S. von Kleist

Cloning of the complete gene for carcinoembryonic antigen: analysis of its promoter indicates a region conveying cell type-specific expression.

Carcinoembryonic antigen (CEA) is a widely used tumor marker, especially in the surveillance of colonic cancer patients. Although CEA is also present in some normal tissues, it is apparently expressed at higher levels in tumorous tissues than in corresponding normal tissues. As a first step toward analyzing the regulation of expression of CEA at the transcriptional level, we have isolated and characterized a cosmid clone (cosCEA1), which contains the entire coding region of the CEA gene. A close correlation exists between the exon and deduced immunoglobulin-like domain borders. We have determined a cluster of transcriptional starts for CEA and the closely related nonspecific cross-reacting antigen (NCA) gene and have sequenced their putative promoters. Regions of sequence homology are found as far as approximately 500 nucleotides upstream from the translational starts of these genes, but farther upstream they diverge completely. In both cases we were unable to find classic TATA or CAAT boxes at their expected positions. To characterize the CEA and NCA promoters, we carried out transient transfection assays with promoter-indicator gene constructs in the CEA-producing adenocarcinoma cell line SW403, as well as in nonproducing HeLa cells. A CEA gene promoter construct, containing approximately 400 nucleotides upstream from the translational start, showed nine times higher activity in the SW403 than in the HeLa cell line. This indicates that cis-acting sequences which convey cell type-specific expression of the CEA gene are contained within this region.

Evaluation of a test system for measuring cytokine production in human whole blood cell cultures

A simple and reproducible method is described for the measurement of mitogen-induced cytokine production in cultures of both human peripheral blood mononuclear cells (PBMC) and whole blood. In the culture supernatants the cytokines interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) were determined by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies. Comparing the PBMC cultures with the whole blood system a good correlation was obtained if the cell number was taken into account. In the post-induction supernatants the cytokine values were found to follow typical kinetic curves. The protocol was evaluated by screening 60 cancer patients with primary disease and 60 healthy controls. A markedly reduced secretion of IFN-gamma and IL-1 alpha was found in the cancer patients compared to controls, although leukocyte and lymphocyte counts were almost identical in both groups.

Localization of CEA, HCG, Lysozyme, alpha-1-antitrypsin, and alpha-1-antichymotrypsin in gastric cancer and prognosis

Carcinoembryonic antigen (CEA),β-human chorionic gonadotropin (HCG), alpha-1-antichymotrypsin (ACT), alpha-1-antitrypsin (AAT) and Lysozyme (LYS) were traced by immunoperoxidase staining in gastric carcinomas. The immunohistological results were evaluated in relation to histological types (WHO and Laurén), stage of disease, grade and survival time. CEA was demonstrated in 96% of the tumours, HCG in 34%, ACT in 78%, AAT in 42%, and LYS in 71%. Comparing the staining patterns of the antigens and the intensity of staining some differences were notable. Except for signet-ring cell carcinomas, all of which were intensively positive, CEA expression decreased significantly with loss of differentiation. This observation was not seen with the other marker substances. None of the tested markers was characteristic for one particular histological type, nor could they be correlated with the tumour stage or grade. The marker positivity of CEA, ACT and LYS was not related to survival time. For HCG only, a correlation between tissue expression and a restricted survival time was established. Patients with AAT positive carcinomas had a significantly better survival probability.

Th1 and Th2 Cytokine Response Patterns in Leukocyte Cultures of Patients with Urinary Bladder, Renal Cell and Prostate Carcinomas

As a decreased production of Th1 cytokines by stimulated peripheral blood leukocytes has recently been shown in patients with various carcinomas, the present study was performed to determine whether these patients also exhibit a Th1/Th2 imbalance compared to healthy controls. We measured the production of the Th1 cytokines IL-2 and IFN-γ as well as the Th2 cytokines IL-4, IL-6 and IL-10 in mitogen-stimulated peripheral blood mononuclear cell (PBMC) cultures of patients with urinary bladder carcinomas (n = 47), prostate carcinomas (n = 111) and renal cell carcinomas (n = 67) as compared to 40 age-matched healthy controls. In the PBMC cultures of the tumor patients, the levels of the Th1 cytokines IL-2 and IFN-γ were lower as compared to the controls. For IFN-γ, the differences were highly significant and in the patients with renal cell carcinomas it could be shown that the values decreased with increasing tumor mass. In contrast, the levels of the Th2 cytokines IL-4, IL-6 and IL-10 were comparable in the PBMC cultures of tumor patients and controls. From these results, it is concluded that there is only a malfunction in Th1 cells but no switch from a Th1 type to a Th2 type cytokine profile in the PBMCs of cancer patients.

Augmentation of natural killer cell activity in vitro against tumor cells by wild plants from Jordan.

Thirteen aqueous extracts prepared from Jordanian plants, that are currently used in traditional medicine to treat various types of cancer, were tested in mice for their ability to augment natural killer (NK) cell function in vitro in generating cytotoxicity against YAC tumor targets. Lymphoid cells at a concentration of 5x10(6)/ml were incubated in medium alone or in medium containing different dilutions of either plant extract or purified interferon alpha for 20 h and tested for NK activity. Maximum NK activity (62. 3%) was obtained at 1:50 dilution of Nigella sativum fresh aqueous extract, 48.5% at 1:100 dilution for Allium sativum (and 38.3% at 1:50 dilution for Onopordum acanthium. Fresh aqueous plant extracts appeared to be more potent than old dried aqueous extract or ethanolic extracts. NK augmentation by plant extracts using nylon wool non-adherent spleen cells was slightly higher than the whole spleen cells.

Impaired cytokine production in whole blood cell cultures of patients with urological carcinomas

The production of the cytokines interferon γ (IFN γ), interleukin-1 α (IL-1 α) and tumor necrosis factor α (TNF α) was investigated in the mitogen-stimulated whole blood cell culture media from 51 patients with urinary bladder carcinomas, 52 patients with renal carcinomas, 31 patients with prostatic carcinomas and 360 healthy controls. The cytokines were measured 4 days after induction by a sensitive enzymo-immunological assay. In the blood cell culture supernatants of the patients with urinary bladder carcinomas significancy lower levels of IFN γ (P≤0.001), IL-2 (P≤0.001) and TNFα (P≤0.05) were found as compared to the controls. Blood cells of patients with renal carcinomas had lower production of IFN γ (P≤0.01), IL-2 (P≤0.001) and IL-1α (P≤0.01), whereas the values of the total group of patients with prostatic carcinomas were not significantly different from those of the controls. Lymphocyte and monocyte counts were almost identical in the control and all tumor patient groups. When the patients with renal carcinomas and prostatic carcinomas were analyzed according to their different clinical stages we could show a gradual depression of the IFN-γ levels, which was related to tumor burden.

Protein analysis of NCA-50 shows identity to NCA cDNA deduced sequences and indicates posttranslational modifications

The amino acid sequence, representing 59% of the protein moiety of NCA-50 (nonspecific crossreacting antigen), has been determined. These data confirm that NCA-50 is the product of the mRNA whose corresponding cDNAs were recently isolated from a human lung (HLC-1), as well as from a colon carcinoma cell line (SW 403) cDNA library. The four cysteine residues detected in the NCA-50 molecule form disulfide bonds. The glycosylation of 7 potential N-glycosylation sites which were analysed, showed pronounced differences. There is strong evidence that NCA-50 is bound to a phosphatidyl-inositol glycan, via an amide linkage to ethanolamine at amino acid position 287, which has replaced the last 24 amino acids.

Increased Plasma Concentrations for Type I and II Tumor Necrosis Factor Receptors and IL-2 Receptors in Cancer Patients

Using enzyme-linked immunoabsorbent assays for the soluble tumor necrosis factor (TNF) receptors type I (p55) and type II (p75) and IL-2 receptor we determined their levels in the plasma of 378 patients with various solid carcinomas, 56 patients with benign tumors, and 241 healthy controls. The plasma concentrations of both TNF receptors as well as the IL-2 receptor were significantly higher in the cancer patients than in the healthy controls, independent of the origin or histology of the tumor. The incidence and the extent of the receptor increase correlated with the extent of the disease. In the patients with benign tumors plasma levels of TNF receptor p75 and IL-2 receptor were not significantly different from the controls.

Expression of an NCA cDNA in NIH3T3 cells yields a 110K glycoprotein, which is anchored into the membrane via glycosyl-phosphatidylinositol

The NCA cDNA, which represents a gene belonging to the CEA family, was inserted into an SV40 early promoter-driven expression vector and used for transfection of mouse NIH/3T3 cells. A cell line, NIH/3T3/KNCA IG7, was selected which expressed a molecule with an apparent molecular weight of 110,000. The mode of membrane attachment of this NCA, which we already proposed to be anchored via glycosyl-phosphatidylinositol, was investigated by treatment of NIH/3T3/KNCA IG7 cells with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Two independent methods, flow cytometry and immunoprecipitation of [3H]-labelled surface glycoproteins, clearly demonstrated that the NCA molecule expressed by NIH/3T3/KNCA IG7 cells is indeed anchored into the membrane via glycosyl-phosphatidylinositol. Furthermore, these results support our previous biochemical data on NCA-50, by unequivocally showing that the NCA cDNA used for transfection encodes an NCA molecule related to NCA-50 and NCA-90.

Comparison of the Effects of Immunosuppressive Factors from Newly Established Colon Carcinoma Cell Cultures on Human Lymphocyte Proliferation and Cytokine Secretion

Tumor cells may influence the host’s immune reactivity by the production of immunosuppressive factors (ISFs). In this study, the effects of ISFs derived from nine polyclonal colorectal carcinoma (CRC) cell lines on PHA-induced lymphocyte proliferation and cytokine secretion was investigated. We found that most of the culture supernatants (8/9) from CRC cell lines contained ISFs, which inhibited T cell proliferation to a variable degree in a dose-dependent manner. Comparison of T cell proliferation in the presence or absence of monocytes showed that monocytes can modulate the effects of tumor-derived ISFs on lymphocyte function. In addition, exposure of activated PBMC to the tumor cell supernatants resulted in an altered secretion of cytokines by these cells, i.e. the secretion of IFN-γ was generally reduced while the secretion of IL-1β, IL-2 and TNF-α was little affected. We further investigated the supernatants’ inhibitory effects on PBMC in respect to the production of prostaglandin E2 (PGE2). It was found that PGE2 was secreted by all tumor cell cultures. Therefore this substance is probably involved in the immunosuppression in vivo. However, the secreted PGE2 was shown not to be solely responsible for the observed suppression of lymphocyte proliferation in vitro. Our results suggest that the secretion of ISF is a common property of CRCs as demonstrated with newly established CRC cell cultures, and therefore this may also be an important immune escape mechanism of colonic carcinomas in vivo.