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C Hanski

Publications and source records attributed to C Hanski.

At least 37 records · Page 2Linked to original sources
PubMed1997

Overexpression or ectopic expression of MUC2 is the common property of mucinous carcinomas of the colon, pancreas, breast, and ovary.

Mucinous carcinomas of the colorectum have been reported to overexpress the intestinal mucin MUC2. The purpose of this study was to determine whether this alteration is shared by mucinous tumours of the ovary, breast, and pancreas. A total of 40 breast carcinomas (22 of mucinous and 18 of ductal invasive type), 39 ovarian adenocarcinomas (16 mucinous, 23 serous), 47 colorectal carcinomas (25 mucinous and 22 non-mucinous), and 41 pancreatic adenocarcinomas (14 mucinous, 27 non-mucinous) were investigated by immunohistochemistry with the anti-MUC2 monoclonal antibody 4F1 and the expression pattern was ranked. MUC2 mucin is expressed in the normal colonic epithelium; in the normal epithelium of the breast, ovary, and pancreas, it was not detectable by immunohistochemistry or by reverse transcriptase-polymerase chain reaction (RT-PCR). In agreement with previous reports, the colonic mucinous carcinomas differed significantly from the non-mucinous carcinomas by strong MUC2 expression. In all mucinous carcinomas of the ovary, breast, and pancreas, de novo expression of the MUC2 gene was observed, which differentiated mucinous and non-mucinous carcinomas of these tissues (P < 0.001). The overexpression or ectopic expression of the MUC2 gene exhibited by mucinous carcinomas of four organs indicates a common genetic lesion associated with the mucinous tumour phenotype.

Adenocarcinoma, Mucinous↗
PubMed1997

Differential stimulation of intestinal mucin secretion by cholera toxin and carbachol.

Cholinergic stimulation triggers the secretion of apically stored, preformed mucin from goblet cells but the pathway of cAMP-stimulated mucin secretion is not known. In this study the effect of cholera toxin on mucin secretion in the human colonic goblet cell line HT-29/B6 was investigated and compared to the action of carbachol. PAS staining of mucin blotted onto nitrocellulose served to quantify the secretion of total mucin. Metabolic labelling was used to evaluate the secretion of newly synthesized mucin. The mucinous nature of the detected material was confirmed with an immunoblot employing a well-characterized polyclonal antibody reacting with MUC2-mucin. Cholera toxin caused a 116-fold increase of intracellular cAMP and strongly stimulated the secretion of both preformed and newly synthesized mucin for more than 20 h. Carbachol only triggered the release of preformed mucin immediately after addition. The secretory response to cholera toxin could be partly inhibited by the protein kinase A inhibitor H8 and the microtubule inhibitor colchicine. The action of carbachol was not affected by these agents. In conclusion, we demonstrate a direct cAMP-dependent effect of cholera toxin on mucin secretion by intestinal goblet cells. In contrast to carbachol, the action of cholera toxin involves de novo synthesis of mucin molecules and microtubule-mediated secretion. There seem to be distinct secretion pathways for muscarinic or cAMP-dependent stimulation of mucin secretion.

Calcium↗
PubMed1997

Carbohydrate recognition on MUC1-expressing targets enhances cytotoxicity of a T cell subpopulation.

The influence of the epithelial mucin MUC1 on T cell-mediated lysis was analysed using lymph node lymphocytes (LNL) from patients with colorectal carcinoma. LNL were stimulated with allogeneic, MUC1-transfected B cells and the bulk cultures were cloned. Alloreactive cytotoxic T cell clones were obtained which preferentially lysed MUC1-expressing targets. The majority was CD4+ and MHC-class II-restricted, and a minor group was CD8+ and MHC-class I-restricted. All the clones expressed CD3 and TCR alpha beta, and were CD56-. The capacity to preferentially kill MUC1-expressing targets was stable in several clones for up to 6 months in culture. The enhancing effect of MUC1 on the lysis was investigated in more detail. It was only seen after inhibition of O-linked glycosylation in the targets. Furthermore, this effect was completely abrogated by the monoclonal antibody 3C9, directed against the Thomsen-Friedenreich antigen (T-antigen, Gal beta 1-3GalNAc bound alpha 1-3 to Ser/Thr) as well as by the soluble disaccharide Gal beta 1-3GalNAc, but not by other similar disaccharides. The authors conclude that in their system the preferential killing of MUC1-expressing targets is due to the recognition of an internal carbohydrate epitope accessible on under-glycosylated MUC1, possibly T-antigen, by an auxiliary receptor molecule on T cells.

Antibodies, Neoplasm↗
PubMed1997

MUC2 gene suppression in human colorectal carcinomas and their metastases: in vitro evidence of the modulatory role of DNA methylation.

The development of the majority of colorectal carcinomas is associated with a diminished expression of the intestinal mucin MUC2 in the tumor cells. The significance and the mechanism of this alteration are not yet known. We sought to determine the molecular basis of this tumor-associated change and to investigate the extent to which it might also relate to metastases. MUC2 gene expression was compared in normal (N), carcinomatous (T), and metastatic tissues (M) from nine patients by immunohistochemistry, in situ hybridization, and Northern blotting. Immunohistochemistry and in situ hybridization showed consistently lower amounts of the expressed protein and mRNA in T and in M than in N; quantitative analysis by Northern blotting confirmed that the differences between MUC2 mRNA expression between N, T, and M were significant, the expression in metastases being less than 5% of that in the normal colonic tissue. The influence of DNA methylation as a possible regulatory mechanism of MUC2 gene expression was tested after the 5' and 3'-regions flanking the first exon of MUC2 were recovered from a genomic DNA library and used as probes in Southern blot. The DNA was isolated from colon carcinoma cell lines expressing MUC2 strongly (LS174T) or moderately (T84) and from that which was nonexpressing (Colo 205), and it was digested with the methylation-sensitive enzyme HpaII. The Southern blot patterns indicated that the increased methylation in the promoter region was concomitant with the decrease of MUC2 mRNA expression. Methylation of the promoter region ligated into a reporter vector suppressed the expression of the luciferase reporter gene in the three investigated cell lines. Furthermore, the expression of MUC2 gene was enhanced by treating the MUC2-expressing colon carcinoma cells with 5-aza-2'-deoxycytidine, a methylation-inhibiting agent. To our knowledge this is the first report to show that: (a) MUC2 gene is strongly suppressed in liver and lymph node metastases of colorectal carcinomas, and (b) suppression of MUC2 gene in colon carcinoma cells in vitro is associated with methylation of the promoter region.

Base Sequence↗
PubMed1996

Molecular characterization of AKAP149, a novel A kinase anchor protein with a KH domain.

The cytosolic cAMP activates in eukaryotic cells several isoforms of cAMP-dependent protein kinase (PKAs) involved in signal transduction. The effects of individual PKA isoforms are determined by their cellular localisation, specified through binding to distinct A Kinase Anchor Proteins (AKAPs). A new member of the AKAP family, a membrane-anchored 903 amino acid long protein, designated AKAP149, is characterized in the present work. It is a putative splicing variant of S-AKAP84 with the important new feature of a RNA-binding motif (KH domain). This domain together with the known characteristics of AKAPs suggests the involvement of AKAP149 in the phosphorylation-dependent regulation of RNA-processing.

A Kinase Anchor Proteins↗
PubMed1996

Low frequency of p53 gene mutation and protein expression in mucinous colorectal carcinomas.

Immunohistochemical data indicate that the frequency of p53 protein overexpression is consistently lower in the mucinous than in the non-mucinous carcinomas of the breast, ovary, pancreas and colon. This peculiar immunohistochemical behavior of the mucinous phenotype could be due to the effect of large amounts of mucus on the staining or to an actual mutation frequency difference between mucinous and non-mucinous carcinomas. This question was investigated on a group of mucinous colorectal carcinomas. DNA was extracted from paraffin sections of 16 human mucinous colorectal carcinomas and the mutation frequency was determined by sequencing of p53 exons amplified in PCR. The expression of p53 protein was determined with the avidin-biotin complex-peroxidase staining procedure and CM-1 antiserum. Twenty-five percent of the tumors, exhibited p53 protein overexpression and in 31% a mutation was detected. Concordance between the two techniques was found in 69% of tumors. Overexpression without mutation was observed in 12% and mutation without overexpression in 19%. G:C --> A:T transitions represented the most frequent lesion (80%), as previously observed in non-mucinous colorectal carcinomas. These data indicate that the mutation pattern in the p53 gene is similar in mucinous and non-mucinous colorectal carcinomas. The low frequency of p53 overexpression in the mucinous phenotype is not due to a mucus effect on the staining but is related to the low mutation frequency of p53 gene. These results lead to the hypothesis that in contrast to the nonmucinous tumors the development of the majority of colonic carcinomas with the mucinous phenotype may be independent from p53 mutations.

Adenocarcinoma, Mucinous↗
PubMed1996

Fucosyltransferase III and sialyl-Le(x) expression correlate in cultured colon carcinoma cells but not in colon carcinoma tissue.

The potential contribution of fucosyltransferases to the overexpression of sialyl-Le(x) antigen was investigated in the colon carcinoma cell line HT-29 and in human colon carcinoma tissue. In HT-29 cells as well as in normal or malignant colonic tissues Fuc-TIII, Fuc-TIV, Fuc-TVI but not Fuc-TV nor Fuc-TVII were detectable after RT-PCR. Sodium butyrate treatment of HT-29 cells increased (to about 200%) and DMSO treatment decreased (to about 20%) the expression of sialyl-Le(x). This modulation of sialyl-Le(x) was concomitant with the analogous increase/decrease of mRNA of Fuc-TIII but not Fuc-TIV. Fuc-TVI was not detectable by Northern blotting in HT-29 cells. In six human colon carcinomas which exhibited strong overexpression of sialyl-Le(x), the expression of Fuc-TIII-mRNA was the same or lower than in the corresponding normal colonic tissue. Thus Fuc-TIII expression may be affecting the expression of the sialyl-Le(x) moiety in HT-29 cells but not in human colon carcinoma tissue.

Antibodies, Monoclonal↗
PubMed1996

Isolation and structural characterization of fucosylated gangliosides with linear poly-N-acetyllactosaminyl chains from human granulocytes.

The isolation and structural characterization of fucosylated neolacto-series gangliosides with linear poly-N-acetyllactosaminyl chains from normal human granulocytes is described. Gangliosides were purified by consecutive use of anion exchange HPLC on Fractogel TMAE-650(S), adsorption and reversed phase HPLC on Nucleosil 50-7 and Nucleosil 7C18 columns, respectively. TLC immunostaining with carbohydrate specific monoclonal antibodies, fast atom bombardment-mass spectrometry (FAB-MS) of the permethylated derivatives and gas chromatography-electron impact mass spectrometry (GC-EIMS) of partially methylated alditol acetates were used for structure elucidations. One ganglioside was identified as sialyl Lewis(x) antigen with nLcOse6Cer core, Neu5-Ac alpha 2-3Gal beta 1-4 (Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc NAc beta 1-3 Gal beta 1-4Glc beta 1-1Cer. Furthermore, monosialylated ceramide deca-, undeca-, dodeca- and tridecasaccharides with three (nLcOse8Cer) and four (nLcOse10Cer) linear lactosaminyl repeats were identified, carring one to three fucoses. The ceramide portions were found to contain C18 sphingosine and predominantly C16:0 fatty acids. All monosialogangliosides were homogenous concerning their terminal alpha 2-3 Neu5Ac-sialylation, but different in their fucosylation status. Beside VI3Neu5Ac, V3Fuc-nLcOse6Cer, in two of the fucosylated polylactosaminyl ganglioside fractions the sialyl Lewis(x) epitope was found, whereas five species expressed the terminal VIM-2 motif. The role of protein linked sialy Lewis(x) epitope of human granulocytes as a ligand for endothelial leukocyte adhesion molecule-1 (ELAM-1; E-selectin) and platelet activation-dependent granule external membrane protein (PADGEM; P-selectin) is well documented. However, the involvement of endothelial cells E-and/or P-selectin mediated cell-cell adhesion via lipid bound sialyl Lewis(x) and/or VIM-2 epitopes on human granulocytes has to be proved in further investigations.

Carbohydrate Sequence↗
PubMed1995

Sialylated O-glycans and L-selectin sequentially mediate myeloid cell rolling in vivo.

Leukocyte rolling precedes firm adhesion and emigration in inflammatory cell recruitment. Both P-selectin, an endothelial lectin that binds to sialylated O-glycans containing sialyl-Lewisx (sLex) on the granulocyte surface, and leukocyte L-selectin have been shown to mediate leukocyte rolling in vivo. Here, we investigate rolling of isolated human neutrophils (PMN), HL-60 promyelocytes, and an L-selectin-transfected cell line (300.19-L) during trauma-induced inflammation in rat mesenteric venules. HL-60 cells, which express no L-selectin but abundant sLex, rolled effectively immediately after abdominal surgery. HL-60 cell rolling was almost completely abolished by pretreatment with sialidase or monoclonal antibody (MoAb) AM-3 recognizing sLex, and was reduced by about 80% by O-sialoglycoprotein-endopeptidase (OSGP). By contrast, 300.19-L cells rolled poorly immediately after surgery but rolled well between 40 and 120 minutes after surgery. Their rolling was completely inhibited by the blocking L-selectin MoAb LAM1-3, but not by a binding control MoAb. PMN express both L-selectin and clustered, sialylated glycoproteins including P-selectin glycoprotein ligand-1 (PSGL-1). PMN showed effective rolling at all times, which was abolished by sialidase or MoAb AM-3 pretreatment during the first 30 minutes after surgery, but not later, when PMN rolling was largely L-selectin-dependent. We conclude that in trauma-induced inflammation, a two-step mechanism accounts for most of myeloid cell rolling, which initially requires O-glycans and subsequently depends on L-selectin function.

Animals↗
PubMed1995

Characterization of the major sialyl-Lex-positive mucins present in colon, colon carcinoma, and sera of patients with colorectal cancer.

The expression of the mucin-bound sialyl-Lewisx epitope is increased in the tissue of most colorectal carcinomas and in the sera of about 30% of tumor patients. In colon cancer, a portion of the sialyl-Lex groups detectable with the monoclonal antibody AM-3 is located on MUC1 (C. Hanski et al., Cancer Res., 53: 4082-4088, 1993). In order to characterize the major colon carcinoma-associated sialyl-Lex-positive glycoprotein components, the tissue- and serum-derived antigens were investigated. The buoyant densities of the sialyl-Lewisx-positive antigens from tumor and normal colonic tissues and from sera of patients with colon carcinoma and healthy donors correspond to that of mucins (1.40 g/ml). The sialyl-Lex-positive mucins purified from both tissues elute under nonreducing conditions in the void volume of a Sepharose CL-2B column, indicating a molecular mass more than 2 x 10(7) daltons. They yield in immunoblot after SDS gel electrophoresis under reducing conditions a main band at an apparent M(r) 880,000. Radioactive labeling revealed that the band at M(r) 880,000 is the major protein component in sialyl-Lewisx-positive mucins both from tumor and normal colonic tissue. In sera of colon carcinoma patients, the sialyl-Lex moiety is also detectable mainly on a M(r) 880,000 glycoprotein band and, additionally, on a M(r) 140,000 molecule as well as on alpha 1-acid glycoprotein. Sera from healthy donors exhibited only a sialyl-Lex-positive glycoprotein with the apparent M(r) 140,000. Sandwich ELISA as well as immunoblots of mucins purified from the colon carcinoma cell line LS174T indicated that the sialyl-Lex moiety migrating in the M(r) 880,000 band is located on MUC2 protein core. Together, these data suggest that sialyl-Lex antigen in colon, colon carcinoma, and the sera of patients with this tumor is located on the MUC2 molecule, consisting of several subunits with an apparent M(r) 880,000, linked via disulfide bridges. The increase of sialyl-Lex expression in colon carcinomas appears to be mainly due to a more frequent transfer of sialyl-Lex moieties onto the mucin core in tumor tissue.

Antibodies, Monoclonal↗
PubMed1995

Is mucinous carcinoma of the colorectum a distinct genetic entity?

Mucinous carcinomas are defined on the basis of the amount of the mucus component in the tumour mass. Apart from this quantitative criterion, a number of clinicopathological parameters (such as localisation, prevalence in different countries and age groups, association with HNPCC and inflammatory processes) and genetic alterations (e.g. frequency of mutation in Ki-ras and p53 genes, level of MUC2 expression) differentiate these tumours from the non-mucinous ones. Since a different set of genetic lesions implies different inducing agents, these observations suggest that there may be a 'mucinous pathway of carcinogenesis'. Further identification of genetic changes characteristic of the mucinous phenotype will help to understand the aetiology of these tumours and possibly establish markers for detection of the high-risk group.

Adenocarcinoma, Mucinous↗
PubMed1994

Identification of two major populations of mucins in respiratory secretions.

Two populations of reduced subunits were present in the mucins purified from pooled normal secretions and asthmatic and chronic bronchitic sputa; their relative level differed between samples. To investigate the nature of this heterogeneity, an asthmatic respiratory mucin preparation from a single individual was reduced and alkylated with 14C-iodoacetamide. This preparation was analyzed by gel filtration, agarose gel electrophoresis, immunoblotting, rate-zonal- and density-gradient centrifugation, and HPLC ion-exchange- and reverse-phase chromatography. Two populations (A and B) of reduced mucin subunits and a high-M(r)protein-rich fraction were identified. Species A has the higher molecular mass, is slowest migrating on agarose electrophoresis, has longer oligosaccharide chains, and expresses the carbohydrate structure sialyl-Le(x). Species B has a lower molecular mass, migrates faster in agarose electrophoresis Species B has a lower molecular mass, migrates faster in agarose electrophoresis, has shorter chains, and does not express sialyl-Le(x). The two subunits have similar but not identical amino acid compositions and 14C-tryptic peptide maps indicating they have different protein cores. The anti-sialyl-Le(x) antibody selectively precipitated subunit A not only from the reduced but also from the nonreduced mucin preparation, demonstrating that subunits A and B are present in different intact mucins.

Amino Acids↗
PubMed1993

Altered glycosylation of the MUC-1 protein core contributes to the colon carcinoma-associated increase of mucin-bound sialyl-Lewis(x) expression.

The mucin carbohydrate epitope sialyl-Le(x), detected with the monoclonal antibody AM-3, is strongly overexpressed in > 90% of human colon carcinomas. We show here that in colon carcinoma one of the mucin cores bearing the sialyl-Le(x) group is MUC-1, whereas sialyl-Le(x) present in normal colon is not detectable on MUC-1. The amounts of MUC-1 core detectable with the monoclonal antibody BC3 in extracts of tumor tissue are 60-180% of those in normal tissue. Two other carbohydrate epitopes located on MUC-1 in mucins from normal and tumor tissue have also been characterized. In contrast to sialyl-Le(x), their expression on MUC-1 is variable and does not correlate with the malignant transformation of colonic mucosa. The transfer of the sialyl-Le(x) group onto the MUC-1 core contributes to the colon carcinoma-associated overexpression of the sialyl-Le(x) epitope.

Antigens↗
PubMed1993

Comparative study of histopathological alterations during intestinal infection of mice with pathogenic and non-pathogenic strains of Yersinia enterocolitica serotype O:8.

Yersinia enterocolitica is an invasive pathogen capable of causing a wide spectrum of gastrointestinal diseases in man. While there is a considerable body of data on the invasiveness of Y. enterocolitica in vitro, little is known about the events in vivo leading to the translocation of the bacteria from the intestinal lumen into the ileal tissue. There is no detailed ultrastructural information describing the course of infection of pathogenic Y. enterocolitica in comparison with an avirulent strain. We compared a virulent plasmid-bearing strain and an isogenic avirulent plasmid-free derivative strain of Y. enterocolitica serotype O:8 at the ultrastructural level, in the established model of murine yersiniosis. At 12 h post-inoculation we found no indications of an active invasion of the intestinal epithelium, although microcolonies of the pathogenic strain were detectable closely under the follicle-associated epithelium of the Peyer's patches. The plasmid-bearing strain of Y. enterocolitica affected the gut-associated lymphoid tissue which was destroyed 36 h post-infection. Unlike the pathogenic strain of Y. enterocolitica, the nonpathogenic plasmid-free strain caused no detectable morphological alterations in the ileal tissue by this time. Morphological evidence is provided that Yersinia does not invade the ileal epithelium in an active manner, as has been observed in vitro, but appears to be transported across the epithelial barrier by M-cells.

Animals↗
PubMed1993

Immunological analysis of the plasmid-encoded proteins from the highly pathogenic Yersinia enterocolitica serogroup O8 and the less pathogenic serogroup O3.

The plasmid-encoded proteins of the pathogenic Yersinia enterocolitica serogroups O3 and O8 were analysed with respect to their immunological relationship. Common epitopes on yersinia outer-membrane proteins (YOPs) and released proteins (RPs) were recognised by orally-induced antisera against living bacteria and by monospecific antisera induced against single RPs of Y. enterocolitica serogroup O8. A major difference between the pathogenic serogroups O3 and O8 was YOP42, which was detected only in the outer membrane of the highly pathogenic O8+ bacteria. The YOP42 may be responsible for the greater virulence of serogroup O8 bacteria.

Animals↗
PubMed1992

Expression of p53 protein in invasive colorectal carcinomas of different histologic types.

BACKGROUND: This study was performed to determine whether morphologic differences of colonic cancer types can be related to different genotypes of these tumors. METHODS: Paraffin sections of 76 human invasive colorectal carcinomas were examined for the overexpression of p53 oncoprotein with the avidin-biotin complex-peroxidase staining procedure and CM-1 antiserum, which detects p53 protein in paraffin-embedded material. The tumors were categorized as mucinous (22 cases), most of which originated from adenomas, and nonmucinous, which were subdivided into carcinomas originating from adenoma-carcinoma sequence (ACS) (29 cases) and de novo (DN) carcinomas (25 cases). RESULTS: Nineteen DN carcinomas (76%), 21 ACS carcinomas (72%), and 8 mucinous carcinomas (36%) exhibited detectable amounts of p53 protein in the tumor cell nuclei. Strong overexpression of p53 protein coincided with a high percentage (> 40%) of stained nuclei in 40% of ACS and 48% of DN carcinomas versus 9% of mucinous tumors. The percentage of stained nuclei, intensity of staining, and distribution of the stained areas did not correlate with the grade of differentiation or the invasive edge of the tumors. Along with nuclear staining of the tumor area, a distinct perinuclear staining of normal epithelial cells adjacent to the tumor was observed in 48% of DN, 7% of ACS, and 9% of mucinous carcinomas. CONCLUSIONS: The current results, in combination with the recently published data on Ki-ras and c-myc alterations, indicate that mucinous carcinomas differ from nonmucinous colorectal carcinomas in their genetic lesions.

Adenoma↗