A, Baeuerle PA, Frank R, Lottspeich F, Mallet J, Huttner WB (1987) The primary structure of human secretogranin I (chromogranin B) : comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region. EMBO J 6:1203-1211 De Maeyer E, De Maeyer-Guignard J (t988) The effects of interferons on cell growth and division. In: De Maeyer E, De MaeyerGuignard J (eds) Interferons and other regulatory cytokines. Wiley, New York, 134-153 Eriksson B, Arnberg H, (Dberg K, Hellman U, Lundqvist G, Wern-
stedt C, Wilander E (1990) A polyclonal antiserum against chromogranin A and B - a new sensitive marker for neuroendocrine tumors. Acta Endocrinol 122:145-155 Fiocca R, Capella C, Buffa R, Fontana P, Solcia E, Hage E, Chance RE, Moody RL (1980) Glucagon-, glicentin- and pancreatic polypeptide-like immunoreactivities in rectal carcinoids and related colorectal cells. Am J Pathol 100:81-92 Fisher-Colbrie R, Frischenschlager I (1985) Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B and enkephalin-containing peptides. J Neurochem 44:1854-1861 Fischer-Colbrie R, Iacangelo A, Eiden LE (1988) Neural and humoral factors separately regulate neuropeptide Y, enkephalin and chromogranin A and B mRNA levels in rat adrenal medulla. Proc Natl Acad Sci USA 85 : 3240-3244 Funa K, Steinholtz L, N6u E, Bergh J (1987) Increased expression of N-myc in human small cell lung cancer biopsies predicts lack of response to chemotherapy and poor prognosis. Am J Clin Pathol 88:216-220 Grimelius L, Wilander E (1980) Silver strains in the study of endocrine cells of the gut and pancreas. Invest Cell Pathol 3:2-12 Icangelo A, Affolter HU, Eiden LE, Herbert E, Grimes M (1986) Bovine chromogranin A: its sequence and the distribution of its messenger RNA in endocrine tissues. Nature 323 : 82-86 Kilpatrick L, Gavine F, Apps D, Phillips J (1983) Biosynthetic relationship between the major matrix proteins of adrenal chromaffin granules. FEBS Lett 164:383-388 Konecki DS, Benedum UM, Gerdes H-H, Huttner WB (1987) The primary structure of human chromogranin A and pancreastatin. J Biol Chem 262:17026-17030 Linnoila RI, Mulshine JL, Steinberg SM, Funa K, Matthews M J, Cotelingam JD, Gazdar AF (1988) Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas. Am J Clin Pathol 90 : 641-652 Lloyd RV, Wilson BS (1983) Specific endocrine tissue marker defined by a monoclonal antibody. Science 222 : 628-630 Lloyd RV, Iacangelo A, Eiden LE, Cano M, Jin L, Grimes M (1989) Chromogranin A and B messenger ribonucleic acids in pituitary and other normal and neoplastic human endocrine tissues. Lab Invest 60:548-556 Oberg K, Funa K, Alto G (1983) Effects of leukocyte interferon on clinical symptoms and hormone levels in patients with midgut carcinoid tumors and carcinoid syndrome. N Engl J Med 309:129-133 Schmid KW, Weiler R, Xu RW, Hogue-Angeletti R, Fischer-Colbrie R, Winkler H (1989) An immunological study on chromogranin A and B in human endocrine and nervous tissues. Histochem J 21:365-373 Simon J-P, Aunis D (1989) Biochemistry of the chromogranin A protein family. Biochem J 262 : 1-13 Smiths AD, Winkler H (1967) Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla. Biochem J 103:483-492 Winkler H, Apps DK, Fisher-Colbrie R (1986) The molecular function of adrenal chromaffin granules : established facts and unresolved topics. Neuroscience 18 : 261-290
Histochemistry © Springer-Verlag 1991
In situ hybridization study of chromogranin A and B mRNA in carcinoid tumors K. Funa 1, B. Eriksson 2, E. Wilander 3, and K. Oberg 1' 2 1 Ludwig Institute for Cancer Research, University Hospital, S-75185 Uppsala, Sweden 2 Department of Internal Medicine, University Hospital, Uppsala 3 Department of Pathology, University Hospital, Uppsala ReceivedAugust 6, 1990 / AcceptedNovember 14, 1990 Summary. The distribution of the mRNAs for chromogranin A and B was analyzed by in situ hybridization with 3~S-labeled oligonucleotide probes in formalinfixed paraffin-embedded carcinoid tumor tissues. All the 15 mid-gut carcinoid tumors examined contained both mRNAs for chromogranin A and B at high level in tumor cells. Sixteen of 18 bronchial carcinoid tumors but only 2 of 5 rectal carcinoid tumors expressed one or both species of chromogranin mRNAs. The same tendency was seen with the argyrophil reaction according to Grimelius where most of the mid-gut tumor cells were uniformly stained, while considerable variation in reactivity was seen in some of the bronchial and rectal carcinoid tumor cells. The sequential sections were stained with a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranins. The expression of the m R N A for chromogranin A on the carcinoid tumors was almost concordant with that of chromogranin B as well as with the chromogranin A protein, whereas almost all tumors stained positively with the polyclonal antibodies. Analyses of m R N A expression of chromogranin A before and after interferon therapy on 4 patients with mid-gut carcinoids indicated an inhibition at pre-translational level. In conclusion, the mRNAs for chromogranin A and B are good markers for the carcinoid tumors, especially of mid-gut origin. Fore-gut, mid-gut and rectal carcinoid tumors are different in their endocrine properties regarding the expression of the chromogranins.
Introduction Chromogranins are acidic secretory proteins which are stored in chromaffin granules of neuroendocrine cells and tumors (Simon and Aunis 1989; Fisher-Colbrie and Frischenschlager 1985). The main component, chromoOffprint requests to: K. Funa
granin A, has been isolated and characterized (Smiths and Winkler 1967) and the amino acid sequence of bovine chromogranin A has been determined (Benedum et al. 1986; Icangelo et al. 1986), Chromogranin A is synthesized as a precursor molecule which is processed by endogenous proteases and gives rise to a family of chromogranin A proteins (Kilpatrick etal. 1983; Winkler et al. 1986). The cDNA cloning of human counterpart revealed that it contains a pancreastatin sequence flanked by sites for proteolytic processing (Konecki et al. 1987). Chromogranin B (secretogranin I) and chromogranin C (secretogranin II) are two other members of the acidic chromogranin protein family that are also found in chromaffin granules in neuroendocrine cells (Fisher-Colbrie and Frischenschlager 1985; Winkler et al. 1986). Chromogranin B cDNA was also cloned and the sequence was shown to have amino acid homology with chromogranin A at their amino acid and carboxyl termini but diverges in the intervening region (Benedum et al. 1987). The biological functions of chromogranins have not yet been established, but they have been proposed to play a regulatory role increasing the storage capacity of various hormones (Simon and Aunis 1989). Chromogranins seem to be differentially processed and regulated (Fischer-Colbrie et al. 1988), and some differences in the distribution of these three proteins have been demonstrated (Schmid et al. 1989). The chromogranins, especially chromogranin A, have been shown to be good markers of neuroendocrine cells and tumors (Eriksson et al. 1990; Linnoila et al. 1988; Lloyd et al. 1989). In this communication, we report the expression pattern of chromogranins A and B in various types of carcinoid tumors examined by in situ hybridization technique using sequence-specific oligonucleotide probes. The results were also compared with the expression of the corresponding proteins determined by immunohistochemistry, using a monoclonal antibody against chromogranin A and a polyclonal antiserum which reacts with both chromogranin A and B.
556
Materials and methods
Materials Formalin-fixed paraffin-embedded tumor tissues consisting of 18 bronchial carcinoid tumors, 15 mid-gut carcinoid tumors and 5 rectal carcinoid tumors were collected (Table 1). In several cases more than one tissue specimen from the same tumor were examined. Seven mid-gut carcinoid tissues (patients 19 25) were from liver metastases which were taken before initiation of interferon (IFN) therapy. The corresponding liver biopsies from 4 patients after 1FN therapy (more than 3 months) were examined (patients 19-22) with the same chromogranin A probe and developed at the same time. Most of the other tissues were derived from primary tumors or regional metastases. The tissues were sectioned at a thickness of about 5 gm and mounted on 3-gamma-propyltriethoxy silane-coated slides (Sigma St. Louis, Mo., USA) to assure the attachment of the tissue to the slides (Belfik et al. 1989).
Probes The sequences of oligonucleotides, used for the probes, were selected so that no cross-hybridization would occur between chromogranin A and B on the one hand and any other sequence on the other, as examined by the Genepro computer program. A 51 base probe complementary to mRNA of human chromogranin A coding for amino acids 104-120 and a 39 base probe to mRNA of human chromogranin B coding for amino acid 413-425 were synthesized by an DNA synthesizer (Applied Biosystems, Foster City, Calif., USA, Model 380B) at the Ludwig Institute in Stockholm and cleaned through Sephadex G25 column (Pharmacia, Uppsala, Sweden). The corresponding sense oligonucleotide probes were also prepared in the same manner. The oligonucleotides were labeled by the 3'-end labeling method. Briefly, ten picomoles of oligonucleotide were incubated with 50 gCi 35S dATP (1300 Ci/mmol), 2 mM COC12, 0.5 mM dithiothreitol, and 20 units of terminal deoxynucleotidyl transferase in 100 mM potassium cacodylate (pH 7.2) for 45 rain at 37° C. The reaction was stopped by adding 400 gl 0.1 M Tris-HC1, 10 mM triethylamine, I mM ethylene-diaminetetraacetic acid (EDTA, pH 7.5). The labeled probes were separated by running the reaction products through NENSORB cartridge (DuPont Scandinavia, Stockholm, Sweden) according to vendors' description.
In situ hybridization The paraffin-embedded sections were deparaffinized by immersing the sections in xylene, graded ethanols and digested with 0.1 mg/ml proteinase K (Sigma) at 37°C for 15 rain before hybridization. The sections were rinsed in 2 x SSC (1 x SSC; 0.15 M sodium chloride and 0.0125 M trisodium citrate) and incubated in 0.25 % acetic anhydride in 0.1 tool/1 triethanolamine for 10 rain. They were then incubated in 0.1 tool/1 Tris-HC1 and 0A tool/1 glycine for 30 rain. They were rinsed in 2 x SSC and prehydridized with 40 ~tl of hybridization mixture consisting of 2 x SSC, 10% dextrane sulphate, 1 x Denhardt's solution (0.20% Ficoll, 0.02% bovine serum aiburain and 0.02% polyvinylpyrrolidone), 50% formamide, 1 mg/ml sheared herring-sperm DNA, and 1 mg/ml Escherichia coli tRNA for 1 h at 40 ° C. The slides were rinsed once in 50% formamide-2 x SSC at 40 ° C and were coated with 10-30 gl of the same hybridization mixture but containing 10 mmoi/1 dithiothreitol and the labeled probe (3 x 105 cpm per section), covered with cover slips, sealed with rubber cement, and incubated at 40 ° C in a humidified chamber overnight. Slides were rinsed in 2 x SSC for 2 h and i x SSC for 1 h at 40 ° C and dehydrated in graded ethanol. Hybridized preparations were autoradiographed with NTB 2 nuclear track emulsion (Eastman Kodak) diluted 1 : 1 with distilled water. After exposure for a week at 4° C, slides were developed in Dektol (Kodak Rochester, NY, USA) developer at 15°C for
4 rain, rinsed in distilled water, fixed for 5 min, soaked in distilled water for 5 min and air-dried. Slides were counterstained with Mayer's hematoxylin and eosin. Control slides included the following. (1) A normal pancreas and a carcinoid tumor section which had previously been shown by immunohistochemistry to contain both chromogranin A and B. (2) The positive tissues were treated with RNase before hybridization in order to see that the probe hybridized with cellular RNA. (3) The hybridization was carried out with addition of 100 times excess cold oligonucleotides for chromogranin A or chromogranin B, respectively, to see that the signals were diminished by the competition for the target mRNA. (4) The tissues which were negative for any of the chromogranins were checked for the presence of cellular mRNA by hybridizing with the RNA probe for human fl-actin (Funa et al. 1987). (5) Finally, a few tissues which were positive for both chromogranin A and B were hybridized with labeled sense oligonucleotide probe for each mRNA. Semiquantitative evaluation of the results was performed independently by two of the authors.
Immunohistochemistry and Grimelius' argyrophil staining The tissue sections were deparaffinized, rehydrated and treated with peroxide in methanol to inactivate endogenous peroxidase. Immunoperoxidase staining was performed using Vectastain ABC kit (Vector Laboratories, Burlingame, Calif., USA) according to the vendor's description and aminoethylcarbazole as chromogen. A mouse monoclonal antibody against chromogranin A (Lloyd and Wilson 1983) and a rabbit polyclonal antiserum which recognizes both chromogranin A and B (Eriksson et al. 1990) were used. In each assay, normal goat serum used for blocking or normal rabbit serum were substituted for primary antibodies as a negative control. All the sections were stained for argyrophil reaction according to Grimelius (Grimelius and Wilander 1980).
Results T h e expression o f c h r o m o g r a n i n s A a n d B m R N A s a n d the c o r r e s p o n d i n g p r o t e i n expressions in the c a r c i n o i d t u m o r tissues are s u m m a r i z e d in Table 1. A l l m i d - g u t c a r c i n o i d t u m o r s (15 cases) expressed m R N A s for b o t h c h r o m o g r a n i n A a n d B (Fig. 1). In one case, I p r i m a r y intestinal t u m o r tissue, 5 r e g i o n a l m e t a s t a s e s as well as 1 o m e n t u m m e t a s t a s i s were e x a m i n e d ( p a t i e n t 27). A l l these tissues s h o w e d s t r o n g signals with m i n o r differences. T h e b i o p s y m a t e r i a l s f r o m liver m e t a s t a s e s (patients 19-25) t a k e n f r o m the p a t i e n t s b e f o r e s t a r t o f I F N t h e r a p y s h o w e d s t r o n g signals for b o t h m R N A species. T h e c h r o m o g r a n i n A m R N A e x p r e s s i o n was c o m p a r e d with the c o r r e s p o n d i n g b i o p s y specimens after I F N thera p y (patients 19-22). A l l f o u r p a t i e n t s s h o w e d r e d u c e d e x p r e s s i o n o f c h r o m o g r a n i n A m R N A o n the liver metastasis o b t a i n e d after I F N t h e r a p y (Fig. 2, Table 1). O n l y 12 o f 18 e x a m i n e d cases o f b r o n c h i a l c a r c i n o i d a n d 1 o f 5 rectal c a r c i n o i d t u m o r s s h o w e d b o t h types o f c h r o m o g r a n i n m R N A . Two b r o n c h i a l c a r c i n o i d (patients 7, 13) a n d one rectal c a r c i n o i d ( p a t i e n t s 3 5 ) s h o w e d o n l y c h r o m o g r a n i n B m R N A , b u t these b r o n chial c a r c i n o i d s (patients 7, 13) s h o w e d i m m u n o r e a c t i v i ty for c h r o m o g r a n i n A. In a n o t h e r two cases o f b r o n c h i al c a r c i n o i d s (patients 15, 17), c h r o m o g r a n i n m R N A (especially c h r o m o g r a n i n A ) was expressed in all t u m o r cells b u t o n l y a focal staining was o b t a i n e d b y i m m u n o h i s t o c h e m i s t r y with b o t h a n t i b o d i e s (Fig. 3).
557 1. Results of in situ hybridization, immunohistochemistry for chromogranin A and B mRNA, and protein in carcinoid tumors, compared with Grimelius' argyrophil reaction
Table
Patient In situ hybridization Immunohistochemistry Grimelius' stailling ChrA ChrB ChrA ChrA + B A. Bronchial carcinoid tumors 1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 t6 17 18
-
. + + ++ ++ -+ + + + + ++ + + +
-
.
. + + ++ ++ + + + + ± ± + + + + ND
-
ND
+ + ++ + + + + + + ± + + ± + ±
+ + ++ + + + ± + + + + ++ + ± ±
ND + ++ ++ ND ++ ++ _+ ++ ++ ++ ND ++ + ++ ++ ++
+
+
+
ND ND ND ND ND ND ND ++ + + + + + + +
ND ND ND ND ND ND ND ++ + ++ + ++ + + +
++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++
ND
± ± ± ± ND
++ ++ ± ++
.
+
+
B. Mid-gut carcinoid tumors
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
++(+)"+ + + +(+) + + + +(+) + + + + ( + ) ND ++ ++ + + ++ ++ ++ ++ ++ ++ + + ++ ++ ++ ++ + + + + + ++
C. Rectal carcinoid tumors
34 35 36 37 38
++
+ -+
"Tissue materials taken after IFN treatment stated within parentheses. Comparison is semiquantitative and not aimed to he performed between different probes or antibodies + + Strong hybridization signals (immunoreaction) on all tumor cells + Positive hybridization signals (immunoreaction) on tumor cells +_ Weak and/or focal hybridization signals (immunoreaction) on tumor cells - No hybridization signals (immunoreaction) ND Not Determined For Grimelius' staining:
+ + More than half of the tumor cells positive + Less than half of the tumor cells positive -t- Rare positive cells No positive cells ND Not Determined
No tumor tissue expressed only chromogranin A m R N A . Normal stromal cells adjacent to tumor cells did not express any of these m R N A s , except neuronal cells in a ganglion which showed chromogranin B m R N A (Fig. 4) and also chromogranin A m R N A at low level (not shown). Normal endocrine ceils, but not exocrine cells in pancreas, expressed the two types of chromogranin m R N A s (not shown). No normal intestinal mucosal cell expressed clear distinct signals for chromogranin m R N A , whereas a few scattered cells expressed the corresponding proteins when determined by immunohistochemistry (not shown). Treatment of the tissue section with RNase before hybridization reduced the signals greatly, indicating that the probe hybridized with cellular R N A . Hybridization with addition of 100 times excess cold oligonucleotides for chromogranin A or B resulted in reduction of the signals by competition for the target m R N A (not shown). Two tissues which were positive for both chromogranin A and B m R N A (patients 15, 26) did not hybridize with sense oligonucleotide probes (not shown). All but one carcinoid tumor tissues were shown to be positive for Grimelius' argyrophil staining. Mid-gut carcinoid tumor were uniformly positive, while in 3 bronchial and 1 rectal carcinoid tumor, only less than half of the tumor cells were positive and 1 rectal carcinoid tumor was completely negative for the reaction (Table 1). This tumor was also negative for m R N A of chromogranin A and B.
Discussion
The carcinoid tumors examined showed an almost complete concordance between chromogranin A and B m R N A expression. The expression of m R N A and protein of chromogranin A also displayed almost complete concordance, except in three cases of bronchial carcinoid tumors where the protein had been detected by immunohistochemistry, but no m R N A having been shown. This might be due to difference in sensitivity, since a single normal mucosal endocrine cell was shown to contain chromogranin immunoreactivity but not distinct hybridization signals for m R N A . Length of half-life and stability of the molecules may also account for the differences. However, in two cases of bronchial carcinoid tumors, the m R N A expression was stronger as well as more homogeneously spread than the protein expression. Immunohistochemistry showed only focal staining in small areas. The reason of the discrepancy is unclear. Chromogranin A and B may function as precursors for biologically active substances and might undergo enzymatic cleavage into smaller molecules. Thus, antigen determinants recognized by these antibodies m a y not be present in the tissue, whereas m R N A s for the precursors was recognized by oligonucleotide probes. However, the high concordance of these two methods indicates that they complement each other quite well in localizing the gene products and proteins in the tissues. We have found that chromogranin A m R N A expression in the mid-gut carcinoids was weaker after initiation
558
Fig. 1 A-D, Microphotographs of mid-gut carcinoid tumor tissue (patient 30). (A) Hybridization with probe against chromogranin A mRNA. (B) A section hybridized with a probe against chromogranin B mRNA. (C) Chromogranin A immunoreactivity with a monoclonal antibody. (D) Immunoreactivity against chromogranin A and B with a polyclonal antiserum in the corresponding area (magnification x 270) Fig. 2 A, B. Microphotographs of liver biopsies from mid-gut carciholds hybridized with chromogranin A probe (patient 22). (A) Tissue section obtained before IFN therapy. (B) Tissue section obtained after IFN therapy (magnification x 270). A few groups of tumor cells are present in fibrotic tissue
Fig. 3 A-D, Microphotographs of bronchial carcinoids (patient 15). (A) Hybridization with probe against chromogranin A mRNA. (B) Immunoreactivity against chromogranin A and B with a polyclonal antiserum in the corresponding area (magnification x 270). (C) An adjacent group of tumor cells expressing the same level of chromogranin A mRNA. (D) The corresponding area showing very weak immunoreactivity with a polyclonal antiserum (magnification x 270) Fig. 4. Microphotograph of neuronaI cells in an intestinal wall ganglion hybridized with chromogranin B probe (magnification x 270)
o f I F N treatment, c o m p a r e d with that before therapy. This finding indicates that I F N might inhibit the hormone production at the transcription level in the t u m o r cells (De Maeyer et al. 1988). This might explain our previous observation of a rather rapid decrease of circulating h o r m o n e levels after IFN-c~ therapy (Oberg et al. 1983).
All mid-gut carcinoid tumors expressed both types of chromogranins, whereas 80% o f bronchial and only 40% of rectal carcinoid tumors expressed one or both m R N A s . Cases of mid-gut carcinoid tumors are also reported to present the highest circulating chromogranin A and B levels (Eriksson et al. 1990). The same tendency was seen with the argyrophil reaction, where most o f
559 the m i d - g u t t u m o r cells were u n i f o r m l y stained, while c o n s i d e r a b l e v a r i a t i o n o f r e a c t i v i t y was seen in s o m e o f the b r o n c h i a l a n d rectal c a r c i n o i d t u m o r cells. M i d - g u t c a r c i n o i d s are h i g h l y d i f f e r e n t i a t e d n e u r o e n docrine tumors with homogenous pattern of hormone p r o d u c t i o n , g r o w t h rate a n d clinical course. R e c t a l carcin o i d t u m o r s , h o w e v e r , r e p r e s e n t a m i x t u r e o f t u m o r subtypes w i t h v a r y i n g degrees o f n e u r o e n d o c r i n e differentiation, a n d they u s u a l l y store m u c h less s e r o t o n i n c o m p a r e d w i t h m i d - g u t c a r c i n o i d ( F i o c c a et al. 1980). It is k n o w n t h a t c h r o m o g r a n i n s f o r m the m a j o r m a t r i x substance o f m a n y different t y p e s o f dense core granules, w h i c h is a c r y t o p l a s m i c s t o r a g e site o f the specific p e p tides a n d b i o g e n i c a m i n e p r o d u c t s o f n e u r o e n d o c r i n e cells. It is also p o s s i b l e t h a t the m a i n p r o t e i n m a t r i x o f the s e c r e t o r y g r a n u l e s o f rectal c a r c i n o i d s consists o f p r o t e i n c o m p o n e n t s t h a t are still unidentified. I n c o n c l u s i o n , the m R N A for c h r o m o g r a n i n A a n d B are g o o d m a r k e r s for c a r c i n o i d t u m o r s , especially those o f m i d - g u t origin. F o r e - g u t , m i d - g u t a n d rectal c a r c i n o i d t u m o r s are different in their n e u r o e n d o c r i n e d i f f e r e n t i a t i o n a n d p r o p e r t i e s r e g a r d i n g the e x p r e s s i o n o f the different c h r o m o g r a n i n s .
Acknowledgements. We thank Mrs. A.-C. Mel6n and Mrs. A. Nagy for skilful technical assistance and Mr. S. Kvist at the Ludwig Institute for Cancer Research in Stockholm for synthesis of the oligonucleotides. This work was partly supported by Swedish Medical Council and Swedish Cancer Society. References Bel/tk K, Funa K, Kelly R, Belfik S (1989) Rapid diagnosis of Aujeszky's disease in pigs by improved in situ hybridization using biotinylated probes on paraffin-embedded tissue sections, J Vet Med B36:10-20 Benedum UM, Baeuele PA, Konecki DS, Frank R, Powell J, Mallet J, Huttner WB (1986) The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells. EMBO J 5:1495-1502 Benedum UM, Lamouroux A, Konecki DS, Rosa P, Hille A, Baeuerle PA, Frank R, Lottspeich F, Mallet J, Huttner WB (1987) The primary structure of human secretogranin I (chromogranin B) : comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region. EMBO J 6:1203-1211 De Maeyer E, De Maeyer-Guignard J (t988) The effects of interferons on cell growth and division. In: De Maeyer E, De MaeyerGuignard J (eds) Interferons and other regulatory cytokines. Wiley, New York, 134-153 Eriksson B, Arnberg H, (Dberg K, Hellman U, Lundqvist G, Wern-
stedt C, Wilander E (1990) A polyclonal antiserum against chromogranin A and B - a new sensitive marker for neuroendocrine tumors. Acta Endocrinol 122:145-155 Fiocca R, Capella C, Buffa R, Fontana P, Solcia E, Hage E, Chance RE, Moody RL (1980) Glucagon-, glicentin- and pancreatic polypeptide-like immunoreactivities in rectal carcinoids and related colorectal cells. Am J Pathol 100:81-92 Fisher-Colbrie R, Frischenschlager I (1985) Immunological characterization of secretory proteins of chromaffin granules: chromogranins A, chromogranins B and enkephalin-containing peptides. J Neurochem 44:1854-1861 Fischer-Colbrie R, Iacangelo A, Eiden LE (1988) Neural and humoral factors separately regulate neuropeptide Y, enkephalin and chromogranin A and B mRNA levels in rat adrenal medulla. Proc Natl Acad Sci USA 85 : 3240-3244 Funa K, Steinholtz L, N6u E, Bergh J (1987) Increased expression of N-myc in human small cell lung cancer biopsies predicts lack of response to chemotherapy and poor prognosis. Am J Clin Pathol 88:216-220 Grimelius L, Wilander E (1980) Silver strains in the study of endocrine cells of the gut and pancreas. Invest Cell Pathol 3:2-12 Icangelo A, Affolter HU, Eiden LE, Herbert E, Grimes M (1986) Bovine chromogranin A: its sequence and the distribution of its messenger RNA in endocrine tissues. Nature 323 : 82-86 Kilpatrick L, Gavine F, Apps D, Phillips J (1983) Biosynthetic relationship between the major matrix proteins of adrenal chromaffin granules. FEBS Lett 164:383-388 Konecki DS, Benedum UM, Gerdes H-H, Huttner WB (1987) The primary structure of human chromogranin A and pancreastatin. J Biol Chem 262:17026-17030 Linnoila RI, Mulshine JL, Steinberg SM, Funa K, Matthews M J, Cotelingam JD, Gazdar AF (1988) Neuroendocrine differentiation in endocrine and nonendocrine lung carcinomas. Am J Clin Pathol 90 : 641-652 Lloyd RV, Wilson BS (1983) Specific endocrine tissue marker defined by a monoclonal antibody. Science 222 : 628-630 Lloyd RV, Iacangelo A, Eiden LE, Cano M, Jin L, Grimes M (1989) Chromogranin A and B messenger ribonucleic acids in pituitary and other normal and neoplastic human endocrine tissues. Lab Invest 60:548-556 Oberg K, Funa K, Alto G (1983) Effects of leukocyte interferon on clinical symptoms and hormone levels in patients with midgut carcinoid tumors and carcinoid syndrome. N Engl J Med 309:129-133 Schmid KW, Weiler R, Xu RW, Hogue-Angeletti R, Fischer-Colbrie R, Winkler H (1989) An immunological study on chromogranin A and B in human endocrine and nervous tissues. Histochem J 21:365-373 Simon J-P, Aunis D (1989) Biochemistry of the chromogranin A protein family. Biochem J 262 : 1-13 Smiths AD, Winkler H (1967) Purification and properties of an acidic protein from chromaffin granules of bovine adrenal medulla. Biochem J 103:483-492 Winkler H, Apps DK, Fisher-Colbrie R (1986) The molecular function of adrenal chromaffin granules : established facts and unresolved topics. Neuroscience 18 : 261-290
