Insulin-like growth factor-I protects colon cancer cells from death factor-induced apoptosis by potentiating tumor necrosis factor alpha-induced mitogen-activated protein kinase and nuclear factor kappaB signaling pathways.

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.

Surface-bound plasmin induces selective proteolysis of insulin-like-growth-factor (IGF)-binding protein-4 (IGFBP-4) and promotes autocrine IGF-II bio-availability in human colon-carcinoma cells.

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.

Up-regulation of insulin/insulin-like growth factor-I hybrid receptors during differentiation of HT29-D4 human colonic carcinoma cells.

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.

Differential secretory polarity of IGFBP-6 vs. IGFBP-2 and IGFBP-4 in human intestinal epithelial cells: is it a way of modulating IGF-II bioavailability towards the IGF-responsive basolateral surface?

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.

Expression of type I, but not type II insulin-like growth factor receptor on both undifferentiated and differentiated HT29 human colon carcinoma cell line.

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.

Potential autocrine role of insulin-like growth factor II during suramin-induced differentiation of HT29-D4 human colonic adenocarcinoma cell line.

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.

In vivo delayed rejection of tumors and inhibition of delayed-type hypersensitivity by HT-29 human colonic adenocarcinoma cell line.

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.

Immunomodulating activities associated with the cytosol fraction of a 3-methylcholanthrene-induced rat fibrosarcoma--II. Association with polyamine complexes.

This paper characterizes the molecular nature of the factors present in cytosol from F-344 rat McFiFi2(s) fibrosarcoma cells (FiCF) which mediate inhibition of PHA-induced lymphoproliferative responses. These are polyamines (spermine/spermidine) conjugated to different protein carriers. Interaction of these complexes with polyamine oxidase (PAO) present in fetal calf or rat serum is responsible of the suppression observed.

Immunomodulating activities associated with the cytosol fraction of a 3-methylcholanthrene-induced rat fibrosarcoma.

Cytosol fraction(s) from McFiFi2(s) fibrosarcoma cells (Fcc), isolated from either cultured cells or solid tumors induced in F344 rats, produced a dose-related inhibition of lymphoproliferative responses to several mitogens, whatever the lymphoid organ or the animal species used as the source of lymphocytes. Only stimulated human lymphocytes were not Fcc inhibited; instead, Fcc was a potent stimulator of their spontaneous proliferation. Fcc cytostatic activity was not effective in various cycling cell lines and was restricted to mitogen-stimulated lymphocytes. Fcc, a primary tumor product, did not induce suppressive cells and was unable to prevent mitogen cell surface binding. However, expression of its modulating effect was accelerated by the simultaneous presence of the mitogen. Moreover, Fcc produced its suppression by interrupting lymphocyte activation at some point within the G0-G1-phase transition. Molecular sieving showed that Fcc contains at least two factors with suppressive (mol wt, approximately 3,000) and stimulatory (mol wt, greater than 5,000) activities, respectively.

Nonspecific inhibitory activity of soluble human colon carcinoma extracts: tentative mechanism of action.

Soluble human colon carcinoma extract(s) (SCE) were potent nonspecific inhibitors of lymphoproliferative responses to mitogens. Inhibition was concomitant with induction in about 35% of cells of morphologic alterations for most of them comparable with the ones observed in mitogen-induced blast cells. Nonetheless, these blastlike cells did not proliferate. SCE did not interfere with mitogen binding to cell receptors. Moreover, SCE was unable to induce or activate suppressor cells, and its primary target cell was the unresponsive lymphoid cell itself. The inhibitory effect of SCE was early and irreversible. The differential activity of SCE can be correlated with an early [3H]uridine uptake, which was inhibited 6 hours later, as seen for the other biochemical parameters of cell activation. Also, SCE altered membrane-bound ATPase activities. Na,K-ATPase was strongly inhibited, whereas Ca2+-dependent and Mg2+-dependent ATPases were stimulated. These observations were discussed as an SCE-lymphocyte plasma membrane interaction translated into differential signals to the intracellular metabolic pathways.

Nonspecific suppressive and cytostatic activities mediated by human colonic carcinoma tissue or cultured cell extract.

We have previously shown that soluble extracts from human colonic carcinoma (SCE) were potent inhibitors of the PHA-induced DNA synthesis of normal allogeneic peripheral lymphocytes. From the present study, SCE appeared to have a broad and nonspecific range of activity. The SCE-suppressive effect was observed whatever the lymphoid organ or the animal species used as a source of stimulated lymphocytes. Moreover, we always obtained a complete inhibition of the lymphoproliferative response to all tested mitogens including PHA, Con A, PWM, and, for animal lymphocytes, LPS. In addition to the suppressive activity on lymphocytes, SCE also inhibited proliferation of cultured human CCL6 embryonic intestine cells and HT29 colonic carcinoma cells, and of cultured rat fibrosarcoma cells. Soluble extracts from HT29 cells (SCCE) were able to mimic the nonspecific suppressive and cytostatic activities of SCE. The suppressive activity was also found in extracts from hepatic metastases (SHME) of a primary colonic tumor. In contrast, soluble extracts from normal liver or nonmalignant colonic mucosa did not interfere with cell proliferation. These data suggest that both SCE and SCCE contain molecular components(s) that can inhibit a wide variety of proliferating cells, including stimulated lymphocytes.