Comprehensive galectin fingerprinting in a panel of 61 human tumor cell lines by RT-PCR and its implications for diagnostic and therapeutic procedures.

PURPOSE: Knowledge about galectin expression by human tumor cells is mainly restricted to galectins-1 and -3. This study was conducted to define the gene expression pattern of all presently known human galectins in tumor cell lines of various histogenetic origin (galectinomics). METHODS: The presence of mRNAs for human galectins-1, -2, -3, -4, -7, -8, and -9 was monitored by RT-PCR analyses in a panel of 61 human tumor cell lines of different origin (breast, colon, lung, brain, skin, kidney, urogenital system, hematopoietic system). RESULTS: The validity of the technique was first confirmed by comparison of RT-PCR data with those obtained by Western blotting and cytofluorometry for galectins-1 and -3 in 18 cell lines. The following detection of a complex pattern of gene expression beyond commonly studied galectins-1 and -3 underscored the need for this fingerprinting. The most abundantly expressed message for a member of this lectin family was galectin-8 with 59 positive cell lines. With the exception of the tested lung tumors, galectin-1 and -3 transcripts were frequently expressed in the cell line panel with differences between individual cases. Positivity for galectins-2 and -4 was confined to a significant fraction of colorectal and neural tumors. Signals for galectin-9, the third known human tandem-repeat-type galectin besides -4 and -8, appeared in colorectal carcinoma cell lines with a frequency similar to that of galectin-4 but with inter-line differences. Its expression was restricted to lines of this tumor type, of the tested ovarian carcinoma, and hematopoietic malignancies. CONCLUSIONS: The results clearly demonstrate that human tumor cells express more mRNA species for galectins than those for galectins-1 and -3. To derive unequivocal diagnostic and prognostic information by immunohistochemistry on galectins with antagonistic impact on growth control and significant influence on cell adhesion, additional monitoring of these so far insufficiently studied family members is essential.

Methylation silencing and mutations of the p14ARF and p16INK4a genes in colon cancer.

The INK4a-ARF locus encodes two tumor suppressor proteins involved in cell-cycle regulation, p16INK4a and p14ARF, whose functions are inactivated in many human cancers. The aim of this study was to evaluate p14ARF and p16INK4a gene inactivation and its association with some clinocopathological parameters in colon cancer. The mutational and methylation status of the p14ARF and p16INK4a genes was analyzed in 60 primary colon carcinomas and 8 colon cancer cell lines. We have identified the first two reported mutations affecting exon 1beta of p14ARF in the HCT116 cell line and in one of the primary colon carcinomas. Both mutations occur within the N-terminal region of p14ARF, documented as important for nucleolar localization and interaction with Mdm2. Tumor-specific methylation of the p14ARF and p16INK4a genes was found in 33% and 32% of primary colon carcinomas, respectively. Methylation of the p14ARF was inversely correlated with p53 overexpression (p = 0.02). p14ARF and p16INK4a gene methylation was significantly more frequent in right-sided than in left-sided tumors (p = 0.02). Methylation of the p14ARF gene occurred more frequently in well-differentiated adenocarcinomas (p = 0.005), whereas the p16INK4a gene was more often methylated in poorly differentiated adenocarcinomas (p = 0.002). The present results underline the role of p14ARF and p16INK4a gene inactivation in the development of colon carcinoma. They suggest that the methylation profile of specific genes, in particular p14ARF and p16INK4a, might be related to biologically distinct subsets of colon carcinomas and possibly to different tumorigenic pathways.

Tumor cell budding and laminin-5 expression in colorectal carcinoma can be modulated by the tissue micro-environment.

Expression of laminin-5 alpha3, beta3 and gamma2 protein subunits was investigated in colorectal adenocarcinomas using immunostaining and confocal microscopy. The laminin-5 heterotrimer was found in basement membranes and as extracellular deposits in tumor stroma. In contrast to the alpha3 subunit, which was under-expressed, the gamma2 and beta3 subunits were detected in the cytoplasm of carcinoma cells dissociating (budding) from neoplastic tubules, suggestive of focal alterations in laminin-5 assembly and secretion. Laminin-5 gamma2 or beta3 subunit-reactive budding carcinoma cells expressed cytokeratins but not vimentin; they did not proliferate and were not apoptotic. Furthermore, expression of laminin-5 gamma2 and beta3 subunits in budding cells was associated with focal under-expression of the E-cadherin-beta-catenin complex. Results from xenograft experiments showed that budding activity in colorectal adenocarcinomas could be suppressed when these tumors grew at ectopic s.c. sites in nude mice. In vitro, cultured colon carcinoma cells, but not adenoma-derived tumor cells, shared the laminin-5 phenotype expressed by carcinoma cells in vivo. Using colon carcinoma cell lines implanted orthotopically and invading the cecum of nude mice, the laminin-5-associated budding was restored, indicating that this phenotype is not only determined by tumor cell properties but also dependent on the tissue micro-environment. Our results indicate that both laminin-5 alpha3 subunit expression and cell-cell cohesiveness are altered in budding carcinoma cells, which we consider to be actively invading. We propose that the local tissue micro-environment contributes to these events.

Protein kinase C isoforms involved in regulation of cell shape and locomotion of human fibrosarcoma HT1080 cells.

The role of protein kinase C (PKC) isoforms in the regulation of cell shape [switch between fibroblast-like and crescent shape (CS)] and of locomotion of human fibrosarcoma HT1080 cells has been investigated. The PKC activator phorbol myristate acetate (PMA) induced the transition of elongated fibroblast-like cells into CS cells and stimulated locomotion. Both responses to PMA were inhibited by the PKC inhibitor Ro 31-8220. Analysis of the time course showed that stimulation of shape changes (formation of CS cells) and locomotor activity (increase in the proportion and speed of locomoting cells) was maximal in the early phase of the response (up to 2.5 hr) and significantly decreased later (15 to 21 hr). CS formation and stimulated locomotion correlated closely with a marked redistribution from the cytosol to the membrane of PKC isoforms alpha, beta1 and gamma in the early phase (0.5 to 2 hr) following activation with PMA. The subsequent reduction of the proportion of CS cells and of cell locomotion correlated with down-regulation of these isoforms in the second phase (16 to 21 hr). In contrast, the total amount and distribution of PKC beta2 remained almost unchanged with 10(-8) M PMA up to 21 hr. Furthermore, changes in shape and locomotion did not correlate with the responses of PKC delta to PMA. Inhibition of PMA-stimulated locomotion by the more specific inhibitor Gö 6976 is consistent with a role of PKC alpha and beta1 in this response. Ro 31-8220 alone induced a moderate down-regulation of PKC isoforms alpha and delta, but it also inhibited the more pronounced down-regulation of these isoforms by PMA. Our results indicate that activation of PKC isoforms alpha, gamma and beta1, but not beta2 or delta, stimulates locomotion and formation of CS cells in human fibrosarcoma HT1080 cells.

Gene expression of galectin-9/ecalectin, a potent eosinophil chemoattractant, and/or the insertional isoform in human colorectal carcinoma cell lines and detection of frame-shift mutations for protein sequence truncations in the second functional lectin domain.

The family of Ca2+-independent galactoside-binding lectins with the beta-strand topology of the jelly-roll, referred to as galectins, is known to mediate and modulate a variety of cellular activities. Their functional versatility explains the current interest in monitoring their expression in cancer research, so far primarily focused on galectin-1 and -3. Tandem-repeat-type galectin-9 and its (most probably) allelic variant ecalectin, a potent eosinophil chemoattractant, are known to be human leukocyte products. We show by RT-PCR with primers specific for both that their mRNA is expressed in 17 of 21 human colorectal cancer lines. As also indicated by restriction analysis, in addition to the expected transcript of 571 bp an otherwise identical isoform coding for a 32-amino acid extension of the link peptide was detected. Positive cell lines differentially expressed either one (7 lines) or both transcripts (10 lines). Sequence analysis of RT-PCR products, performed in four cases, allowed to assign the standard transcript to ecalectin in the case of SW480 cells and detected two point mutations in the insert of the link peptide-coding sequence in WiDr and Colo205. Furthermore, this analysis identified the insertion of a single nucleotide into the coding sequence generating a frame-shift mutation, an event which has so far not been reported for any galectin. This alteration encountered in both transcripts of the WiDr line and the isoform transcript of Colo205 cells will most likely truncate the protein part within the second (C-terminal) carbohydrate recognition domain. Our results thus reveal the presence of mRNA for a galectin-9-isoform or a potent eosinophil chemoattractant (ecalectin) or a truncated version thereof with preserved N-terminal carbohydrate recognition domain in established human colon cancer cell lines.

Interleukin-6 stimulates clonogenic growth of primary and metastatic human colon carcinoma cells.

Interleukin-6 (IL-6) is a pleiotropic cytokine which exerts biological activities on various cell types including neoplastic cells. We have investigated the biological effect of IL-6 and the expression of IL-6 receptors (IL-6R) on human colorectal carcinoma cell lines. Isreco-1 was derived from the primary site of a colon cancer while Isreco-2 and Isreco-3 were established from a liver and peritoneal metastasis of the same patient. IL-6 stimulated colony formation in methylcellulose of Isreco-1 cells to 150% (P < 0.05). The effect was even more pronounced on the metastatic Isreco-2 line where colony numbers in the presence of IL-6 were enhanced up to four-fold (P < 0.0001) in a dose-dependent fashion. An anti-IL-6 antibody completely abolished this growth stimulatory effect of IL-6. RT-PCR analysis revealed transcripts for IL-6Ralpha and gp 130 in these cell lines. Experiments with additional cell lines confirmed the general expression of gp130 but showed limited expression of the IL-6Ralpha chain. Surprisingly, about half of the cell lines tested expressed IL-6 mRNA at low levels which was not translated into protein. Our results suggest that IL-6 can potently stimulate anchorage-independent growth of some colorectal carcinoma cells. This stimulation appears to occur through a paracrine mechanism.

LAT2, a new basolateral 4F2hc/CD98-associated amino acid transporter of kidney and intestine.

Glycoprotein-associated amino acid transporters (gpaAT) are permease-related proteins that require heterodimerization to express their function. So far, four vertebrate gpaATs have been shown to associate with 4F2hc/CD98 for functional expression, whereas one gpaAT specifically associates with rBAT. In this study, we characterized a novel gpaAT, LAT2, for which mouse and human cDNAs were identified by expressed sequence tag data base searches. The encoded ortholog proteins are 531 and 535 amino acids long and 92% identical. They share 52 and 48% residues with the gpaATs LAT1 and y(+)LAT1, respectively. When mouse LAT2 and human 4F2hc cRNAs were co-injected into Xenopus oocytes, disulfide-linked heterodimers were formed, and an L-type amino acid uptake was induced, which differed slightly from that produced by LAT1-4F2hc: the apparent affinity for L-phenylalanine was higher, and L-alanine was transported at physiological concentrations. In the presence of an external amino acid substrate, LAT2-4F2hc also mediated amino acid efflux. LAT2 mRNA is expressed mainly in kidney and intestine, whereas LAT1 mRNA is expressed widely. Immunofluorescence experiments showed colocalization of 4F2hc and LAT2 at the basolateral membrane of kidney proximal tubules and small intestine epithelia. In conclusion, LAT2 forms with LAT1 a subfamily of L-type gpaATs. We propose that LAT1 is involved in cellular amino acid uptake, whereas LAT2 plays a role in epithelial amino acid (re)absorption.

Bombesin stimulates adhesion, spreading, lamellipodia formation, and proliferation in the human colon carcinoma Isreco1 cell line.

The neuropeptide bombesin and its mammalian homologue, gastrin-releasing peptide (GRP), enhance proliferation in some but not all human tumor cell lines. The pathophysiological relevance of the bombesin/GRP receptor (GRP-R), which is expressed in 30% of human colon tumor cell lines and in 24-40% of native tumors, has not been clearly assessed at this time. We studied the effects of bombesin in the recently characterized human colon carcinoma Isreco1 cell line. Competitive reverse transcription-PCR showed a high GRP-R mRNA level in Isreco1 cells, and binding studies confirmed the expression of bombesin/GRP-subtype receptors (Kd = 0.42 nM; Bmax = 18,000 sites/cell). Exposure to bombesin resulted in an increase of intracellular calcium concentrations. Bombesin (1 nM) induced cell spreading at 24 h (21.7+/-1.6% versus 6.4+/-0.8% in control cells; P<0.01) and markedly increased the formation of lamellipodia. In addition, adhesion of Isreco1 cells to collagen I-coated culture dishes was stimulated in the presence of 1 nM bombesin (69+/-6% versus 42+/-1% in control cells; P<0.01). Finally, bombesin significantly increased [3H]thymidine uptake by Isreco1 cells in a dose-dependent manner, with a first significant response at 0.1 nM and a maximal effect at 100 nM bombesin (192.2+/-9.7% of control). These results clearly indicate that bombesin exerts morphological, adhesive, and proliferative effects on Isreco1 cells, suggesting that expression of the bombesin/GRP-R may contribute to the malignant properties of colon carcinoma cells.

APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

Differential expression of laminin-5 subunits and integrin receptors in human colorectal neoplasia.

Cell-matrix interactions contribute to regulating the adhesion, growth, migration, and differentiation of epithelial intestinal cells. Alterations in matrix components and their cellular receptors have been found in tumours but their specific roles remain unclear. The tissue patterns of laminin-5 and alpha 3, beta 3 and gamma 2 subunits, as well as those of the alpha 3, alpha 6, beta 1, and beta 4 integrin chains, were determined by immunofluorescence on frozen sections of 12 colorectal mucosal samples from four patients, 15 adenomas, 29 adenocarcinomas, and eight metastases. Distinct patterns of laminin-5 and integrin expression were found along the mucosa-adenoma, and adenoma-carcinoma transitions. Expression of basement membrane laminin-5 and subunits was continuous and gradient-like in normal mucosa, enhanced at the periphery of adenomas, and discontinuous in places in carcinomas and metastases. Decrease of the alpha 3 integrin chain was found in adenomas, together with that of alpha 6 and beta 4 chains in carcinomas. A subpopulation of carcinoma cells dissociating (budding) from neoplastic tubules was found to accumulate the laminin-5 beta 3 gamma 2 heterodimer in the cytoplasm, with progressive loss of surface integrin expression. These results suggest that in colorectal cancer, an abnormal expression of laminin-5 subunits and integrin chains may identify a subset of carcinoma cells prone to invade focally and to contribute to disease aggressiveness.

The orphan receptor CRF2-4 is an essential subunit of the interleukin 10 receptor.

The orphan receptor CRF2-4 is a member of the class II cytokine receptor family (CRF2), which includes the interferon receptors, the interleukin (IL) 10 receptor, and tissue factor. CRFB4, the gene encoding CRF2-4, is located within a gene cluster on human chromosome 21 that comprises three interferon receptor subunits. To elucidate the role of CRF2-4, we disrupted the CRFB4 gene in mice by means of homologous recombination. Mice lacking CRF2-4 show no overt abnormalities, grow normally, and are fertile. CRF2-4 deficient cells are normally responsive to type I and type II interferons, but lack responsiveness to IL-10. By approximately 12 wk of age, the majority of mutant mice raised in a conventional facility developed a chronic colitis and splenomegaly. Thus, CRFB4 mutant mice recapitulate the phenotype of IL-10-deficient mice. These findings suggest that CRF2-4 is essential for IL-10-mediated effects and is a subunit of the IL-10 receptor.

Alterations in plasminogen activation correlate with epithelial cell dysplasia grading in colorectal adenomas.

Proteases are important for neoplastic invasion but a specific role for the plasminogen activator system in the progression of colorectal epithelial dysplasia to adenomatous lesions remains unclear. Consecutive tissue cryosections of 51 adenomas, 49 distant mucosa samples and five mucosa samples from control subjects were histopathologically analysed for dysplasia grade and tissue type, urokinase plasminogen activator levels and plasminogen activator inhibitor type 1 (PAI-1) using immunosorbent methods. Plasminogen activation and urokinase-mediated proteolytic activity levels were assessed using in situ zymography. Plasminogen activation and tissue-type activator levels were lower in adenomas than in mucosae (P < 0.001). PAI-1 concentration and urokinase levels were higher in adenomas than in mucosae (P < 0.001 and P < 0.001 respectively). In adenomas, urokinase concentration increased in parallel with PAI-1, but only the urokinase levels correlated with the dysplasia grade (P < 0.01). Thus, the alterations in plasminogen activation correlated with epithelial cell dysplasia grading. In the mucosa to adenoma transition, a marked decrease in tissue-type plasminogen activator occurred. In adenomas, this decrease was accompanied by a concomitant increase in urokinase and PAI-1. The urokinase level only continued to rise in parallel with the dysplasia grade. Resulting protease-antiprotease imbalance in high-grade dysplasia may represent the phenotypic change associated with malignant transformation and invasive behaviour.

T-kininogen present in the liver of old rats is biologically active and readily forms complexes with endogenous cysteine proteinases.

We have previously reported an increase in T-kininogen mRNA levels in the liver of ageing Sprague-Dawley rats. T-Kininogen functions both as a precursor to the vasoactive peptide T-kinin, and as a potent and specific inhibitor of cysteine proteinases. Under normal physiological conditions, the majority of cysteine proteinases are found intracellularly and we have shown that a significant proportion of T-kininogen also accumulates intracellularly in the liver of old rats. Therefore, our aim was to determine whether or not this T-kininogen is biologically active as an inhibitor of cysteine proteases. Titration of whole liver extracts indicates that old rats do indeed contain a 4-fold higher level of cysteine proteinase inhibitory activity than younger counterparts. Using gel permeation chromatography in conjunction with an enzyme inhibitor assay, we show that this difference is mainly due to the presence of a low level of free biologically active T-kininogen. However, Western blot analysis of the gel permeation chromatography fractions demonstrate that most of the intrahepatic T-kininogen is found as enzyme-inhibitor complexes. Alkaline inactivation of the cysteine proteinase component of these complexes leads to the release of biologically competent free T-kininogen. These findings are discussed with regard to the possible mechanisms responsible for the accumulation of T-kininogen within the aged rat liver.

Differential display cloning identifies motility-related protein (MRP1/CD9) as highly expressed in primary compared to metastatic human colon carcinoma cells.

Differential display cloning was performed to analyze genes that are differentially expressed in matched primary and metastases-derived human colon carcinoma cell lines. This led to the identification of PMA16, a gene identical to the previously cloned motility-related protein gene (MRP1/CD9). Northern and Western blot analyses of cell lines, as well as immunostaining of tissue sections from the original tumor surgical samples, confirmed that MRP1/CD9 was highly expressed at the primary site, compared to the low levels of expression in metastases. We also demonstrated that primary colon cancer cells displayed a significantly higher migration potential, compared to metastasis-derived cells. Antibodies directed against MRP1/CD9 largely prevented cell migration in vitro, but they did not influence cell adhesion. Thus, differential display cloning has allowed for the identification of MRP1/CD9, a motility-related gene product, which may regulate the metastatic phenotype of human colon cancer.

Abnormal T lymphocyte development induced by targeted overexpression of IkappaB alpha.

A role in thymic maturation for factors of the NF-kappaB family has long been suspected, but not yet proven. Transgenic mice with a lymphocyte-specific defect in NF-kappaB activation were produced by targeted expression of human IkappaB alpha. The thymic cellularity of these mice was significantly decreased. The proportion of mature, TCRhigh thymocytes of the alphabeta lineage was reduced, and the remaining TCRhigh population contained an unusually high proportion of double-positive cells. This defect in maturation resulted in a transgene dose-dependent reduction in peripheral T lymphocytes, with the CD8 lineage being more severely affected. These data provide direct evidence for the involvement of NF-kappaB/Rel family proteins in late stages of T lymphocyte development, coincident with positive and negative selection.

Genetic heterogeneity in sporadic colorectal adenomas.

The majority of colorectal cancers develop from adenomatous polyps under the influence of factors that are still poorly understood. Tumourigenesis is generally considered a multistep process in which multiple genetic alterations occur, eventually reflected in abnormalities of the cellular DNA content. Macroscopical features such as tumour size and tumour architecture (tubular, tubulovillous, or villous) are correlated wit the chance of malignancy in the lesion. Grade of dysplasia can be considered an indicator for the level of progression of the adenoma towards invasive carcinoma. These characteristics were correlated with the presence or absence of K-ras mutations and the DNA ploidy in a prospective study performed on 46 large sporadic colorectal adenomas resected by endoscopy. DNA ploidy and K-ras mutations were analysed in two samples taken at distant sites in the adenomas. Aneuploidy was present in 12 adenomas (26 per cent) and K-ras mutations occurred in 26 (57 per cent). A highly significant correlation was found between aneuploidy and adenoma size, architecture, and grade of dysplasia. The presence of K-ras mutations was significantly correlated only with the size of the adenomas. The proportion of adenomas with aneuploidy and/or a K-ras mutation increased when two samples were analysed instead of one. This observation suggests that the prevalence of genetic mutations and of aneuploidy is probably underestimated, as generally only one sample is investigated. No correlation was observed between K-ras mutations and ploidy. This study demonstrates the presence of genetic heterogeneity in colorectal adenomas and supports the notion that K-ras mutation is an early event, while aneuploidy is a late event in the adenoma-carcinoma sequence.

Role of integrins and evidence for two distinct mechanisms mediating human colorectal carcinoma cell interaction with peritoneal mesothelial cells and extracellular matrix.

Peritoneal carcinomatosis involves a series of events including tumor cell interactions with mesothelial cells and the extracellular matrix (ECM). We have studied the adhesive and invasive properties of four human colorectal carcinoma cell lines (Co115, HT29, SW480, SW620) confronted in vitro with a human mesothelial cell monolayer or with the ECM proteins collagen IV, laminin-1, fibronectin, tenascin-C and vitronectin. Quantitation was achieved following staining of tumor cells with the calcein-AM fluorescent dye. We found that all four cell lines rapidly adhered to a mesothelial cell monolayer. This adhesion event was not inhibitable by anti-integrin and anti-CD44 antibodies. Following initial attachment, the SW480 and SW620 cells invaded the mesothelial cell monolayer more aggressively than HT29 and Co115 cells. All cell lines adhered to ECM proteins with each one exhibiting an individual adhesion pattern. Adhesion to matrix was completely integrin-dependent. When tested in an invasion assay, HT29 and Co115 cells crossed Matrigel-coated filters while SW480 and SW620 cells did not. This invasion was inhibited by anti-beta 1 integrin antibodies. Taken together, our results demonstrate that the initial colorectal tumor cell-mesothelial cell interaction occurs through an integrin-independent mechanism while adhesion to matrix proteins and invasion through Matrigel are integrin-dependent events. Furthermore, the different invasive capacity of SW480 and SW620 versus HT29 and Co115 cells upon interaction with a mesothelial cell monolayer or Matrigel suggests that these two invasion events may be mediated by distinct mechanisms.

Plasmin-induced proteolysis of tenascin-C: modulation by T lymphocyte-derived urokinase-type plasminogen activator and effect on T lymphocyte adhesion, activation, and cell clustering.

Proteolysis and remodeling of the extracellular matrix occur physiologically in processes such as tissue morphogenesis and repair and may participate in the regulation of complex cell functions, including proliferation and differentiation. While matrix degradation appears to be relevant to T lymphocyte migration through tissues, little is known about whether degraded matrix affects T lymphocyte function. We have studied the interaction between T lymphocytes and tenascin-C (TN-C), a matrix protein we have previously reported to inhibit T lymphocyte activation, in the context of plasmin-induced degradation. Here we report that plasmin efficiently cleaves TN-C. Peripheral blood T lymphocytes stimulated with phorbol ester, anti-CD28, or anti-CD3 Ab, induce, within 24 to 48 h, a strong plasminogen-dependent proteolysis of TN-C. We demonstrate that stimulated T lymphocytes activate plasminogen by secreting the urokinase-type plasminogen activator (u-PA). Plasminogen activation by T lymphocyte-derived u-PA occurs efficiently in fluid phase in the absence of cells. We investigate the consequences of plasmin-induced proteolysis on three of the effects of TN-C in relation to lymphocyte functions. Plasmin proteolysis converts TN-C from a nonadhesive into an adhesive substrate for T lymphocytes and abolishes its aggregating activity on PBMC. In contrast, the inhibitory effect of TN-C on T lymphocyte activation remains unaffected. These observations demonstrate that stimulated T lymphocytes induce plasminogen-dependent proteolysis of TN-C by secreting u-PA and suggest that proteolysis of TN-C may represent a mechanism by which to regulate some of its effects on T lymphocyte functions.

In situ stromal expression of the urokinase/plasmin system correlates with epithelial dysplasia in colorectal adenomas.

An increase of urokinase-type plasminogen activator (uPA) and a decrease of tissue-type PA (tPA) have been associated with the transition from normal to adenomatous colorectal mucosa. Serial sections from 25 adenomas were used to identify PA-related caseinolytic activities by in situ zymography, blocking selectively uPA or tPA. The distribution of uPA, tPA, and type 1 PA inhibitor mRNAs was investigated by nonradioactive in situ hybridization, and the receptor for uPA was detected by immunostaining. Low- and high-grade epithelial cell dysplasia was mapped histologically. Results show that 23 of 25 adenomas expressed uPA-related lytic activity located predominantly in the periphery whereas tPA-related activity was mainly in central areas of adenomas. In 15 of 25 adenomas, uPA mRNA was expressed in stromal cells clustered in foci that coincided with areas of uPA lytic activity. The probability of finding uPA mRNA-reactive cells was significantly higher in areas with high-grade epithelial dysplasia. uPA receptor was mainly stromal and expressed at the periphery. Type 1 PA inhibitor mRNA cellular expression was diffuse in the stroma, in endothelial cells, and in a subpopulation of alpha-smooth muscle cell actin-reactive cells. These results show that a stromal up-regulation of the uPA/plasmin system is associated with foci of severe dysplasia in a subset of colorectal adenomas.

Modulation of the plasminogen activation system by inflammatory cytokines in human colon carcinoma cells.

Inflammation may promote malignant invasion by enhancing cancer cell-associated proteolysis. Here we present the effect of inflammatory cytokines on the plasminogen activation system of eight human colon carcinoma cell lines. Tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) increased in several, but not all, cell lines the production of urokinase-type plasminogen activator (uPA), tissue-type PA (tPA) and plasminogen activator inhibitor type 1 (PAI-1) as analysed by zymography, enzyme immunoassays and Northern analysis. Interleukin 6 (IL-6) had no effect. uPA receptor (uPAR) mRNA levels were also upregulated. However, each individual cell line responded differently following exposure to TNF-alpha or IL-1 beta. For example, there was a dose-dependent up-regulation of uPA and PAI-1 in SW 620 cells, whereas increased uPA production in SW 1116 cells was not accompanied by an increase in PAI-1. The TNF-alpha stimulatory effect was blocked by anti-TNF-alpha Fab fragments. All cell lines expressed both types of TNF receptor mRNAs, whereas no transcript for TNF-alpha, IL-1 beta, IL-6, IL-6 receptor or the IL-1 receptors was found. Our results demonstrate that TNF-alpha and IL-1 beta stimulate the plasminogen activation system in tumour cell but the responses differed even in cells derived from the same tissue origin.