Cancer Letters. 62 (1992) 23 - 33 Elsevier Scientific Publishers Ireland
23 Ltd.
Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth Stephen
Baghdiguiana,
Bernard
Verriera, Corinne
Gerard”
and Jacques
Fantinib
“INSERM U270. Facultd de Me’decine, Secteur Nord, Bd. Pierre Dramard. 13326 Marseille Cedex U322. Campus Universitaire de Luminy. BP 33, 13273 Marseille, Cedex 9 (France) (Received (Accepted
15 and blNSERM
30 July 1991) 10 October 1991)
Summary The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of ‘251-IGF-I binding was obtained in the presence of 25 pg/ml suramin. Moreover, the drug was able to dissociate ‘?-IGF previously bound to its cell surface receptor. Affinity labeling experiments demonstrated that ‘251-lGF-I bound to a polypeptide of 140 kDa. When
HT29-D4 cells were cultured in the presence of 10 pg/ml of a-lR3, a monoclonal antibody directed against the binding site of IGF-1, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with (r-lR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show fhat IGF-1 significantly increasCorrespondence to: Jacques Fantini, INSERM U322. Campus Universitaire de Luminy, BP 33,13273 Marseille, Cedex 9.
France. 0304.3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
ed the initial rate of glucose uptake by HT29-D4 cells, while we haue previous/y shown that suramin decreased glucose consumption. From these data we conclude that IGF-1 secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differen-
tiation.
insulin-like Keywords: suramin; autocrine loop; man colon cancer cells
growth factor I; differentiation; hu-
Introduction Suramin is a polysulfonated naphthylurea that has been used for more than 50 years in the treatment of onchoceriasis and trypanosomiasis [ 11. Recently, the drug attracted a renewed interest because of its antineoplastic activity in humans [Z - 41. We have shown that suramin was a potent inducer of differentiation of the human colonic adenocarcinoma cell clone HT29-D4. This process of differentiation Ireland Ltd
24
was associated with a decrease of glucose utilization and lactate production [5]. Because suramin has been demonstrated to interfere with the cellular binding of a variety of growth factors [8 - 111 which can control some key steps in carbohydrate metabolism [12], we have hypothesized that the drug could act by blocking an autocrine loop involving such growth factors. Two indirect arguments support this hypothesis: (i) the effect of suramin on HT29-D4 cell differentiation was observed in a serum-free medium [ 131, (ii) the differentiation process of HT29-D4 cells was shown to be controlled by the concentration of glucose in the culture medium [14]. However, definitive proofs for the mechanism of action of suramin were lacking. In this report we demonstrate that a chronic treatment of HT29-D4 cells in the presence of monoclonal antibodies which block the binding of IGF-I to its receptor induced the differentiation process of these cells. Furthermore, we demonstrate that suramin, at the doses used to trigger HT29-D4 cell differentiation, blocks the binding of IGF-I. These data clearly establish a link between suramin effect on differentiation and its ability to interfere with an autocrine loop involving IGF-I and its receptor. Materials
and Methods
Materials Suramin was obtained from Specia (Paris, France), prepared as a sterile stock solution of 100 mg/ml in distilled water, and stored at - 20°C. The anti-CEA mouse IgG2a antibody (Biosys, CompiGgne, France) is directed against epitope 4930 on the human CEA. It does not cross-react with NCA-1, NCA-2 and BGP1. The anti-transferrin receptor monoclonal antibody was from Sera-Lab (Paris, France). The anti-IGF-I receptor monoclonal antibody a-IR3 (purchased from Oncogene Science, Manhaset, N.Y .) is directed against the binding site of IGF-I. Fluorescent antibodies against mouse IgG were from Immunotech. (Marseil-
le, France). IGF-I was purchased from Boehringer (Mannheim) . Disuccinimidyl suberate (DSS) was purchased from Pierce Chemical 1251-IGF-I was purchased from Company. Amersham or home labelled using the chloramine T method and purified by high performance liquid chromatography using a C-18 reverse-phase column for separation. Cell culture HT29-D4 cells [16] were routinely grown in Dulbecco’s Modified Minimum Eagle’s Medium (DMEM) containing 25 mM glucose and supplemented with 10% fetal bovine serum (FBS). Treatment of HT29-D4 with (r-IR3 antibody was performed in 24 multiwell plates (Nunc, Roskilde, Denmark). Cells were seeded at a density of 5 x lo4 cells per square centimeter. Fresh medium containing ~1R3 at the indicated dilution was added 48 h after seeding and replaced every day. lmmunofluorescence microscopy Cells were grown in Labtek chambers (Nunc, Roskilde, Denmark) in the presence or absence of (r-IR3 (10 pg/ml), then rinsed five times with phosphate-buffer saline (PBS) and processed for immunofluorescence as previously described [13]. In some instances, the tight junctions of differentiated cells were disrupted by a 1 h incubation in a Ca*+-free medium according to Godefroy et al. [17], before the indirect immunofluorescence staining was performed. This treatment allows free access of antibodies to the basolateral membrane domain. Transmission electron microscopy Cells were fixed in situ with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), for 1 h, washed 10 min in the same buffer containing 6.84% saccharose, post-fixed in 1% ‘osmium tetroxide, then dehydrated in ethanol and embedded in Epon. Sections were cut perpendicularly to the plane of the cell layer and observed with a Philips CM 10 Electron Transmission Microscope.
25
‘251-lGF-l binding to cells in monolayer-s HT29-D4 cells were seeded into 12-well tissue culture clusters in DMEM with 10% FBS. The medium was removed 36 h later and the cells were rinsed with serum free DMEM and re-fed with this medium. The binding assay was carried out 12 h later. Cultures were washed once with warmed (37OC) Earle Hepes buffered binding medium (pH 7.4) and incubated with the same medium containing 100 pg/ml of suramin for 10 min at 37OC. Cultures were washed two times with ice-cold binding medium containing 0.1% BSA and incubated for various times or with graded concentrations of ‘251-IGF-I, in the absence or presence of suramin, on a ice-bath. At the end of the incubation, cells were washed three times with ice-cold binding medium containing 0.1% BSA, then dissolved in 1 M NaOH and the radioactivity determined in a LKB gamma spectrometer. Values are the mean of duplicate experiments. Standard errors were always below 5% of mean values. Experiments were performed at least two times with identical results. Affinity cross-linking study Cell cultures were treated exactly as those used in binding studies. Cells were incubated for 1.5 h at O°C with iz51-IGF-I in binding medium containing 0.1% BSA. To end ‘251-IGFI binding, cells were washed three times with binding medium and incubated with the crosslinking agent disuccinimidyl suberate (DSS, 1 mM) for 20 min at room temperature. This solution was removed and cells were solubilized (65 mM Tris - Cl, 10% glycerol, 3% SDS, 5% mercaptoethanol, 0.003% Bromophenol blue) and boiled for 5 min. Electrophoresis was performed on 7.5% polyacrylamide slab gel with a 3% stacking gel and autoradiographed using RX Fuji films. Uptake of S-O-methyl-~-glucose Cells were washed once for 5 min in Earle Hepes medium free of glucose containing 100 pg/ml of suramin and once in the same medi-
Fig. 1. Effect of mAb (r-IR3 on HT29-D4 cells. (A) Morphology of untreated HT29-D4 cells 10 days after seeding. (B) Morphology of HT29-D4 cells treated for 10 days with mAb cx-IR3 10 pg/ml. In this case the cells formed a regular epithelial monolayer.
Bar: 50 pm
26
27
Fig. 3.
lmmunofluorescence staining of HT.29.D4 cells grown for 10 days in the absence (A - C) or in the presence (D-F) of mAb (r-1R3 10 pg/ml. When grown in the absence of o-lR3, HT29-D4 cells were not polarized and a uniform staining was obtained with a-IR3 (A), anti-CEA (B) and anti-transferrin receptor (C) monoclonal antibodies. In contrast, o-IRS-treated HT29-D4 were stained only with the anti-CEA monoclonal antibody (D) since the tight junctions prevented the access of anti-transferrin receptor (E) to the basolateral membrane. After a l-h treatment in Cazf -free medium, tight junctions were opened and basolateral membranes were uniformly stained with anti-transferrin receptor antibody (F) Bar: 20 pm.
Fig. 2.
Transmission electron micrographs of HT29-D4 cells cultured for 10 days in the presence of a-IRS 10 pg/ml. (A) General view of the epithelial monolayer. Bar: 5 pm. (B) A typical differentiated HT29D4 cell. Bar: 2 pm. (C) Apical part of two adjacent HT29-D4 cells showing a tight junction and a desmosome. Bar: 1 pm. (D) Apical part of a differentiated HT29-D4 cell displaying a functional lysosomal apparatus. Bar: 2 pm. (E) Mitochondria with well-developed cristae characteristic of HT29-D4 cells cultured in the presence of mAb (r-IR3. Bar: 2 pm. bb, brush border; d, desmosome; g, Golgi apparatus; ly, lysosomes; m, mitochondria; n, nucleus; tj, tight junction.
28
urn free of suramin. Cells were incubated 15 min in the absence or presence of 30 nM IGFI. Uptake of 3-O-methyI-D-[l-3H]glucose (3-OMG) was started by the addition of 5 mM 3-O-methyl-D-glucose (1 &i/ml). After 3 min, the incubation medium was discarded and the cell layer washed three times with ice cold 100 mM Tris - Cl (pH 7.4). Cells were disrupted with 0.1 N NaOH and the radioactivity was determined using a Packard fl counter. Results were expressed in nmol 3-OMG/ mg of cellular protein f S.D. Results
Effect of anti-IGF-I receptor antibody on HT29-D4 cell growth and morphology Treatment of HT29-D4 cell with the antiIGF-I antibody a-IR3 resulted in a dosedependent decrease in cell density. With a concentration of 20 pg/ml, confluency was
0
20
40
Time F@ 4.
60
60
100
never obtained and the maximum density reached was 2 x lo5 ceIls/cm2. At a concentration of 10 pg/ml, the antibody allowed the formation of a confluent monolayer (Fig. 1B) with a main density of 3.5 x lo5 cells/cm2. Untreated cells grew as densely packed multilayers (Fig. 1A) with a cell yield of 9 x lo5 celIs/cm2. The morphologic change induced by the antibody (Fig. 1B) was similar to the one induced by suramin (see Fig. 2B in Ref. 5). At the ultrastructural level, HT29-D4 cells treated with (r-IR3 formed a polarized monolayer constituted of regularly arranged cells (Fig. 2A, B) showing the typical features of enterocytes: an apical brush border with densely packed microvilli (Fig. 2C, D),the uniform presence of tight junctions which separate the apical and basolateral membrane domain (Fig. 2C), a functional lysosomal system in the supranuclear region (Fig. 2D), and the pre-
120
(min)
Time-course of ‘251-1GF-Ibinding to HT29-D4 cells in the absence (9 or presence 0
of suramin (100 pg/ml) .
29
sence of numerous mitochondria developed cristae (Fig. ZE).
with well
Polarized expression of apical and basolateral markers It is widely admitted that the definition of epithelial differentiation includes the correct sorting of apical and basolateral proteins to their respective membrane domains. To check this biochemical degree of differentiation in CYIR3 treated HT29-D4 cells, we have studied by immunofluorescence the membrane localization of carcinoembryonic antigen (CEA) (an apical marker) and the transferrin receptor (a basolateral marker). Untreated HT29-D4 cells showed a uniform labeling of CEA (Fig. 3B) and transferrin receptors (Fig. 3C). After 10 days of treatment with the antibody (Y-IR3, CEA was found exclusively localized on the apical side of the cells, giving a punctated fluorescence corresponding to the microvilli of the brush border (Fig. 3D). In contrast, transferrin receptors could not be revealed on the apical membrane domain of differentiated cells (Fig. 3E). The localization of this marker was shown to be exclusively basolateral, because strong labeling was obtained after disrupting the tight junctions by a treatment with a Ca’+-lacking medium (Fig. 3F). It should be noted that a-IR3 labeled virtually all HT29-D4 cells (Fig. 3A). blocks the binding of ‘251-IGF-l lz51-IGF-I binding to HT29-D4 cells at 0°C was time-dependent (Fig. 4). A plateau was reached after 90 min of incubation at OOC. At this time, displacement of ‘251-IGF-I by graded concentrations of cold IGF-I showed (Fig. 5A) that half maximal effect was obtained for 0.6 nM. Non-saturable binding was obtained for 10 nM and represented 20% of total binding. Further characterization of IGF-I binding sites on HT29-D4 cells was obtained by affinity labeling with ‘251-IGF-I and the cross-linker disuccinimidyl suberate and subsequent solubilization of the cells. When analysed by SDSPAGE, solubilized cells revealed a 140-kDa labeled band. Addition of lOO~g/ml of suraSuramin
El
@I@-14OKd
,001 A
,Ol
1 ,l IGF.1 (nM)
10
100 B
Fig. 5. Specificity of IGF-I binding sites at the surface of HT29-D4 cells. (A) Binding of ‘251-IGF-I in the presence of increasing concentrations of unlabeled IGF-I. (B) Affinity labeling of IGF-I receptors. Cells were incubated with ‘251-IGF-I, washed, treated with 1 mM cross-linker DSS, solubilized and submitted to SDSPAGE as described in Materials and Methods. A single band was observed with an apparent mol. wt. of 140 kDa.
min at the onset of the time course of the binding abolished ‘251-IGF-I binding (Fig. 4). This inhibition was concentration dependent with an half-maximal effect for 25 pg/ml (Fig. 6). Addition of suramin (100 pg/ml) to cells previously bound with ‘251-IGF-I specifically dis-placed the hormone from the cells (Fig. ,7). After 20 min incubation with the drug only 30% of the labeled hormone remained bound to the cells. Effect of IGF-1 on glucose uptake Because the process of differentiation induced by suramin is associated with a decreased glucose consumption [5], we have studied the effect of IGF-I on the uptake of glucose in HT29-D4 cells. The initial rate of influx of 3-OMG, a nonmetabolizable glucose analog, was followed to determine the effect of IGF-I on glucose uptake. A weak, but significant increase of
0 0
I
I
I
I
50
100
150
200
Suramin Fis. 6.
Dose-response
(pglml)
curve of increasing
concentrations
of suramin
on ‘251-IGF-I binding to HT29-D4
3-OMG influx was observed
ceils.
in the presence
of
30 nM IGF-I (Table I).
Discussion The establishment response, whereby
of an autocrine growth a tumor cell both ex-
Table 1. Effect of IGF-I on the uptake HT29-D4 cells.
5
Effector (nM)
nmol 3-OMG/mg * S.D.
Control IGF-I 30
7.56 12.46
20-
0! 0
I
I
I
I
I
I
lo
20
30
40
50
60
Time
(min)
Fig. 7. Suramin-induced dissociation of ‘251-IGF-I bound to HT29-D4 cells. ‘251-IGF-I was incubated for 90 min with HT29-D4 cells. Specific binding of radioligand alone was 7% of added radioactivity. Cells were subsequently treated (U) or not (0) by 100 Kg/ml suramin.
of 3-OMG
in
protein
zt 0.89 zt 0.58’
Cells were washed once in the presence of suramin (lOO~g/mI), once in Earle Hepes medium free of suramin then incubated 15 min in the absence or presence of 30 nM IGFI. 3-OMG uptake was then measured for 3 min. ‘0.001 < P < 0.01.
31
by a mitogenic presses, and is stimulated growth factor, is now recognized to contribute to the tumorigenicity of neoplastic cells [18]. The secretion of growth-promoting activities by HT29 cells suggests the involvement of autocrine growth factors in the autonomous proliferation of these cells [15,19,20]. Indeed, HT29 cells are able to proliferate in a defined serum-free medium containing no added growth factors and the addition of suramin in these cultures reduces the incorporation of [ 3H]thymidine [2 1). Suramin is a polyanionic compound which has been shown to bind tightly to a wide range of growth factors [8 - 111. In the presence of suramin, these factors are no longer able to bind to their cell surface receptors and this explains the growth inhibitory activity of the drug against a number of tumoral cell lines in vitro [11,22]. Therefore, suramin has been widely used in studies dealing with autocrine loops in vitro [23 - 251. When added to the glucose-containing medium of the human colic adenocarcinoma cell clone HT29-D4, suramin inhibited cell growth, glycolytic activity and triggered enterocyte like differentiation of these cells [5]. From these data we speculated that autocrine growth factors, which stimulate glycolysis, prevented the cells from differentiating; blocking these factors with suramin leaded to cell differentiation [5,6,13]. Yet the nature of such growth factors remained to be elucidated. Among the growthpromoting factors identified in HT29 culture supernatants, insulin-like growth factor I has retained our attention because of its potential effect on glucose uptake and metabolism [ 121. By fractionating a conditioned medium from HT29 cells, Culouscouet al. [19] have purified a mitogenic factor, exhibiting an IGF-I competing activity, which is recognized by antiIGF-I antibodies. Moreover, competition radioligand binding experiments using ‘251-IGF-I demonstrated the presence of IGF-I receptors on HT29 cells 1261. The results we report here clearly demonstrate that: (i) suramin inhibits binding of 1251IGF-I to HT29-D4 cells, with a half-maximal
inhibition for a concentration of the drug of 25 pg/ml (25% of the dose necessary to induce cell differentiation). This result is in agreement with a recent report demonstrating an inhibition of IGF-I binding and IGF-I stimulated proliferation by suramin in osteosarcoma cells [27]. (ii) Suramin is also able to dissociate ‘251-IGF-I previously bound to its receptors. Similar results were reported for interleukin-2 [lo], platelet-derived growth factor [8] and autocrine factors secreted by HT29 [20]. (iii) IGF-I-binding sites expressed by HT29-D4 ceils have a similar structure than IGF-I receptors characterized in human colonic epithelium [28] and in the kidney [29] as demonstrated by affinity labeling experiments. In particular, the molecular size of the cross-linked band (140 kDa) is consistent with the o-subunit of the IGF-I receptor [30] and we can totally exclude a possible association of IGF binding proteins to the HT29-D4 ceI1 surface [ 15). (iv) Chronic treatment of HT29-D4 cells with a monoclonal antibody recognizing the binding site of IGF-I on IGF-I receptor [15] induces an inhibition of proliferation. (v) Treatment with this antibody is as efficient as suramin to induce the enterocyte-like differentiation of HT29-D4 cells, and in both cases an inhibition of glucose uptake and glycolytic activity was observed
151’ From these data we conclude that the proliferation of HT29-D4 cells is controlled by an autocrine loop involving IGF-I and its receptor. Inhibition of IGF-I stimulated proliferation of these cells by suramin or specific monoclonal antibodies has the same consequence on the organization of HT29-D4 cells as a polarized differentiated monolayer. We are aware that the mechanism of inhibition of cell differentiation by IGF-I remains to be elucidated. In this respect, the striking property of insulin and may be of IGF to induce a marked decrease of the transepithelial resistance of intestinal epithelial cell lines [31] should be taken into account. Acknowledgements We thank the Association
pour la Recherche
32
contre le Cancer (grant to B.V.) and the expert technical assistance of Olivier Dekay, JeanClaude Bugeia and Mireille Renard. References 1
Hawking, F. (1978) Suramin: with special reference to onchoceriasis. Adv. Pharmacol. Chemother., 15, 289-322.
2
Stein, C.A., LaRocca, R.V., Thomas, R., McAtee, N. and Myers, C.E. (1988) Suramin: an anticancer drug with a unique mechanism of action. J. Clin. Oncol., 7, 499 - 508.
3
Cooper, M., LaRocca, R., Stein, C. and Myers, C. (1989) Pharmacokinetic monitoring is necessary for the safe use of suramin as an anticancer drug. Proc. Am. Assoc. Cancer Res., 30, 242.
4
Allolio. B., Jaursch-Hancke, C., Reincke, M., Arlt, W.. Metzler, V. and Winkelmann, W. (1989) Treatment of metastazing adrenal carcinoma with suramin. Dtsch. Med. Wscht., 114, 381-384.
5
Fantini, J., Rognoni, J.B., Roccabianca, M., Pommier G. and Marvaldi J. (1989) Suramin inhibits cell growth and glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT 29-D4. J. Biol. Chem..
6
264, 10282 - 10286. Fantini, J., Rognoni, J.B., Verrier, B., Lehmann, M., Roccabianca. M., Mauchamp, J. and Marvaldi, J. (1990) Suramin-treated HT 29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with the same cells grown in the absence of glucose. Eur. J. Cell Biol., 51, llO- 119.
7
8
9
10
11
Baghdiguian, S., Nickel, P., Marvaldi, J. and Fantini, J. (1990) A suramin derivative induces enterocyte-like differentiation of human colon cancer cells without lysosomal storage disorder. Anti-Cancer Drugs, 1, 59-66. Hosang, M. (1985) Suramin btnds to platelet-derived growth factor and inhibits its biological activity. J. Cell Biochem., 29, 265 - 273. Kopp. R. and Pfeiffer, A. (1990) Suramin alters phosphoinositide synthesis and inhibits growth factor receptor binding in HT 29 cells. Cancer Res.. 50, 6490 - 6496. Mills, G.B., Zhang, N., May, C., Hill. M. and Chung, A. (1990) Suramin prevents binding of interleukin 2 to its cell surface receptor: a possible mechanism for immunosuppression. Cancer Res., 50, 3036 - 3042. Coffey Jr., R.J., Leof, E.B., Shipley. G.D. and Moses,
13
14
15
Fantini, J., Marvaldi, J. and Pommier G. (1990) Production of insulin-like growth factor R (IGF-II) and different forms of IGF-binding proteins by HT.29 human colon carcinoma cell line. J. Cell. Physiol., 143, 405-415. 16
17
18
19
20
21
132, 143 - 148. Chan, C.P. and Krebs, E.G. (1986) Effects of growth factors on carbohydrate metabolism in cultured mammalian cells, In: Mechanisms of Insulin Action, pp. 13-32. Editors: P. Belfrage, J. Donner and P. Stralfors. Elsevier Science Publishing Co., Inc.. New York.
Fantini, J., Abadie, B., Tirard, A., Remy, L., Ripen, J.P.. El Battari, A. and Marvaldi, J. (1986) Spontaneous and induced dome formation by two clonal populations derived from a human adenocarcinoma cell line. J. Cell Sci., 83, 235 - 249. Godefroy, O., Huet. C., Blair, L.A.C., SahuquilloMerino, C. and Louvard, D. (1988) Differentiation of a clone isolated from the HT 29 cell line: polarized distribution of histocompatibility antigens (HLA) and transferrin receptors. Biol. Cell, 63, 41- 55. Cross, M. and Dexter, T.M. (1991) Growth factors in development, transformation and tumorigenesis. Cell, 64. 271-280. Culouscou, J.M., Remacle-Bonnet, M., Garrouste, F., Marvaldi, J. and Pommier. G. (1987) Simultaneous production of IGF-I and EGF competing growth factors by HT-29 human colon cancer cell line. Int. J. Cancer, 40, 646 - 652. Culouscou, J.M., Garrouste, F., Remacle-Bonnet, M., Bettetini, D., Marvaldi, J. and Pommier. G. (1988) Autocrine secretion of a colorectum-derived growth factor by HT.29 human colon carcinoma cell line. Int. J. Cancer, 42, 895-901. Forgue-Lafitte, M.E., Condray, A.M., Breant, B. and Mester, J. (1989) Proliferation of the human colon carcinoma cell line HT 29: autocrine growth and deregulated expression of the c-myc oncogene. Cancer Res., 49, 6566-6571.
22
Spigelman, Z., Dowers, A., Kennedy, S., Disorbo, D.. O’Brien, M., Barr, R. and McCaffrey, R. (1987) Antiproliferative effects of suramin on lymphoid cells. Cancer Res., 47, 4694-4698.
23
Huang, S.S. and Huang, J.S. (1988) Rapid turnover of the platelet-derived growth factor receptor in sistransformed cells and reversal by suramin. J. Biol. Chem.. 263, 1260812618. Fleming, T.P., Matsui, T., Molloy, C.J., Robbins, K.C. and Aaronson, S.A. (1989) Autocrine mechanism for v-sis transformation requires cell surface localization of internally activated growth factor receptors. Proc. Natl. Acad. Sci. U.S.A., 86, 8063-8067.
J.L. (1987) Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J. Cell. Physiol., 12
Fantini, J., Verrier, B., Robert, C., Pit, P., Pichon, J., Mauchamp, J. and Marvaldi, J. (1990) Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT 29-D4 in serum-free medium. Exp. Cell Res., 189, 109- 117. Rabenandrasana, C., Baghdiguian, S., Roccabianca. M., Brunet, M., Marvaldi, J. and Fantini, J. (1990) The concentration of glucose in the culture medium determines the effect of suramin on the growth and differentiation of the human colonic adenocarcinoma cell clone HT 29-D4. Cancer Lett., 53, 109- 115. Culouscou, J.M., Remacle-Bonnet, M., Garrouste, F.,
24
33
25
26
27
28
without augmentation of glucose transport. Biochim Biophys. Acta, 972. 60-68. Pollak. M. and Richard, M. (1990) Suramin blokade of insulin-like growth factor l-stimulated proliferation of
30
growth factors in human colonic epithelium. Gastroenterology, 98, 703 - 707. Conti. F.G., Elliot. S.J., Striker, L.J. and Striker, G.E. (1989) Binding of Insulin-like growth factor-l by glomerular endothelial and epithelial cells: further evidence for IGF-I action in the renal glomerulus. Biochem. Biophys. Res. Commun., 163, 952 - 958. Massague, J. and Czech, M.P. (1982) The subunit struc-
human osteosarcoma cells. J. Natl. Cancer Inst., 82, 1349 - 1352. Rouyer-Fessard, C., Gammeltoft, S. and Laburthe, M. (1990) Expression of two types of receptor for Insulinlike
31
ture of two I and II and 01. Chem.. McRoberts.
Yayon, A. and Klagsbrun. M. (1990) Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: reversal by suramin. Proc. Natl. Acad. Sci. U.S.A., 87, 5346 - 5350. Franklin, C.C.. Chin, P.C., Turner, J.T. and Kim, H.D. (1988) Insulin regulation of glucose metabolism in HT 29 colonic adenocarcinoma cells: activation of glycolysis
29
distinct receptors for insulin-like growth factor their relationship to the insulin receptor. J. Bi257. 5038-5045. J.A., Aranda, R., Riley, N. and Kang. H.
(1990) Insulin regulates the paracellular permeability of cultured intestinal epithelial cell monolayers. J. Clin. Invest., 85, 1127- 1134.
23 Ltd.
Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth Stephen
Baghdiguiana,
Bernard
Verriera, Corinne
Gerard”
and Jacques
Fantinib
“INSERM U270. Facultd de Me’decine, Secteur Nord, Bd. Pierre Dramard. 13326 Marseille Cedex U322. Campus Universitaire de Luminy. BP 33, 13273 Marseille, Cedex 9 (France) (Received (Accepted
15 and blNSERM
30 July 1991) 10 October 1991)
Summary The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of ‘251-IGF-I binding was obtained in the presence of 25 pg/ml suramin. Moreover, the drug was able to dissociate ‘?-IGF previously bound to its cell surface receptor. Affinity labeling experiments demonstrated that ‘251-lGF-I bound to a polypeptide of 140 kDa. When
HT29-D4 cells were cultured in the presence of 10 pg/ml of a-lR3, a monoclonal antibody directed against the binding site of IGF-1, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with (r-lR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show fhat IGF-1 significantly increasCorrespondence to: Jacques Fantini, INSERM U322. Campus Universitaire de Luminy, BP 33,13273 Marseille, Cedex 9.
France. 0304.3835/92/$05.00 Printed and Published
0 1992 Elsevier Scientific Publishers in Ireland
ed the initial rate of glucose uptake by HT29-D4 cells, while we haue previous/y shown that suramin decreased glucose consumption. From these data we conclude that IGF-1 secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differen-
tiation.
insulin-like Keywords: suramin; autocrine loop; man colon cancer cells
growth factor I; differentiation; hu-
Introduction Suramin is a polysulfonated naphthylurea that has been used for more than 50 years in the treatment of onchoceriasis and trypanosomiasis [ 11. Recently, the drug attracted a renewed interest because of its antineoplastic activity in humans [Z - 41. We have shown that suramin was a potent inducer of differentiation of the human colonic adenocarcinoma cell clone HT29-D4. This process of differentiation Ireland Ltd
24
was associated with a decrease of glucose utilization and lactate production [5]. Because suramin has been demonstrated to interfere with the cellular binding of a variety of growth factors [8 - 111 which can control some key steps in carbohydrate metabolism [12], we have hypothesized that the drug could act by blocking an autocrine loop involving such growth factors. Two indirect arguments support this hypothesis: (i) the effect of suramin on HT29-D4 cell differentiation was observed in a serum-free medium [ 131, (ii) the differentiation process of HT29-D4 cells was shown to be controlled by the concentration of glucose in the culture medium [14]. However, definitive proofs for the mechanism of action of suramin were lacking. In this report we demonstrate that a chronic treatment of HT29-D4 cells in the presence of monoclonal antibodies which block the binding of IGF-I to its receptor induced the differentiation process of these cells. Furthermore, we demonstrate that suramin, at the doses used to trigger HT29-D4 cell differentiation, blocks the binding of IGF-I. These data clearly establish a link between suramin effect on differentiation and its ability to interfere with an autocrine loop involving IGF-I and its receptor. Materials
and Methods
Materials Suramin was obtained from Specia (Paris, France), prepared as a sterile stock solution of 100 mg/ml in distilled water, and stored at - 20°C. The anti-CEA mouse IgG2a antibody (Biosys, CompiGgne, France) is directed against epitope 4930 on the human CEA. It does not cross-react with NCA-1, NCA-2 and BGP1. The anti-transferrin receptor monoclonal antibody was from Sera-Lab (Paris, France). The anti-IGF-I receptor monoclonal antibody a-IR3 (purchased from Oncogene Science, Manhaset, N.Y .) is directed against the binding site of IGF-I. Fluorescent antibodies against mouse IgG were from Immunotech. (Marseil-
le, France). IGF-I was purchased from Boehringer (Mannheim) . Disuccinimidyl suberate (DSS) was purchased from Pierce Chemical 1251-IGF-I was purchased from Company. Amersham or home labelled using the chloramine T method and purified by high performance liquid chromatography using a C-18 reverse-phase column for separation. Cell culture HT29-D4 cells [16] were routinely grown in Dulbecco’s Modified Minimum Eagle’s Medium (DMEM) containing 25 mM glucose and supplemented with 10% fetal bovine serum (FBS). Treatment of HT29-D4 with (r-IR3 antibody was performed in 24 multiwell plates (Nunc, Roskilde, Denmark). Cells were seeded at a density of 5 x lo4 cells per square centimeter. Fresh medium containing ~1R3 at the indicated dilution was added 48 h after seeding and replaced every day. lmmunofluorescence microscopy Cells were grown in Labtek chambers (Nunc, Roskilde, Denmark) in the presence or absence of (r-IR3 (10 pg/ml), then rinsed five times with phosphate-buffer saline (PBS) and processed for immunofluorescence as previously described [13]. In some instances, the tight junctions of differentiated cells were disrupted by a 1 h incubation in a Ca*+-free medium according to Godefroy et al. [17], before the indirect immunofluorescence staining was performed. This treatment allows free access of antibodies to the basolateral membrane domain. Transmission electron microscopy Cells were fixed in situ with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), for 1 h, washed 10 min in the same buffer containing 6.84% saccharose, post-fixed in 1% ‘osmium tetroxide, then dehydrated in ethanol and embedded in Epon. Sections were cut perpendicularly to the plane of the cell layer and observed with a Philips CM 10 Electron Transmission Microscope.
25
‘251-lGF-l binding to cells in monolayer-s HT29-D4 cells were seeded into 12-well tissue culture clusters in DMEM with 10% FBS. The medium was removed 36 h later and the cells were rinsed with serum free DMEM and re-fed with this medium. The binding assay was carried out 12 h later. Cultures were washed once with warmed (37OC) Earle Hepes buffered binding medium (pH 7.4) and incubated with the same medium containing 100 pg/ml of suramin for 10 min at 37OC. Cultures were washed two times with ice-cold binding medium containing 0.1% BSA and incubated for various times or with graded concentrations of ‘251-IGF-I, in the absence or presence of suramin, on a ice-bath. At the end of the incubation, cells were washed three times with ice-cold binding medium containing 0.1% BSA, then dissolved in 1 M NaOH and the radioactivity determined in a LKB gamma spectrometer. Values are the mean of duplicate experiments. Standard errors were always below 5% of mean values. Experiments were performed at least two times with identical results. Affinity cross-linking study Cell cultures were treated exactly as those used in binding studies. Cells were incubated for 1.5 h at O°C with iz51-IGF-I in binding medium containing 0.1% BSA. To end ‘251-IGFI binding, cells were washed three times with binding medium and incubated with the crosslinking agent disuccinimidyl suberate (DSS, 1 mM) for 20 min at room temperature. This solution was removed and cells were solubilized (65 mM Tris - Cl, 10% glycerol, 3% SDS, 5% mercaptoethanol, 0.003% Bromophenol blue) and boiled for 5 min. Electrophoresis was performed on 7.5% polyacrylamide slab gel with a 3% stacking gel and autoradiographed using RX Fuji films. Uptake of S-O-methyl-~-glucose Cells were washed once for 5 min in Earle Hepes medium free of glucose containing 100 pg/ml of suramin and once in the same medi-
Fig. 1. Effect of mAb (r-IR3 on HT29-D4 cells. (A) Morphology of untreated HT29-D4 cells 10 days after seeding. (B) Morphology of HT29-D4 cells treated for 10 days with mAb cx-IR3 10 pg/ml. In this case the cells formed a regular epithelial monolayer.
Bar: 50 pm
26
27
Fig. 3.
lmmunofluorescence staining of HT.29.D4 cells grown for 10 days in the absence (A - C) or in the presence (D-F) of mAb (r-1R3 10 pg/ml. When grown in the absence of o-lR3, HT29-D4 cells were not polarized and a uniform staining was obtained with a-IR3 (A), anti-CEA (B) and anti-transferrin receptor (C) monoclonal antibodies. In contrast, o-IRS-treated HT29-D4 were stained only with the anti-CEA monoclonal antibody (D) since the tight junctions prevented the access of anti-transferrin receptor (E) to the basolateral membrane. After a l-h treatment in Cazf -free medium, tight junctions were opened and basolateral membranes were uniformly stained with anti-transferrin receptor antibody (F) Bar: 20 pm.
Fig. 2.
Transmission electron micrographs of HT29-D4 cells cultured for 10 days in the presence of a-IRS 10 pg/ml. (A) General view of the epithelial monolayer. Bar: 5 pm. (B) A typical differentiated HT29D4 cell. Bar: 2 pm. (C) Apical part of two adjacent HT29-D4 cells showing a tight junction and a desmosome. Bar: 1 pm. (D) Apical part of a differentiated HT29-D4 cell displaying a functional lysosomal apparatus. Bar: 2 pm. (E) Mitochondria with well-developed cristae characteristic of HT29-D4 cells cultured in the presence of mAb (r-IR3. Bar: 2 pm. bb, brush border; d, desmosome; g, Golgi apparatus; ly, lysosomes; m, mitochondria; n, nucleus; tj, tight junction.
28
urn free of suramin. Cells were incubated 15 min in the absence or presence of 30 nM IGFI. Uptake of 3-O-methyI-D-[l-3H]glucose (3-OMG) was started by the addition of 5 mM 3-O-methyl-D-glucose (1 &i/ml). After 3 min, the incubation medium was discarded and the cell layer washed three times with ice cold 100 mM Tris - Cl (pH 7.4). Cells were disrupted with 0.1 N NaOH and the radioactivity was determined using a Packard fl counter. Results were expressed in nmol 3-OMG/ mg of cellular protein f S.D. Results
Effect of anti-IGF-I receptor antibody on HT29-D4 cell growth and morphology Treatment of HT29-D4 cell with the antiIGF-I antibody a-IR3 resulted in a dosedependent decrease in cell density. With a concentration of 20 pg/ml, confluency was
0
20
40
Time F@ 4.
60
60
100
never obtained and the maximum density reached was 2 x lo5 ceIls/cm2. At a concentration of 10 pg/ml, the antibody allowed the formation of a confluent monolayer (Fig. 1B) with a main density of 3.5 x lo5 cells/cm2. Untreated cells grew as densely packed multilayers (Fig. 1A) with a cell yield of 9 x lo5 celIs/cm2. The morphologic change induced by the antibody (Fig. 1B) was similar to the one induced by suramin (see Fig. 2B in Ref. 5). At the ultrastructural level, HT29-D4 cells treated with (r-IR3 formed a polarized monolayer constituted of regularly arranged cells (Fig. 2A, B) showing the typical features of enterocytes: an apical brush border with densely packed microvilli (Fig. 2C, D),the uniform presence of tight junctions which separate the apical and basolateral membrane domain (Fig. 2C), a functional lysosomal system in the supranuclear region (Fig. 2D), and the pre-
120
(min)
Time-course of ‘251-1GF-Ibinding to HT29-D4 cells in the absence (9 or presence 0
of suramin (100 pg/ml) .
29
sence of numerous mitochondria developed cristae (Fig. ZE).
with well
Polarized expression of apical and basolateral markers It is widely admitted that the definition of epithelial differentiation includes the correct sorting of apical and basolateral proteins to their respective membrane domains. To check this biochemical degree of differentiation in CYIR3 treated HT29-D4 cells, we have studied by immunofluorescence the membrane localization of carcinoembryonic antigen (CEA) (an apical marker) and the transferrin receptor (a basolateral marker). Untreated HT29-D4 cells showed a uniform labeling of CEA (Fig. 3B) and transferrin receptors (Fig. 3C). After 10 days of treatment with the antibody (Y-IR3, CEA was found exclusively localized on the apical side of the cells, giving a punctated fluorescence corresponding to the microvilli of the brush border (Fig. 3D). In contrast, transferrin receptors could not be revealed on the apical membrane domain of differentiated cells (Fig. 3E). The localization of this marker was shown to be exclusively basolateral, because strong labeling was obtained after disrupting the tight junctions by a treatment with a Ca’+-lacking medium (Fig. 3F). It should be noted that a-IR3 labeled virtually all HT29-D4 cells (Fig. 3A). blocks the binding of ‘251-IGF-l lz51-IGF-I binding to HT29-D4 cells at 0°C was time-dependent (Fig. 4). A plateau was reached after 90 min of incubation at OOC. At this time, displacement of ‘251-IGF-I by graded concentrations of cold IGF-I showed (Fig. 5A) that half maximal effect was obtained for 0.6 nM. Non-saturable binding was obtained for 10 nM and represented 20% of total binding. Further characterization of IGF-I binding sites on HT29-D4 cells was obtained by affinity labeling with ‘251-IGF-I and the cross-linker disuccinimidyl suberate and subsequent solubilization of the cells. When analysed by SDSPAGE, solubilized cells revealed a 140-kDa labeled band. Addition of lOO~g/ml of suraSuramin
El
@I@-14OKd
,001 A
,Ol
1 ,l IGF.1 (nM)
10
100 B
Fig. 5. Specificity of IGF-I binding sites at the surface of HT29-D4 cells. (A) Binding of ‘251-IGF-I in the presence of increasing concentrations of unlabeled IGF-I. (B) Affinity labeling of IGF-I receptors. Cells were incubated with ‘251-IGF-I, washed, treated with 1 mM cross-linker DSS, solubilized and submitted to SDSPAGE as described in Materials and Methods. A single band was observed with an apparent mol. wt. of 140 kDa.
min at the onset of the time course of the binding abolished ‘251-IGF-I binding (Fig. 4). This inhibition was concentration dependent with an half-maximal effect for 25 pg/ml (Fig. 6). Addition of suramin (100 pg/ml) to cells previously bound with ‘251-IGF-I specifically dis-placed the hormone from the cells (Fig. ,7). After 20 min incubation with the drug only 30% of the labeled hormone remained bound to the cells. Effect of IGF-1 on glucose uptake Because the process of differentiation induced by suramin is associated with a decreased glucose consumption [5], we have studied the effect of IGF-I on the uptake of glucose in HT29-D4 cells. The initial rate of influx of 3-OMG, a nonmetabolizable glucose analog, was followed to determine the effect of IGF-I on glucose uptake. A weak, but significant increase of
0 0
I
I
I
I
50
100
150
200
Suramin Fis. 6.
Dose-response
(pglml)
curve of increasing
concentrations
of suramin
on ‘251-IGF-I binding to HT29-D4
3-OMG influx was observed
ceils.
in the presence
of
30 nM IGF-I (Table I).
Discussion The establishment response, whereby
of an autocrine growth a tumor cell both ex-
Table 1. Effect of IGF-I on the uptake HT29-D4 cells.
5
Effector (nM)
nmol 3-OMG/mg * S.D.
Control IGF-I 30
7.56 12.46
20-
0! 0
I
I
I
I
I
I
lo
20
30
40
50
60
Time
(min)
Fig. 7. Suramin-induced dissociation of ‘251-IGF-I bound to HT29-D4 cells. ‘251-IGF-I was incubated for 90 min with HT29-D4 cells. Specific binding of radioligand alone was 7% of added radioactivity. Cells were subsequently treated (U) or not (0) by 100 Kg/ml suramin.
of 3-OMG
in
protein
zt 0.89 zt 0.58’
Cells were washed once in the presence of suramin (lOO~g/mI), once in Earle Hepes medium free of suramin then incubated 15 min in the absence or presence of 30 nM IGFI. 3-OMG uptake was then measured for 3 min. ‘0.001 < P < 0.01.
31
by a mitogenic presses, and is stimulated growth factor, is now recognized to contribute to the tumorigenicity of neoplastic cells [18]. The secretion of growth-promoting activities by HT29 cells suggests the involvement of autocrine growth factors in the autonomous proliferation of these cells [15,19,20]. Indeed, HT29 cells are able to proliferate in a defined serum-free medium containing no added growth factors and the addition of suramin in these cultures reduces the incorporation of [ 3H]thymidine [2 1). Suramin is a polyanionic compound which has been shown to bind tightly to a wide range of growth factors [8 - 111. In the presence of suramin, these factors are no longer able to bind to their cell surface receptors and this explains the growth inhibitory activity of the drug against a number of tumoral cell lines in vitro [11,22]. Therefore, suramin has been widely used in studies dealing with autocrine loops in vitro [23 - 251. When added to the glucose-containing medium of the human colic adenocarcinoma cell clone HT29-D4, suramin inhibited cell growth, glycolytic activity and triggered enterocyte like differentiation of these cells [5]. From these data we speculated that autocrine growth factors, which stimulate glycolysis, prevented the cells from differentiating; blocking these factors with suramin leaded to cell differentiation [5,6,13]. Yet the nature of such growth factors remained to be elucidated. Among the growthpromoting factors identified in HT29 culture supernatants, insulin-like growth factor I has retained our attention because of its potential effect on glucose uptake and metabolism [ 121. By fractionating a conditioned medium from HT29 cells, Culouscouet al. [19] have purified a mitogenic factor, exhibiting an IGF-I competing activity, which is recognized by antiIGF-I antibodies. Moreover, competition radioligand binding experiments using ‘251-IGF-I demonstrated the presence of IGF-I receptors on HT29 cells 1261. The results we report here clearly demonstrate that: (i) suramin inhibits binding of 1251IGF-I to HT29-D4 cells, with a half-maximal
inhibition for a concentration of the drug of 25 pg/ml (25% of the dose necessary to induce cell differentiation). This result is in agreement with a recent report demonstrating an inhibition of IGF-I binding and IGF-I stimulated proliferation by suramin in osteosarcoma cells [27]. (ii) Suramin is also able to dissociate ‘251-IGF-I previously bound to its receptors. Similar results were reported for interleukin-2 [lo], platelet-derived growth factor [8] and autocrine factors secreted by HT29 [20]. (iii) IGF-I-binding sites expressed by HT29-D4 ceils have a similar structure than IGF-I receptors characterized in human colonic epithelium [28] and in the kidney [29] as demonstrated by affinity labeling experiments. In particular, the molecular size of the cross-linked band (140 kDa) is consistent with the o-subunit of the IGF-I receptor [30] and we can totally exclude a possible association of IGF binding proteins to the HT29-D4 ceI1 surface [ 15). (iv) Chronic treatment of HT29-D4 cells with a monoclonal antibody recognizing the binding site of IGF-I on IGF-I receptor [15] induces an inhibition of proliferation. (v) Treatment with this antibody is as efficient as suramin to induce the enterocyte-like differentiation of HT29-D4 cells, and in both cases an inhibition of glucose uptake and glycolytic activity was observed
151’ From these data we conclude that the proliferation of HT29-D4 cells is controlled by an autocrine loop involving IGF-I and its receptor. Inhibition of IGF-I stimulated proliferation of these cells by suramin or specific monoclonal antibodies has the same consequence on the organization of HT29-D4 cells as a polarized differentiated monolayer. We are aware that the mechanism of inhibition of cell differentiation by IGF-I remains to be elucidated. In this respect, the striking property of insulin and may be of IGF to induce a marked decrease of the transepithelial resistance of intestinal epithelial cell lines [31] should be taken into account. Acknowledgements We thank the Association
pour la Recherche
32
contre le Cancer (grant to B.V.) and the expert technical assistance of Olivier Dekay, JeanClaude Bugeia and Mireille Renard. References 1
Hawking, F. (1978) Suramin: with special reference to onchoceriasis. Adv. Pharmacol. Chemother., 15, 289-322.
2
Stein, C.A., LaRocca, R.V., Thomas, R., McAtee, N. and Myers, C.E. (1988) Suramin: an anticancer drug with a unique mechanism of action. J. Clin. Oncol., 7, 499 - 508.
3
Cooper, M., LaRocca, R., Stein, C. and Myers, C. (1989) Pharmacokinetic monitoring is necessary for the safe use of suramin as an anticancer drug. Proc. Am. Assoc. Cancer Res., 30, 242.
4
Allolio. B., Jaursch-Hancke, C., Reincke, M., Arlt, W.. Metzler, V. and Winkelmann, W. (1989) Treatment of metastazing adrenal carcinoma with suramin. Dtsch. Med. Wscht., 114, 381-384.
5
Fantini, J., Rognoni, J.B., Roccabianca, M., Pommier G. and Marvaldi J. (1989) Suramin inhibits cell growth and glycolytic activity and triggers differentiation of human colic adenocarcinoma cell clone HT 29-D4. J. Biol. Chem..
6
264, 10282 - 10286. Fantini, J., Rognoni, J.B., Verrier, B., Lehmann, M., Roccabianca. M., Mauchamp, J. and Marvaldi, J. (1990) Suramin-treated HT 29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with the same cells grown in the absence of glucose. Eur. J. Cell Biol., 51, llO- 119.
7
8
9
10
11
Baghdiguian, S., Nickel, P., Marvaldi, J. and Fantini, J. (1990) A suramin derivative induces enterocyte-like differentiation of human colon cancer cells without lysosomal storage disorder. Anti-Cancer Drugs, 1, 59-66. Hosang, M. (1985) Suramin btnds to platelet-derived growth factor and inhibits its biological activity. J. Cell Biochem., 29, 265 - 273. Kopp. R. and Pfeiffer, A. (1990) Suramin alters phosphoinositide synthesis and inhibits growth factor receptor binding in HT 29 cells. Cancer Res.. 50, 6490 - 6496. Mills, G.B., Zhang, N., May, C., Hill. M. and Chung, A. (1990) Suramin prevents binding of interleukin 2 to its cell surface receptor: a possible mechanism for immunosuppression. Cancer Res., 50, 3036 - 3042. Coffey Jr., R.J., Leof, E.B., Shipley. G.D. and Moses,
13
14
15
Fantini, J., Marvaldi, J. and Pommier G. (1990) Production of insulin-like growth factor R (IGF-II) and different forms of IGF-binding proteins by HT.29 human colon carcinoma cell line. J. Cell. Physiol., 143, 405-415. 16
17
18
19
20
21
132, 143 - 148. Chan, C.P. and Krebs, E.G. (1986) Effects of growth factors on carbohydrate metabolism in cultured mammalian cells, In: Mechanisms of Insulin Action, pp. 13-32. Editors: P. Belfrage, J. Donner and P. Stralfors. Elsevier Science Publishing Co., Inc.. New York.
Fantini, J., Abadie, B., Tirard, A., Remy, L., Ripen, J.P.. El Battari, A. and Marvaldi, J. (1986) Spontaneous and induced dome formation by two clonal populations derived from a human adenocarcinoma cell line. J. Cell Sci., 83, 235 - 249. Godefroy, O., Huet. C., Blair, L.A.C., SahuquilloMerino, C. and Louvard, D. (1988) Differentiation of a clone isolated from the HT 29 cell line: polarized distribution of histocompatibility antigens (HLA) and transferrin receptors. Biol. Cell, 63, 41- 55. Cross, M. and Dexter, T.M. (1991) Growth factors in development, transformation and tumorigenesis. Cell, 64. 271-280. Culouscou, J.M., Remacle-Bonnet, M., Garrouste, F., Marvaldi, J. and Pommier. G. (1987) Simultaneous production of IGF-I and EGF competing growth factors by HT-29 human colon cancer cell line. Int. J. Cancer, 40, 646 - 652. Culouscou, J.M., Garrouste, F., Remacle-Bonnet, M., Bettetini, D., Marvaldi, J. and Pommier. G. (1988) Autocrine secretion of a colorectum-derived growth factor by HT.29 human colon carcinoma cell line. Int. J. Cancer, 42, 895-901. Forgue-Lafitte, M.E., Condray, A.M., Breant, B. and Mester, J. (1989) Proliferation of the human colon carcinoma cell line HT 29: autocrine growth and deregulated expression of the c-myc oncogene. Cancer Res., 49, 6566-6571.
22
Spigelman, Z., Dowers, A., Kennedy, S., Disorbo, D.. O’Brien, M., Barr, R. and McCaffrey, R. (1987) Antiproliferative effects of suramin on lymphoid cells. Cancer Res., 47, 4694-4698.
23
Huang, S.S. and Huang, J.S. (1988) Rapid turnover of the platelet-derived growth factor receptor in sistransformed cells and reversal by suramin. J. Biol. Chem.. 263, 1260812618. Fleming, T.P., Matsui, T., Molloy, C.J., Robbins, K.C. and Aaronson, S.A. (1989) Autocrine mechanism for v-sis transformation requires cell surface localization of internally activated growth factor receptors. Proc. Natl. Acad. Sci. U.S.A., 86, 8063-8067.
J.L. (1987) Suramin inhibition of growth factor receptor binding and mitogenicity in AKR-2B cells. J. Cell. Physiol., 12
Fantini, J., Verrier, B., Robert, C., Pit, P., Pichon, J., Mauchamp, J. and Marvaldi, J. (1990) Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT 29-D4 in serum-free medium. Exp. Cell Res., 189, 109- 117. Rabenandrasana, C., Baghdiguian, S., Roccabianca. M., Brunet, M., Marvaldi, J. and Fantini, J. (1990) The concentration of glucose in the culture medium determines the effect of suramin on the growth and differentiation of the human colonic adenocarcinoma cell clone HT 29-D4. Cancer Lett., 53, 109- 115. Culouscou, J.M., Remacle-Bonnet, M., Garrouste, F.,
24
33
25
26
27
28
without augmentation of glucose transport. Biochim Biophys. Acta, 972. 60-68. Pollak. M. and Richard, M. (1990) Suramin blokade of insulin-like growth factor l-stimulated proliferation of
30
growth factors in human colonic epithelium. Gastroenterology, 98, 703 - 707. Conti. F.G., Elliot. S.J., Striker, L.J. and Striker, G.E. (1989) Binding of Insulin-like growth factor-l by glomerular endothelial and epithelial cells: further evidence for IGF-I action in the renal glomerulus. Biochem. Biophys. Res. Commun., 163, 952 - 958. Massague, J. and Czech, M.P. (1982) The subunit struc-
human osteosarcoma cells. J. Natl. Cancer Inst., 82, 1349 - 1352. Rouyer-Fessard, C., Gammeltoft, S. and Laburthe, M. (1990) Expression of two types of receptor for Insulinlike
31
ture of two I and II and 01. Chem.. McRoberts.
Yayon, A. and Klagsbrun. M. (1990) Autocrine transformation by chimeric signal peptide-basic fibroblast growth factor: reversal by suramin. Proc. Natl. Acad. Sci. U.S.A., 87, 5346 - 5350. Franklin, C.C.. Chin, P.C., Turner, J.T. and Kim, H.D. (1988) Insulin regulation of glucose metabolism in HT 29 colonic adenocarcinoma cells: activation of glycolysis
29
distinct receptors for insulin-like growth factor their relationship to the insulin receptor. J. Bi257. 5038-5045. J.A., Aranda, R., Riley, N. and Kang. H.
(1990) Insulin regulates the paracellular permeability of cultured intestinal epithelial cell monolayers. J. Clin. Invest., 85, 1127- 1134.
