Expression and interaction of different catenins in colorectal carcinoma cells.

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.

Changes of E-cadherin and beta-catenin in human and mouse intestinal tumours.

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.

Different expression of some molecular markers in sporadic cancer of the left and right colon.

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.

Different morphological changes of two colorectal adenocarcinoma cell lines after sodium butyrate treatment.

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.

Changes of E-cadherin and beta-catenin levels during induced differentiation of colorectal carcinoma cells.

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.

Rapamycin-resistant phosphorylation of the initiation factor-4E-binding protein (4E-BP1) in v-SRC-transformed hamster fibroblasts.

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.

Activity of glycogen synthase kinase-3beta is down-regulated during transient differentiation of human colon cancer HT-29 cells.

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.

Are intestinal tumours in Apc+/-mice a suitable model of colorectal carcinoma in man?

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.

Angiotensin II receptors on colorectal carcinoma cells.

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.

Differences of alkaline phosphatase and arginase activities in human colorectal carcinoma cell lines.

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.

Tumour vaccines expressing IL-2, CD80, and IL-2 plus CD80 gene.

Experiments were designed to investigate immunogenicity and therapeutic efficacy of tumour vaccines constructed by transfection of poorly immunogenic murine sarcoma Mc12 with synergistic CD80 and IL-2 genes. Immunization/challenge experiments demonstrated that both, IL-2(+) and IL-2(+) plus CD80(+) live cell vaccines can exert an immunizing stimulus, the IL-2(+) plus CD80(+) vaccine being superior to the IL-2(+) vaccine. Live CD80(+) Mc12 cell vaccine could not be tested, since the vaccine was highly tumorigenic in the doses required for immunization. Preimmunization with IL-2(+) and IL-2(+) plus CD80(+) vaccines induced regressions of a proportion of the parental Mc12 challenge inocula after their temporary growth. Areas of necrosis and extensive infiltration with Mac1(+) and CD4(+) leukocytes have been observed in the regressing sarcomas. When the therapeutic efficacy of the irradiated CD80(+), IL-2(+), and mixed CD80(+) plus IL-2(+) vaccines was compared, it was found that the insertion of the IL-2, but not CD80 gene alone was efficient. The mixed IL-2(+) plus CD80(+) tumour vaccine was able to protect and prolong survival of a higher proportion of mice than the IL-2(+) tumour vaccine.

Changes in c-src activity in a human colon cancer cell line during differentiation.

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.

Epidermal growth factor receptors in Rous sarcoma virus-transformed cells of different hosts.

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.

Activity of PDGF (A) gene in the human cell line derived from a renal carcinoma metastasis.

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.

Stable line of turkey bone marrow cells transformed by the myelocytomatosis virus strain MC31.

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.

Avian bone-derived cell line infected with osteopetrosis virus pts-56.

Cell culture derived from embryonal turkey bone was infected with osteopetrosis virus pts-56. After 13 passages the morphology of the infected cells was changed and later a cell line was established. Some features of this cell line are described and compared with a non-infected cell culture.

Avian nephroblastomas induced by a retrovirus (MAV-2) lacking oncogene. II. Search for common sites of proviral integration in tumour DNA.

To demonstrate the existence of a common site of integration in independent tumour clones, restriction mapping of the vicinity of integrated MAV-2 proviruses in nephroblastoma DNA was performed, using Southern hybridization with an MAV-2 specific probe U3(pAT). The results have shown that (1) nephroblastomas are of semiclonal origin. (2) Nephroblastoma cells contain an average of 5 clonally located integrated proviruses per diploid genome; they do not contain any detectable amount of non-integrated proviruses. (3) In the DNA from independent nephroblastoma clones, there appear at an increased frequency Tth111I fragments of 14.6 and 17.8 kb that hybridize with the U3(pAT) probe. Considering a random selection of integration sites, such coincidence is of little probability. Thus we suppose that these fragments represent a common site(s) of integration with an MAV-2 proviral insert. Two hypotheses concerning possible mechanisms of nephroblastoma induction are discussed: proto-oncogene insertional activation and anti-oncogene insertional inactivation.

A new cell line, GS, derived from a human renal cell carcinoma.

A new cell line, GS, of human renal cell carcinoma was established and characterized. It was derived from a metastasis of a human renal cell carcinoma, which appeared 6 years after nephrectomy. The GS cells exhibit basic characteristics of renal cell carcinoma: epithelial cell character, PAS and glycogen positivity, typical ultrastructural features. The cells have a pseudotriploid stemline with a modal number of 75 chromosomes and two marker chromosomes. GS cells formed neither colonies in soft agar nor transplantable tumours in nude mice but produced a factor(s) stimulating growth and colony forming activity of indicator cells.

Analysis of epidermal growth factor and epidermal growth factor receptor expression in human renal carcinoma cell cultures.

In cells derived from two human renal carcinomas only the precursor form of epidermal growth factor (EGF) was found. The binding assay revealed a high level of EGF receptor expression in both cell types tested. However, these receptors are not involved in the growth activity of the cells under in vitro conditions used. The source of DNA synthesis-stimulating activity found in conditioned media of the cells tested is discussed with respect to possible participation of TGF beta.

Biological activity of proviral DNA isolated from cells infected with MAV-2(0) virus.

The ability of DNA isolated from avian myeloblastosis-associated virus MAV-2(0)-infected fibroblasts to induce the synthesis of virus upon transfection of susceptible but not resistant cells was demonstrated. The virus obtained, when inoculated into 12-day-old embryos, led to the manifestation of osteopetrosis after prolonged incubation and to the induction of nephroblastomas in some cases. Positive transfection with DNA from the non-virus producing myeloblast cell line was not detected on bone marrow cells and fibroblasts preinfected with tdB77-C virus. After transfection with DNA from virus-producing myeloblasts, the reproduction of non-transforming virus was observed. This virus did not induce myeloblastosis upon infection of chickens but induced nephroblastomas on rare occasions.