Generation of cytotoxic T lymphocytes in vitro. IV. Functional activation of memory cells in the absence of DNA synthesis.

Re-exposure of day 14 mixed leukocyte culture (MLC) cells to the original stimulating alloantigens (secondary response) has previously been shown to result in significant proliferation and in rapid reappearance of high levels of cytolytic T-lymphocyte (CTL) activity within the next 4 days. Moreover, evidence has been presented that CTL precursor cells in day 14 MLC populations, while they derived from cells were large at peak of the primary response (day 4) were themselves small lymphocytes which developed into large CTL after restimulation. In this study, inhibition of DNA synthesis by cytosine arabinoside (ARA-C) was used to investigate whether CTL formation could be dissociated from proliferation during the secondary response. It was found that within the first 24 h after restimulation (a) CTL activity increased 6-to-20-fold, (b) 60-70% of the small T lymphocytes became medium- to large-sized cells, and (c) both events were independent of DNA synthesis. By using two successive cell separations by velocity sedimentation at unit gravity, before and after stimulation of day 14 MLC cells for 24 h in the presence or absence of ARA-C, direct evidence was obtained that small CTL precursor cells developed into large CTL, irrespective of DNA synthesis. The presence of ARA-C for periods longer than 24 h inhibited any further increase in CTL activity, in contrast to a parallel increase in lytic activity and cell number from day 1 to day 4 in control restimulated cultures. Taken together with the finding that 90% of the medium- and large-sized lymphoid cells in control restimulated cultures underwent DNA synthesis within 24 h, these results thus suggest that during a secondary MLC response there is initially a differentiation step leading to the formation of CTL which, although it can be clearly dissociated from DNA synthesis, is under normal conditions followed by proliferation of these effector cells.

T cell antigen receptor expression in athymic (nu/nu) mice. Evidence for an oligoclonal beta chain repertoire.

The expression of T cell antigen receptors (TCR) in congenitally athymic (nude) mice has been investigated. Lymph node T cells from 4-5-mo-old athymic mice expressed full-length transcripts for the TCR alpha and beta chains at a level two-to three-fold lower than normal littermate (nu/+) controls. Low levels of expression of TCR protein at the surface of a proportion of nude T cells was demonstrated by staining with monoclonal antibodies KJ16-133 and F23.1 (directed against protein products of a family of TCR beta chain variable region genes known as V beta 8). Immunoprecipitation studies confirmed that F23.1 reacted with a similar molecular species on nude and nu/+ T cells. Studies with individual nude mice revealed a striking heterogeneity in the proportion of T cells expressing KJ16/F23.1 that was not seen in normal animals. This heterogeneity correlated with the expression of mRNA specific for V beta 8 but not with total expression of full-length beta chain transcripts. Analysis of Lyt-2+ and L3T4+ T cell subsets in individual nude mice further demonstrated that F23.1 expression was frequently associated with only one subset, and several cases were seen in which all L3T4+ cells expressed F23.1. In contrast, a similar (and constant) proportion of Lyt-2+ or L3T4+ T cells expressed F23.1 in control mice. Southern blotting of Hind III-digested DNA from nude T cells with a C beta probe revealed a more restricted pattern of TCR beta chain rearrangements than was seen for normal T cells. Taken together, these data provide compelling evidence that TCR gene rearrangement and expression can occur extrathymically. Furthermore, they suggest a model according to which the restricted functional repertoire of T cells previously observed in individual nude mice results from an oligoclonal expansion of T cells that have randomly rearranged and expressed TCR beta chain genes.

Specific antibody within lymphoid germinal center cells of mice after primary immunization with horseradish peroxidase: a light and electron microscopic study.

The appearance in mice of specific antibody within newly formed germinal centers in lymph nodes was demonstrated by light and electron microscopy after regional primary antigenic stimulation with horseradish peroxidase (HRP). Lymphoid germinal center cells containing anti-HRP antibody in the perinuclear space and in the cytoplasm were detected from 17 to 26 days after antigenic stimulation. Extracellular anti-HRP antibody within germinal centers, localized between dendritic reticular cells and lymphoid elements, could not be found before the appearance of intracellular antibody. These findings strongly suggest antibody formation by lymphoid germinal center cells. Both antigen and corresponding antibody persisted in intercellular spaces up to 35 days after primary stimulation. The concomitant presence in a given lymph node of germinal centers which are positive or negative with regard to specific antibody provide evidence in favor of monospecificity of individual centers. The mechanisms of antigen-trapping within germinal centers are discussed in the light of the present observations.

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.

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.

Mouse L cells expressing human prourokinase-type plasminogen activator: effects on extracellular matrix degradation and invasion.

A cosmid (cos pUK0322) harboring the complete human urokinase-type plasminogen activator (u-PA) gene and Geneticin resistance as a selectable marker was isolated from a human genomic library and characterized. After transfection of cos pUK0322 into mouse L cells and selection, several plasminogen activator (PA)-expressing clones were obtained and one (LuPA) was chosen for additional study. The PA expressed was identical to human pro-u-PA in enzymatic, electrophoretic, and antigenic properties. The expression of PA was stable over 50 population doublings. The regulation of the transfected gene was studied by treatment of the cells with various hormones and other effectors. Expression of PA activity was inhibited fivefold by dexamethasone and stimulated two- to threefold by agonists of the adenylate cyclase dependent pathway of signal transduction, such as dibutyryl cyclic AMP and cholera and pertussis toxins. The modulation of PA activity was associated with corresponding changes in mRNA steady-state levels. The phenotypic changes associated with pro-u-PA expression were analyzed in vitro by degradation of 3H-labeled extracellular matrix (ECM), invasion of a matrigel basement membrane analogue, and by light and electron microscopy. LuPA cells and reference HT-1080 fibrosarcoma cells, in contrast to control Lneo cells transfected with the neomycin resistance gene, degraded the ECM and invaded the matrigel basement membrane. Matrix degradation correlated with the modulation of pro-u-PA gene expression as it was inhibited by dexamethasone and promoted by dibutyryl cyclic AMP. Inhibition of PA or plasmin using anti-u-PA IgG or aprotinin prevented ECM degradation and invasion. These results demonstrate that u-PA expression alone is sufficient to confer to a cell an experimental invasive phenotype.

Establishment of two rabbit mammary epithelial cell lines with distinct oncogenic potential and differentiated phenotype after microinjection of transforming genes.

The goal of this work was to establish an assay for transformation of epithelial cells. Two epithelial cell lines were obtained after microinjecting transforming genes into primary rabbit mammary secretory cells. The cell lines were analyzed for their oncogenic potential and for the maintenance of a differentiated phenotype. A fully transformed cell line, which retained epithelial cell organization, was obtained by coinjecting simian virus 40 DNA and the activated human c-Ha-ras gene. The proliferation rate of these cells was high, with a doubling time of 16 h. Their growth was anchorage independent, and they had lost contact inhibition. The cells were tumorigenic in nude mice, but had no metastatic potential. Both microinjected DNAs were efficiently transcribed and translated, in contrast to the casein genes, which were expressed in primary cells but not in the transformed cell line. An immortalized cell line established after injection with simian virus 40 DNA alone was characterized by a moderate rate of proliferation with a doubling time of approximately 30 h. The growth of these cells was contact inhibited and anchorage dependent. The cells were not tumorigenic in nude mice. The viral DNA was expressed during early passages, as shown by the presence of the large T antigen in cell nuclei, but not at later passages. A high number of lactogenic hormone receptors were found associated with the cell surface. Despite the presence of these receptors, no induction of genes coding for milk proteins was observed after addition of prolactin. These data demonstrate that this assay system can be used to assess the immortalizing and transforming potential of candidate oncogenes in epithelial cells.

Human primary colon carcinomas xenografted into nude mice. II. Modulation of tumor plasminogen activator activity by the host tissue environment.

The characterization and quantitation of plasminogen activators (PAs) expressed by human colon carcinoma cell lines and primary colon carcinomas inoculated into nude outbred (nu/nu) mice, either as subcutaneous or gut-implanted (GI) xenografts, were discussed. The two colon carcinoma cell lines used in this study, Col 112 (moderately differentiated) and Col 115 (poorly differentiated), differ in their PA expression, the former being a urinary-type PA and the latter being a tissue-type PA producer. Both cell lines demonstrate a positive correlation between tumor invasiveness and measured PA activity; subcutaneous xenografts growing as noninvasive pseudobenign tumor masses were associated with low levels of PA activity, whereas GI xenografts exhibiting invasive growth expressed higher PA activity. Furthermore, coinoculation of Col 115 tumor cells sc and GI in the same host induced high levels of PA activity in subcutaneous xenografts, suggesting a stimulatory effect of the GI xenograft on subcutaneous xenograft PA expression. Also, purified murine plasminogen was demonstrated to represent an efficient substrate for tumor-secreted human PA.

Human primary colon carcinomas xenografted into nude mice. I. Characterization of plasminogen activators expressed by primary tumors and their xenografts.

Analysis was made of plasminogen activator (PA) activities present in 0.125% Triton X-100 extracts of human primary colon carcinomas and of their respective serial subcutaneous xenografts in nude mice. A correlation between tumor invasiveness and PA expression was observed in that primary tumors exhibiting clearly invasive growth patterns demonstrated high concentrations of PAs while subcutaneous xenografts, exhibiting noninvasive pseudobenign growth, contained very low levels of PA activity. The decrease in fibrinolytic activity observed in subcutaneous xenografts was not due to an increase in inhibitors of fibrinolytic activity. Immunologic characterization of PAs in tumor extracts showed that over 90% of human PA activity was of the urokinase type. Furthermore, tumor-derived urokinase was shown to be present in a proenzyme form. It was resistant to diisopropyl fluorophosphate (DFP) and was not inhibited by purified PA inhibitor. However, after its activation into urokinase by plasmin, it was completely inhibited by DFP and PA inhibitor.

Establishment of a cell line (Co-115) from a human colon carcinoma transplanted into nude mice.

A human colon carcinoma cell line, Co-115, has been established in vitro from solid xenografts maintained in nude mice and subcultured for 95 passages. Co-115 cells grow in vitro as tightly packed, epithelial-like colonies, have a doubling time of about 36 hr, have a relatively low plating efficiency in agar, and release significant amounts of carcinoembryonic antigen to the culture medium. Their epithelial nature has been confirmed by ultrastructural examination. The injection of Co-115 cells into nude mice reinduced the formation of solid tumor masses that could be retransplanted and showed a morphology comparable of that of the original xenograft.

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.

Oxygenation and differentiation in multicellular spheroids of human colon carcinoma.

Oxygenation and development of necrosis were evaluated in multicellular spheroids of poorly differentiated (HT29) and moderately well-differentiated (Co112) human adenocarcinoma of the colon. Spheroids were grown in vitro under well-controlled oxygen and nutrient conditions in spinner flasks up to sizes of 2800-micron diameter after 5 wk of culture. Morphological studies showed that the Co112 spheroids contained pseudoglandular structures with lumen, very similar to the characteristics of the original tumor specimen from the patient and to the cells when grown as xenograft tumors in nude mice. Microelectrodes were used to measure the oxygen tension (PO2) profile within individual spheroids at different stages of growth. Histological sections through the centers of spheroids were measured to determine the thickness of the viable rim of cells surrounding spheroid necrotic centers in order to estimate the size of the severely hypoxic zone of cells by comparison with the PO2 profiles of the same spheroids. The data demonstrate significant differences between these two human colon tumor spheroid systems. Both spheroid types exhibited steep PO2 gradients at relatively small sizes of less than 600-micron diameter, but for any given size in this range, the more differentiated Co112 spheroids were more hypoxic. Although severe hypoxia (PO2, less than 10 mm of Hg) was present in both spheroid types at larger sizes, there was a significant difference in the central PO2 values which were between 5 and 10 mm of Hg in large Co112 spheroids but remained at or close to 0 mm of Hg in large HT29 poorly differentiated human colon tumor spheroids. The presence of pseudoglandular structures and lumen in the Co112 spheroids was associated with changes in the shape of PO2 profiles. Such profiles have not previously been seen in other poorly differentiated human or rodent tumor spheroids. Furthermore, the PO2 profiles of both of these human tumor spheroid types were often continuously curving with a very shallow gradient in the inner edge of the viable rim of cells surrounding the necrotic center. Regulation of oxygen consumption and/or diffusion in these inner regions of human spheroids could produce these continuously curving PO2 gradients.

Responsiveness of three newly established human colorectal cancer cell lines to transforming growth factors beta 1 and beta 2.

We have established 3 new human colorectal cancer cell lines (LS411N, LS513, and LS1034) from clinical biopsy samples. These lines are tumorigenic and grow s.c. as adenocarcinomas in nude mouse xenografts. Specific marker chromosomes are observed in each line. Carcinoembryonic antigen is expressed at the surface of all 3 lines, but with marked quantitative differences. Indeed, less than 10% of the cells from the HT-29 line used as a reference express carcinoembryonic antigen while more than 90% of the LS1034 cells do so. LS513 and LS1034 consistently express HLA class I antigens and intercellular adhesion molecule 1 which are not detected at the surface of the LS411N cells. No expression of HLA class II antigens DR, DQ, and DP has been measured on any of the lines. All three lines grow well in 5% fetal calf serum medium without addition of exogenous growth factors. The LS1034 line has been adapted to growth in serum-free conditions and exhibits increased clonogenicity when cells are seeded in serum-free methylcellulose medium, as compared with medium containing 5% fetal calf serum. The LS513 and LS1034 lines have proved to be of particular interest since they respond to the growth-inhibitory action of TGF-beta 1 and TGF-beta 2 in both liquid and semisolid medium. Both factors were, at pM concentrations, equipotent inhibitors of LS1034 cell proliferation. In contrast, higher concentrations of TGF-beta 1 are inhibitory for proliferation of LS513 cells, whereas TGF-beta 2 has no effect on the growth of these cells in liquid assay. On this basis, using appropriate anti-TGF-beta 1 and anti-TGF-beta 1 IgY, we developed a bioassay for TGF-beta 1 and TGF-beta 2. Two of the three lines have indeed been shown to produce latent-TGF-beta 1 activity.

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.

Latency of cathepsin B secreted by human colon carcinoma cells is not linked to secretion of cystatin C and is relieved by neutrophil elastase.

The lysosomal cysteine proteinase cathepsin B is shown to be secreted by ten human colon carcinoma cell lines and to accumulate in culture media as a latent enzyme. The cell lines also secrete a physiological inhibitor of cathepsin B, cystatin C. A significant correlation was found between secretion of the latent enzyme and the inhibitor (r = 0.755, P < 0.01). The aim of the present study was to modulate the respective secretion of the two antagonists to test whether or not latency of cathepsin B was due to the concomitant secretion of the inhibitor. SW480 colon carcinoma cells were treated with the acidotropic agent ammonium chloride, phorbol 12-myristate 13-acetate, and the inflammatory cytokines TGF-beta, TNF-alpha, and IL-1 beta. Ammonium chloride significantly increased latent cathepsin B levels without affecting the constitutive secretion of cystatin C. Phorbol 12-myristate 13-acetate induced a 4- to 5-fold increase in secreted latent cathepsin B, but did not alter significantly the accumulation of cystatin C in media. The cytokines, TGF-beta, TNF-alpha, and IL-1 beta, had no major effect on the expression of these two antagonists. Latent cathepsin B released from human carcinoma cells could be efficiently activated by neutrophil elastase at neutral pH. It is concluded that latent cathepsin B is a true proenzyme rather than an enzyme-inhibitor complex. In addition, our data from neutrophil elastase activation experiments indicate that a proteolytic system for activation of the tumor cell-secreted latent enzyme may exist in vivo.

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.

Cellular growth and metabolic adaptations to nutrient stress environments in tumor microregions.

Heterogeneity of cell subpopulation growth was significantly modulated by different oxygen and glucose environments and necrosis in multicellular tumor spheroids of rodent and human origin. PO2 profiles within spheroids measured with microelectrodes showed major differences associated with different oxygen and glucose supply conditions, indicating important interactions of these two substrates affecting oxygen consumption rates and cellular viability. Cellular interactions in association with the development of growth quiescence and differentiation changed oxygen consumption rates and slopes of PO2 profiles within spheroids. Protein synthesis in monolayer cells in culture was severely inhibited when exposed to extreme hypoxia, but certain proteins were synthesized at increased rates. Many of these oxygenated regulated proteins can also be induced by glucose deprivation. The data demonstrate cellular and subcellular changes in tumor models in vitro because of variations in oxygen and glucose supply. Many of these changes would be expected to occur in tumor microregions in vivo and could have important consequences for therapeutic responsiveness.

Detection of rare circulating human colon tumor cells in a nude mouse xenograft model.

A highly sensitive allele specific polymerase chain reaction was developed and applied to the detection of K-ras mutation in human colon tumor cells both in the blood and in tissues. An experimental model of human colon carcinoma cells, carrying a GAT mutation in the 12th codon of the K-ras gene, and grafted into nude mice has been selected for evaluating the occurrence of cells in the course of disseminating into the host. We have found tumor cells circulating in the blood starting 37 days following subcutaneous primary implantation. Occasional micrometastatic deposits could be detected in lymph node draining the xenograft, but no tumor cells were found in lungs and mediastinum. In this experimental model, our results indicate that the mere presence of tumor cells in the blood does not imply the full accomplishment of the multi-step metastatic process.

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.

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.