Can cryoglobulins interfere with the measurement of IgM antiphosphatidylethanolamine antibodies by ELISA?

Clinical manifestations of the antiphospholipid antibody syndrome (APS) have been recently related to the presence of phosphatidylethanolamine antibodies (aPE). However, it is well known that some molecules such as cryoglobulins, immunoglobulins that undergo a reversible precipitation at low temperatures, may interfere with biological assays. With this in view, we report the case of a patient with APS who was positive for both IgM aPE and type III cryoglobulinemia. Moreover, we show for this patient a potential implication of aPE in the cryoprecipitate formation. To further analyze the potential association between cryoglobulins and aPE, and also the possible consequences for aPE assay, we selected 55 patients according to positivity for both IgM aPE and cryoglobulinemia. Determination of IgM aPE levels was made before and after removal of cryoprecipitate from the serum. Of the 55 selected patients, 52 (95%) presented no significant difference for IgM aPE levels before and after cryoprecipitation. These results were ascertained whatever the aPE levels and clinical status of the patient. Taken together, our results indicate that cryoprecipitation does not interfere in most cases (95%) with the dosage of IgM aPE. Thus, IgM aPE do not appear to be involved in the formation of the cryoprecipitate.

Prevention of cytokine-induced apoptosis by insulin-like growth factor-I is independent of cell adhesion molecules in HT29-D4 colon carcinoma cells-evidence for a NF-kappaB-dependent survival mechanism.

We have previously established that insulin-like growth factor (IGF)-I, -II and insulin exert a strong protective effect against tumor necrosis factor-alpha (TNF)-induced apoptosis in interferon-gamma (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-cadherin-mediated 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 signal-related 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-kappaB activation induced a complete reversal of the IGF-I anchorage-independent protective effect. Correspondingly, IGF-I markedly enhanced the TNF- and IFN/TNF-induced NF-kappaB-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-kappaB 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.

Activation of protein tyrosine kinases by Coxiella burnetii: role in actin cytoskeleton reorganization and bacterial phagocytosis.

Coxiella burnetii, the agent of Q fever, is an obligate intracellular microorganism that grows in monocytes/macrophages. The internalization of virulent organisms by monocytes is lower than that of avirulent variants and is associated with actin cytoskeleton reorganization. We studied the activation of protein tyrosine kinases (PTKs) by C. burnetii in THP-1 monocytes. Virulent organisms induced early PTK activation and the tyrosine phosphorylation of several endogenous substrates, including Hck and Lyn, two Src-related kinases. PTK activation reflects C. burnetii virulence since avirulent variants were unable to stimulate PTK. We also investigated the role of PTK activation in C. burnetii-stimulated F-actin reorganization. Tyrosine-phosphorylated proteins were colocalized with F-actin inside cell protrusions induced by C. burnetii, and PTK activity was increased in Triton X-100-insoluble fractions. In addition, lavendustin A, a PTK inhibitor, and PP1, a Src kinase inhibitor, prevented C. burnetii-induced cell protrusions and F-actin reorganization. We finally assessed the role of PTK activation in bacterial phagocytosis. Pretreatment of THP-1 cells with lavendustin A and PP1 upregulated the uptake of virulent C. burnetii but had no effect on the phagocytosis of avirulent organisms. Thus, it is likely that PTK activation by C. burnetii negatively regulates bacterial uptake by interfering with cytoskeleton organization.

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.

IGF-I/IGFBPs system response to endotoxin challenge in sheep.

Endotoxin (LPS), a membrane component of gram-negative bacteria produces multiple endocrine and metabolic effects that mimic those seen in acute sepsis. It induces species-dependent alterations of the growth hormone (GH) axis that may participate in the shift of the metabolism towards catabolic events. Humans and sheep show increased GH secretion in response to LPS, as opposed to rats, which have been the most studied. The purpose of our work was to evaluate the effects in intact rams of an acute intravenous administration of a high dose of LPS on the insulin-like growth factor (IGF)-I/IGF-binding proteins (IGFBPs) system and to analyse the temporal relationship of GH axis changes with those of several hormonal and metabolic parameters such as somatostatin, cortisol, insulin, and glucose. LPS induced a late moderate decrease of total IGF-I plasma levels following a 5-h steady-state period (-26.6+/-4. 2%, P<0.05, 9 h after LPS), despite a biphasic and sustained increase of GH secretion in the same animals (2.48+/-0.39 ng/ml 2 h after LPS and 2.7+/-0.37 ng/ml 5 h after LPS vs 0.77+/-0.10 before LPS; Briard et al. 1998a). Western ligand blot analysis in IGFBPs showed an early short-lasting increase in IGFBP-1 (188.8+/-39% P<0. 05, 3 h after LPS). No significant change was seen for either IGFBP-2, -3 or -4. We observed a marked and sustained increase in cortisol (128.18+/-7.21 ng/ml 3 h after LPS, vs 21.17+/-4.22 before LPS). Insulin also increased (27.69+/-3.90 microU/ml 3 h after LPS, vs 13.48+/-1.69 before LPS) and its burst coincided with that of IGFBP-1. Moderately decreased IGF-I and increased IGFBP-1 plasma levels contrasted with the sustained increase in GH secretion that we recently described, thereby suggesting that endotoxin causes a state of resistance to GH. This may be exacerbated by reduced IGF-I bioavailability and/or action, and which may participate in the pathophysiology of the catabolic state seen in sepsis. The temporal analysis of hormone responses suggests that endotoxin-induced alterations of the IGF-I/IGFBPs system may involve the prolonged and substantial somatostatin rise that we recently demonstrated, together with an increase in glucocorticoid and cytokine as more generally assumed.

Integrins and E-cadherin cooperate with IGF-I to induce migration of epithelial colonic cells.

Although the detailed mechanisms of cell migration remain largely unknown, it is now clear that growth factors and cell adhesion molecules are crucial for this process. We have shown that type I insulin-like growth factor (IGF-I) promotes migration of human colonic tumour cells. Since morphological analysis suggested an involvement of adhesion molecules, we have now examined the role of integrins (cell-matrix adhesion molecules) and E-cadherin/catenins complex (cell-cell adhesion molecules) in the IGF-I-induced migration. Using a monolayer wounding assay, we have determined that, except for alpha2beta1, all of the integrins expressed in HT29-D4 cells are involved in the induced cell migration. Immunofluorescence studies revealed that upon IGF-I stimulation the integrins reorganized at the leading edge of migrating cells. We also demonstrate that E-cadherin is involved in cell migration. A rapid tyrosine phosphorylation of E-cadherin and beta-catenin was detected upon IGF-I stimulation. Tyrosine phosphorylation was associated with reduced membranous expression of E-cadherin and promotion of cell motility, suggesting a regulation of the E-cadherin/catenins complex. This effect can be reversed by incubating cells with tyrosine kinase inhibitors. Taken together, our results suggest that IGF-I promotes colonic cell migration through reorganization of integrin receptors and through modulation of E-cadherin/catenins complex function.

Protein kinases C-gamma and -delta are involved in insulin-like growth factor I-induced migration of colonic epithelial cells.

BACKGROUND & AIMS: The mechanisms by which epithelial cells migrate during the repair of damaged colonic mucosa are poorly understood. This study investigated the insulin-like growth factor I (IGF-I) signaling pathway leading to HT29-D4 human colonic epithelial cell line migration. METHODS: IGF-stimulated cell migration was determined using a wound model in the presence or absence of kinase inhibitors. Activation of protein kinase C (PKC) was determined by immunodetection. RESULTS: IGF-I and insulin induce cell migration without affecting cell proliferation through their cognate receptors. Des(1-3)-IGF-I, a truncated analogue of IGF-I, was more potent than IGF-I, suggesting that IGF-binding proteins secreted in the medium modulated IGF-I-induced cell migration. Inhibition of phosphatidylinositol 3-kinase, PKC, and mitogen-activated protein kinases eliminated cell restitution. Long-term exposure of cells to phorbol myristate acetate caused the depletion of PKC-delta and -gamma and prevented also IGF-I-induced cell motility. IGF-I also induced activation of PKC-delta and -gamma only. CONCLUSIONS: IGF-I stimulates colonic restitution through the activation of multiple signaling pathways including activation of phosphatidylinositol 3-kinase, PKC-delta and -gamma, and mitogen-activated protein kinases.

Tau protein is involved in the apoptotic process induced by anti-microtubule agents on neuroblastoma cells.

Paclitaxel and docetaxel are potent anti-microtubule and antimitotic agents that induce apoptosis in bone marrow-derived cells and epithelial cells. This study examined apoptosis induced by anti-microtubule agents in the neuroblastoma SK-N-SH cell line with a special focus on tau protein which is one of the main Microtubule-Associated- Proteins (MAPs) in neuronal cells. In time, treatment with 1 microM paclitaxel successively induced formation of bundles, then pseudo-asters concomitantly with mitotic block and phosphorylation of bcl-2 (48 h), then phosphorylation of tau and externalization of phosphatidylserine at the early phase of apoptosis (72 h) and finally DNA fragmentation (96 h). Similar results were obtained with 0.5 microM vinorelbine. Paclitaxel induced a lower increase in tau phosphorylation in differentiated SK-N-SH/RA+ cells which are less sensitive to apoptosis. Moreover, doxorubicin whose mechanism of action is independent of microtubules also induced immunostaining of tau at 72 h treatment. In conclusion, our results on neuroblastoma cells show that overexpression of hyperphosphorylated tau is involved in the apoptotic process induced by anti-microtubule agents and may be extended to others cytostatic drugs. Thus, tau protein may play a role in the cellular events observed in neuroblastoma cells undergoing apoptosis.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) proteolysis in patients with colorectal cancer: possible association with the metastatic potential of the tumor.

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.

Study of serum big-insulin-like growth factor (IGF)-II and IGF binding proteins in two patients with extrapancreatic tumor hypoglycemia, using a combination of Western blotting methods.

Extrapancreatic tumor hypoglycemia (EPTH) is associated with increased amounts of high-molecular-weight precursor forms of insulin-like growth factor (IGF)-II ('big-IGF-II') that have a primary role in the pathophysiology of hypoglycemia. In the present study, using Western ligand and immunoblotting methods, we investigated IGF-binding proteins (IGFBPs), IGFBP-3 proteolysis and big-IGF-II in pre- and postoperative serum from two patients with EPTH due to benign pleural fibroma. In the preoperative serum, IGFBP-3 was reduced and IGFBP-2 was increased compared with that from an age-matched healthy control. IGFBP-3 proteolysis was dramatically reduced in one patient, whereas no major alteration was observed in the other (9% and 120% of control serum, respectively). IGFBPs progressively returned to a subnormal pattern in postoperative serum, whereas IGFBP- 3 proteolysis remained greater than in preoperative serum in both patients at days 14 and 90 after surgery. High-molecular-weight forms of IGF-II predominate in EPTH serum (65% and 57% of total IGF-II immunoreactivity in patients 1 and 2, respectively, compared with 2 5% in control serum). Two forms, of molecular mass 10 and 12 kDa ('standard big-IGF-II') were present in both EPTH and control sera, whereas two additional forms, of molecular mass 15 and 18 kDa ('big big-IGF-II') were observed in EPTH sera only. Big big-IGF-II represented 72% and 55% of total high-molecular-weight forms of IGF-II in the two EPTH sera, respectively. All big forms of IGF-II disappeared from the serum as early as 6 h after surgery. This study shows that combination of simple Western blotting methods, available routinely in most laboratories, should prove useful in providing reliable physiopathological information in EPTH.

Deficient processing and activity of type I insulin-like growth factor receptor in the furin-deficient LoVo-C5 cells.

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.

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.

Serum factors inhibit melanoma cell surface expression of type I and type II IGF receptors.

We have identified one class of IGF-I-binding sites and two classes of IGF-II-binding sites at the surface of the melanoma cell line IGR39. By means of affinity labeling with 125I-IGF-I, 290-300 kDa form was characterized. Using 125I-IGF-II, a 270 kDa polypeptide was labeled, corresponding to the type II IGF receptor. In the two serials of experiments, the order of potency in inhibiting 125I-IGF-I or 125I-IGF-II labeling of IGF-related peptides and alpha IR3, an antibody directed against type I receptor alpha subunit, was the same as in competition experiments. When IGR39 cells were cultured in a serum-free medium, the number of both high affinity IGF-II and IGF-I binding sites was increased, by 8- and 5-fold respectively, without any significant change in Kd values. In both culture conditions, we found IGFBP-2, -3 -4 and a 30 kDa form which Mr was consistent with IGFBP-5 or -6. Except for IGFBP-2, the amount of secreted IGFBPs was modified depending on culture conditions: in conditioned medium from cells cultured with 10% FCS, the amount of IGFBP-3 or -4 was higher, and the amount of the 30 kDa IGFBP was lower when compared to conditioned medium from cells cultured in serum-free medium.

Cell polarity of the insulin-like growth factor system in human intestinal epithelial cells. Unique apical sorting of insulin-like growth factor binding protein-6 in differentiated human colon cancer cells.

In this study, we have used enterocyte-like differentiated HT29-D4 human colonic carcinoma cells cultured in a glucose-free medium (HT29-D4-GAL cells) on semi-permeable supports in order to investigate the polarity of the insulin-like growth factor (IGF) system. We report that these cells secrete endogenous IGF-II predominantly (66%) from the basolateral cell surface where type I IGF receptors are almost all (> 96%) localized. HT29-D4-GAL cells also secrete IGF-binding protein (IGFBP) -2, -4, and -6 as evidenced by Western ligand and immunoblot analyses of conditioned medium. IGFBP-2 and IGFBP-4 are secreted primarily into the basolateral side (71 and 87%, respectively), whereas IGFBP-6 is targeted to the apical surface (76%) as a possible consequence of an active sorting. Finally, HT29-D4-GAL cells are found to display responses to IGF-II added to the basolateral but not the apical membrane side in terms of intracellular tyrosine phosphorylation and long-term stimulation of amino acid uptake. This study indicates (a) that IGF-II is potentially capable of autocrine regulation on the basolateral side of HT29-D4-GAL cell, and (b) that IGFBP-6 has a unique pattern of secretory polarity. It supports the concept that a differential sorting of the various forms of IGFBPs might play a modulatory role in the maintenance of a functional polarity in the differentiated HT29-D4-GAL 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.

Surface distribution of the EGF receptor during differentiation of the human colon carcinoma cell line HT29-D4.

The clone HT29-D4 can be induced to differentiate into enterocyte-like cells, by simply removing glucose from culture medium. In this report, we used the HT29-D4 model to study the membrane segregation of the EGF receptor on epithelial intestinal cells. Differentiated and undifferentiated cells displayed a single class of EGF binding sites with similar dissociation constants. However, differentiation of HT29-D4 led to a 3-fold decrease in the total number of EGF binding sites, while the number of IGF-I binding sites was unchanged. Fifteen percent of EGF receptors present on differentiated HT29-D4 cells were localized in the apical surface, whereas 98% of IGF-I receptors were segregated to the basolateral domain. By covalent cross-linking experiments using 125I-EGF and by immunoprecipitation with an anti-EGF receptor antibody, we have characterized the HT29-D4 EGF receptor as a Mr = 165,000 protein in both differentiated and undifferentiated cells. Apical EGF receptors were functional, as evidenced by their ability to be internalized in response to EGF binding. Thus, intact and functional EGF receptors are present at the apical surface of differentiated HT29-D4 cells, suggesting the presence of EGF receptors on the apical domain of enterocytes.

Alterations in serum levels of insulin-like growth factors and insulin-like growth-factor-binding proteins in patients with colorectal cancer.

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.

des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells.

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.