Detection of the DCC gene product in normal and malignant colorectal tissues and its relation to a codon 201 mutation.

Protein expression of the putative tumour-suppressor gene DCC on chromosome 18q was evaluated in a panel of 16 matched colorectal cancer and normal colonic tissue samples together with DCC mRNA expression and allelic deletions (loss of heterozygosity, LOH). Determined by a polymerase chain reaction (PCR)-LOH assay, 12 of the 16 (75%) cases were informative with LOH occurring in 2 of the 12 cases. For DCC mRNA, transcripts could be detected in all analysed normal tissues (eight out of eight) by RT-PCR, whereas 6 of the 15 tumours were negative. DCC protein expression, investigated by immunohistochemistry using the monoclonal antibody 15041 A directed against the intracellular domain, was homogeneously positive in all normal tissue samples. In tumour tissues, no DCC protein was seen in 11 out of 16 samples (69%). For the DCC codon 201, we found a loss of a wild-type codon sequence caused by mutation or LOH in at least 8 out of 15 cases (53%) compared with the corresponding normal tissue. DCC protein expression was undetectable in eight of the nine tumours missing both wild-type codons. Only one of the five tumours with retained DCC protein expression had no detectable wild-type codon 201. In addition, 9 out of 15 normal tissue specimens were mutated in codon 201. In two out of three cases with homozygous wild-type codons in peripheral blood lymphocyte (PBL) DNA, mutations were already observed in the tumour adjacent normal colonic mucosa. We conclude that DCC immunostaining should be introduced in the clinicopathological routine because of its strong correlation with the known prognostic markers 18q LOH and mutation of codon 201.

alpha(1,3)Fucosyltransferase expression in E-selectin-mediated binding of gastrointestinal tumor cells.

E-selectin recognizes the oncofetal antigen sialyl-Lewis X, which is highly expressed in adenocarcinoma. Five alpha(1,3)fucosyltransferases (FT) have been cloned that confer cell-surface expression of sialyl-Lewis X on transfected cells. We show here that 12/18 gastrointestinal-tumor cell lines bind specifically to immobilized E-selectin and that in sialyl-Lewis-X-positive cells binding is inhibited with a monoclonal antibody against sialyl-Lewis X. Using RT-PCR, we determined the expression of the alpha(1,3)fucosyltransferases III, IV, V, VI and VII in gastrointestinal tumor cells. Transcripts of FT IV and FT VII are abundantly expressed in all tested cells. Therefore no single fucosyltransferase could be correlated with the expression of sialyl-Lewis X and the ability of the tumor cells to bind to E-selectin. The data suggest that in gastrointestinal-tumor cells sialyl-Lewis X is necessary but not sufficient for E-selectin binding.

Elevated serum E-selectin in patients with liver metastases of colorectal cancer.

E-selectin, an endothelial cell adhesion molecule, mediates the initial step of leucocyte adhesion to activated vascular endothelium. The soluble isoform of E-selectin promotes angiogenesis in rat cornea. In the present study, we investigated whether leucocyte adhesion and angiogenesis are also involved in tumour progression and metastasis of colorectal cancer. Therefore, we determined the level of circulating soluble E-selectin in serum samples of 38 patients with colorectal cancer; 20 patients with non-metastatic and 18 patients with metastatic disease. Median levels of soluble E-selectin were found to be significantly higher in metastatic tumour disease (88.7 ng/ml, range 25-203 ng/ml) than in healthy controls (34.9 ng/ml, range 15-59 ng/ml, P = 0.01), in patients with primary tumours or with local recurrences (39.5 ng/ml, range 22-100 ng/ml). Furthermore, there was no correlation with the serum level of C-reactive protein, fibrinogen or tumour necrosis factor alpha suggesting that the elevation of E-selectin is independent of inflammation in tumour patients. Therefore, we propose that elevated soluble E-selectin may reflect increased neovascularisation in metastatic tumour tissue.

The interleukin-12 subunit p40 specifically inhibits effects of the interleukin-12 heterodimer.

The recently discovered cytokine interleukin (IL)-12 is a heterodimeric protein of two disulfide-bonded subunits of 35 and 40 kDa. IL-12 has multiple effects on T cells and natural killer (NK) cells. In particular it appears to be a major factor for the development of cellular immunity. So far activity of the single subunits alone has not been described, however their expression is regulated independently. In this report we demonstrate for the first time that the mouse IL-12 subunit p40 (IL-12p40) specifically antagonizes the effects of the IL-12 heterodimer in different assay systems. The proliferation of mouse splenocytes activated by phorbol ester and IL-12 was inhibited by IL-12p40, whereas the proliferation induced by phorbol ester and IL-2 was not affected. Furthermore, the synthesis of interferon (IFN)-gamma by mouse splenocytes activated with IL-2 and IL-12 was suppressed by IL-12p40. Purified mouse splenic CD4+ T cells produced IFN-gamma upon activation with plate-bound anti-CD3 monoclonal antibody which was enhanced more than tenfold in the presence of IL-12. In this system IL-12p40 inhibited only the enhancement caused by IL-12 but not IFN-gamma synthesis of CD4+ T cells stimulated with anti-CD3 alone. Moreover, IL-12p40 inhibited the effects of IL-12 on differentiated T helper type 1 (Th1) cells. IFN-gamma production by Th1 cells induced in a T cell receptor-independent way by macrophages and IL-2 or macrophages and IL-12 was greatly reduced by IL-12p40 providing evidence for the endogenous synthesis of IL-12 in the Th1 cell, macrophage and IL-2 co-cultures. The specificity of inhibition was clearly demonstrated in the homotypic aggregation assay of Th1 cells. Incubation of Th1 cells with either IL-2 and IL-12 or IL-2 and tumor necrosis factor induces LFA-1/ICAM-1-dependent aggregation. Only IL-2 + IL-12 but not IL-2 + tumor necrosis factor-induced aggregation was inhibited in a dose-dependent manner by IL-12p40. Thus, the IL-12 subunit p40 appears to be a specific inhibitor for the IL-12 heterodimer.

The Xenopus B1 factor is closely related to the mammalian activator USF and is implicated in the developmental regulation of TFIIIA gene expression.

The Xenopus laevis TFIIIA promoter contains a motif that has been implicated in promoter activation in late-stage oocytes and contains the sequence (-269) CACGTG (-264). A cDNA encoding a protein (B1) that binds to this element has been cloned from X. laevis and Xenopus borealis ovarian cDNA libraries. We show that this protein is a member of the helix-loop-helix family of regulatory proteins and contains 80% sequence identity with the human adenovirus major late transcription factor (MLTF or USF). A survey of B1 protein expression during oogenesis and embryogenesis revealed both oocyte-specific and somatic cell-specific B1 protein-DNA complexes. Immunological data, RNA blot analysis, and proteolytic clipping band shift assays indicated that these complexes most likely represent altered forms of a single B1 polypeptide. Implications for TFIIIA gene regulation during development are discussed.

A Nuclear Factor Recognizing a Positive Regulatory Upstream Element of the Antirrhinum majus Chalcone Synthase Promoter.

A positive regulatory element directing maximal expression of the Antirrhinum majus chalcone synthase promoter was characterized by protein-DNA-interaction studies and cis deletion analysis. The positive regulatory element consists of a 47 base pair direct repeat between positions -564 and -670 and provides three binding sites for nuclear protein factors from Nicotiana tabacum and Antirrhinum majus. Oligonucleotide competition assays revealed that the same factor(s) interact(s) with all three binding sites. Transient expression of chimeric chalcone synthase-neomycin phosphotransferase II genes in parsley protoplasts demonstrated that both halves of the 47 base pair repeat element are required for its in vivo function. A possible role of redundant binding sites for the positive regulatory function of the 47 base pair repeat element is discussed.

A CACGTG motif of the Antirrhinum majus chalcone synthase promoter is recognized by an evolutionarily conserved nuclear protein.

In the chalcone synthase gene of Antirrhinum majus (snapdragon), 150 base pairs of the 5' flanking region contain cis-acting signals for UV light-induced expression. A nuclear factor, designated CG-1, specifically recognizes a hexameric motif with internal dyad symmetry, CACGTG, located within this light-responsive sequence. Binding of CG-1 is influenced by C-methylation of the CpG dinucleotide in the recognition sequence. CG-1 is a factor found in a variety of dicotyledonous plant species including Nicotiana tabacum, A. majus, Petunia hybrida, Arabidopsis thaliana, and Glycine max. CACGTG motifs contained within trans-acting factor recognition sites in various other plant promoters can interact with CG-1. In addition, the binding site of the human adenovirus major late transcription factor USF can compete for CG-1 binding to the chalcone synthase promoter. This suggests an evolutionary conservation of trans-acting factor recognition sites involved in divergent mechanisms of gene control.

Positive and negative regulation of the gene for transcription factor IIIA in Xenopus laevis oocytes.

Expression of the positively acting 5S gene-specific transcription factor, TFIIIA, is regulated during development, with highest levels of mRNA and protein occurring during oogenesis. By analysis of TFIIIA promoter mutants microinjected into late stage Xenopus oocytes, we have determined DNA sequences required for the transcription of this gene and we have identified proteins that bind to these regulatory sequences. A negative element lies between positions -306 and -289. Three positive-acting sequences are located between positions -289 and -253, -250 and -173, and -144 and -101. Gel shift analyses of TFIIIA promoter fragments incubated with Xenopus oocyte extracts have identified two DNA-protein complexes. One complex, designated B1, requires sequences within the promoter region extending from -271 to -253 while the second complex, designated B2, involves promoter sequences from -235 to -221. The protein involved in formation of the B1 complex has been found to be related to the human adenovirus major late transcription factor, USF.

Immunochemical detection of the Thomsen-Friedenreich antigen (T-antigen) on platelet plasma membranes.

Platelet plasma membranes were found to possess the disaccharide beta-D-galactosyl (1-3)-N-acetyl-D-galactosamine which was measured by gas chromatography after release by alkaline borohyride treatment and desialylation. Immunological evidence using the specific lectins from Arachis hypogoea and Agaricus bisporus and an anti-T serum confirmed the presence of this disaccharide, the immunodominant group of the Thomsen-Friedenreich antigen (T-antigen). This receptor was only found after prior neuraminidase treatment indicating that it is normally a cryptic antigen, i.e. masked by sialic acid in the native membrane. Evidence for a second receptor with terminal N-acetylgalactosamine was obtained using the lectin from Helix pomatia. The binding of myxovirus and the lectins from Phaseolus vulgaris (PHA) and Canavalia ensiformis (Con A) to platelet membrane was also demonstrated. The implication of the T-antigen in elimination of the platelets and its role in the haemolytic-uraemic syndrome is discussed.

Metabolic properties of human platelet membranes. II. Thrombin-induced phosphorylation of membrane lipids and demonstration of phosphorylating enzymes in the platelet membrane.

Washed human platelets have been labeled with either 32Pi or glycerol-1-14C and the distribution of the label in the phospholipids determined. 32Pi was introduced primarily into polyphosphoinositides, i.e. di- and triphosphoinositide, whereas the label from glycerol which indicates de novo synthesis of lipid molecules did not appear in these phospholipids. In the course of thrombin-induced aggregation and release the phosphate incorporation into phosphatic acid, di- and triphosphoinositide was rapidly stimulated in parallel to the platelet reaction. The incorporation of glycerol did not change under the same conditions. It is concluded that phosphoinositides with rapid incorporation of phosphate groups are not as rapidly synthesized de novo and presumably form a separate phospholipid pool in the platelets. Only the phosphorylating reactions are stimulated by the thrombin aggregation. The necessary enzymes for these reactions, namely diglyceride kinase, phosphatidylinositol kinase, and phosphatidylinositol-phosphate kinase all can be shown to be associated with a well characterized platelet membrane fraction.