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We have previously established that insulin-like growth factor (IGF)-I,-II and insulin exert a strong protective effect against tumor necrosis factor-α (TNF)-induced apoptosis in interferon-γ (IFN)-sensitized HT29-D4 human colon carcinoma cells. In this study, we report that this effect was still operative when cells were cultured in the absence of integrin- and E-cadherinmediated cell-extracellular matrix and cell-cell interactions. In this model, IGF-I did not activate the focal adhesion kinase, whereas it induced tyrosine phosphorylation of the insulin receptor substrate-1 and activation of the extracellular signalrelated kinase 1 and 2, p38, phosphatidylinositol 3'-kinase and protein kinase B/Akt. However, the use of specific inhibitors indicated that these pathways did not play a role in the adhesion-independent IGF-I anti-apoptotic signal. In contrast, inhibition of the NF-κB activation induced a complete reversal of the IGF-I anchorage-independent protective effect. Correspondingly, IGF-I markedly enhanced the TNF- and IFN/TNFinduced NF-κB-dependent interleukin-8 production. Our results provide evidence that IGF-I induces resistance against cytokine-induced cell death even in the absence of cell adhesion-mediated signaling. NF-κB appears to be a key mediator of this anti-apoptotic effect that should contribute to the resistance of colon cancer cells to immune-destruction during metastasis. [ABSTRACT FROM AUTHOR]

Noxo1, the organizing element of the Nox1-dependent NADPH oxidase complex responsible for producing reactive oxygen species, has been described to be degraded by the proteasome. We mutated a D-box in Noxo1 to express a protein with limited degradation and capable of maintaining Nox1 activation. Wild-type (wt) and mutated Noxo1 (mut1) proteins were expressed in different cell lines to characterize their phenotype, functionality, and regulation. Mut1 increases ROS production through Nox1 activity affects mitochondrial organization and increases cytotoxicity in colorectal cancer cell lines. Unexpectedly the increased activity of Noxo1 is not related to a blockade of its proteasomal degradation since we were unable in our conditions to see any proteasomal degradation either for wt or mut1 Noxo1. Instead, D-box mutation mut1 leads to an increased translocation from the membrane soluble fraction to a cytoskeletal insoluble fraction compared to wt Noxo1. This mut1 localization is associated in cells with a filamentous phenotype of Noxo1, which is not observed with wt Noxo1. We found that mut1 Noxo1 associates with intermediate filaments such as keratin 18 and vimentin. In addition, Noxo1 D-Box mutation increases Nox1-dependent NADPH oxidase activity. Altogether, Nox1 D-box does not seem to be involved in Noxo1 degradation but rather related to the maintenance of the Noxo1 membrane/cytoskeleton balance.

The Microtubule-Associated Protein Tau is expressed in several cancers, including low-grade gliomas and glioblastomas. We have previously shown that Tau is crucial for the 2D motility of several glioblastoma cell lines, including U87-MG cells. Using an RNA interference (shRNA), we tested if Tau contributed to glioblastoma in vivo tumorigenicity and analyzed its function in a 3D model of multicellular spheroids (MCS). Tau depletion significantly increased median mouse survival in an orthotopic glioblastoma xenograft model. This was accompanied by the inhibition of MCS growth and cell evasion, as well as decreased MCS compactness, implying N-cadherin mislocalization. Intracellular Signaling Array analysis revealed a defective activation of the PI3K/AKT pathway in Tau-depleted cells. Such a defect in PI3K/AKT signaling was responsible for reduced MCS growth and cell evasion, as demonstrated by the inhibition of the pathway in control MCS using LY294002 or Perifosine, which did not significantly affect Tau-depleted MCS. Finally, analysis of the glioblastoma TCGA dataset showed a positive correlation between the amount of phosphorylated Akt-Ser473 and the expression of MAPT RNA encoding Tau, underlining the relevance of our findings in glioblastoma disease. We suggest a role for Tau in glioblastoma by controlling 3D cell organization and functions via the PI3K/AKT signaling axis.

The NADPH oxidase proteins catalyse the formation of superoxide anion which act as signalling molecules in physiological and pathological processes. Nox1-dependent NADPH oxidase is expressed in heart, lung, colon, blood vessels and brain. Different strategies involving Nox1 inhibition based on diphenylene iodonium derivatives are currently tested for colorectal cancer therapy. Here, after peptides screening on Nox1-dependent NADPH oxidase assay in HT-29 cells, we identify a peptide (referred to as NF02), cell-active, that potently block Nox1-dependent reactive oxygen species generation. Study of DEPMPO adduct formation by electron paramagnetic resonance showed that NF02 has no superoxide scavenging activity and no impact on cellular reactive oxygen species-producing enzymes such xanthine oxidase. NF02 was not cytotoxic, inhibited reactive oxygen species production of reconstituted Nox1/Noxo1/Noxa1 complex in HEK293 and did not decrease Nox2 dependent cellular NADPH oxidase reactive oxygen species production. Finally, NF02 inhibited cell migration and invasion of colorectal cancer cells which is consistent with the described impact of Nox1 inhibitors on cell migration. NF02 peptide is a new NADPH oxidase inhibitor specific for Nox1 over Nox2 and xanthine oxidase which might represent a useful Nox1 tool with potential therapeutic insights., (Copyright © 2016 Elsevier B.V. All rights reserved.)

α6β4 integrin is the main component of hemidesmosomes (HD) that stably anchor the epithelium to the underlying basement membrane. Epithelial cell migration requires HD remodelling, which can be promoted by epidermal growth factor (EGF). We previously showed that extracellular nucleotides inhibit growth factor-induced keratinocyte migration. Here, we investigate the effect of extracellular nucleotides on α6β4 integrin localisation in HD during EGF-induced cell migration. Using a combination of pharmacological inhibition and gene silencing approaches, we found that UTP activates the P2Y2 purinergic receptor and Gαq protein to inhibit EGF/ERK1/2-induced cell migration in keratinocytes. Using a keratinocyte cell line expressing an inducible form of the Raf kinase, we show that UTP inhibits the EGF-induced ERK1/2 pathway activation downstream of Raf. Moreover, we established that ERK1/2 activation by EGF leads to the mobilisation of α6β4 integrin from HD. Importantly, activation of P2Y2R and Gαq by UTP promotes HD formation and protects these structures from EGF-triggered dissolution as revealed by confocal analysis of the distribution of α6β4 integrin, plectin, BPAG1, BPAG2 and CD151 in keratinocytes. Finally, we demonstrated that the activation of p90RSK, downstream of ERK1/2, is sufficient to promote EGF-mediated HD dismantling and that UTP does not stabilise HD in cells expressing an activated form of p90RSK. Our data underline an unexpected role of P2Y2R and Gαq in the inhibition of the ERK1/2 signalling pathway and in the modulation of hemidesmosome dynamics and keratinocyte migration.

Insulin-like growth factor-I (IGF-I) activation of phosphoinositol 3-kinase (PI3K) is an essential pathway for keratinocyte migration that is required for epidermis wound healing. We have previously reported that activation of Galpha((q/11))-coupled-P2Y(2) purinergic receptors by extracellular nucleotides delays keratinocyte wound closure. Here, we report that activation of P2Y(2) receptors by extracellular UTP inhibits the IGF-I-induced p110alpha-PI3K activation. Using siRNA and pharmacological inhibitors, we demonstrate that the UTP antagonistic effects on PI3K pathway are mediated by Galpha((q/11))-and not G((i/o))-independently of phospholipase Cbeta. Purinergic signaling does not affect the formation of the IGF-I receptor/insulin receptor substrate-I/p85 complex, but blocks the activity of a membrane-targeted active p110alpha mutant, indicating that UTP acts downstream of PI3K membrane recruitment. UTP was also found to efficiently attenuate, within few minutes, the IGF-I-induced PI3K-controlled translocation of the actin-nucleating protein cortactin to the plasma membrane. This supports the UTP ability to alter later migratory events. Indeed, UTP inhibits keratinocyte spreading and migration promoted by either IGF-I or a membrane-targeted active p110alpha mutant, in a Galpha(q/11)-dependent manner both. These findings provide new insight into the signaling cross-talk between receptor tyrosine kinase and Galpha((q/11))-coupled receptors, which mediate opposite effects on p110alpha-PI3K activity and keratinocyte migration.

Dynamic crosstalk between cell adhesion molecules, extracellular matrix and soluble informative factors is essential for cancer cell migration and invasion. Here, we investigated the mechanisms by which the E-cadherin/catenin complex and alpha v integrin can modulate insulin-like growth factor-I (IGF-I)-induced cell migration. Human colon mucosa, human colon cancer cell lines, HT29-D4 and HCT-8 derivatives that differ in their expression of alpha-catenin, were used as models. Interactions between E-cadherin, alpha v integrin and IGF-I receptor (IGF-IR) were analyzed by coimmunoprecipitation and immunolocalization experiments. The impact of these interactions on cell mobility was determined by haptotaxis assays. We report that alpha v integrin, E-cadherin and IGF-IR form a ternary complex in both cultured cancer cells and human normal colonic mucosa. alpha-Catenin regulates the scaffolding of this complex. IGF-IR ligation by IGF-I induces the disruption of the complex and the relocalization of alpha v integrin from cell-cell contacts to focal contact sites. This perturbation is correlated with the observed increase in cell migration. These results suggest that regulation of the alpha v integrin/E-cadherin/IGF-IR scaffolding is essential for the modulation of cell mobility. Its alteration could be of major importance to sustain alterations in cell adhesion that occur during cancer cell invasion and metastasis., ((c) 2007 Wiley-Liss, Inc.)

Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y2 nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y2 receptor activation also induces a dramatic dismantling of the actin network, the loss of alpha3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of alpha(v) integrins and focal contact protein distribution. In addition, activation of P2Y2R prevents growth factor-induced phosphorylation of Erk(1,2) and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G alpha(q/11) by siRNA, or the expression of a constitutively active G alpha(q/11) mutant (Q209L) show that activation of G alpha(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.

In the tumor microenvironment, autocrine/paracrine loops of insulin-like growth factors (IGFs) contribute to cancer cell survival. However, we report here that IGF-I can send contradictory signals that interfere with cell death induced by different ligands of the tumor necrosis factor (TNF) superfamily. IGF-I protected human colon carcinoma cells from TNF-alpha-induced apoptosis, but it enhanced the apoptotic response to anti-Fas antibody and TNF-related apoptosis inducing ligand stimulation. This proapoptotic effect of IGF-I, observed in several but not all tested colon cancer cell lines, was mediated via the phosphatidylinositol 3'-kinase (PI3K)/Akt pathway. Furthermore, IGF-I receptors (IGF-IR) were located in and out of membrane lipid rafts and were tyrosine autophosphorylated in response to IGF-I. However, disruption of rafts by acute cholesterol depletion shifted IGF-IR to non-raft domains, abolished the IGF-I-mediated proapoptotic effect, and inhibited the IGF-I-dependent IRS-1 and Akt recruitment into and phosphorylation/activation within lipid rafts. Replenishing cell membranes with cholesterol reversed these effects. Activation of extracellular-regulated kinase-1/2 and p38 mitogen-activated protein kinase, which convey the IGF-I anti-apoptotic effect, occurred independently of lipid rafts. Thus, we propose that segregation of IGF-IR in and out of lipid rafts may dynamically regulate the pro- and anti-apoptotic effects of IGF-I on apoptosis induced by TNF superfamily members.

Apoptosis is a crucial mechanism to eliminate harmful cells in which growth factors and cytokines are key regulators. In HT29-D4 cells, a model of human colon carcinoma, IFNgamma presensitization is essential to induce an apoptotic response to TNFalpha whereas it only slightly enhances TRAIL-induced apoptosis. To compare the transcriptional profiles induced by TNFalpha and TRAIL and their regulation by IFNgamma, we optimized a cDNA array analysis on targeted signaling pathways and confirmed the gene expression modulations by comparative RT-PCR. Although the two TNFSF ligands induced a same strong up-expression of pro-apoptotic Bax gene, the expression of anti-apoptotic Bcl-xL gene was more strongly up-regulated in TNFalpha- than in TRAIL-stimulated cells. Thus, TRAIL but not TNFalpha induced apoptotic mitochondrial cascade as highlighted by cytochrome c release into cytosol. IFNgamma presensitization of TRAIL-stimulated cells did not induce any change in cytochrome c release, suggesting that the increase of IFNgamma/TRAIL-induced apoptosis is independent of this pathway. In contrast, IFNgamma pretreatment prevented Bcl-xL gene up-expression in TNFalpha-stimulated cells and allowed cytochrome c release. Thus, we hypothesize that the Bcl-xL/Bax ratio can block the apoptotic response in TNFalpha-stimulated cells but allows cell death initiation when it is altered by a crosstalk between IFNgamma presensitization and TNFalpha induced signalings.

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.

The limited proteolysis of insulin-like growth factor (IGF)-binding protein (IGFBP)-3 is a key event in the regulation of endocrine bioavailability of IGFs. Here, we investigated IGFBP-3 and IGFBP-3 proteolysis in serum from patients with colorectal cancer both before and at different times following surgery. In vivo IGFBP-3 proteolysis, estimated by immunoblot analysis of IGFBP-3 fragments in serum, and in vitro IGFBP-3 protease activity of serum, estimated by a 125I-IGFBP-3 degradation assay, allowed us to identify 2 groups of patients (IGF-M vs. IGF-NM) with respect to their status for mobilizing the IGF system. In IGF-M patients, in vivo and in vitro IGFBP-3 proteolysis were significantly elevated (156% and 181% of the age-matched control pool, respectively) and accompanied by a decrease in intact IGFBP-3 (38% of the control pool). The IGFBP-3 proteolytic processing was further increased in response to surgical ablation of the tumor (mean increase 45-55%), then gradually returned to levels comparable with controls. In contrast, IGF-NM patients exhibited a minimal alteration of in vitro IGFBP-3 protease activity and even an inhibition of in vivo IGFBP-3 proteolysis, whereas intact IGFBP-3 was unaltered when compared with controls. Moreover, this pattern was not further significantly altered in response to the surgical stress. None (0/6) of the IGF-M patients vs. 70% (5/7) of the IGF-NM patients developed a metastatic disease (median duration of follow-up 26 months). Neither elevated amounts of pro-IGF-II nor presence of detectable IGFBP-3 protease inhibitors in the circulation could explain the observed suppression of IGFBP-3 proteolytic processing in IGF-NM patients. These results indicate that inhibition of IGFBP-3 proteolysis and invasive properties of cancer cells are related in colorectal cancer patients.

To investigate endoproteolytic processing of the type I insulin-like growth factor receptor (IGF-IR), we have examined its structure and activity in the furin-deficient LoVo-C5 cell line. Immunoprecipitation experiments using the monoclonal anti-IGF-IR antibody (alpha-IR3) showed that LoVo-C5 cells expressed a major high molecular mass receptor (200 kDa) corresponding to the unprocessed alpha/beta pro-receptor. A small amount of successfully cleaved alpha/beta heterodimers was also produced, indicating a residual endoproteolytic cleavage activity in these cells. In vitro, a soluble form of recombinant furin was able to cleave the pro-IGF-IR (200 kDa) into alpha-subunit (130 kDa) and beta-subunit (97 kDa). Measurement of IGF binding parameters in LoVo-C5 cells indicated a low number of typical type I IGF-binding sites (binding capacity, 5 x 10(3) sites/cell; Kd, 1.9 nM for IGF-I and 7.0 nM for IGF-II). These findings in LoVo-C5 contrast with those in HT29-D4 cells, which have active furin, and where IGF-IR (2.8 x 10(4) sites/cell) was fully processed. Moreover, the 200-kDa pro-IGF-IR of LoVo-C5 was unable to induce intracellular signaling, such as beta-subunit tyrosine autophosphorylation and insulin-related substrate-1 tyrosine phosphorylation. Flow immunocytometry analysis using alpha-IR3 antibody indicated that LoVo-C5 cells expressed 40% more receptors than HT29-D4 cells, suggesting that in LoVo-C5 cells only the small amount of mature type I IGF-IR binds IGFs with high affinity. To provide evidence for this idea, we showed that mild trypsin treatment of living LoVo-C5 cells partially restored alpha/beta cleavage of IGF-IR, and greatly enhanced (6-fold) the IGF-I binding capacity of LoVo-C5 cells, but did not restore IGF-IR signaling activity. Moreover, LoVo-C5 cells were totally unresponsive to IGF-I in terms of cell migration, in contrast to fully processed IGF-IR-HT29-D4 cells. Our data indicate that furin is involved in the endoproteolytic processing of the IGF-IR and suggest that this posttranslational event might be crucial for its ligand binding and signaling activities. However, our data do not exclude that other proprotein convertases could participate to IGF-IR maturation.

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.

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.

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.

It has been reported that insulin-like growth factor (IGF) II is associated with human primary colorectal tumors and colon-carcinoma cell lines. Here, we examine alterations in circulating levels of IGFs and IGF binding proteins (IGFBPs) in patients with colorectal carcinoma, and compare them to age- and nutrition-adjusted references. We report (i) an increase in serum IGF-II concentrations (about 2-fold), whereas IGF-I concentrations are regarded as normal when aging is taken into account; (ii) an apparent increase in serum IGFBP-3 levels when compared to those of healthy elderly subjects, IGFBP-3 only being detected in the 150-kDa IGFBP ternary complex as in normal serum; (iii) abnormally elevated serum IGFBP-2 levels taking into account the apparent concentrations of IGFBP-3. This simultaneous elevation of IGFBP-3 and IGFBP-2 in the serum of patients with colorectal tumors appears to be unique in that it reflects a break in the inverse relationship between the serum IGFBP-3 and IGFBP-2 levels that is observed in normal and in several physiopathological conditions. Moreover, it enables a distinction to be made between 76.5% (13/17) of patients with colorectal carcinoma and normal adults, age-related healthy aged and malnourished patients. We propose that the disturbed serum IGFBP profile observed in the patients with colorectal cancer may be a consequence of oversecretion of IGF-II by the tumor cells. The usefulness of IGFs and IGFBPs as potential colorectal tumor-associated metabolic markers should be further investigated.

HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.

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.

The HT-29 human colon cancer cell line has previously been shown to secrete high amounts of insulin-like growth factor II (IGF-II). The recent demonstration that soluble IGF-II/mannose 6-phosphate receptor was present in fetal serum prompted us to search for a release of type-II IGF receptor by these human colonic carcinoma cells. Serum-free conditioned medium from the HT-29 cell line was gel filtered on Sephadex G-200. There was significant binding of [125I]IGF-II to the void volume fractions in addition to binding to the 40-kDa IGF-binding protein (IGF-BP) fractions. Competitive binding studies using [125I]IGF-II and the void volume pool showed a pattern typical of the type-II receptor. It exhibited a high affinity for IGF-II (KD = 0.4 nM), but had a low affinity for IGF-I (KD = 6.8 nM), and no detectable affinity for insulin. Additional evidence was provided by affinity cross-linking of [125I]IGF-II to the same high-molecular-weight material which demonstrated a major specific band at 250 kDa after reduction of disulfide bonds. In contrast, the type-I IGF receptor was undetectable. The extracellular type-II IGF receptor was not a significant carrier for IGF-II since virtually all IGF-II secreted by HT-29 cells was associated with IGF-BP. The presence of a soluble IGF-II/mannose 6-phosphate receptor in the culture medium from colonic cancer cells suggests that it may play an important role in tumor pathogenesis.

The serum-free medium conditioned by the human colon cancer cell line HT-29 contains insulin-like growth factors (IGF) that are entirely complexed to binding proteins (IGF-BP). Gel filtration in acid conditions of the cell-conditioned medium permits separation of IGF-BP from two molecular forms of IGF of 15,000 and 7,500 Mr. As determined by ligand blotting, IGF-BP are heterogeneous and constituted of three molecular forms of 31,000, 28,000, and 26,000 Mr. Using IGF-I and IGF-II radioreceptor assays, IGF-I radioimmunoassay (RIA), and competitive protein-binding assay specific for IGF-II, it is shown that the IGF-type eluting in 15 K and 7.5 K position from gel filtration is restricted to IGF-II. Its concentration is approximately 6 ng/10(6) HT-29 cells with 60% present as a high-molecular-weight form of IGF-II. This large 15 K IGF molecule is devoided of any IGF-binding activity and might represent incomplete processing of pro-IGF-II peptide. By contrast, the level of IGF-I detected by RIA is barely measurable and considered negligible (0.57 pg/10(6) HT-29 cells). Although these IGF-II-like peptides exhibit a growth-promoting activity on FR3T3 fibroblasts, they cannot stimulate, as recombinant IGF-I or IGF-II, 3H-thymidine incorporation into DNA of HT-29 cells, whatever the experimental conditions used. Finally, we have shown that IGF binding is restricted predominantly to the basolateral domain of the cell membrane by using HT-29-D4 clonal cells, derived from the parental HT-29 cell line, maintained in a differentiated state by culture in a medium in which glucose is replaced by galactose.

The conditioned medium from the HT-29 human colonic adenocarcinoma cell line contains a potent mitogenic activity that can markedly stimulate the proliferation of both rat and human fibroblasts in the absence of serum. Fractionation of conditioned medium on Bio-Gel P-100 shows that HT-29 cells simultaneously produce 2 different types of endogenous growth factors. The first one (molecular mass of 35, 8 and 5.5 kDa) exhibits an IGF-I competing activity which is positively correlated to mitogenic activity. This mitogen is recognized by anti-IGF-I antibodies but is resistant to reducing agents. It is distinct from IGF-II, insulin and PDGF. The second one (molecular mass of 40- and 20-kDa) is able to displace EGF binding to its receptor. This factor is immunologically recognized by anti-EGF antibodies but with a lower affinity as compared to EGF. This suggests that this endogenous HT-29-growth factor is related to but distinct from native EGF. Although more active in radioreceptor assay than in radioimmunoassay, the EGF-competing factor is distinct from TGF alpha or beta since it is unable to induce anchorage-independent growth of NRK or FR3T3 target cells in the presence or absence of exogenous EGF. Moreover, free functional EGF receptors are available at the HT-29 cell surface.

Rat peritoneal cells can bind immunoglobulin-coated sheep red cells (IGSRC), glutaraldehyde-treated sheep red cells (GSPC), Leishmania, latex beads, and autologous thymocytes in a serum-deprived medium. When macrophages were plated on plastic Petri dishes, their ability to bind thymocytes and GSRC was decreased ninefold and fourfold, respectively, as compared to macrophages adhering to glass coverslips. However, the binding of IGSRC, Leishmania, and latex was not significantly dependent on the nature of the surface where peritoneal cells were plated. Sequential adhesion to plastic and glass did not reveal any cell subpopulation adhering only to one substrate. The ability of plastic-bound macrophages to bind thymocytes or GSRC was not restored after a 16 hr culture. Hence, some cell properties may be strikingly dependent on the nature of the surface where these cells are plated.

The generation of superoxide anion (O2-) by human blood monocytes in response to stimulation by either phorbol myristate acetate (PMA) or opsonized Zymosan was greatly enhanced (range: 100-200% according to donor) by prior exposure of the peripheral blood mononuclear cells (PBM) to human colonic adenocarcinoma cells (HT 29 line) or their conditioned culture medium (DMEM-HT 29). This priming effect was observed after 5 hr and persisted for up to 15 hr of contact between PBM and endotoxin-free DMEM-HT 29. Beyond this time, primed monocytes gradually lost this ability. However, they maintained a higher capacity (about 100%) to produce O2- when compared to controls. DMEM-HT 29-induced monocyte priming requires that the tumor-active substance(s) act(s) on 2 target cells: first, on non adherent mononuclear cells (NA-PBM) to induce cytokine production and, second, on the monocyte itself. Priming activity was also found in conditioned medium from FR3T3 embryonic fibroblasts but not in conditioned medium from HT 29 repolarized cells (by culture in glucose-free medium) or from non-tumorous human colonic mucosa explants.

The human colon cancer cell line HT-29 produces a growth factor (CRDGF; Mr = 25,000) which inhibits EGF binding to a wide variety of different normal and tumoral cell types in culture. Scatchard analysis of EGF binding shows that CRDGF induces a decrease in EGF receptor affinity. In contrast, EGF binding to any of the human colorectal cancer cell lines tested, i.e., HT-29, HT-29 (clone D4), HRT-18 or CAL-14, remains unaltered in the presence of exogenous CRDGF. However, the inhibitory effect of CRDGF becomes apparent on HT-29 cells after overnight exposure of these to suramin (at 37 degrees C). A short exposure to suramin (1 hr at 4 degrees C) or a mild acid washing of HT-29 cells can partially restore the inhibitory activity of CRDGF. These observations suggest that the action of suramin results in an unmasking of substantial levels of CRDGF receptors on HT-29 cells. Scatchard analysis of EGF binding on suramin-treated HT-29 cells shows that CRDGF inhibits EGF binding by decreasing EGF receptor affinity, as previously observed with the non-colonic cell types. A similar unmasking of CRDGF receptors is observed when the other colorectal cell lines are exposed to suramin. These results provide evidence for a model in which the colorectal cell lines have the property of secreting a unique growth factor that binds to its receptor by an autocrine mechanism.

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.

Generation of superoxide anion (O2-) by human polymorphonuclear leukocytes (PMNs) in response to stimulation by opsonized zymosan was enhanced about 100% by prior exposure of the PMNs to human colonic adenocarcinoma cells (HT-29 cell line) or their conditioned culture medium. In addition, HT-29 cells produced substances that had an appreciable chemokinetic activity on PMNs. These tumor-secreted substances appeared to act directly on the PMNs rather than indirectly by interacting with nonadherent mononuclear cells, e.g., lymphocytes. Such a priming activity to display enhanced production of O2- was also found in conditioned medium from F344 rat FR3T3 embryonic fibroblasts but not in conditioned medium from HT-29 repolarized cells (by culture in galactose-containing medium) or from nontumorous human colonic mucosa explants. Such active substances may be important in the host-tumor relationship and, therefore, in the outcome of tumor growth.