The growing galectin network in colon cancer and clinical relevance of cytoplasmic galectin-3 reactivity

BACKGROUND/AIM Human lectins translate sugar-encoded signals of cell surface glycoconjugates into biological effects, and this is what is known for the adhesion/growth-regulatory galectins. In addition, the multifunctional members of this group can be intracellular, binding to distinct proteins. The presence of galectins and galectin reactivity were exemplarily studied in the present article. MATERIALS AND METHODS We combined immuno- and lectin histochemical monitoring in colon cancer on tissue arrays. RESULTS Intracellular presence of galectins-7 and -9 in colon cancer is detected, extending the previously known set of five expressed lectins this tumor type. The assumed significance of intracellular galectin presence, e.g. for an interplay with BCL2, β-catenin, oncogenic KRAS or synexin, is underscored by respective staining with labeled galectin-3. Statistical significance was obtained for galectin-3 staining with respect to tumor differentiation (p=0.0376), lymph node metastasis (p=0.0069) and lymphatic invasion (p=0.0156). Survival was correlated to staining, galectin-3 reactivity indicating a favorable prognosis (p=0.0183), albeit not as an independent marker. No correlation to KRAS/BRAF status was detected. CONCLUSION These results encourage further testing of labeled human galectins as probes and immunohistochemical fingerprinting instead of measuring single or few activities, in colon cancer and other tumor types.

Construct Validity of the Pig Intestine Model in the Simulation of Laparoscopic Urethrovesical Anastomosis

Introduction: Laparoscopic training models are increasingly important in urology to allow trainees to improve their laparoscopic skills prior to going to the operating room. For a training model to be valid, it must correlate with performance in a real case. The model must also discriminate between experienced and inexperienced subjects. [See PDF for complete abstract]

Does colon cancer ever metastasize to bone first? a temporal analysis of colorectal cancer progression.

BACKGROUND: It is well recognized that colorectal cancer does not frequently metastasize to bone. The aim of this retrospective study was to establish whether colorectal cancer ever bypasses other organs and metastasizes directly to bone and whether the presence of lung lesions is superior to liver as a better predictor of the likelihood and timing of bone metastasis. METHODS: We performed a retrospective analysis on patients with a clinical diagnosis of colon cancer referred for staging using whole-body 18F-FDG PET and CT or PET/CT. We combined PET and CT reports from 252 individuals with information concerning patient history, other imaging modalities, and treatments to analyze disease progression. RESULTS: No patient had isolated osseous metastasis at the time of diagnosis, and none developed isolated bone metastasis without other organ involvement during our survey period. It took significantly longer for colorectal cancer patients to develop metastasis to the lungs (23.3 months) or to bone (21.2 months) than to the liver (9.8 months). Conclusion: Metastasis only to bone without other organ involvement in colorectal cancer patients is extremely rare, perhaps more rare than we previously thought. Our findings suggest that resistant metastasis to the lungs predicts potential disease progression to bone in the colorectal cancer population better than liver metastasis does.

Characterization of the cytochrome P-450 drug metabolism system of LS174T human colon tumor cell line

The human colon tumor cell line, LS174T, has been shown to have four major components of the drug metabolizing system; cytochrome b$\sb5$ reductase, cytochrome b$\sb5$, cytochrome P450 reductase and cytochrome P450, by activity measurements, spectral studies and antibody cross-reactivity. Cytochrome P450IA1 is induced by benzanthracene in these cells as shown by activity with the specific substrate, ethoxyresorufin, cross-reactivity with rabbit antibodies to rat IA1, and by a hybridizing band on a Northern blot to a rat IA1 probe.^ Further, this system has proven responsive to various inducers and conditions of growth. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 $\mu$mol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b$\sb5$ per milligram and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone treatment showed a consistent, but not always significant increase in the NADPH and NADH cyt c reducing activity and benzanthracene treatment an increase in the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5mM) caused a significant decrease in the specific activity of all enzyme contents and activities tested.^ Finally, the cytochrome b$\sb5$ to cytochrome P450, by the coordinate induction of the cytochrome b$\sb5$ pathway by P450 inducers, by the high ratio of NADH to NADPH ethoxycoumarin deethylase activity in uninduced cell microsomes, and by the increase in NADH and NADPH ethoxycoumarin deethylase activity when the microsomes were treated with potassium cyanide, a desaturase inhibitor. ^

The development and characterization of two types of chronic responses in irradiated mouse colon

The hypothesis to be tested is that there are two distinct types of chronic responses in irradiated normal tissues, each resulting from damage to different cell populations in the tissue. The first is a sequala of chronic epithelial depletion in which the tissue's integrity cannot be maintained, i.e. a "consequential" chronic response. The other response is due to cell loss in the connective tissue and/or vascular stroma, i.e. a "primary" chronic response. The purpose of this study was to test the hypothesis in the murine colon by first, establishing a model of each chronic response and then, by determining whether the responses differed in timing of expression, histology, and expression of specific collagen types. The model of late damage used was colonic obstructions/strictures induced by a single dose of 27 Gy ("consequential" response) and two equal doses of 14.75 Gy (t = 10 days) ("primary" response). "Consequential" lesions appeared as early as 5 weeks after 27 Gy and were characterized by a deep mucosal ulceration and a thickened fibrotic serosa containing excessive accumulations of collagen types I and III. Both types were commingled in the scar at the base of the ulcer. Fibroblasts were synthesizing pro-collagen types I and III mRNA 10 weeks prior to measurable increases in collagen. A significant decrease in the ratio of collagen types I:III was associated with the "consequential" response at 4-5 months post-irradiation. The "primary" response, on the other hand, did not appear until 40 weeks after the split dose even though the total dose delivered was approximately the same as that for the "consequential" response. The "primary" response was characterized with an intact mucosa and a thickened fibrotic submucosa which contained excessive amounts of only collagen type I. An increased number of fibroblasts were synthesizing pro-collagen type I mRNA nearly 25 weeks before collagen type I levels were increased. The "primary" response lesion had a significantly elevated collagen type I:III ratio at 10-13 months post-irradiation. These data show a clear difference between the two chronic response and suggest that not all chronic responses share a common pathogenesis, but depend on the cell population in the tissue that is damaged. ^

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

Analysis of galectin expression and identification of endogenous ligands in a human colon carcinoma cell line

The 14.5 kDa (galectin-1) and 31 kDa (galectin-3) lectins are the most well characterized members of a family of vertebrate carbohydrate-binding proteins known as the galectins. Evidence has been obtained implicating these galectins in events as diverse as cell-cell and cell-extracellular matrix interactions, growth regulation, transformation, differentiation, and programmed cell death. In the present study, sodium butyrate was found to be a potent inducer of galectin-1 in the KM12 human colon carcinoma cell line. Prior to treatment with butyrate this cell line expresses only galectin-3. These cells were utilized as an in vitro model system to study galectin expression as well as that of their endogenous ligands. The initial phase of this project involved the examination of the induction of galectin-1 by butyrate at the protein level. These studies indicated that galectin-1 induction by butyrate was relatively rapid reaching nearly maximal levels after only 24 hours. Additionally, the induction was found to be reversible upon the removal of butyrate and to precede the increase in expression of the well characterized differentiation marker, carcinoembryonic antigen (CEA). The second phase of this project involved the characterization of potential glycoprotein ligands for galectin-1 and galectin-3. This work demonstrated that the polylactosaminoglycan-containing glycoproteins laminin, CEA, and the lysosome-associated glycoproteins-1 and -2 (LAMPs-1 and -2) are capable of serving as ligands for both galectin-1 and -3. The third phase of this project involved the analysis of the induction of the galectin-1 promoter by butyrate. Through the analysis of deletion constructs transiently transfected into KM12 cells, the region of the galectin-1 promoter mediating a high level of induction by butyrate was localized primarily within a proximal portion of the promoter containing a CCAAT element and an Sp1 binding site. The CCAAT-binding activity in the KM12 nuclear extracts was subsequently dentified as NF-Y by gel shift analysis. These studies suggest that: (1) the galectins may be involved in modulating adhesive interactions in human colon carcinoma cells through the binding of several polylactosaminoglycans shown to play a role in adhesion and (2) high level induction of the galectin-1 promoter by butyrate can proceed through a discreet, proximal element containing an NF-Y-binding CCAAT box and an Sp1 site. ^

Analysis of pp60$\sp{{\rm c}{-}src}$ and pp62$\sp{{\rm c}{-}yes}$ intracellular protein tyrosine kinase function in colon tumor cell growth regulation using herbimycin A, a tyrosine kinase specific inhibitor

Two approaches were utilized to investigate the role of pp60c-src activation in growth control of model colon tumor cell lines. The first approach involved analysis of pp60c-src activity in response to growth factor treatment to determine if transient activation of the protein was associated with ligand induced mitogenic signal transduction as occurs in non-colonic cell types. Activation of pp60c-src was detected using colon tumor cell lysates after treatment with platelet derived growth factor (PDGF). Activation of pp60c-src was also detected in response to epidermal growth factor (EGF) treatment using cellular lysates and intact cells. In contrast, down-regulation of purified pp60c-src occurred after incubation with EGF-treated EGFr immune complexes in vitro suggesting additional cellular events were potentially required for the stimulatory response observed in intact cells. The results demonstrated activation of pp60c-src in colon tumor cells in response to PDGF and EGF which is consistent with the role of the protein in mitogenic signal transduction in non-colonic cell types.^ The second approach used to study the role of pp60c-src activation in colonic cell growth control focused on analysis of the role of constitutive activation of the protein, which occurs in approximately 80% of colon tumors and cell lines, in growth control. These studies involved analysis of the effects of the tyrosine kinase specific inhibitor Herbimycin A (HA) on monolayer growth and pp60c-src enzymatic activity using model colon tumor cell lines. HA induced dose-dependent growth inhibition of all colon tumor cell lines examined possessing elevated pp60c-src activity. In HT29 cells the dose-dependent growth inhibition induced by HA correlated with dose-dependent pp60c-src inactivation. Inactivation of pp60c-src was shown to be an early event in response to treatment with HA which preceded induction of HT29 colon tumor cell growth inhibition. The growth effects of HA towards the colon tumor cells examined did not appear to be associated with induction of differentiation or a cytotoxic mechanism of action as changes in morphology were not detected in treated cells and growth inhibition (and pp60c-src inactivation) were reversible upon release from treatment with the compound. The results suggested the constitutive activation of pp60c-src functioned as a proliferative signal in colon tumor cells. Correlation between pp60c-src inactivation and growth inhibition was also observed using HA chemical derivatives confirming the role of tyrosine kinase inactivation by these compounds in inhibition of mitogenic signalling. In contrast, in AS15 cells possessing specific antisense mRNA mediated inactivation of pp60c-src, HA-induced inactivation of the related pp62c-yes tyrosine kinase, which is also activated during colon tumor progression, was not associated with induction of monolayer growth inhibition. These results suggested a function for the constitutively activated pp62c-yes protein in colon tumor cell proliferation which was different from that of activated pp60c-src. (Abstract shortened by UMI.) ^

Complement dependent early immunological responses during ex vivo xenoperfusion of hCD46/HLA-E double transgenic pig forelimbs with human blood.

BACKGROUND Besides α1,3-galactosyltransferase gene (GGTA1) knockout, several transgene combinations to prevent pig-to-human xenograft rejection are currently being investigated. In this study, the potential of combined overexpression of human CD46 and HLA-E to prevent complement- and NK-cell-mediated xenograft rejection was tested in an ex vivo pig-to-human xenoperfusion model. METHODS α1,3-Galactosyltransferase knockout heterozygous, hCD46/HLA-E double transgenic (transgenic) as well as wild-type pig forelimbs were ex vivo perfused with whole, heparinized human and autologous pig blood, respectively. Blood samples were analyzed for the production of porcine and/or human inflammatory cytokines as well as complement activation products. Biopsy samples were examined for deposition of human and porcine C3b/c, C4b/c, and C6 as well as CD62E (E-selectin) and CD106 (VCAM-1) expression. Apoptosis was measured in the porcine muscle tissue using TUNEL assays. Finally, the formation of thrombin-antithrombin (TAT) complexes was measured in EDTA plasma samples. RESULTS No hyperacute rejection was seen in this model. Extremity perfusions lasted for up to 12 h without increase in vascular resistance and were terminated due to continuous small blood losses. Plasma levels of porcine cytokines IL1β, IL-6, IL-8, IL-10, TNF-α, and MCP-1 as well as human complement activation markers C3a (P = 0.0002), C5a (P = 0.004), and soluble C5b-9 (P = 0.03) were lower in blood perfused through transgenic as compared to wild-type limbs. Human C3b/c, C4b/c, and C6 as well as CD62E and CD106 were deposited in tissue of wild-type limbs, but significantly lower levels (P < 0.0001) of C3b/c, C4b/c, and C6 deposition as well as CD62E and CD106 expression were detected in transgenic limbs perfused with human blood. Transgenic porcine tissue was protected from xenoperfusion-induced apoptosis (P < 0.0001). Finally, TAT levels were significantly lower (P < 0.0001) in transgenic limb as compared to wild-type limb xenoperfusions. CONCLUSION Transgenic hCD46/HLA-E expression clearly reduced humoral xenoresponses since all, the terminal pathway of complement activation, endothelial cell activation, muscle cell apoptosis, inflammatory cytokine production, as well as coagulation activation, were all downregulated. Overall, this model represents a useful tool to study early immunological responses during pig-to-human vascularized xenotransplantation in the absence of hyperacute rejection.

Effects of butyrate on the colon cancer cell phenotype-chemosensitization and upregulation of proteins implicated in cancer progression

Colon cancer is the second leading cause of cancer mortality in the U.S. Surgery is the only truly effective human colon cancer (HCC) therapy due to marked intrinsic drug resistance. The inefficacy of therapies developed for metastatic HCC suggests that advances in colon cancer chemoprevention and chemotherapy will be needed to reduce HCC mortality. The dietary fiber metabolite butyrate (NaB) is a candidate cancer chemopreventive agent that inhibits growth, promotes differentiation and stimulates apoptosis of HCC cells. Epidemiological and experimental studies suggest that dietary fiber protects against the development of HCC, however, recent large prospective trials have not found significant protection. ^ The first central hypothesis of this dissertation project is that the diversity of phenotypic changes induced by NaB in HCC cells includes molecular alterations that oppose its chemopreventive action and thereby limit its efficacy. We investigated the effect of NaB on the expression/activity of epidermal growth factor receptor (EGFR) and cyclooxygenase-2 (COX-2) in HCC HT29 cells. NaB treatment induced a 13-fold increase in EGFR expression in concert with its chemopreventive action in vitro, i.e., induction of growth suppression and G1 arrest, apoptosis and a differentiated phenotype. NaB-induced EGFR was active based on multiple lines of evidence. The EGFR was: (1) heavily phosphorylated at Tyrosine (P-Tyr); (2) associated with the cytoskeleton; (3) localized at the cell surface, and activated in response to EGF; and (4) NaB treatment of the cells induced activation of the EGFR effector Erk1/2. NaB treatment also induced a 7-fold increase in COX-2 expression. The NaB-induced COX-2 was active based on significantly increased PGE2 production. ^ The second central hypothesis is that NaB treatment would render HCC cells more chemosensitive to chemotherapy agents based on the increased apoptotic index induced by NaB. NaB treatment chemosensitized HT29 cells to 5-FU and doxorubicin, despite increases in the expression of P-glycoprotein and a related drug resistance protein (MRP). ^ These results raise the intriguing possibility that the chemopreventive effects of fiber may require concomitant treatment with EGFR and/or COX-2 inhibitors. Similarly, NaB may be a rational drug to combine with existing chemotherapeutic agents for the management of advanced HCC patients. ^

EXPRESSION OF TUMOR ASSOCIATED ANTIGENS ON HUMAN COLON CARCINOMA CELLS (HUMAN COLON CANCER, MONOCLONAL ANTIBODY)

Human colon cancer cells, LS180 and 174T, exhibit monoclonal antibody (mAb) 1083-17-1A and 5E113 defined tumor associated antigens. By radioimmunoassay, LS180 cells expressed the highest amount of mAb1083 defined antigens among the cell lines tested. Another mAb, 5E113, competed with mAb1083 for binding to LS180 cells, suggesting that both mAbs might bind onto identical (or adjacent) epitopes. By Scatchard analysis, about one million copies of the epitopes were present on LS180 colon cancer cells. The affinity of mAb1083 binding to the cells was 2.97 x 10('10) M('-1); the Sipsian heteroclonality value of mAb1083 was 0.9, thus approximating a single clone of reactive antibody. The qualitative studies showed that the epitopes were probably not carbohydrate because of their sensitivity to proteinases and not to mixed glucosidases and neuraminidase. The tunicamycin homologue B(,2) inhibited the incoporation of ('3)H-labeled galactose but not uptake of ('35)S-labeled methionine, nor expression of monoclonal antibody defined antigens providing further evidence to exclude the possibility of carbohydrate epitopes. There was evidence that the epitope might be partially masked in its "native" conformation, since short exposure or low dose treatment with proteases increased mAbs binding. The best detergent for antigen extraction, as detected by dot blotting and competitive inhibition assays, was octylglucoside at 30 mM concentration. Three methods, immunoprecipitation, Western blotting and photoaffinity labeling, were used to determine the molecular nature of the antigens. These results demonstrated that the antibody bound both 43 K daltons (KD) and 22 KD proteins.^ An in vitro cell-mediated immune approach was also used to attempt identifying function for the antigens. The strategy was to use mAbs to block cytotoxic effector cell killing. However, instead of blocking, the mAb1083 and 5E113 showed strong antibody-dependent cell-mediated cytotoxicities (ADCCs) in the in vitro xenoimmune assay system. In addition, cytotoxic T lymphocytes (CTLs), natural killer cells, and K cell activity were found. Since even the F(ab')2 fragment of mAbs did not inhibit the cytolytic effect, the mAbs defined antigens may not be major target molecules for CTLs. In summary, two molecular species of tumor antigen(s) were identified by mAbs to be present on colon tumor cell lines, LS180 and LS174T. (Abstract shortened with permission of author.) ^

A new sulfonamide resistance gene (sul3) in Escherichia coli is widespread in the pig population of Switzerland

A new gene, sul3, which specifies a 263-amino-acid protein similar to a dihydropteroate synthase encoded by the 54-kb conjugative plasmid pVP440 from Escherichia coli was characterized. Expression of the cloned sul3 gene conferred resistance to sulfamethoxazole on E. coli. Two copies of the insertion element IS15Delta/26 flanked the region containing sul3. The sul3 gene was detected in one-third of the sulfonamide-resistant pathogenic E. coli isolates from pigs in Switzerland.