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Aberrant signalling activities of beta-catenin, originally identified as a component of cell-adhesion complexes, are now considered to be an important factor in colorectal carcinogenesis. However, recently it was shown that also gamma- as well as p120 catenins have a dual role either in cell adhesion or in affecting some gene activation. Therefore, the levels and interactions of these three catenins in human colorectal carcinoma cell lines were analysed. A great heterogeneity in the expression of all catenins tested was found in colorectal carcinoma cell lines HT29 and LS174T. Detailed analysis of beta-catenin interactions was done. GST-APC fragment-fused proteins were used to absorb beta-catenin and its complexes from cell lysates. Similarly, the E-cadherin binding capacity of the residual pool of beta-catenin was analysed using the GST-ECT construct. It was found that the level of beta-catenin does not necessarily depend either on the APC or beta-catenin gene mutations and that co-precipitation of beta-, gamma-, and p120 catenins is not limited to cells that express E-cadherin.

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An immunohistochemical analysis of E-cadherin and beta-catenin was performed in human colorectal cancer as well as in surrounding normal intestinal tissue. We also analysed the expression of these two cell adhesion proteins in transgenic Apc1638N mice as a model of human familial adenometous polyposis syndrome. In the normal intestinal mucosa of both species, E-cadherin and beta-catenin were localized along the lateral plasma membranes of epithelial cells. In intestinal tumour cells, however, they were also present in the cytoplasm. The expression of both proteins was reduced in human and mouse tumours. The pattern of their distribution was frequently heterogenous with strongly positive cells in a mosaic of negative ones. Further, E-cadherin and beta-catenin expression did not correlate to the Duke's staging of tumours and therefore neither can be used as prognostic criteria.

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The expression of cytoplasmic c-erbB2, epidermal growth factor receptor (EGFR), proliferating cell nuclear antigen (PCNA) and dipeptidylpeptidase IV (DPP IV) was significantly higher in sporadic cancer of the right than of the left colon. In addition, cytoplasmic c-erbB2 displayed the same difference in the adjacent (less than 2 cm) and distant (more than 5 cm from the tumour margin) mucosa. The findings cannot be related to Dukes staging. It is suggested that different ontogenic development of the right (from the midgut) and the left (from the hindgut) colon may be a possible explanation. Therefore, data on the expression of different molecular markers in colorectal cancer and surrounding mucosa should always be supplemented by data on tumour location.

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The principal aim of this study was to determine whether the sodium butyrate, cell differentiation-inducing compound, induces identical morphological changes in two colorectal adenocarcinoma cell lines which exhibit the different changes in the alkaline phosphatase activity after treatment with this agent. Ultrastructural analysis showed that these two cell lines possessed different sensitivity to the presence of sodium butyrate. Particularly different changes were observed in the chromatin structure of the cell lines tested. Our study demonstrates that sodium butyrate initiates cell differentiation, modifies the cell components, but the characteristics and extent of this modification depends on the cell line used.

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Following the finding of a great increase in cell-cell adhesion in several colorectal carcinoma cell lines after induced differentiation, the expression of E-cadherin-catenin complexes was analyzed. The sensitivity of cell lines to the differentiation induced by sodium butyrate differed. Nevertheless, all cells growing for 5 days in the medium containing 2 mM sodium butyrate changed their morphology and adherent properties. The expression of E-cadherin and catenins participating in its function were analyzed. A significant increase in E-cadherin level after butyrate treatment was found in HT29 and LS174T cell lines only. However, a high decrease in beta-catenin level was detected in all cell lines treated with butyrate. Further analysis showed regulation of beta-catenin at the level of mRNA.

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Increased phosphorylation of the translational repressor protein 4E-BP1 was found in the cell line derived from the tumor induced in Syrian hamster by Rous sarcoma virus (RSV). This was accompanied by its dissociation from the complex with initiation factor eIF4E. The ribosomal S6 protein kinase p70S6k is supposed to be regulated by the same or a closely related rapamycin-sensitive signalling pathway to that which modulates 4E-BP1. Phosphorylation and activity of p70S6k were found to be also increased in RSV-transformed H19 cells that express significantly higher amounts of the Src protein (p60src) relative to the non-transformed hamster fibroblasts NIL-2. The increased activity and phosphorylation of p70S6k were blocked by rapamycin, indicating that the rapamycin-sensitive pathway is involved in its regulation in v-src-transformed hamster fibroblasts. In agreement with this, rapamycin reduced the expression of elongation factor eEF1alpha (whose translation is regulated by a rapamycin-sensitive mechanism thought to involve p70S6k) and did not affect the production of a housekeeping protein, alpha-tubulin, in these cells. Synthesis of Src protein was also inhibited in cells treated with rapamycin. However, treatment of cells with a concentration of rapamycin sufficient to completely inhibit the activity and phosphorylation of p70S6k resulted in only partial de-phosphorylation of 4E-BP1 and its re-association with eIF4E in the transformed cells, indicating that additional rapamycin-insensitive mechanisms/pathways are implicated in the control of 4E-BP1 phosphorylation in RSV-transformed hamster fibroblasts. Over-expression of eIF4E favours cell proliferation and can lead to a transformed phenotype, while over-expression of 4E-BP1 has the opposite effect. The altered signalling to the phosphorylation of 4E-BP1 in RSV-transformed cells, which leads to its dissociation from eIF4E and thus relief of inhibition of eIF4E function, may therefore represent an important regulatory mechanism in malignant cell growth.

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The increased phosphorylation and activity of protein kinase B (PKB/Akt) was found early upon butyrate treatment of HT-29 cells with a potent differentiating agent, sodium butyrate. It was accompanied by the increased phosphorylation of glycogen synthase kinase-3 (GSK-3) and the inhibition of the activity of GSK-3beta to catalyze phosphorylation of its substrate, translation initiation factor eIF2B. Phosphorylation of PKB and GSK-3 in HT-29 cells was reduced by wortmannin, the inhibitor of phosphatidylinositol-3' kinase (PI3'-kinase), which is upstream activator of PKB and GSK-3 in the intracellular signalling. Modulation of the activity and phosphorylation of these protein kinases during transient in vitro differentiation of HT-29 cells indicates that control of the PI3'-kinase/PKB-dependent signalling pathway may be implicated very early in the changes of malignant phenotype of HT-29 cells.

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In snap frozen sections of the duodenum, jejunum, ileum, the right and left colon of APC+/-mice mucosubstances, activities of brush border glycosidases and proteases, immunoreactivity of sucrase and activities of some enzymes of pericellular proteolysis were studied. Multiple adenomas (tubular or tubulovillous) the numbers of which decreased in the aboral direction occurred in the small intestine. Two tubulovillous adenomas with dysplastic nuclei but with no invasion were found in the right colon. The morphological and histochemical findings resembled those of human colorectal tumours. Activities of brush border enzymes and sucrase immunoreactivity were decreased to various extent or were not present at all. The findings fluctuated even within the same section. Activities of enzymes of pericellular proteolysis were slightly increased in comparison with non affected mucosa. This model is suitable and deserves further studies.

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To test the hypothesis that the block of polyprotein precursor processing and particle formation in RSV-transformed mammalian cells is due to a low level of pr76gag expression, rat tumor cell lines with different amounts of precursor molecules were used. The wild-type forms of pr76gag have been expressed at a high level by use of SV40-based vector and thirty-two stable transfected cell clones were isolated. The gag protein expression was detected in the cell lysate by immunoblotting. Untransfected cells released no proteins that could be detected by immunoprecipitation with anti-RSV serum. Membrane-enclosed gag precursor-polyprotein molecules and infectious virus particles from different stably transfected clones have been found in the medium. Both immature and mature virions of type C morphology were directly detected by transmission electron microscopy. Surprisingly, virus-like particles of morphology similar to mature type C retroviruses were found enclosed within intracellular membranes in a stably transfected nonproducing clone.

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The presence of angiotensin II receptors was found on cells of three colorectal carcinoma cell lines. The binding assays with 125I-labelled angiotensin II and ligands specific for angiotensin AT1 or AT2 receptors showed that angiotensin receptors on colorectal cancer cells are mostly of the AT2 type. The binding capacity of tumor cells was not significantly changed by butyrate-induced differentiation.

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The transport and localization of env-proteins of ts1 virus (a paralytogenic temperature-sensitive mutant of Moloney murine leukemia virus) in infected cells of the TB cell line have been studied at the ultrastructural level. It was found that the envelope precursor polyprotein gPr80-env of ts1 was inefficiently transported out of the endoplasmic reticulum at the restrictive temperature. It was speculated that inefficient transport correlates with inefficient processing of gPr80env into gp70 and Prp15E and leads to paralytic disease.

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Remarkable differences in the levels of alkaline phosphatase and arginase were found in colorectal cell lines tested. In HT-29 cells, which are extremely sensitive to the induction of cell differentiation, very low levels of arginase were detected. On the other hand, high levels of arginase were present in cell lines derived from highly malignant tumours. Both findings support a prognostic significance of arginase activity in colorectal carcinomas.

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Sodium butyrate is a potent agent inducing transient differentiation of colorectal carcinoma cell line HT29. Besides an increase in the level of alkaline phosphatase and morphological changes, this differentiation is followed by a great reduction of kinase activity of the c-src gene product (pp60(c-src)), combined with a sharp decrease in binding of pp60(c-src) to GST-src SH2+SH3 fusion proteins. This finding suggests that the cause of the reduction of pp60(c-src) kinase activity could be an inactive conformation of the pp60(c-src) molecule in sodium butyrate-treated HT29 cells.

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It was found that epidermal growth factor (EGF) binding on cells transformed by Rous sarcoma virus (RSV) differed in mice and hamsters. While EGF binding was considerably reduced in mouse tumours, the binding activity of hamster cells did not change after RSV transformation.

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A high level of c-myc gene expression was found to be a constant feature of v-src transformation. The c-myc gene product was analyzed in quail embryo cells transformed by different mutants of Rous sarcoma virus (RSV) that were temperature-sensitive with respect to various parameters of v-src function. The high-level expression of c-myc proved not to be temperature-sensitive: at both permissive (35 degrees C) and non-permissive (41 degrees C) temperatures, the same high levels of c-myc gene product were found for all RSV mutants tested. This result, in agreement with earlier evidence for a v-src-induced proliferative stimulus which was unaffected by ts mutants at the non-permissive temperature, shows that certain v-src functions have not yet been fully characterized.

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PDGF-like activity was investigated in conditioned media of cell cultures derived from 4 human renal carcinomas. Transient production of PDGF-like factor was found only in the cell line derived from a subcutaneously growing metastasis. Further analysis of this cell line showed an increase of PDGF (A) gene activity in one cellular clone.

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Two methods for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines were compared: the direct culture test and the DNA staining method employing bisBenzimide (Hoechst No. 33258). Contamination by different species of mycoplasma was found in 39% samples tested. It is recommended to use both techniques for a reliable detection of mycoplasma contamination.

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We describe immunofluorescence detection of the vimentin epitope, recognized by monoclonal antibody VI-01, in chromatin structures of eukaryotic cell nuclei and chromosomes. The approach used is based on increased sensitivity of 5-bromodeoxyuridine (BrdU)-substituted DNA to UV irradiation-induced crosslinking of DNA with proteins in vivo, by which the proteins interacting with chromosomal DNA can be immunovisualized in situ.

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Attempts were made to characterize cells of the LSTC-SF2 line by scanning electron microscopy and transmission electron microscopy on the ultrastructural level. The virus-transformed cells are of oval, slightly elongated shape with an undulating surface. The cell nucleus is well outlined, poor in heterochromatin but with a strongly developed nucleolus. The cytoplasm is not rich in organelles except for an abundance of mitochondria with dense granules that are often found in them. With high-resolution autoradiography the DNA synthesis sites were identified mainly in proximity to the nuclear membrane and in the perinuclear spaces. The cells under study can be regarded as immature forms of the blood series and most likely as precursors of cells of the granulocyte or monocyte series.

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A stable bone marrow-derived cell line LSTC-SF2, producing transforming avian acute leukaemia virus, was established. The cell line has been maintained in culture for over 4 years, and 450 passages can be frozen and easily recovered in viable form. The cells grow rapidly in monolayers with multilayer clumps, have low serum requirements, display specific morphology, form colonies in semisolid medium and release transforming MC31 virus. A helper virus was also detected by the reverse transcriptase assay.

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