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.

Enhancement of production of superoxide anion by human polymorphonuclear leukocytes exposed to products of the HT-29 human colonic adenocarcinoma cell line.

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.

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.

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.

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.

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.

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.

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.

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.

Type-II insulin-like growth-factor receptor in conditioned medium from HT-29 human colon carcinoma cell line.

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.

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.

Non-specific binding by macrophages: evaluation of the influence of medium-range electrostatic repulsion and short-range hydrophobic interaction.

Rat peritoneal macrophages can bind glutaraldehyde-treated human rat cells (GHRC), but not normal human red cells (HRC). When the surface charge of HRC was reduced by treating them with neuraminidase (to remove some negative sialic acid residues) or coating them with polylysine (a positively charged polymer), no substantial binding to macrophages was obtained. However, a similar reduction of the surface charge of GHRC resulted in sevenfold enhancement of their binding of macrophages. All erythrocyte batches were tested with a phase partition technique: only glutaraldehyde-treated cells were found hydrophobic. It is concluded that: i) Short range hydrophobic interactions are responsible for macrophage-GSRC adhesion. ii) Medium range electrostatic repulsion may substantially hamper any close approach of the macrophage and particle surface in physiological conditions.

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.

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.

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.

Nonspecific binding by macrophages: different modulation of adhesive properties of rat peritoneal cells after plating on a glass or a plastic 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.

Concanavalin-A-mediated thymocyte agglutination: a model for a quantitative study of cell adhesion.

This report describes a quantitative study of the agglutination of rat thymocytes with concanavalin A (ConA). The probability that two ConA-coated cells remain bound after centrifugation was determined over a wide range of lectin concentrations. The minimal force required to separate agglutinated cells and the number of ConA molecules bound per cell were measured in similar experimental conditions. Agglutinated cells were examined by electron microscopy to estimate the area of membrane involved in adhesion. The dependence of agglutination on cell metabolism was studied: cold (4 degrees C), sodium azide (15 mM) and cytochalasin B (10 micrograms/ml) inhibited thymocyte adhesion. The importance of lateral movements of ConA molecules was assayed by measuring the adhesion of ConA-coated glutaraldehyde-fixed thymocytes to untreated cells: substantial binding occurred, but at a reduced level relative to untreated cells. A mathematical analysis of experimental data allowed the following conclusions. (1) At least 10(3) ConA bonds were involved in cross-linking two bound cells, which required the lectin molecules to be concentrated in the binding area, at least when low ConA concentrations (0.5 microgram/ml or less) were used. (2) The dependence of the binding probability on lectin concentration was fairly linear when the latter was small, which implied that the limiting step in cell-cell adhesion was the formation of a bond between a single ConA molecule and a ligand on the other cell. (3) The mean intercellular-contact time for the formation of this first bond was about 10 S for high concentrations of ligand (8 micrograms/ml). It was possible to fit the above data into a physically consistent quantitative model of cell adhesion.

Enhancement of production of superoxide anion by human monocytes exposed to products of HT 29 human colonic adenocarcinoma cell line.

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.

Simultaneous production of IGF-I and EGF competing growth factors by HT-29 human colon cancer line.

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.