Growth-regulatory effect of gastrin on human colon cancer cell lines is determined by protein kinase a isoform content

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Regulatory Peptides 53 (1994)61-70

Growth-regulatory effect of gastrin on human colon cancer cell lines is determined by protein kinase a isoform content R i c h a r d J. B o l d , S c o t t A l p a r d , J i n I s h i z u k a , C o u r t n e y M . T o w n s e n d , J r . * , J a m e s C. T h o m p s o n Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0533, USA Received 7 March 1994; revised version received 6 June 1994; accepted 14 June 1994

Abstract

Cell growth is regulated by various peptide growth factors through receptor-linked multiple intracellular signal-transduction pathways, such as the cyclic adenosine monophosphate (cAMP) pathway, cAMP activates cAMPdependent protein kinase A (PKA) either to stimulate or inhibit cell growth. The effect on growth is determined by the presence of two isoforms of the regulatory (R) subunit of PKA; activation of Rx~-type PKA leads to stimulation of growth, activation of Rixa-type inhibits cell growth. We determined whether the effect of gastrin on the growth of human colon cancer cells is determined by cell-specific content of PKA. We utilized two human colon cancer cell lines: LoVo, growth of which is stimulated by gastrin, and HCT116, growth of which is inhibited by gastrin. Activation of both types of PKA with 8-Br-cAMP mimicked the regulation of growth by gastrin; preferential activation of Rna-type PKA with 8-CI-cAMP inhibited growth of both cell lines. LoVo cells possess the predominantly R~ isoform of PKA at the mRNA and protein level; HCT116 cells possess predominantly the R~ia-type PKA. The cAMP-mediated regulation of growth (either stimulatory or inhibitory) by gastrin on these human colon cancer cells was determined by the predominant isoform of PKA. Key words: Cyclic AMP; Regulatory subunit; Colon cancer

1. Introduction

The effect of various peptide hormones on cell growth requires specific interaction with a cell surface receptor and subsequent transduction of an intracellular signal, or 'second message'. The produc* Corresponding author. Fax: + 1 (409) 7725611. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 5 4 - 2

tion of 3',5'-cyclic adenosine monophosphate (cAMP) by adenylyl cyclase has been shown to be an important regulatory agent in cell growth and differentiation [ 1-3]. We and others have reported that various gastrointestinal hormones are able either to stimulate or to inhibit the growth of different types of cells through activation of the cAMP pathway [4-8]. Delineation of the mechanism through which

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R.J. Bold et al. / Regulatory Peptides 53 (1994) 61-70

a single signal-transduction pathway, such as the cAMP pathway, either stimulates or inhibits growth of cells is critically important in order to further understand the mechanisms of cellular growth. ChoChung et al. [7,8], have reported that such dual effects on growth are respectively controlled by two isoforms of the cAMP-dependent protein kinase A (PKA), which is composed of a catalytic unit and either the type I or type II regulatory subunits (R h and Rn~). The activation of Rl~-type has been reported to correlate with stimulation of growth, while an increase in activity of the Rntrtype has reported to correlate with inhibition of growth [ 8-13 ]. However, both types of PKA may be present in the same cell; therefore, the growth response to activation of the cAMP pathway should be determined by the predominant isoform of PKA. We have previously shown that gastrin activates adenylyl cyclase to increase intracellular levels of cAMP in three human colon cancer cell lines: LoVo, COLO 320 and HCT116 [14]. We have also shown that gastrin stimulates the growth of LoVo and COLO 320 cells while inhibiting the growth of HCT116 cells [14,15]. This dual growth-regulatory effect of gastrin may be related to cell-specific content of the regulatory subunits of PKA. Two membrane-permeable cAMP analogs have been developed to allow for receptor-independent activation of each isoform of PKA. 8-bromo-cAMP (8-BrcAMP) is capable of activating both RI~- and RHtr type of PKA to the same extent and therefore simulating the effect of hormone-induced activation of the cAMP pathway [16-18]. 8-chloro-cAMP (8-ClcAMP) preferentially activates Rll/rtype PKA and therefore unmasks the intracellular content of this isoform if a significant amount of R~-type PKA exists [19-20]. Use of these two cAMP analogs in growth studies will allow for a physiologic determination of PKA content: if 8-Br-cAMP leads to stimulation while 8-CI-cAMP leads to inhibition, the predominant PKA isoform is R~-type; however, if both 8-Br-cAMP and 8-CI-cAMP lead to inhibition of growth, the predominant isoform is RH/rtype.

The genes for both types of the regulatory subunit have been identified and cloned, which will allow for analysis of cellular mRNA transcript content by Northern blot analysis [21,22]. Polyclonal antibodies have been developed which allow for determination of intracellular protein content utilizing Western blotting techniques. In this study, we compared the effects of gastrin, 8-Br-cAMP and 8-CI-cAMP on the growth of two human colon cancer cell lines, LoVo and HCT116. We have examined cellular mRNA transcript levels and isoform protein content of the RI~- and RH/r type subunits of PKA. We performed these studies in order to determine whether the effect ofgastrin on growth of these human colon cancer cell lines is determined by the cell-specific presence of the PKA isoform.

2. Materials and Methods 2. I. Cell culture

The tissue culture cell lines employed in this study were LoVo and HCT116 human colon cancer cells (American Type Culture Collection, Rockville, MD). LoVo cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco Laboratories, Grand Island, NY) supplemented with 5 ~o fetal calf serum (FCS; Hyclone Laboratories, Logan, UT). H C T l l 6 cells were cultured in McCoy's medium (Gibco) also containing 5 ~o FCS. Both cell cultures were maintained in a humidified incubator containing 95 ~o air and 5 °/o CO2 at 37 ° C. To avoid changes of cell characteristics produced by prolonged culture, only cells from specific passages were used for all studies (passage 25-35 for LoVo and passage 3-10 for HCTll6). 2.2. Measurement of cAMP levels

LoVo and HCT116 cells were cultured in 6-well dishes (5.10 5 cells/well) with medium containing

R.J. BoM et al. / Regulatory Peptides 53 (1994) 61-70

either 1~o FCS (LoVo) or no FCS (HCTll6) for 3 days. Cells were washed and incubated in 1 ml of oxygenated Krebs Ringer's bicarbonate buffer (KRBB, pH 7.4) containing 10 mM Hepes, 0.1 bovine serum albumin (BSA; RIA grade, Sigma Chemical Co., St. Louis, MO), 2.5 mM glucose, and 0.1 mM 3-isobutyl-l-methylxanthine (Sigma). After 5, 15 and 30 min incubation in the presence of synthetic human gastrin-17 (G-17; nonsulfated, Bachem, Torrance, CA), cells were extracted with 10~o trichloroacetic acid and neutralized with an excess of CaCO 3 [23]. After acetylation of samples with acetic anhydride and triethylamine (1:2, v/v), intracellular levels of cAMP were measured by radioimmunoassay (Amersham, Arlington Heights, IL).

2.3. Cell growth studies LoVo and HCT116 cells were cultured in 12-well plates (1.10 4 cells/well) using the appropriate culture medium supplemented with 5~o FCS. Two days later, medium was replaced with fresh medium containing either 1~ heat-inactivated FCS (HIFCS; heat-inactivation by heating to 54 °C for 30 min) for LoVo cells or 0.1 ~o HIFCS for HCT116 cells. G-17, 8-Br-cAMP (Sigma) or 8-CI-cAMP (a generous gift from Dr. Yoon Cho-Chung, National Institutes of Health, Bethesda, MD) was added at the time of medium change. Cells were collected from the culture medium, detached from dishes with trypsin (0.25~o, Gibco) and diluted 10-fold with Isoton ® (Curtin Matheson Scientific, Houston, TX) on the 2nd, 4 th, 6th or 8 TM day after addition of test agents. Cells were counted by a Coulter counter (Coulter Electronics, Inc., Hialeah, FL). Low serum conditions allowed for the detection of moderate changes in proliferative rate by prolonging the basal doubling time (approx. 2-fold longer than in 5~o FCS). HIFCS was used to inactivate enzymes that may be capable of producing toxic adenosine metabolites from the halogenated cAMP analogs [20].

63

2.4. Northern blot analysis for RI~ and RH¢ mRNA levels LoVo and HCT116 cells were cultured in 150 mm culture dishes (2.106 cells/dish) for 3 days in appropriate medium containing 5 ~o FCS. Total RNA was harvested using RNAzol B ® (Cinna/Biotec Laboratories, Inc., Houston, TX) and precipitated with isopropanol and subsequently washed with 75~o ethanol. Polyadenylated (poly(A) ÷ ) RNA was selected from the total RNA by oligo(dT) cellulose (Collaborative Research Inc., Bedford, MA) column chromatography. 20 /~g aliquots of mRNA were electrophoresed through a denaturing 1.25~o agarose gel. Size-fractionated mRNA was then transferred to a nitrocellulose membrane by capillary action. [32p]dATP (New England Nuclear Research Products, Boston, MA)-labeled probes (cDNA) were synthesized using the random-primer technique (Stratagene Cloning Systems, La Jolla, CA). Plasmids containing the cDNAs for the Rt~ and RIIa genes were kindly provided by Dr. Yoon Cho-Chung (National Institute of Health, Bethesda, MD). Complementary DNA probes corresponding to the gene cyclophilin (1B 15) were also synthesized using the random-primer technique. This gene is constitutively expressed in these cells and was used to control for RNA loading [24]. Hybridization and posthybridization washes were performed as described previously [25]. After the washes were completed, the membranes were blotted dry and exposed to XAR-5 X-ray film (Eastman Kodak, Rochester, NY) in the presence of an intensifying screen at -70°C. The hybridization signals on the blots were analyzed quantitatively using a Bio Image Visage 60 densitometer (Bio Image, Ann Arbor, MI).

2.5. Western blot analysis for Rl~ and RHt~ protein levels LoVo and HCT116 cells were cultured in 150 mm culture dishes (2.106 cells/dish) for 3 days in appro-

R.J. Bold et al. / Regulatoo' Peptides 53 (1994) 61-70

64

priated medium containing 5~o FCS. Cells were washed three times in ice-cold phosphate-buffered saline (Gibco) and gently harvested by scraping with a rubber policeman. The cells were pelleted and resuspended in 500 gl of buffer '10' (20 mM Tris-HC1, pH 7.4; 10 mM NaC1; 5 mM MgC12; 0.5~o sodium deoxycholate; 1~o NP-40; 0.1 mM phenylmethylsulfonyl fluoride; 0.5 mg/ml soybean trypsin inhibitor; 0.4 mg/ml aprotinin; 0.2 mM leupeptin). Cells were lysed by passing through a 20 gauge needle five times and subsequently centrifuged (15,000 rpm, 15 min at 4°C). The supernatant containing the soluble cytoplasmic proteins was collected and the protein concentration determined by the method of Lowry using BSA as a standard [26]. 100/~g of proteins are loaded onto a 10~o sodium dodecylsulfate-polyacrylamidegel (SDS-PAG) and separated by electrophoresis. Size-fractionated proteins were then transferred to a PVDF membrane (Bio-Rad, Hercules, CA) by electro-transfer [27]. When the transfer was complete, the membrane was

a LoVo 175

° t

50.

70

with IBMX (0.1 mM)

lO-

o~ E 125

E 100 o.

All growth studies, measurements of cAMP levels and Northern blot analyses were repeated at least three separate times. Growth studies and measurements of cAMP levels were performed using multiwell plates and groups of 6 wells were used for each point of analysis. Data from individual experiments

10 "12 M

A°

o

2.6. Statistical analysis

b HCT116

with IBMX (0.1 mM)

"~ 150

soaked in NTE-NP40 buffer (12 g Tris-HCl, pH 7.5; 17.5 g NaC1; 2 ml NP-40; 1.7 g Na-EDTA/I) and subsequently incubated in NTE-NP40 buffer containing 3~o BSA to block the nonspecific sites. A polyclonal rabbit antibody which recognizes both R I~ and Rn~ (1:1000 dilution, kindly provided by Dr. Stein Doskeland, University of Bergen, Bergen, Norway) was added [28]. Bound antibody was detected by adding [125I]-protein-A (New England Nuclear). The blot was washed with NTE-NP40 buffer and the radioactivity detected by autoradiography using XAR-5 X-ray film (Kodak).

0"7 M

I/~ V7

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"a1°'8

I " ~ Control 3 0 ~ - - - ~ - - , ~ - - - r - - r - ~ ~ G 10 -9 M 0 5 10 15 20 25 30 Time (rain)

Fig. 1. Levelof cAMP followingadministrationof various doses of G-17 (G) to LoVocells (a) or HCT116cells (b).

R.J. Bold et al. / Regulatory Peptides 53 (1994) 61-70

a

~

2.0 x

b

LoVo

2.2

1.8 1.6.

.Q

E

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65

8th day

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ol

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;on12 11 10 9 8 7 6

Days of Culture

Gastrin-17 (-log M)

Fig. 2. Growth curve (a) or cell number after 8th day of treatment (b) following administration of various doses of G-17 to LoVo cells. ( * P < 0 . 0 5 vs. control)

were analyzed by analysis of variance (ANOVA) and subsequent Newman-Keuls multiple regression analysis. Significance was assumed for a P value less than 0.05. Representative data from each study are shown as m e a n + s t a n d a r d error of the mean (S.E.M.).

3. Results G-17 increased intracellular levels of cAMP in LoVo and HCT116 cells in a nonlinear fashion (Fig. 1). The time of maximal stimulation of cAMP production by G-17 was different between these two

b

a

HCT116 35

8th day Control~

30 x

25 20

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Days of Culture

[ I

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Gastrin-17 (-log M)

Fig. 3. Growth curve (a) or cell number after 8th day of treatment (b) following administration of various doses of G-17 to H C T l l 6 cells. ( * P < 0 . 0 5 vs. control)

R.J. Bold et al. / Regulatory Peptides 53 (1994) 61-70

66

HCT116

LoVo .k -I-

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0.0 Fig. 4. Growth regulatory effect of 8-Br-cAMP (25 ~tM) AND 8-CI-cAMP (25/~M) on LoVo (left) or HCT116 (right) cells after 8 days of treatment. (*P<0.05 vs. control)

cell lines (5 min for H C T l l 6 cells and 15 min for LoVo cells). In LoVo cells, peak increases of cAMP levels by G-17 were observed at 10-12 M (66~/o increase) and 10 -8 M (61~o increase). In H C T l l 6 cells, peak increases by G-17 were observed at 10 - t0 M (43~o increase) and 10 - 6 M (76~o increase).

a

Growth of LoVo cells was significantly stimulated by G-17 in a nonlinear fashion (Fig. 2). In contrast, growth of H C T l l 6 cells was significantly inhibited by G-17 in a similar fashion (Fig. 3). Effective doses of G-17 were different between the two cell lines; however, the effective doses of cAMP production

b

LoVo 2,00-

HCT116

1110-

A

m o

1.75-

T

T

x

.,~ 1.50 Z

I

..T_ qr

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987,

0 8 Fig. 5. Dose dependent growth inhibition of8-CI-cAMP on LoVo (a)or HCTll6cells (b)after 8 days oftreatment. (*P<0.05 vs. control)

R.J. Bold et al. / Regulatory Peptides 53 (1994) 61-70

were also the doses which regulated cell growth. In LoVo cells, stimulation of growth by G-17 was observed at 10- 12 M (18~o increase compared to control) and 10 -8 M (24Fo increase). In H C T l l 6 cells, inhibition of growth by G-17 was observed at 10- 10 M (23~o decrease) and 10 - 6 M (36Fo decrease). Activation of both types of PKA with 8-Br-cAMP mimicked the effect ofgastrin on both cell lines; that is, 8-Br-cAMP stimulated the growth of LoVo cells while it inhibited the growth of H C T l l 6 cells (Fig. 4). Conversely, activation of the Rixtrtype PKA

¢O ,p,,

O > 0

..I

'P" I-0 -!-

67

with 8-CI-cAMP inhibited the growth of both cell lines (Fig. 4). The growth-inhibitory effect of 8-ClcAMP on HCT116 cells was similar to the inhibitory effect of either 8-Br-cAMP or G-17. Furthermore, the inhibition of growth by 8-CI-cAMP was dosedependent in both cell lines; however, 8-CI-cAMP was more potent in inhibiting growth of HCT116 cells (inhibition of growth at doses > 1 #M) than in LoVo cells (inhibition of growth at doses > 10 FM) (Fig. 5). Northern blot analysis showed that LoVo cells have more mRNA encoding the Ri~-type PKA than does H C T l l 6 (Fig. 6). Conversely, HCT116 cells possess more mRNA for the Rntrtype PKA than do LoVo cells (Fig. 6). It appears that the disparate effect of both G-17 and 8-Br-cAMP on growth is due to the relative amount of Rl~-type PKA when compared to the amount of Riltrtype PKA. The Western blot data for protein levels of both Ri~-type and R~itrtype PKA supports this conclusion (Fig. 7). Both cell lines have an approximately equal amount of the R]=-type subunit; however, HCT116

RI~

O > O

.-I

v= IO

"I-

R.I 3 4-- RIll3 (52 kDa) ~-- RI~ (48 kDa) L~

,,--1B15

Fig. 6. Northern blot analysis of R ~ and Rnt~ m RNA levels (1B 15 as loading control) in LoVo and HCT116 cells.

Fig. 7. Western blot analysis of RI= and Rnt~ protein levels in LoVo and HCT116 cells.

68

R.J. Bold et al. / Regulatory Peptides 53 (1994) 61-70

cells have much more RHtrtype subunit than does LoVo.

4. Discussion

We have demonstrated that G-17 stimulates production of cAMP in both LoVo and HCT116 human colon cancer cell lines. Furthermore, G-17 regulates growth of these two cell lines but with an opposite result; G-17 stimulates growth of LoVo cells while G-17 inhibits the growth of H C T l l 6 cells. The effect ofgastrin on both cAMP production and growth was biphasic with a mid-dose nadir. This is consistent with the recent report that the CCK-B/gastrin receptor may simultaneously exist in multiple affinity states within the same cell [29]. We have previously demonstrated that the effects of gastrin on cAMP production and growth-regulation can be specifically blocked by a CCK-B/gastrin receptor antagonist and therefore it appears that gastrin exerts its growth regulatory effect through receptorspecific activation of adenylyl cyclase and cAMP production [ 14]. Furthermore, the effect of gastrin on growth of these cells is modest (20-30 ~o change), though this is typical of the mitogenic effect observed on non-synchronous, non-quiescent cancer cells [301 One postulated mechanism for a dual effect of cAMP on growth of various types of cells has been at the level of PKA, which may exist in one of two isoforms [8-10,31]. The Ri~-type PKA will lead to stimulation of growth upon activation while the Rx~fftype PKA will inhibit growth when activated [9-11,32-34]. To investigate this possibility, we used analogs of cAMP which are either nonselective in activation of PKA (8-Br-cAMP) or selective in activation of only Rii~-type PKA (8-CI-cAMP). 8-Br-cAMP mimics the effect of G-17 on growth of both of these cell lines, demonstrating that regulation of the cAMP signal-transduction system is capable of altering the growth rate of these two cell lines. However, activation of only the Rn/rtype PKA with

8-CI-cAMP inhibits growth of both cell lines. These findings suggest that both cell types have the RH/r type isoform of PKA but, in LoVo cells, there must be much more R~-type PKA, because generalized increases of cAMP lead to stimulation of growth. That is, the stimulatory effect of the Rl~-type PKA overwhelms the inhibitory effect of the Rntrtype PKA. In HCT116 cells, the predominant isoform of PKA appears to be the R~-type, since generalized cAMP activation with 8-Br-cAMP yields the same inhibitory effect as does the selective RH~-type activation with 8-CI-cAMP. Findings with both Northern and Western blots confirm the conclusions that the regulatory effect of growth by activation of the cAMP signal-transduction system by G-17 is determined by the relative ratio of the two isoforms of PKA. Although the exact level of either mRNA or protein appears to vary between these two cell lines, HCT116 clearly has much more Rli~-type PKA compared to R~-type; LoVo cells have much more Rt~-type PKA when compared to Riitrtype. The disparate growth effects observed when the cAMP system is activated by G-17 may be explained by the relative proportion of the regulatory subunit of the cAMP-dependent protein kinase. Determination of the growth response to activation of the cAMP signal-transduction cannot be determined without specific examination of the PKA isoform content [35]. Selective activation of the Rntrtype PKA by 8-C1cAMP leads to inhibition of growth regardless of the predominant isoform of PKA present. Therefore, this compound may be useful in the development of new treatments of various neoplasms [36-39]. Furthermore, with the advent of novel gene therapy techniques, altering the PKA isoform content may also be a way to alter the growth rate of neoplastic cells [7,40-42]. Inhibition of growth of any cell type upon activation of the cAMP pathway may be achieved through either the introduction of additional Rntrtype PKA or the elimination of excessive R~-type PKA.

R.J. Bold et al. / Regulatory Peptides 53 (1994) 61-70

Acknowledgements We wish to thank Jell Hsieh and Chu-chi Wang for their technical assistance and Gene Dockal, Daryl Janes, Steve Schuenke, Karen Martin, and Bob Todd for their assistance in the preparation of this manuscript. This study was supported by grants from the American Cancer Society (CB-571), the National Institutes of Health (5R37 DK 15241, PO1 DK 35608) and the Walls Medical Foundation. References [ I] Bentley, J.K. and Beavo, J.A., Regulation and function of cyclic nucleotides, Curr. Opin. Cell. Biol., 4 (1992) 233-240. [ 2] Roesler, W.J., Vandenbark, G.R. and Hanson, R.W., Cyclic AMP and induction of eukaryotic gene transcription, J. Biol. Chem., 263 (1988) 9063-9066. [ 3] Hardie, D.G., Roles of protein kinases and phosphatases in signal transduction. In J. Roberts, C. Kirk and M. Venis (Eds.), Hormone Perception and Signal Transduction in Animals and Plants, Symposia of Society Experimental Biology, 44 (1990) 241-255. [ 4] lshizuka, J., Martinez, J., Townsend, C.M. Jr. and Thompson, J.C., The effect of gastrin on growth of human stomach cancer cells, Ann. Surgery, 215 (1992) 528-535. [ 5] Townsend, C.M. Jr., Ishizuka, J. and Thompson, J.C., Gastrin trophic effects on transplanted colon cancer cells, In J.H. Walsh (Ed.), Gastrin, New York, Raven Press, 1993, pp. 407-417. [ 6] Kim, S.W., Beauchamp, R.D., Townsend, C.M. Jr. and Thompson, J.C., Vasoactive intestinal polypeptide inhibits c-myc expression and growth of human gastric carcinoma cells, Surgery, 110 (1991) 270-276. [ 7] Cho-Chung, Y.S., Clair, T., Tortora, G., Yokoszki, H. and Pepe, S., Suppression of malignancy targeting the intracellular signal transducing proteins of cAMP: the use of siteselective cAMP analogs, antisense strategy, and gene transfer, Life Sci., 48 (1991) 1123-1132. [ 8] Cho-Chung, Y.S., Role of cyclic AMP receptor proteins ingrowth, differentiation, and suppression of malignancy: new approaches to therapy, Cancer Res., 50 (1990) 70937100. [ 9] Yasui, W., Sumiyoshi, H., Ochiai, A., Mikimasa, Y. and Tahara, E., Type I and II cyclic adenosine 3':5'monophoshate-dependent protein kinase in human gastric mucosa and carcinomas, Cancer Res., 45 (1985) 1565-1568. [10] Taylor, S.S., Knighton, D.R., Zheng, J., Eyck, L.F.T. and

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[21]

[22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

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R.J, Bold et al. ,' Regulatoo" Peptides 53 (1994) 61-70

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