Molecular and CellularEndocrinology, 14 (1979) 123- 130 0 Elsevier/North-Holland Scientific Publishers, Ltd.

123

INSULINBINDINGBYACELLLINE(HT 29)DERIVEDFROMHUMAN COLONICCANCER M.E. FORGUE-LAFITTE,

A. HORVAT * and G. ROSSELIN

Unit6 de Recherches de Diab&ologie et d’ktudes Radio-Immunologiques des Hormones Prot&ques, U.55 (Institut National de la Sank et de la Recherche Mhdicale), H6pital Saint-Antoine, 184, rue du Faubourg Saint-Antoine, 75571 Paris Cedex 120 (France) Received 10 October 1978; accepted 12 March 1979

Insulin binding was demonstrated in cultured HT 29 cells originating from a human colon carcinoma. At 37” and in complete medium, the binding of [1251]insulin (l-4 X lo-” M) reaches a maximum in 40 min and the cell associated radioactivity remains constant for at least 4 h. No degradation of the hormone is observed under these conditions. The binding is proportional to the number of cells and its pH optimum is 7.8. In the presence of excess insulin 50% of the [ ’ 251]insulin is dissociated from the complex after 10 min. At equilibrium, insulin binding is specific: proinsulin is 25 times less potent than native insulin in competing with [ 1251]insulin and related polypeptide hormones are inactive. Scatchard analysis indicates two classes of binding sites (1400 sites/cell of “high affinity” e.g. 4.7 X 10’ M-' , and 20 000 sites of “low affinity” e.g. 4 X 10’ M-l). The binding of insulin to this non-target cell shows the same kinetic characteristics and specificity as found for insulin in its target cells, except that HT 29 cells do not degrade the hormone. The problem of the correlation between insulin

binding and a biological effect in these cells remains to be elucidated. Keywords: receptors; insulin degradation; target cells; transformed cells; tissue culture.

Early studies of the interaction of hormones with receptors were limited to “target tissues” of the hormone. Thus, the biological effect of insulin was studied in fat, muscle, liver and mammary cells (Levine et al., 1950; Rodbell, 1964; Morgan et al., 1964; Rivera, 1964; Crofford et al., 1965; Jiirgens et al., 1965; Wagle et al., 1973). Similarly, the kinetics of binding of insulin were primarily determined in isolated cells or membranes from these tissues (Crofford et al., 1970; Freychet et al., 1971; Cuatrecasas, 1971; Kono and Barham, 1971; Hammond et al., 1972; Olefsky et al., 1974; Freychet et al., 1974; Forgue and Freychet, 1975). More recently, it has become evident that there is a wide variety of cells to which insulin can bind (Posner et al., 1974; Kahn, 1975; Roldan et al., 1976). The availability

* Present address: Department 44106 (U.S.A.).

of Medicine, Case Western Reserve University, Cleveland, Ohio

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A. Horvat, G. Rosselin

of cultures of intestinal cells (Fogh and Trempe, 1975), cells hitherto not considered to be targets for insulin, offered us a system to determine if specific insulin receptors exist on such cells and, if so, their possible significance. Insulin in the whole animal increases the conversion of glucose to glycogen, fatty acids and triglycerides and accelerates protein and RNA synthesis (Randle et al., 1966). In the diabetic rat, insulin restores a normal rate of growth (Younger et al., 1966). Similarly, insulin is a growth-promoting factor for tissues and cells in culture (Gey and Thalhimer, 1924;Temin, 1967; Baseman et al., 1974;Mandel and Pearson, 1974; Hollenberg and Cuatrecasas, 1975; Holley, 1975; Osborne et al., 1976; M&tone and Piatigorsky, 1977). However, there are only few reports on the kinetics and quantitative aspects of hormone-receptor interactions in tissue culture (Gavin et al., 1972; Hollenberg and Cuatrecasas, 1973; Thomopoulos et al., 1976; Rechler and Podskalny, 1976; Roldan et al., 1976; Raizada and Perdue, 1976; Osborne et al., 1978; Petersen et al., 1978). In this paper we report our findings on the biochemical characterization of insulin-binding sites on HT 29 cells grown in culture. HT 29 cells were originally isolated from a human carcinoma of the colon by Fogh and Trempe (1975) and recently it was demonstrated that these cells possess receptors for a polypeptide hormone, the vasoactive intestinal peptide (Laburthe et al., 1978).

MATERIALS AND METHODS chemicals

[‘251]Insulin was a generous gift of Dr. Freychet, INSERM U.145, Faculte de Medecine (Pasteur), 06100 Nice (France). Monocomponent pork insulin, crystalline beef insulin, pork proinsulin and glucagon were obtained from NOVO,Novoallee, DK-2880 Bagsvaerd (Denmark). Human growth hormone (hGH) was obtained from the service of Prof. Dray, Institut Pasteur Paris. Dulbecco’s modified Eagle’s medium (196 G), fetal calf serum, Ca*+ and Mg’+-free phosphate-buffered saline (PBS), trypsin-EDTA solution and penicillin-streptomycin solutions were purchased from Grand Island Biological Co., 3 Washington Road, Paisley, Scotland. Bovine serum albumin (BSA) fraction V, was obtained from Miles Laboratories, Elkhart, IN 46514 (U.S.A.). Culture flasks and plastic labware were purchased from Corning Co., Corning, NY 14830 (U.S.A.). All chemicals were of reagent grade. Doubly distilled water was used throughout all experiments. Cell culture

HT 29 cells were routinely grown in plastic culture flasks in Dulbecco’s modified Eagle’s medium containing 100 ug/ml penicillin and 100 &ml streptomycin supplemented with 10% fetal calf serum and equilibrated with 10% CO2 in air. The cells were passaged once a week using a mixture of 0.05% trypsin and 0.02% EDTA, centrifuged and washed with PBS before seeding. The medium was changed twice a week.

Insulin binding by a cell line from human cotonic cancer

12s

Bindingassays Culture flasks, 25 cm’, were seeded at least 48 h prior to the binding experiments to allow the cells to overcome membrane damage consequent to the action of trypsin. The binding assay was performed essentialIy as described by Hollenberg and Cuatrecasas (1975). CeIIs were incubated in the flasks with 2 mi of growth medium at 37” under the different conditions described in the legends to the figures. At the end of the incubation period, the cells were rinsed 4 times with 2 ml of icecold Krebs-Ringer phosphate buffer (KRP), pH 7.4 containing 0.1% bovine serum albumin. They were then allowed to disintegrate in 1 ml of 0.2 M NaOH at 37” for 1 h. The viscous solution was transferred into plastic tubes, the flask was sub~quently rinsed twice with 1.0 and 0.5 ml of 0.2 M NaOH and the combined solutions of NaOH were counted in a Packard autogamma scint~lation spectrometer with a 60% counting efficiency. Results are presented as specific binding, i.e. the difference between total binding and nonspecific binding. Total binding is the measured radioactivity with labeled hormone only, while nonspecific binding is the recorded radioactivity in the presence of a large excess of unlabeled hormone, 1.6 X lo-’ M. AII assays were performed in duplicate. Cells were counted in a hemocytometer and the cell viability determined by the trypan blue exclusion method of Phillips (1973). Degradation of insulin under conditions of the binding assay was determined in the cell supernatant by the ability of the labeled hormone to rebind to rat-liver plasma membranes as described by Frey. chet et al. (1972a). Briefly, after incubation of the cells with [“25X]insulinthe medium was removed. the flasks with the attached cells were washed twice with PBS2% BSA, pH 7.8. The combined medium and washes were centrifuged for 5 min at 500 Xg to eliminate the detached cells and celI debris. The supernatant was then assayed for binding to isolated rat-liver plasma membranes.

RESULTS As shown in Fig. 1, left panel, binding of insulin to HT 29 cells is a time-dependent process. Binding is initially fast. Half-maximal binding is achieved after 8-10 min of incubation at 37”, and an apparent equilibrium is attained after 30640 min. The cell-associated radioacti~ty then remains constant for at least 4 h. The stability of the hormone-receptor compiex suggests a possibIe lack of degradation of labeled hormone at the cell surface. To test this, we assayed the supernatants of cell cultures from Fig. I for the presence of labeled hormone able to bind to isolated liver plasma membranes (Freychet et al., 1972a). The results of such an experiment supported the suggestion. In two separate experiments more than 96% of the labeled hormone was rebound to isolated plasma membranes. Thus, ~~“I]insuIin is not degraded by HT 29 cells for at Ieast 4 h at 37”. The dissociation of labeled insulin from HT 29 cells at 37’ is shown in the right panel of Fig. 1. The initial rate of dissociation in the presence of native insulin is

ME. F~y~~-~a~:~e, A. Horvat, G. Rosselin

126

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Fig. 1. Left: Time course of insulin binding to HT 29 cells. Subcon~uent cultures {S X 106 ~~s/~ks) were incubated with 2.2 X lo- lo M [’ 2sI]insulin for the times indicated on the abscissa, at pH 7.8 and 37”. Specific binding was determined as described in Materials and Methods except that the cells were collected by scraping from the fIask with a rubber policeman. Nonspecifjc binding increased slightly in the first 20 min and remained co srant thereafter. It amounted to 20-25% of total binding, Each point on the curve represents the average of duplicate determinations. Right: Dissociation of bound [ I2 sIJinsulin. Cells (6.2 X lOa) were incubated for 40 min at 37O with 2.6 X 10WIOM [ 12sIJinsulin and the specific binding

determined (see Materials and Methods). This corresponds to 100% on the ordinate. To duplicate flasks 1.6 X lo-’ M unlabeled insulin was added and the incubation contmued for various lengths af time as indicated on the abscissa. Fig.2. Binding of [t2’ IJinsulin as a function of the number of ceils. Cells were seeded at different densities and 48 h after insulin binding, and in parallel flasks, the number of cells were determined as described under Methods. The concen~ation of [ ‘251]insulin was 1.2 X lo-” M and the time of incubation was 60 min.

very rapid. Half of the bound hormone is released in 6-7 min and 35% more within 30 min of incubation at 37”. 15% of the label remained associated with HT 29 cells after 1 h of incubation. The relation of insulin binding to the number of cells per flask is shown in Fig. 2. The results indicate a linear relationship between the amount of insulin bound and the number of cells between 1 X 1Oe and 10 X lo6 cells/flask. All experiments reported here were performed with cell numbers within this range. Fig. 3 shows the pH dependence of insulin binding to HT 29 cells. The optimum pH was found to be at 7.8. This experiment was performed at 24” to minimize the effect of the metabolic acti~ty of the cells on the pH of the medium during the 60min incubation. Under the conditions of the assay (see legend to Fig. 3) cell viability remained unchanged at ah pHs. Concentration dependence of insulin binding was determined by adding increasing concentrations of unlabeled insulin (10”’ to lo-* M) to fixed concentrations of [ ‘2sI]inslllin (l-4 x 10 -lo M) in the incubation mixture under the optimal conditions observed for binding (Fig. 4). Native insulin reduced tracer binding in a continuous dose-dependent fashion, half-maximal inhibition being obtained at about 2 X IO-’ M. Scatchard analysis (Scatchard, 1949) of the binding

Fig. 3. Binding of [‘“’ r@kYurinas B function of pM. C&s (53 X 10”) were h3cubaatedwith media containing 25 ml++HEFES ~~-~-h~d~ax~et~~~~~~~e~~~~e~-~~~~~e s&phonic acid) adjusted to the pH indicated on the absciSSa,far f h at 26. The conoentr&ion of [’ 25EJ$nsulin was 5 X 10W1’M. The pH of the media was determined just prior to the start of the reaction, a$ well as after the 1-h incubation time. Nonspecific binding was practically the same at all pHs.

Fig. 4. CanGentration dependence of insulin binding (one typical experiment). 7.7 X IO6 cells were incubated with 4 X 10-lo M ~12si~~su~n in the absence (100% on ar~~?e~ ruld presence of increasing concentrations of unlabeled insulin for 40 min at pH 7.8 and 57”. Insert: Scatchard plot obtained from the binding data.

data (see insert in Fig. 4) gives a curvilinear plot similar to those obtained in binding studies of insulm to various target issues, Curvilinear Scatchard plots can be interpreted as representing two classes of &dependent binding sites it, as suggested by De Meyts et al, (1973), negative cooper&iv&y. If we assume that there are two independent binding sites, the affinity constant for the high affinity site is 4.7 X lO* M”’ and for the low affmity site, 4 X 107 M”. The corresponding numbers of sites/cell are 1400 and 20 000 respectively, In that sense, recent reports (Krupp and ~v~gst~~~ 1978; Sahyoun et al,, 1978) show the presence of two sofubilized ~~~-b~d~g proteins. The specificity of binding was tested in competition experiments between Iabeled insulin (1-4 X lQ_‘e M), unIabeled insulin (5 X lO*” to lo-’ M) and proinsulin (10”’ to lo-’ M). 50% of the labeled hormone was displaced with 5 X lo-* M proinsulin indicating that the ability of proinsulin to bind to I-IT 29 cells was onIy abaut 4% that of native insulin. GIucagon (l@ M) and human growth hormones (5 X lV7 M) did not compete with [f251finsuB_nfor the binding site.

DISCUSSIaN The aim of this work was to chara~ter~~ the insulin receptor in a ~~~~orna cefl HT 29, which in its normal variant, the intestinal epithelial cell (Fogh and Txempe, 1975) is not considered to be a target tissue for insulin. These cells retain the capacity to bind insulin as do such other cultured cells asfibroblasts, transformed human

128

ME. Forgue-Lafitte,

A. Horvat, G. Rosselin

lymphocytes and human breast cancer cells (Ho~enberg and Cuatrecasas, 19’7.5; Gavin et al., 1972; Osborne et al., 1978). In view of our ultimate goal to relate, if possible, the presence of insulin receptors to a function in these cells, the binding characteristics were investigated under normal growth conditions, i.e., in the presence of the complete medium and at 37’. The basic characteristics of the insulin receptor proved similar with those described in other systems, with respect to affinity (Posner, 1974; Olefsky et al., 1974; Beck-Nielsen et al., 1977), pH dependence (Freychet et al., 1972b; Gavin et al., 1973; Olefsky and Reaven, 1974; Thomopoulos et al., 1976; Osborne et al., 1978) and specificity (Freychet et al., 1971). However, when surface receptors of cultures cells are studied problems in methodology must be considered. If one uses detached cells, it is impossible to determine the degree of damage the cell may suffer. On the other hand, since the active surface of attached cells may be smalIer than that of detached cells (Stoker and Piggot, 1974), quantitative analysis using attached cells may be inaccurate, in studies of the insulin receptor in cultured cells (Hollenberg and Cuatrecasas, 1975; Rechler and Podskalny, 1976; Raizada and Perdue, 1976; Thomopoulos et al., 1976; PoIlet et al., 1977; Petersen et al., 1978) both detached and attached cells have been used. We studied the binding of insulin to attached cells. The binding isotherm (Fig. 1) indicates ,a great stability of the hormone-receptor complex. At 37” most investigators have reported a very rapid association of the hormone followed by a decline of the cell-associated radioactivity (Gammeltoft and Gliemann, 1973; Freychet et al., 1974; Raizada and Perdue, 1976). Rechler and Podskalny (1976) working with detached human fibrobla~s in culture observed no steady state at 37” and the nonspecific binding amounted to more than 80% of the total binding. In our system nonspecific binding did not amount to more than 25 30% of total binding at equilibrium. Differences in the stability of the bound hormone could be due to the damage caused by trypsinization of cultures cells, or collagenase treatment in the case of hepatocytes and adipocytes. It should be emphasized that morphologically undetectable damage to the cell membrane can result in altered biological properties, such as permeability changes. Raizada and Perdue (1976), working with attached chick-embryo fibroblasts, reported a 50% decrease in the radioactivity bound after 30 min incubation and they also observed that 24% of the labeled hormone was degraded in the medium. The role of degradation in the biological activity of insulin could be as spoilt as b~d~g itself. The recent work of Osborne et al. (1978) is in good agreement with our finding. These authors, working with several human breast-cancer ceils, reported a direct correlation between the responsiveness of the strain to insulin and its ability to degrade the hormone. In our system and in the conditions of the binding studies, we were unable to detect any degradation of the labeled hormone. The problem of the correlation between insulin binding, receptor concentration or receptor affinity and a biological effect of insulin in HT 29 remains to be elucidated. It is likely that the presence of insulinbinding sites in NT 29 cells indicates the persistence of receptors that are already present in intestinal epithelial cells from which it originates, as was demonstrated

Insulin binding by a cell line from human colonic cancer

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for other receptors in that cell (VIP, secretin, glucagon, Laburthe et al., 1978). Indeed we have found the presence of insulin receptors in normal rat intestinal epithehal cells (Forgue-Lafitte et al., in preparation). However, no effect of insulin has thus far been demonstrated in intestinal cells, more particularly on the hexokinase activity (Shakespeare et al., 1969). Our unpublished studies indicate that insulin has no stimulatory effect on glucose oxidation; however the absence of insulin effect on glucose metabolism in those cells does not exclude other biological effects of insulin: indeed, when the culture medium is deprived of serum, insulin at concentrations as low as lo-’ M is able to stimulate cell growth. The relationship of insulin receptors and growth of this transformed cell line is now under investigation in our laboratory.

ACKNOWLEDGEMENT We express our thanks to Marie-Claude Chamblier for her excellent technical assistance. This work was supported by INSERM (ASR No. 2), CNRS and DGRST (Grant No. 7916).

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