supplied by V. Mutt (Stockholm, Sweden), synthetic secretin. (porcine) by E. .... zero was taken as. 100%. Table 1. Effect ofa phosphodiesterase inhibitor (iBuMeXan) on .... (Table 2). The addition of a fl-adrenergic antagonist, pro- pranolol .... Freychet, P., Rosselin, G., Rancon, F., Fouchereau, M. & Broer,. Y. (1974) Horm.
Proc. Natl. Acad. S$c. USA Vol. 75, No. 6, pp. 2772-2775, June 1978
Cell Biology
Vasoactive intestinal peptide: A potent stimulator of adenosine 3':5'-cyclic monophosphate accumulation in gut carcinoma cell lines in culture* (hormone action/receptor/prostaglandin/catecholamine/malignant digestive cell)
M. LABURTHEt, M. ROUSSETt, C. BOISSARDt, G. CHEVALIERt, A. ZWEIBAUMt, AND G. ROSSELINt tUnit6 de Recherche de Piab6tologie et d'Etudes Radio-Immunologiques des Hormones Proteiques (INSERM, U.55-CNRS, ERA 494), H6pital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France; and *Groupe de Recherche sur l'Immunologie de la Diff6renciation (INSERM, U. 178), Hopital Broussais, 96 Rue Didot, 75674 Paris Cedex 14, France
Communicated by Rosalyn S. Yalow, March 1, 1978
ABSTRACT Vasoactive intestinal peptide (VIP) is a potent and efficient stimulator of adenosine 3':5'-cyclic monophosphate (cAMP) accumulation in a human colon carcinoma cell line, HT 29. cAMP accumulation is sensitive to a concentration of VIP as low as 3 X 10-12 M. Maximum VIP-induced cAMP levels were observed with 10-9 M VIP and are about 200 times above the basal levels. Half-maximum cAMP production was obtained at 3 X 10-1° M VIP. 125I-Labeled VIP was found to bind to HT 29 cells; this binding was competitively inhibited by concentrations of unlabeled VIP between 10-10 and 10-7 M. Half-maximum inhibition of binding was observed with 2 X 10-9 M VIP. Secretin also stimnulated cAMP accumulation in HT 29 cells, but its effectiveness was 1/1000 that of VIP. The other peptides tested at 10-7 M, s6ch as insulin, glucagon, bovine pancreatic polypeptide, somatostatin, oetapeptide of cholecystokinin, neurotensin, and substance P, did not stimulate cAMP accumulation. Prostaglandin El and catecholamines stimulated cAMP production but were 1/2.3 and 1/5.5 as efficient as VIP, respectively. Another malignant cell line from the gut, the human rectal tumor cell line HBRT 18, is also sensitive to VIP. In HRT 18 cells, VIP stimulated cAMP accumulation with a maximal effect at .10-8 M; half-maximum stimulation was observed at about 10-9 M. These, results demonstrate the presence of VIP receptors in two malignant human intestinal cell lines (UT 29 and HRT 18) in culture and provide a unique model for studying the action of VIP on cell proliferation. Vasoactive intestinal peptide (VIP) was initially isolated from the gut (1) and later found in the central and peripheral nervous systems (2, 3). Its biological effects seem to be mediated via adenosine 3':5'-cyclic monophosphate (cAMP) production: VIP stimulates adenylate cyclase in plasma membrane preparations from fat cells (4),' liver (4, 5), and exocrine pancreas (6), and also increases cAMP accumulation in adipose cells (7), pancreatic acinar cells (8), and epithelial cells from intestinal mucosa (9). Several findings recently reviewed (10) suggest the existence of a relationship between proliferation and transformation of cells and a modification of their cAMP content. Many tumors or cultured cells responded to catecholamines and/or prostaglandins (11), but few of them have been shown to have an increased cAMP content upon polypeptide hormone treatment: some adrenal tumors respond to adrenocorticotropin (12), melanoma cells to melanocyte-stimulating hormone (13), hepatoma cells in stationary culture to glucagon (14), and differentiated thyroid carcinoma to thyroid-stimulating hormone (15). This paper presents evidence that a malignant cell line (HT 29) derived from a human colon carcinoma (16) exhibits considerable sensitivity to vasoactive intestinal peptide.
MATERIALS AND METHODS Hormones and Chemicals. Porcine VIP was generously supplied by V. Mutt (Stockholm, Sweden), synthetic secretin (porcine) by E. Wunsch (Munich, West Germany), synthetic octapeptide of cholecystokinin by M. Ondetti (Squibb, United States), and bovine pancreatic polypeptide by R. E. Chance (Lilly, United States). Porcine insulin and glucagon were obtained from Novo, synthetic neurotensin and substance P (bovine) from Beckman, and synthetic somatostatin from Serono (West Germany). Isoproterenol, epinephrine, norepinephrine, propranolol, 3-isobutyl-1-methylxanthine (iBuMeXan), and prostaglandins El and E2 were purchased from Sigma Chemical Company. Other prostaglandins were gifts of F. Dray (Institut Pasteur, Paris). Cell Culture and Preparation. The human colon carcinoma cell line HT 29 was established in 1964 (16) by J. Fogh (Sloan Kettering Institute, NY). The cells were routinely grown in plastic flasks (Corning) at 370, in an atmosphere of 90% air/10% CO2. The culture medium was Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Gibco). Cells were cultured for a week in 75-cm2 flasks until they reached 75-80% of confluence. They were then harvested with 0.05% trypsin (Gibco 1:250 lot 859211)/0.53 mM EDTA in phosphate-buffered saline (0.13 M) Ca2+ and Mg2+ free, pH 7.2. For experimental studies, 3-4 X 106 cells were transfered into 150-cm2 flasks in 30 ml of culture medium. The medium was changed on day 4, and 3 days later, cells were collected with the buffer described above, without trypsin. Under such conditions, the cells were in exponential growth phase. The average yield was 30 X 106 cells per flask. The cells were washed three times in Krebs-Ringer phosphate buffer, pH 7.4, containing 2% (wt/vol) bovine serum albumin (Fraction V, Pentex) for further studies. Cell viability, as tested by their ability to exclude trypan blue, was 90%. HT 29 cells were free of contamination with mycoplasma, as determined by autoradiography after incubation with [3H]thymidine. The cells retained the same number of chromosomes and had the same blood group A marker as initially described in ref. 16. Tumors induced into nude mice showed the morphological characteristics of a differentiated colon carcinoma and exhibited the colon polymorphic antigen WZ (17). Analytical Procedure. cAMP was measured by the raAbbreviations: VIP, vasoactive intestinal peptide; 125I-VIP, 125I-labeled VIP; cAMP, adenosine 3':5'-cyclic monophosphate; iBuMeXan, 3
isobutyl-1-methylxanthine.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisemwnt" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
* Presented in part at the 11th Annual Meeting of the Federation of European Biochemical Societies, Copenhagen, Denmark, August 15-20, 1977.
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Proc. Natl. Acad. Sci. USA 75 (1978)
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Table 1. Effect of a phosphodiesterase inhibitor (iBuMeXan) on basal and VIP-induced cAMP accumulation in HT 29 cells*
VIP, nM 0 3
iBuMeXan, mM 0
0.1
0.2
0.5
1.0
1.5 0.6 3.8 0.1 378 34 913 15
6.4 0.2 8.0 1.0 7.0 0.2 927 ± 14 1083 87 1045 51 All results are expressed as pmol of cAMP/106 cells. Each value is the mean + SEM of triplicate determinations. * Cells were incubated at 150 for 30 min as described in the legend to
Fig. 1.
Time, min FIG. 1. Time course of cAMP accumulation in HT 29 cells after stimulation by VIP, 3 X 10-9 M (Left) or 10-11 M (Right) at 15° (@*-) and 300 (O -0). Cells (2.5 X 106/ml) were incubated under continuous agitation (150 oscillations/min) in 500 ,l of Krebs-Ringer phosphate buffer containing 2% (wt/vol) bovine serum albumin and 0.2 mM iBuMeXan at pH 7.5. VIP was added at time zero, after 10 min preincubation. At the end of incubation, 2.5 ml of methanol were added to stop the reaction process and extract cAMP (20). Basal levels of cAMP are represented by black squares (15°) and open squares (30°) on the right, and symbolized by a dotted line on the left. ----
dioimmunological method (18) by technique of Steiner et al. (19) with slight modifications (20). Binding studies were carried out with VIP labeled with 125I (125I-VIP) at a specific activity of 300 Ci/g (average 0.5 atom of iodine per molecule) as reported elsewhere (4, 21). Binding of 125I-VIP to cells was measured by the method previously described in this laboratory for the binding of labeled insulin and glucagon to rat liver cells (22). Data are reported as specific binding; this is obtained by subtracting from the total that amount of labeled peptide that was not displaced by an excess of native peptide (4 ,gM). The integrity of 125I-VIP in the incubation medium after exposure to HT 29 cells was tested by its ability to bind to liver plasma membranes as described (23).
0
0
~0 C
:3 0 .0
50
6 a.
-
.
|
3`0 1 2 3 Time, hr FIG. 2. Time course of inactivation of 125I-VIP exposed to HT 29 cells. 125I-VIP was incubated at concentrations of 3 X 10-9 M (Left) or 10-11 M (Right) at 150 (@-*) and 300 (O -0) under conditions indicated in Fig. 1. At the end of the incubation the reaction was stopped by transferring 200-Ml aliquots of the incubation medium into 200 Ml of ice-cold Krebs-Ringer phosphate buffer containing 1% (wt/vol) bovine serum albumin. After 5 min of centrifugation in a Beckman microfuge, aliquots of the supernatant were taken at the indicated time points. The integrity of 125I-VIP after exposure to cells was tested by its ability to bind to liver plasma membranes (23): 350-Ml aliquots of the same supernatant were incubated for 30 min at 300 with 150 Ml of Krebs-Ringer phosphate containing 2% (wt/vol) bovine serum albumin, 200 ;Lg of bacitracin, and 120 Mg of liver plasma membrane proteins. Binding of 125I-VIP at time zero was taken as 100%. 0
1
2
-
----
RESULTS Interaction of VIP with HT 29 Cells. VIP elicited a considerable accumulation of cAMP in HT 29 cells under all experimental conditions tested. The rate and the extent of cAMP accumulation were dependent on time, temperature, and VIP concentration. The time course of cAMP accumulation was studied at 15° and 300 for two concentrations of VIP, in the presence of a phosphodiesterase inhibitor (Fig. 1). At low, 10-11 M (Fig. 1, right), and at high, 3 X 10`9 M (Fig. 1, left), VIP concentrations, the initial rate of cAMP accumulation was higher at 300 than at 15°. At 10-1" M VIP, the maximal cAMP level reached was higher at 15° than at 300, whereas it was the reverse at 3 X 10-9 M VIP. These observations may reflect differences between the two concentrations of VIP in the temperature dependence of VIP degradation. Indeed, it was observed (Fig. 2) that the inactivation of VIl exposed to HT 29 cells was greater at 10-11 M VIP (Fig. 2, right) than at 3 X 10-9 M VIP (Fig. 2, left) and was also greater at 300 than at 150. The extent of cAMP accumulation in HT 29 cells was dependent on the presence of phosphodiesterase inhibitor. Maximum basal and VIP-induced cAMP levels were observed from 0.2 mM to 1 mM iBuMeXan (Table 1), but cAMP accumulation was also strongly stimulated when the inhibitor was omitted. The dose-effect of VIP in HT 29 cells was studied at 15° in order to reduce the inactivation of VIP during the incubation and in the presence of phosphodiesterase inhibitor to amplify the cAMP accumulation. VIP at 3 X 10-12 M significantly (P < 0.01) stimulated cAMP accumulation above basal level (Fig. 3, inset). Maximal stimulation (about 200 times basal levels) was observed with 10-9 M VIP (Fig. 3). Half-maximal stimulation was elicited by 3 X 10-10 M VIP. Thus, VIP appeared to be a very potent and efficient agent in stimulating the accumulation of cAMP in HT 29 cells. The extent of VIP-induced increase in cAMP levels was considerable and the sensitivity to VIP very high in HT 29 cells as compared to other cellular systems in which receptors to VIP had been demonstrated, i.e., epithelial intestinal cells (9), pancreatic acinar cells (8), adipose cells (7), and liver cells (23). In HT 29 cells we have demonstrated the presence of receptors for VIP (Fig. 4). Indeed, the binding of 125I-VIP to HT 29 cells was competitively inhibited by native VIP in the concentration range of 10-10-10-7 M. Half-inhibition of the initial binding of 125I-VIP (i.e., in the absence of native VIP) was observed with 2 X 10-9 M native VIP, indicating the high affinity of VIP to HT 29 cells. Experiments that are not shown here indicated that among the different substances tested, only secretin competed with 125I-VIP in binding to HT 29 cells (23). However, to obtain the competitive displacement of 50% of bound 125I-VIP, 1000 times as much secretin was needed than VIP. Effect of Other Substances on cAMP Accumulation in HT 29 Cells. Other peptides isolated from the gastroenteropancreatic system have been tested with respect to their effect on
Cell Biology: Laburthe et al.
2774
Proc. Natl. Acad. Sci. USA 75 (1978) Table 2. Effect of various substances on cAMP accumulation in HT 29 cells*
Substances added None VIP Secretin 0
Concentration, cAMP accumulated, M pmol/106 cells 4.8 i 1.1
10-9
1075 + 30
10-9 10-8
4.9 + 0.8 8.6 + 0.3 100 3 326 31 916 i 23
1o-7
2 X 10-7
500
10-6
0
1o10-
10-11
10-9
10-8
VIP concentration, M
FIG. 3. Dose-effect of VIP on cAMP accumulation in HT 29 cells. Cells (2.5 X 106/ml) were incubated at 150 for 30 min. cAMP was determined as described in the legend to Fig. 1. The effect of very low concentrations of VIP is magnified in the Inset. Each point is the mean I SEM of triplicate determinations.
cAMP accumulation in HT 29 cells. Secretin was also effective in stimulating cAMP accumulation (Table 2). cAMP levels maximally induced by secretin were similar to those obtained with VIP; however, about 1000 times as much secretin as VIP was needed to obtain a similar stimulation of cAMP accumulation. Thus, dose-effect of cAMP production (Table 2), together 100
E
E .R
' 0~
50
_
10
Isoproterenol Isoproterenol + propranolol
10-5 10-5 l0-5
197 i 24 6.3 + 0.5
Epinephrine Epinephrine + propranolol
l0-5 l0-5 l0-5
56 i 3 7.8 + 2.4
Norepinephrine
l0-5
31 + 4
Prostaglandin El
0-5
475 + 17
Each value is the mean i SEM of triplicate determinations. * Cells were incubated at 150 for 30 min in the presence of 0.2 mM iBuMeXan, under conditions described in the legend to Fig. 1.
with the binding studies with secretin (23), suggested that this hormone acts through the VIP receptor. Native peptides, such as insulin, glucagon, or bovine pancreatic polypeptide, and synthetic peptides, such as somatostatin, the octapeptide of cholecystokinin-pancreozymin, neurotensin, and substance P, did not change cAMP concentration when tested at 10-7 M. Catecholamines stimulated cAMP accumulation in HT 29 cells (Table 2). The addition of a fl-adrenergic antagonist, propranolol, completely inhibited the effect of isoproterenol and epinephrine; furthermore the order of potency (isoproterenol > epinephrine > norepinephrine) indicated the presence of a (32 receptor (25). Among the prostaglandins tested (at 10-5 M), i.e., prostaglandin of the E (El, E2), A (Al, A2), B (B1, B2), and F (Fla, F2a) series, only E1 (shown in Table 2) was effective in stimulating cAMP accumulation. The effectiveness of prostaglandin E1 in stimulating cAMP production has been also described in normal (26) and tumoral (26, 27) human colon. Interaction of VIP with Another Malignant Intestinal Cell Line. VIP was thus the most potent and efficient substance for raising cAMP levels in HT 29 cells. Further investigations indicated that this human colon adenocarcinoma cell line was not the only cell line sensitive to VIP. Indeed, a human rectal tumor cell line (HRT 18) also exhibited a large increase of cAMP levels when stimulated by VIP (Fig. 5). VIP at 10-8 M elicited maximum stimulation of cAMP accumulation (i.e., about 20 times above basal level) in HRT 18 cells. Half-maximum stimulation was observed for about 10-9 M VIP. Thus, HRT 18 cells also respond to VIP, although the extent of response and their sensitivity to VIP appeared to be lower than in HT 29 cells.
3876152
VI P concentration, nM
FIG. 4. Competitive inhibition of the specific binding of 126I-VIP to HT 29 cells. 125I-VIP at 3 X 10-11 M and unlabeled VIP at the concentrations indicated were incubated for 3 hr at 150 in 500 Al of Krebs-Ringer phosphate buffer containing 2% (wt/vol) bovine serum albumin with 1.7 X 106 cells. Bound VIP was separated from free by centrifugation as described (24). Results are expressed as percent of maximum binding, i.e., the binding of 125I-VWP in the absence of native VIP. The nonspecific binding has been subtracted (see Materials and Methods). Initial binding was 39.7 1.2% of the total 125I-VIP.
DISCUSSION This work shows the presence of receptors for VIP in the cultured human colonic carcinoma cell line HT 29. Among the different compounds tested, VIP appears unique in its ability to stimulate a considerable production of cAMP in these cells. Rat epithelial intestinal cells and HT 29 are remarkably similar in several ways: they both have binding sites for VIP (9, 29); the hierarchy of the effectors in stimulating cAMP accumulation
Cell Biology: Laburthe et al.
Proc. Nati. Acad. Sci. USA 75 (1978)
2775
supported by the Institut National de la Sante et de la Recherche Medicale (Grant 77 15 31) and by the D6legation Generale a la Recherche Scientifique et Technique (Grant 77 7 0465). 1. Said, S. I. & Mutt, V. (1970) Science 169, 1217-1218. 2. Said, S. I. & Rosenberg, R. N. (1976) Science 192,907-908. 3. Larsson, L. I., Fahrenkrug, J., Schaffalitsky de Muckadell, O., Sundler, F., Hakanson, R. & Rehfeld, J. H. (1976) Proc. Natl. Acad. Sci. USA 73,3197-3200. 4. Bataille, D., Freychet, P. & Rosselin, G. (1974) Endocrinology
o 150
0
E
. 100
5.
50
6.
7. 50
8.
t
'
10-' lo-, i0-9 10-7 VIP concentration, M FIG. 5. Dose-effect of VIP on cAMP accumulation in human rectal tumor cells (HRT 18). Conditions of cell culture were in general as indicated under Materials and Methods for HT 29 cells. HRT 18 cells were free of contamination by mycoplasma, retained the same number of chromosomes, and had the same blood group H marker as initially described by Tompkins et al. (28). Tumors induced in nude mice showed the morphological characteristics of a differentiated rectal tumor and exhibited the colon polymorphic antigen WZ (17). Experiments with HRT 18 were conducted as described for HT 29 in Fig. 3. Each point is the mean SEM of triplicate determina0
:1
tions.
follows the same pattern in HT 29 cells and in enterocytes (29): VIP > secretin > prostaglandin > catecholamine; and compounds that are ineffective in HT 29 cells have no effect in enterocytes either (29). Thus, human HT 29 cells possess a cAMP stimulation pattern that in many respects resembles that of normal epithelial intestinal cells and is completely different
from that observed in other digestive cells, such as liver (30) and pancreatic acinar cells (8). The presence of VIP receptors in malignant intestinal cells is not a result of the transfer of cells into tissue culture since these receptors have also been characterized in human colon adenocarcinoma obtained after resection of the tumor (27). Thus, it may be suggested that VIP receptors are retained during the malignant transformation in vvo as well as during the transfer of cells into tissue culture. The sensitivity to VIP of the human rectal tumor cell line HRT 18 shows that the presence of VIP receptors in malignant intestinal cells is not exceptional. A variety of cells have been shown to be sensitive to arrest of growth by cAMP (see table 3 in ref. 10). Because VIP is the most potent and efficient effector of cAMP production in normal and malignant intestinal cells (HT 29) and because it is very abundant in the gut (2, 21), it will be of interest to investigate whether VIP regulates normal and malignant cell growth in gut mucosa. In that respect, malignant intestinal cells in culture, such as HT 29, provide a unique tool for studying the effect of VIP on cell proliferation. In addition to those who gave us hormones (Materials and Methods), we would like to thank Dr. A. Horvat for critical review of the manuscript, D. Hui Bon Hoa for her excellent technical assistance, and D. Lhenry for her careful preparation of the manuscript. This work was
9. 10.
11. 12. 13.
95,713-721. Desbuquois, B., Laudat, M. H. & Laudat, P. (1973) Biochem. Blophys. Res. Commun. 53, 1187-1194. Milutinovic, S., Schulz, L. & Rosselin, G. (1976) Biochim. Biophys. Acta 436, 113-127. Frandsen, E. W. & Moody, A. J. (1973) Horm. Metab. Res. 5, 196-199. Robberecht, P., Conlon, T. P. & Gardner, J. D. (1976) J. Blol. Chem. 251,4635-4639. Laburthe, M., Besson, J., Hui Bon Hoa, D. & Rosselin, G. (1977) C. R. Acad. Sci. Paris 284,2139-2142. Makman, M. H., Morris, S. A. & Ho Sam Ahn (1977) in Growth, Nutrition and Metabolism of Cells in Culture, eds. Rothblat, G. H. & Cristofalo, V. J. (Academic, New York), Vol. 3, pp. 295-34. Chlapowski, F. J., Kelly, L. A. & Butcher, R. W. (1975) Adv. Cyclic Nucleotide Res. 6, 245-338. Schimmer, B. R. (1972) J. Biol. Chem. 247,3134-3138. Pawelek, J., Wong, G., Sansone, M. & Morowitz, J. (1973) Yale J. Biol. Med. 46, 430-443.
14. Makman, M. H. (1971) Proc. Natl. Acad. Sci. USA 68, 21272130. 15. Abe, Y., Ichikawa, Y., Homma, M., Ito, K. & Mimura, T. (1977) Lancet ii, 506. 16. Fogh, J. & Trempe, G. (1975) in Human Tumor Cells In Vitro, ed. Fog, J. (Plenum, New York), pp. 115-159. 17. Zweibaum, A., Oriol, R., Dausset, J., Marcelli-Ilarge, A., Ropartz, S. & Lanset, S. (1975) Tissue Antigens 4, 121-128. 18. Yalow, R. S. & Berson, S. A. (1960) J. Clin. Invest. 39, 11571175. 19. Steiner, A. L., Pagliera, A. S., Chase, L. R. & Kipnis, D. M. (1972) J. Biol. Chem. 247,1106-1113. 20. Rosselin, G., Freychet, P., Fouchereau, M., Rangon, F. & Broer, Y. (1974) Horm. Metab. Res. 5,78-86. 21. Laburthe, M., Bataille, D. & Rosselin, G. (1977) Acta Endocrinol.
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22. Freychet, P., Rosselin, G., Rancon, F., Fouchereau, M. & Broer, Y. (1974) Horm. Metab. Res. 5,72-78. 23. Laburthe, M., Bataille, D., Rousset, M., Besson, J., Broer, Y., Zweibaum, A. & Rosselin, G. (1978) in Proceedings of the Membrane Proteins Section of the 11th FEBS Meeting, Copenhagen, eds., Nicholls, P., Moller, J. V., Leth, P. & Moody, A. J. (Pergamon, Oxford, England), Vol. 45, pp. 271-290. 24. Rodbell, M., Krans,M. J., Pohl, S. L. & Birnbaumer, L. (1971) J. Biol. Chem. 246, 1861-1871. 25. Lands, A. M., Arnold, A., Mc Auliff, J. P., Luderena, F. P. & Brown, T. G. (1967) Nature 214,597-598. 26. De Rubertis, F. R., Chayoth, R. & Field, J. B. (1976) J. Clin. Invest. 57, 641-649. 27. Dupont, C., Amiranoff, B., Laburthe, M. & Rosselin, G. (1978) C.R. Acad. Sci. Paris 286,209-212. 28. Tompkins, W. A. F., Watrach, A. M., Schnale, J. D., Schultz, R. M. & Harris, J. A. (1974) J. Natl. Cancer Inst. 52, 1101-1106. 29. Laburthe, M., Dupont, C., Besson, J. & Rosselin, G. (1978) Gastroenterol. Clin. Biol. 2, 219 (abstr.). 30. Sutherland, E. W. & Rall, T. W. (1957) J. Am. Chem. Soc. 79, 3608.
