Colonic cancer cell (HT29) adhesion to laminin is altered by differentiation: Adhesion may involve galactosyltransferase

The effects of cell differentiation on cell adhesion to laminin were studied using the human colon tumor cell line, HT29. HT29 cells were induced to differentiate either by glucose deprivation (HT29glc- vs HT29glc+) or by 2 mM butyrate (HT29glc-+B+). Adhesion was assayed after incubating cell suspensions in microtiter wells previously coated with laminin or other substrates. HT29glc+ cells adhered preferentially to laminin over BSA, fibronectin, and ovalbumin. The adhesion to laminin was greater than 50% of maximum within 15 min. HT29glc- cell adhesion to laminin was consistently lower than that for HT29glc+ or HT29glc+B+ cells. alpha-Lactalbumin (ALA), a modifier of galactosyltransferase (GT) substrate specificity, caused a significant reduction (greater than 50%) in HT29glc+ cell adhesion to laminin when ALA was added to the adhesion incubation mixture. Addition of glucose+ALA to the suspension restored adhesion to laminin. Ovalbumin, a GT substrate, increased adhesion of HT29glc+ and HT29glc- cells to laminin, but lactose, a GT product, did not. The data show that undifferentiated HT29 cells adhere preferentially to laminin over fibronectin and collagen IV and that differentiation of HT29 cells reduces adhesion to laminin. In addition, the data imply that cell adhesion to laminin may be mediated by factors that also modify galactosyltransferase activity.     

Parathyroid hormone-related protein overexpression in the human colon cancer cell line HT-29 enhances adhesion of the cells to collagen type I

The present experiments examined the potential ability of parathyroid hormone-related protein (PTHrP) to influence growth of the human colon cancer cell HT-29 and the ability of the cell to adhere to several extracellular matrix (ECM) proteins found in normal tissues. Addition of PTHrP analogs, PTHrP (1-34), PTHrP (67-86), or PTHrP (107-139), to HT-29 cells in culture did not influence cell growth or the adhesion of the cells to wells coated with fibronectin, laminin, or collagen type I. Likewise, in HT-29 cells induced to overexpress PTHrP by stable transfection with PTHrP cDNA, compared to vector-transfected control HT-29 cells, no effect on cell growth occurred. However, in the transfected cells, the increased production of PTHrP significantly enhanced cell adhesion to type I collagen but not to fibronectin or laminin. The results raise the possibility that PTHrP might play a role in colon tumor invasion and metastasis by influencing cell adhesion to specific extracellular matrix proteins.     

The role of the arginine-glycine-aspartic acid-directed cellular binding to type I collagen and rat mesenchymal cells in colorectal tumour differentiation

The relationship between the adhesion of five human colorectal carcinoma cell lines to extracellular matrix (ECM) proteins, namely type I collagen, type IV collagen, fibronectin, laminin and basement membrane extract (Matrigel), and the ability of these cells to express morphological differentiation when grown in a basement membrane extract (Matrigel) or on normal rat mesenchymal cells has been examined. Two cell lines, SW1222 and HRA-19, organised into glandular structures, with well-defined polarity when cultured on both substrata as well as in three-dimensional (3D) collagen gel culture as previously shown. The remaining three cell lines (SW620, SW480 and HT29) grew as loose aggregates or as they would normally grow on tissue culture plastic. Addition to the culture medium of a hexapeptide, containing the cell-matrix recognition sequence arginine-glycine-aspartic acid (RGD), inhibited attachment and glandular formation of SW1222 and HRA-19 when these cells were grown on living mesenchymal cells, but not in Matrigel. The morphological differentiation of HRA-19 cells in 3D-collagen was also inhibited by the same RGD-containing peptide, as previously shown for SW1222 cells. Attachment of the remaining three cell lines was inhibited on mesenchyme but not in Matrigel, further supporting the specificity of the peptide effect on epithelial-mesenchymal binding. In conclusion we have shown that colorectal tumour cells are able to bind ECM proteins and that the cellular binding is an essential step in the induction of the morphological differentiation seen on living mesenchymal cells, in basement membrane extracts and in type I collagen gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential properties of attachment of human fibroblasts to various extracellular matrix proteins

Human diploid fibroblasts (TIG-3) were shown to attach and spread onto substrata coated with collagen, fibronectin, laminin and vitronectin. The cell attachment to these proteins required divalent cations. Mg2+ stimulated the cell attachment to all the proteins, while Ca2+ alone was not effective for the attachment to collagen and laminin. A mild trypsin treatment had prevented cells from attaching to the laminin, while it had no effect on the attachment to the other proteins. The fibronectin fragment, which retained cell binding activity, inhibited the cells from attaching and spreading onto fibronectin, but it did not cause any inhibition on the other proteins. The synthetic peptide GRGDSP inhibited the cells from attaching and spreading onto fibronectin and vitronectin, while it did not cause any inhibition on collagen and laminin. In attempts to isolate distinct receptors for these proteins, we were able to purify proteins very similar to the fibronectin and vitronectin receptors of human placenta. Based on the differential properties of the attachment of TIG-3 cells to these proteins and biochemical data, we indicate that human diploid fibroblasts have distinctive binding sites (receptors) for collagen, fibronectin, laminin and vitronectin.     

Human colon carcinoma cells use multiple receptors to adhere to laminin: involvement of alpha 6 beta 4 and alpha 2 beta 1 integrins.

In this study, we used clone A, a human colon carcinoma cell line, to characterize those integrins that mediate colon carcinoma adhesion to laminin. Monoclonal antibodies specific for the human beta 1 subunit inhibited clone A adhesion to laminin. They also precipitated a complex of surface proteins that exhibited an electrophoretic behavior characteristic of alpha 2 beta 1 and alpha 3 beta 1. A monoclonal antibody specific for alpha 2 (PIH5) blocked clone A adhesion to laminin, as well as to collagen I. An alpha 3-specific antibody (P1B5) had no effect on clone A adhesion to laminin, even though it can block the adhesion of other cell types to laminin. Thus, the alpha 2 beta 1 integrin can function as both a laminin and collagen I receptor on clone A cells. Although these cells express alpha 3 beta 1, an established laminin receptor, they do not appear to use it to mediate laminin adhesion. In addition, the monoclonal antibody GoH3, which recognizes the alpha 6 integrin subunit, also inhibited carcinoma adhesion to laminin but not to fibronectin or collagen I. This antibody precipitated the alpha 6 subunit in association with the beta 4 subunit. There was no evidence of alpha 6 beta 1 association on these cells. In summary, the results obtained in this study indicate that multiple integrin alpha subunits, in association with two distinct beta subunits, are involved in colon carcinoma adhesion to laminin. Based on the behavior of alpha 3 beta 1 and alpha 2 beta 1, the results also suggest that cells can regulate the ability of a specific integrin to mediate adhesion.     

细胞外间质-整合素在牵张刺激心肌细胞肥大中的作用

应用牵张刺激培养细胞的模型,观察原原、纤维连接蛋白、层粘连素对牵张刺激心肌细胞肥大的影响,探讨细胞外间质－融洽纱受体在超负荷心肌肥大的跨膜信号传导机制中的作用。发现,胶原、纤维连接蛋白、层粘连素明显有助于培养心肌细胞的贴壁、伸展。牵张刺激后,胶原、纤维连接蛋白基质组心肌细胞的＾３Ｈ－亮氨酸掺入率和心肌细胞表面积均显著大于对照组,而层粘连素组无显著变化;可溶性纤维连接蛋白、ＲＧＤ肽均可显著抑制牵张刺     

细胞外间质-整合素在牵张刺激心肌细胞肥大中的作用

应用牵张刺激培养细胞的模型 ,观察胶原、纤维连接蛋白、层粘连素对牵张刺激心肌细胞肥大的影响 ,探讨细胞外间质 -整合素受体在超负荷心肌肥大的跨膜信号传导机制中的作用。结果发现 ,胶原、纤维连接蛋白、层粘连素明显有助于培养心肌细胞的贴壁、伸展。牵张刺激后 ,胶原、纤维连接蛋白基质组心肌细胞的 3H -亮氨酸掺入率和心肌细胞表面积均显著大于对照组 ,而层粘连素组无显著变化 ;可溶性纤维连接蛋白、RGD肽均可显著抑制牵张刺激诱导的培养心肌细胞 (胶原为粘附基质 )的3H -亮氨酸掺入率升高和心肌细胞表面积增大 ,而层粘连素无明显作用。结果表明 ,特异的细胞外间质 -整合素在超负荷心肌肥大机制中发挥了跨膜信号传导作用。     

Amyloid precursor-like protein 2 promotes cell migration toward fibronectin and collagen IV.

Previous studies have established that in response to wounding, the expression of amyloid precursor-like protein 2 (APLP2) in the basal cells of migrating corneal epithelium is greatly up-regulated. To further our understanding of the functional significance of APLP2 in wound healing, we have measured the migratory response of transfected Chinese hamster ovary (CHO) cells expressing APLP2 isoforms to a variety of extracellular matrix components including laminin, collagen types I, IV, and VII, fibronectin, and heparan sulfate proteoglycans (HSPGs). CHO cells overexpressing either of two APLP2 variants, differing in chondroitin sulfate (CS) attachment, exhibit a marked increase in chemotaxis toward type IV collagen and fibronectin but not to laminin, collagen types I and VII, and HSPGs. Cells overexpressing APLP2-751 (CS-modified) exhibited a greater migratory response to fibronectin and type IV collagen than their non-CS-attached counterparts (APLP2-763), suggesting that CS modification enhanced APLP2 effects on cell migration. Moreover, in the presence of chondroitin sulfate, transfectants overexpressing APLP2-751 failed to exhibit this enhanced migration toward fibronectin. The APLP2-ECM interactions were also explored by solid phase adhesion assays. While overexpression of APLP2 isoforms moderately enhanced CHO adhesion to laminin, collagen types I and VII, and HSPGs lines, especially those overexpressing APLP2-751, exhibited greatly increased adhesion to type IV collagen and fibronectin. These observations suggest that APLP2 contributes to re-epithelialization during wound healing by supporting epithelial cell adhesion to fibronectin and collagen IV, thus influencing their capacity to migrate over the wound bed. Furthermore, APLP2 interactions with fibronectin and collagen IV appear to be potentiated by the addition of a CS chain to the core proteins.     

Expression and adhesive properties of basement membrane proteins in cerebral capillary endothelial cell cultures

Previous studies have indicated the importance of basement membrane components both for cellular differentiation in general and for the barrier properties of cerebral microvascular endothelial cells in particular. Therefore, we have examined the expression of basement membrane proteins in primary capillary endothelial cell cultures from adult porcine brain. By indirect immunofluorescence, we could detect type IV collagen, fibronectin, and laminin both in vivo (basal lamina of cerebral capillaries) and in vitro (primary culture of cerebral capillary endothelial cells). In culture, these proteins were secreted at the subcellular matrix. Moreover, the interaction between basement membrane constituents and cerebral capillary endothelial cells was studied in adhesion assays. Type IV collagen, fibronectin, and laminin proved to be good adhesive substrata for these cells. Although the number of adherent cells did not differ significantly between the individual proteins, spreading on fibronectin was more pronounced than on type IV collagen or laminin. Our results suggest that type IV collagen, fibronectin, and laminin are not only major components of the cerebral microvascular basal lamina, but also assemble into a protein network, which resembles basement membrane, in cerebral capillary endothelial cell cultures.     

Retinoic acid modulates attachment of mouse fibroblasts to laminin substrates

The effect of retinoic acid treatment on cell attachment to plastic substrates precoated with fibronectin, gelatin, laminin, and type IV collagen was investigated. Both retinoic acid-treated and control cells attached efficiently to fibronectin or gelatin substrates without any significant difference. In contrast, retinoic acid-treated cells attached to laminin or type IV collagen substrates, while control cells showed little or no attachment. The minimal effective concentration of retinoic acid for pretreatment to yield a significant increase in the attachment assay was higher than 10(-8) M. The attachment of retinoic acid-treated cells to laminin substrates reached a maximum at 60 min, while that to type IV collagen substrates had a time lag and did not reach a maximum by 60 min. The effect of retinoic acid treatment reached a maximum at 2 days and was partly reversible. These results suggest that retinoic acid may increase NIH/3T3 cell adhesion through an effect on laminin receptors. Other mouse fibroblast lines, 3T3-Swiss, 3T6-Swiss, Balb/3T3, and Balb/3T12-3 (spontaneously transformed Balb/3T3), responded to retinoic acid treatment in a manner similar to that of NIH/3T3 cells. However, the virus-transformed Balb/3T3 lines, SV-T2 and M-MSV, showed significant attachment to laminin substrates without retinoic acid treatment, and retinoic acid did not affect or slightly decreased the cell attachment to laminin substrates.     

Identification of asparagine-linked oligosaccharides involved in tumor cell adhesion to laminin and type IV collagen

MDW4, a wheat germ agglutinin-resistant nonmetastatic mutant of the highly metastatic murine tumor cell line called MDAY-D2 has previously been shown to attach to fibronectin and type IV collagen, whereas MDAY- D2 and phenotypic revertants of MDW4 attached poorly to these substrates. The increased adhesiveness of the mutant cells appeared to be closely related to a lesion in cell surface carbohydrate structures. In an effort to identify the carbohydrates involved in cell attachment, glycopeptides isolated from mutant and wild-type cells as well as from purified glycoproteins were tested for their ability to inhibit the attachment of MDW4 cells to plastic surfaces coated with fibronectin, laminin, or type IV collagen. The addition of mannose-terminating glycopeptide to the adhesion assay inhibited MDW4 cell attachment to type IV collagen. In contrast, a sialylated poly N-acetyllactosamine- containing glycopeptide, isolated from wheat germ agglutinin-sensitive MDAY-D2 cells but absent in MDW4 cells, inhibited MDW4 attachment to laminin. None of the glycopeptides used in this study inhibited attachment of MDW4 cells to fibronectin-coated plastic. Peptide N- glycosidase treatment of the cells to remove surface asparagine-linked oligosaccharides inhibited MDW4 adhesion to type IV collagen, but not to laminin, and the same treatment of the wheat germ agglutinin- sensitive cells enhanced attachment to laminin. Tumor cell attachment to, and detachment from, the sublaminal matrix protein laminin and type IV collagen are thought to be important events in the metastatic process. Our results indicate that tumor cell attachment to these proteins may be partially modulated by the expression of specific oligosaccharide structures associated with the cell surface.     

Evidence for the presence of an integrin cell adhesion receptor on the oolemma of unfertilized human oocytes.

The Arg-Gly-Asp peptide (RGD), contained in several extracellular matrix proteins such as fibronectin, laminin, vitronectin, and collagen, is a tripeptide that plays a role as a recognition sequence in many cell-to-cell and cell-to-matrix adhesion mechanisms, through its interaction with several receptors of the integrin family. We previously described the ability of the oolemma of hamster oocytes to bind GRGDTP coupled to the surface of activated immunobeads and demonstrated that RGD-containing oligopeptides inhibit the adhesion of human and hamster spermatozoa to zona-free hamster oocytes and their subsequent penetration. In the present experiments, we show, utilizing immunobeads coated with an RGD-containing peptide (PepTiteTM 2000), that the oolemma of unfertilized human eggs is also able to recognize this adhesion sequence. The binding of PepTiteTM 2000-coated immunobeads to the oolemma was inhibited by the oligopeptide GRGDTP as well as by fibronectin and laminin. When immunobeads were prepared with a PepTiteTM concentration of 10 micrograms/ml, GRGDTP 150 micrograms/ml, laminin 80 micrograms/ml, and fibronectin 60 micrograms/ml inhibited bead rosetting on the egg surface. These data suggest that a specific binding moiety for RGD is present on the human egg surface. The binding of fibronectin to the oolemma was also demonstrated by the rosetting of immunobeads coupled with antifibronectin antibody to human oocytes after their exposure to 1 mg/ml free fibronectin. Such binding of fibronectin to the oolemma could be inhibited by coincubation with a monoclonal antibody directed against the cell adhesion fragment of fibronectin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of multiple cell adhesion receptors for collagen and fibronectin in human fibrosarcoma cells possessing unique alpha and common beta subunits

Using monoclonal antibody technology and affinity chromatography we have identified four distinct classes of cell surface receptors for native collagen on a cultured human fibrosarcoma cell line, HT-1080. Two classes of monoclonal antibodies prepared against HT-1080 cells inhibited adhesion to extracellular matrix components. Class I antibodies inhibited cell adhesion to collagen, fibronectin, and laminin. These antibodies immunoprecipitated two noncovalently linked proteins (subunits) with molecular masses of 147 and 125 kD, termed alpha and beta, respectively. Class II antibodies inhibited cell adhesion to native collagen only and not fibronectin or laminin. Class II antibodies immunoprecipitated a single cell surface protein containing two noncovalently linked subunits with molecular masses of 145 and 125 kD, termed alpha and beta, respectively. The two classes of antibodies did not cross-react with the same cell surface protein and recognized epitopes present on the alpha subunits. Pulse-chase labeling studies with [35S]methionine indicated that neither class I nor II antigen was a metabolic precursor of the other. Comparison of the alpha and beta subunits of the class I and II antigens by peptide mapping indicated that the beta subunits were identical while the alpha subunits were distinct. In affinity chromatography experiments HT-1080 cells were extracted with Triton X-100 or octylglucoside detergents and chromatographed on insoluble fibronectin or native type I or VI collagens. A single membrane protein with the biochemical characteristics of the class I antigen was isolated on fibronectin-Sepharose and could be immunoprecipitated with the class I monoclonal antibody. The class I antigen also specifically bound to type I and VI collagens, consistent with the observation that the class I antibodies inhibit cell adhesion to types VI and I collagen and fibronectin. The class II antigen, however, did not bind to collagen (or fibronectin) even though class II monoclonal antibodies completely inhibited adhesion of HT-1080 cells to types I and III-VI collagen. The class I beta and II beta subunits were structurally related to the beta subunit of the fibronectin receptor described by others. However, none of these receptors shared the same alpha subunits. Additional membrane glycoprotein(s) with molecular mass ranges of 80-90 and 35-45 kD, termed the class III and IV receptors, respectively, bound to types I and VI collagen but not to fibronectin. Monoclonal antibodies prepared against the class III receptor had no consistent effect on cell attachment or spreading, suggesting that it is not directly involved in adhesion to collagen-coated substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Adhesion properties of human bladder cell lines with extracellular matrix components: the role of integrins and glycosylation

Integrin subunits present on human bladder cells displayed heterogeneous functional specificity in adhesion to extracellular matrix proteins (ECM). The non-malignant cell line (HCV29) showed significantly higher adhesion efficiency to collagen IV, laminin (LN) and fibronectin (FN) than cancer (T24, Hu456) and v-raf transfected (BC3726) cell lines. Specific antibodies to the alpha(2), alpha(5) and beta(1) integrin subunits inhibited adhesion of the non-malignant cells, indicating these integrin participation in the adhesion to ECM proteins. In contrast, adhesion of cancer cells was not inhibited by specific antibodies to the beta(1) integrin subunit. Antibodies to alpha(3) integrin increased adhesion of cancer cells to collagen, LN and FN, but also of the HCV29 line with collagen. It seems that alpha(3) subunit plays a major role in modulation of other integrin receptors especially in cancer cells. Differences in adhesion to ECM proteins between the non-malignant and cancer cell lines in response to Gal and Fuc were not evident, except for the v-raf transfected cell line which showed a distinct about 6-fold increased adhesion to LN on addition of both saccharides. N-Acetylneuraminic acid inhibited adhesion of all cell lines to LN and FN irrespective of their malignancy.     

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECM components.     

Inhibition of adhesion of hepatocellular carcinoma cells to basement membrane components by receptor competition with RGD- or YIGSR-containing synthetic peptides

A micropipette technique was used to investigate the effects of four synthetic peptides, YIGSR, CDPGYIGSR, RGDS and GRGDTP, on the adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen/laminin/fibronectin coated surfaces. Adhesion of HCC cells to laminin was found to be YIGSR- or CDPGYIGSR-dependent while that to fibronectin and type IV collagen was RGDS- or GRGDTP-dependent. The reduction in adhesion strengths of HCC cells was slight to moderate (up to 55%), and was dependent on the peptide concentration. The decrease in adhesion strengths was reversed by an increase in ligand coating concentration and was compromised by prolonged interaction of the cells with the surfaces. These results suggested that the inhibition was due to competitive retardation rather than to a blockade of adhesion strengthening. A simple asymptotic function was adopted to fit the correlation between the mean of cell adhesion strengths and peptide concentration within defined concentration ranges. Regression analysis showed that cell adhesion strengths appeared to approach a plateau with increasing concentration of the inhibitory peptides, which was not always uniform over the entire concentration range tested. Further reduction in adhesion strengths was observed at higher peptide concentrations. It is suggested that the constants obtained by fitting over a low peptide concentration range might be kinetically representative of the inhibition during early events of adhesion or attachment.     

A Collagen-Binding S-Layer Protein in Lactobacillus crispatus

Two S-layer-expressing strains, Lactobacillus crispatus JCM 5810 and Lactobacillus acidophilus JCM 1132, were assessed for adherence to proteins of the mammalian extracellular matrix. L. crispatus JCM 5810 adhered efficiently to immobilized type IV and I collagens, laminin, and, with a lower affinity, to type V collagen and fibronectin. Strain JCM 1132 did not exhibit detectable adhesiveness. Within the fibronectin molecule, JCM 5810 recognized the 120-kDa cell-binding fragment of the protein, while no bacterial adhesion to the amino-terminal 30-kDa or the gelatin-binding 40-kDa fragment was detected. JCM 5810 but not JCM 1132 also bound (sup125)I-labelled soluble type IV collagen, and this binding was efficiently inhibited by unlabelled type IV and I collagens and less efficiently by type V collagen, but not by laminin or fibronectin. L. crispatus JCM 5810 but not L. acidophilus JCM 1132 also adhered to Matrigel, a reconstituted basement membrane preparation from mouse sarcoma cells, as well as to the extracellular matrix prepared from human Intestine 407 cells. S-layers from both strains were extracted with 2 M guanidine hydrochloride, separated by electrophoresis, and transferred to nitrocellulose sheets. The S-layer protein from JCM 5810 bound (sup125)I-labelled type IV collagen, whereas no binding was seen with the S-layer protein from JCM 1132. Binding of (sup125)I-collagen IV to the JCM 5810 S-layer protein was effectively inhibited by unlabelled type I and IV collagens but not by type V collagen, laminin, or fibronectin. It was concluded that L. crispatus JCM 5810 has the capacity to adhere to human subintestinal extracellular matrix via a collagen-binding S-layer.     

Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10(-9) and 10(-7) M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.     

PTH-related protein enhances LoVo colon cancer cell proliferation, adhesion, and integrin expression

Parathyroid hormone-related protein (PTHrP) has been localized in human colon cancer tissue and cell lines. Tumor cell adhesion to extracellular matrix (ECM) proteins plays a major role in the invasion and metastasis of tumor cells, and is mediated via integrin subunits. The LoVo human colon cancer cell line was used as a model system to study the effects of PTHrP on cell proliferation and adhesion to ECM proteins found in normal liver. Clones of LoVo cells engineered to overexpress PTHrP by stable transfection with a PTHrP cDNA showed enhanced cell proliferation vs. control (empty vector-transfected) cells. PTHrP-overexpressing cells also showed significantly higher adhesion to collagen type I, fibronectin, and laminin, and enhanced expression of the [symbol: see text] integrin subunits. These results indicate that PTHrP may play a role in colon cancer invasion and metastasis by increasing cell proliferation and adhesion to the ECM via upregulation of proinvasive integrin expression.     

Binding of hyaluronan to plasma fibronectin increases the attachment of corneal epithelial cells to a fibronectin matrix

We wished to determine whether hyaluronan would affect the attachment of epithelial cells to extracellular matrix proteins. Multiwell tissue culture plates were coated with human plasma fibronectin, laminin, or collagen type IV (0.01–10.0 μg/ml). Single-cell suspensions of rabbit corneal epithelial cells were placed in the wells, and after 45 minutes incubation the cells adhering to the matrix proteins were stained and counted. Cells attached to all three types of proteins. Preincubation of the matrix proteins with hyaluronan (0.1–1.0 mg/ml) significantly increased the number of cells attached to the fibronectin matrix, but it did not increase the numbers of cells attached to laminin or collagen type IV. Hyaluronidase inhibited this stimulatory effect. Glycosaminoglcyans other than hyaluronan (chondroitin sulfate, keratan sulfate, or heparan sulfate) failed to increase the numbers of attached cells. Treatment of the fibronectin matrix with monoclonal antibodies against the cell-binding domain of fibronectin (FN12–8 or FN30–8, 0.03–0.3 mg/ml, for 1 hour), before or after hyaluronan treatment, significantly decreased the numbers of attached cells. Monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal (FN9–1), however, significantly decreased the number of attached cells only when this antibody treatment preceded the hyaluronan treatment. Preincubation of the cells with hyaluronan had no effect; preincubation with GRGDSP (1 mg/ml), a synthetic peptide that blocks the cell surface receptor for fibronectin, significantly decreased cell attachment whether the fibronectin matrix was treated with hyaluronan or not. Further studies demonstrated that monoclonal antibody against the fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin prevented radiolabeled hyaluronan from binding to fibronectin; likewise, the isolated N-terminal fragment, coupled with Sepharose 4B, bound to hyaluronan in columns. We conclude that hyaluronan binds to a fibrin- and heparin-binding domain at the N-terminal of plasma fibronectin and facilitates the attachment of epithelial cells. © 1994 wiley-Liss, Inc.