RESUMEN
Resistance of cancer cells against apoptosis induced by death factors contributes to the limited efficiency of immune- and drug-induced destruction of tumors. We report here that insulin and insulin-like growth factor-I (IGF-I) fully protect HT29-D4 colon carcinoma cells from IFN-gamma/tumor necrosis factor-alpha (TNF) induced apoptosis. Survival signaling initiated by IGF-I was not dependent on the canonical survival pathway involving phosphatidylinositol 3'-kinase. In addition, neither pp70(S6K) nor protein kinase C conveyed IGF-I antiapoptotic function. Inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) with the MAPK/ERK kinase inhibitor PD098059 and MAPK/p38 with the specific inhibitor SB203580 partially reversed, in a nonadditive manner, the IGF-I survival effect. Inhibition of nuclear factor kappaB (NF-kappaB) activity by preventing degradation of the inhibitor of NF-kappaB (IkappaB-alpha) with BAY 11-7082 also blocked in part the IGF-I antiapoptotic effect. However, the complete reversal of the IGF-I effect was obtained only when NF-kappaB and either MAPK/ERK or MAPK/p38 were inhibited together. Because these pathways are also those used by TNF to signal inflammation and survival, these data point to a cross talk between IGF-I- and TNF-induced signaling. We further report that TNF-induced IL-8 production was indeed strongly enhanced upon IGF-I addition, and this effect was totally abrogated by both MAPK and NF-kappaB inhibitors. The IGF-I antiapoptotic function was stimulus-dependent because Fas- and IFN/Fas-induced apoptosis was not efficiently inhibited by IGF-I. This was correlated with the weak ability of Fas ligation to enhance IL-8 production in the presence or absence of IGF-I. These findings indicate that the antiapoptotic function of IGF-I in HT29-D4 cells is based on the enhancement of the survival pathways initiated by TNF, but not Fas, and mediated by MAPK/p38, MAPK/ERK, and NF-kappaB, which act in concert to suppress the proapoptotic signals. In agreement with this model, we show that it was possible to render HT29-D4 cells resistant to Fas-induced apoptosis provided that IGF-I and TNF receptors were activated simultaneously.
Asunto(s)
Apoptosis/fisiología , Factor I del Crecimiento Similar a la Insulina/farmacología , Interferón gamma/toxicidad , Factor de Necrosis Tumoral alfa/toxicidad , Adenocarcinoma , Anticuerpos Monoclonales/farmacología , Antígenos CD/fisiología , Apoptosis/efectos de los fármacos , Neoplasias del Colon , Fragmentación del ADN , Humanos , Interleucina-8/biosíntesis , Receptores del Factor de Necrosis Tumoral/fisiología , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Proteínas Recombinantes , Células Tumorales CultivadasRESUMEN
Limited proteolysis of insulin-like-growth-factor (IGF)-binding proteins (IGFBPs) represents a key process to modulate IGF bio-availability at the cellular level. In human colon carcinomas, urokinase-type plasminogen activator (u-PA) produced by stroma cells can bind to cancer-cell-associated u-PA receptor (u-PAR), and then catalyze the conversion of plasminogen (Pg) into plasmin (Pm). We therefore investigated the interplay between the IGF and Pm systems in the HT29-D4 human colon-carcinoma-cell model. HT29-D4 cells secreted IGF-II totally complexed to IGFBP-2, IGFBP-4 and IGFBP-6. Approximately 15% of IGFBP-4 was associated with the extracellular matrix. HT29-D4 cells produced neither u-PA- nor IGFBP-specific proteases. However, activation of Pm at the HT29-D4 cell surface obtained by the sequential addition of exogenous u-PA and Pg to mimic the stromal complementation induced selective proteolysis targeted to IGFBP-4 only (>95%). IGFBP-2 and IGFBP-6, though sensitive to proteolysis by soluble Pm, were not altered by cell-bound Pm. IGFBP-4 proteolysis yielded 18- and 14-kDa immunoreactive fragments which were not detectable by Western ligand blotting, indicating that they bound IGF-II with poor affinity. Release of IGF-II from IGF-II-IGFBP complexes after IGFBP-4 proteolysis by cell-bound Pm was indicated by the observation that approximately 20% of the 125I-IGF-II initially associated with endogenous IGFBP in reconstituted complexes was transferred to HT29-D4 cell-surface IGF-I receptors. These results suggest that IGFBP-4 proteolysis by cell-bound Pm can promote autocrine/paracrine IGF-II bio-availability in colon-cancer cells. This may have important consequences on the behavior of cancer cells at the interface between stroma and malignant cells in carcinomas of the colon in vivo.
Asunto(s)
Adenocarcinoma/metabolismo , Neoplasias del Colon/metabolismo , Fibrinolisina/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Aprotinina/farmacología , Western Blotting , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/metabolismo , Endopeptidasas/análisis , Endopeptidasas/metabolismo , Matriz Extracelular/metabolismo , Humanos , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Factor II del Crecimiento Similar a la Insulina/análisis , Plasminógeno/farmacología , Células Tumorales CultivadasRESUMEN
To assess the autocrine function of insulin-like growth factor II (IGF-II) in the balance of proliferation and differentiation in HT29-D4 human colonic cancer cells, we studied the expression of IGF-I receptors (IGF-IR) and insulin receptors (IR) in relation to the state of cell differentiation. IGF-IR and IR were expressed in both undifferentiated and enterocyte-like differentiated HT29-D4 cells. IGF-IR had two isoforms with a 97-kDa and a 102-kDa beta-subunit. In addition, HT29-D4 cells expressed hybrid receptors (HR) formed by the association of two alphabeta heterodimers from both IR and IGF-IR. HR were evidenced through 1) inhibition of IGF-I binding by the B6 anti-IR antibody and 2) immunoprecipitation with the alpha-IR3 anti-IGF-IR antibody, which revealed an additional 95-kDa IR beta-subunit that disappeared when the heterotetrameric receptor was dissociated by disulfide reduction into alphabeta heterodimers before immunoprecipitation. Like IGF-IR, HR had a high affinity for IGF-I (Kd, approximately 1.5 nM), but did not bind insulin significantly; the latter interacted with the native IR only (Kd, approximately 4 nM). In the differentiated HT29-D4 cell monolayer, all receptor species were strongly polarized (>97%) toward the basolateral membrane. Moreover, HT29-D4 cell differentiation was accompanied by an approximately 2-fold increase in the number of IR, whereas the number of IGF-I-binding sites was unaltered. However, in differentiated HT29-D4 cells, approximately 55% of the latter were involved in HR vs. approximately 20% in undifferentiated HT29-D4 cells. Thus, HT29-D4 cell differentiation is characterized by an up-regulation (approximately 3-fold) of the level of HR coupled to a down-regulation (approximately 40%) of the level of native tetrameric IGF-IR. Alterations were induced early during the cell differentiation process, i.e. 5 days postconfluence, and remained unchanged for at least 21 days. Taken together, these results suggest that the IGF-II autocrine loop in HT29-D4 cells may trigger distinct signaling pathways if it activates native IGF-IR, which predominate in undifferentiated cells, or if it activates HR, which are up-regulated in differentiated cells.
Asunto(s)
Diferenciación Celular , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Unión Competitiva , Reactivos de Enlaces Cruzados , Citometría de Flujo , Células HT29 , Humanos , Técnicas de Inmunoadsorción , Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Radioisótopos de YodoRESUMEN
We have examined the polarity of the IGF system in differentiated HT29-D4 colonic epithelial cells cultured on permeable supports. Type I IGF receptors (approximately 30,000 per cell; Kd approximately 1 nM) are highly polarized (> 97%) in the basolateral membrane, and this figure does not change whatever the stage of post-confluent differentiation. In early differentiated cells, i.e., up to day 7 post-confluence, IGF-II, IGFBP-2, IGFBP-4 (> 96%) and IGFBP-6 (approximately 85%) are recovered in the basolateral medium. In contrast, in well differentiated cells, e.g. at day 23, a differential distribution of the IGFBPs secretory pathways is observed: IGFBP-2 and IGFBP-4 continue to be predominantly secreted from the basolateral surface whereas IGFBP-6 is almost all (> 96%) targeted towards the apical surface. As a result, IGF-II is secreted in equal quantities in both apical and basolateral compartments. Since the constitutive secretory pathway in intestine epithelial cells is known to be basolateral only, it is suggested that the IGFBP-6 apical release results from an active sorting. In addition, IGFBP-6 secretory level is down-regulated (approximately 60% decrease) whereas those of IGFBP-2 and IGFBP-4 remain constant during the differentiation process. Although speculative, we suggest that this IGFBPs differential secretory sorting could regulate the IGFBPs basolateral secretory profile, that in turn could ensure a fine tuning of IGF-II autocrine bioavailability towards the IGF-responsive basolateral membrane of the colonic epithelial cells.
Asunto(s)
Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 4 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Proteína 6 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor II del Crecimiento Similar a la Insulina/metabolismo , Mucosa Intestinal/metabolismo , Membrana Basal/metabolismo , Disponibilidad Biológica , Western Blotting , Antígeno Carcinoembrionario/metabolismo , Diferenciación Celular , Células HT29 , Humanos , Intestinos/citología , Receptor IGF Tipo 1/metabolismoRESUMEN
The HT29 human colonic carcinoma cell line secretes insulin-like growth factor (IGF)-II. We have examined these cells for expression of IGF receptors. Competitive binding assays as affinity cross-linking experiments using 125I-IGF-II fail to reveal type II IGF receptors at the cell surface. In contrast, cross-linking studies with either 125I-IGF-I or 125I-IGF-II reveal an M(r) 135,000 protein that follows a peptide binding specificity characteristic of the alpha-subunit of the type I IGF receptor. However, 125I-IGF-II binding to this receptor is not inhibited at 4 C by alpha IR-3, a monoclonal antibody to the type I IGF receptor. Analysis of the competitive binding curves with each one of these radioligands suggests that HT29 cells express both a classical type I IGF receptor (about 6,000/cell; KdIGF-I = 0.48 nmol) and a variant one whose 125I-IGF-II binding is not blocked by alpha IR-3 (about 15,000/cell; KdIGF-II = 4.0 nmol). Endocytosis studies of specific cell-bound 125I-IGF-I or 125I-IGF-II suggest that ligand interaction with the classical, but not the variant, binding site is only able to induce receptor internalization. An identical IGF receptors pattern is observed with HT29-D4 clonal cells induced to differentiate by culture in a glucose-free medium.
Asunto(s)
Carcinoma/metabolismo , Neoplasias del Colon/metabolismo , Receptores de Superficie Celular/metabolismo , Marcadores de Afinidad , Anticuerpos Monoclonales , Unión Competitiva , Carcinoma/patología , Diferenciación Celular , Neoplasias del Colon/patología , Humanos , Receptores de Superficie Celular/clasificación , Receptores de Somatomedina , Somatomedinas/metabolismo , Temperatura , Células Tumorales CultivadasRESUMEN
Suramin, a drug that binds to several types of growth factors, has been previously shown to induce the enterocyte-like differentiation of HT29-D4 human colonic adenocarcinoma cells, suggesting that growth factors are involved in such a process. Undifferentiated HT29-D4 cells release insulin-like growth factor II (IGF-II) into the culture medium that is totally complexed to heterogeneous IGF binding proteins (IGFBP) expressing high affinities for this growth factor (Kda = 0.02 nM and Kdb = 1.4 nM). These complexes do not allow IGF-II to bind to HT29-D4 cell surface type I IGF receptors, as evidenced by using 125I-IGF-II-IGFBP complexes. However, the addition of 40-100 micrograms/ml suramin, i.e., concentrations identical to the ones that are able to induce HT29-D4 cell differentiation, induces the release of IGF-II from IGF-II-IGFBP complexes, thereby allowing IGF-II to bind to the cell surface receptors. At such concentrations, suramin is indeed unable to alter IGF-II binding to HT29-D4 cells, a capacity that is observed only for concentrations higher than 200 micrograms/ml. Thus, suramin might have the unusual capacity to allow the establishment of an IGF-II autocrine loop involved in HT29-D4 cell differentiation. Consistent with this hypothesis is the fact that exogenously applied IGF-I (2.5 micrograms/ml) or agonist monoclonal antibody alpha IR-3 (2.5 micrograms/ml), which can bypass IGFBP present in the culture medium, induces part of HT29-D4 cell differentiation that is characterized by an important carcinoembryonic antigen release and the induction of numerous intercellular cysts with microvilli.
Asunto(s)
Diferenciación Celular/fisiología , Factor II del Crecimiento Similar a la Insulina/fisiología , Suramina/farmacología , Adenocarcinoma/ultraestructura , Antígeno Carcinoembrionario/análisis , Proteínas Portadoras/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular , Neoplasias del Colon/ultraestructura , Humanos , Proteína 2 de Unión a Factor de Crecimiento Similar a la Insulina , Factor II del Crecimiento Similar a la Insulina/metabolismo , Cinética , Microscopía Electrónica , Células Tumorales CultivadasRESUMEN
Products secreted by HT-29 human colonic adenocarcinoma cells (DMEM-HT-29) mediated strong suppressive activity of in vitro lymphoproliferative responses to several mitogens. In vivo administration of DMEM-HT-29 both inhibited the afferent limb of delayed-type hypersensitivity against the Mc FiFi2(s) syngeneic fibrosarcoma and delayed the rejection of these tumor cells by immunized animals. Transfer experiments prior or after cell fractionation did not demonstrate suppressor cells induced by DMEM-HT-29. This suggests that DMEM-HT-29 produces its effect by directly interacting with macrophage and/or T cells at the sensitization stage of the antitumor immune response.