Eva Pocsik

Interferon‐γ induces cell surface expression for both types of tumor necrosis factor receptors

Interferons are known to potentiate various biological effects of tumor necrosis factor (TNF). Recently, two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80), primarily expressed by epithelial cells and myeloid cells, respectively, have been identified. In the present report, we examined the effect of interferon‐γ (IFN‐γ) on each type of TNF receptor. Our results indicate that IFN‐γ induces TNF receptors on both myeloid (e.g. HL‐60) and epithelial cells (e.g. HeLa). Furthermore, by using antibodies specific to each type of receptor, we demonstrate that both TNF receptors are equally inducible by IFN‐α, IFN‐β and IFN‐γ. Thus, the increase in TNF receptors by interferons may play a role in their synergistic cellular response.

The pattern of cytokine gene expression in human colorectal carcinoma.

Systemic and local cytokine environment may modulate the immunogenicity of colorectal cancer cells, and affect anti-tumor immune functions of tumorinfiltrating lymphocytes. We therefore investigated cytokine mRNA expression patterns in tumors and peripheral blood mononuclear cells (PBMC) from patients with colorectal adenocarcinoma. IL-2, IFN-γ, tumor necrosis factor-α (TNF-α), IL-4, IL-6, IL-8, IL-10 and IL-1β mRNAs in single cell suspension of freshly isolated colorectal cancer tissue were studied by RT-PCR. Frequencies of cytokine gene expression were compared to those in normal colonic mucosa from tumor patients. The frequencies of IL-2, IFN-γ, IL-4 and IL-10 gene expression were also determined in peripheral blood mononuclear cells from patients with colorectal adenocarcinoma and compared to those of healthy individuals. Tumor samples were more frequently positive for IFN-γ, IL-2, TNF-α and IL-10 gene expression than normal mucosa (p=0.0001, p=0.0118, p=0.001 and p<0.0001, respectively). Frequencies of IL-2 and TNF-α gene expressions were significantly higher in tumors with a diameter <5 cm, than in those with a diameter >5 cm. The genes for IL-6, IL-1β and IL-8 were commonly expressed in both tumor tissue and normal colonic mucosa. IFN-γ transcripts were detected in more PBMC samples from patients with colorectal cancer than those from normal controls (p=0.0449). Thus, colorectal cancer tissue is characterized by a specific pattern of cytokine gene expression. It is likely that multiple interactions between pro- and anti-inflammatory cytokines regulate tumor growth and the functional activity of tumor-infiltrating lymphocytes.

Detection of drug-induced apoptosis by flow cytometry after alkaline extraction of ethanol fixed cells

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs.

pp60ν-src kinase overexpression leads to cellular resistance to the antiproliferative effects of tumor necrosis factor

While some tumor cells are sensitive to the antiproliferative effects of tumor necrosis factor (TNF), others are resistant. The molecular basis for cellular resistance to TNF is not completely understood. Previously we have shown that transfection of cells with an oncogene HER2/neu/erb B2, a receptor tyrosine kinase, leads to resistance to the anticellular effects of TNF [(1988) Proc. Natl. Acad. Sci. USA 85, 5102‐5106]. In the present study, we demonstrate that the overexpression of another oncogenic tyrosine kinase, pp60 v‐src also induces resistance to TNF. In contrast to HER2, however, pp60 v‐src transfection of cells did not lead to down‐modulation of TNF receptors but rather to decreased intracellular glutathione levels. The pp60 v‐src ‐induced cellular resistance to TNF could be abrogated by interferon‐γ. Thus, these results indicate that the resistance of certain tumors to TNF may also be due in part to the overexpression of pp60 v‐src oncogene.

Transfection of cells with transforming growth factor‐α leads to cellular resistance to the antiproliferative effects of tumor necrosis factor

Tumor necrosis factor (TNF) is a growth‐modulatory cytokine that inhibits the growth of certain cell lines, stimulates the growth of some, and has no effect on the growth of still others. The molecular basis for this differential regulation of growth by TNF is not understood. We postulate that the growth of normal or tumor cells is determined by the balance between growth‐stimulatory and ‐inhibitory signals. In the present study, we demonstrate that the transfection of cells with the transforming growth factor (TGF)‐α gene induces resistance to TNF. Colon carcinoma cell lines that express elevated levels of TGF‐α were also found to be resistant to this cytokine. Exogenous addition of the growth factor was also effective in decreasing the antiproliferative effects of TNF. Transfection of cells with the TGF‐α gene led to downmodulation of TNF receptors but an increase in intracellular glutathione levels. Thus, these results support our hypothesis that expression of growth factors by certain tumor cells can lead to resistance to antiproliferative agents such as TNF.

Suppression of antiproliferative effects of tumor necrosis factor by transfection of cells with human platelet‐derived growth factor B/c‐sis gene

The growth of cells is determined by the balance between growth‐stimulatory and growth‐inhibitory signals. In the present study, we demonstrate that the transfection of NIH 3T3 cells with a platelet‐derived growth factor (PDGF‐B/c‐sis) gene induces resistance to the anticellular effects of tumor necrosis factor (TNF). Human tumor cell lines that express elevated levels of c‐sis (e.g. epidermoid carcinoma, A‐431) are also TNF resistant, whereas those that express no significant levels of this gene (e.g. breast adenocarcinoma, MCF‐7) are TNF sensitive. Transfection of cells with the c‐sis gene leads to down‐modulation of TNF receptors and also a decrease in intracellular glutathione levels. Thus, our results demonstrate that over‐expression of PDGF‐B/c‐sis by certain tumor cells can lead to their protection from the anticellular effects of TNF.

Detection of Activation Antigens on Chronic Lymphocytic Leukaemia Cells.

The peripheral blood mononuclear cells of patients with chronic lymphocytic leukaemia were characterized by the presence of a variety of cell surface differentiation antigens. The cells of 20 patients were found to be of B-cell phenotype when studied with antibodies directed against CD19, CD20, HLA-DR and sIg. Furthermore, a significant percentage of the cells gave a positive reaction with the monoclonal antibody to CD5. On the other hand, the CLL-cells did not express the CD21 antigen (C3d receptor, EBV receptor). We studied in parallel the presence of various activation antigens using 19 monoclonal antibodies grouped into 7 clusters (CD25, CD30, CD40, CD69, CD70, CD39, CD71). A significantly higher percentage of the CLL cells expressed activation antigens than lymphocytes from healthy controls. The percentage of CD3/HLA + DR + cells, compared to the healthy control lymphocytes was not increased in the CLL patients, and the activated cells in CLL were found to have characteristics of B-cells. Based on these results, we suggest that the CLL cells, like the cells in Hodgkins disease and T-cell lymphoma, are not resting, but activated B-cells or the neoplastic abberrants of activated cells.

Transfection of cells with basic fibroblast growth factor and Kaposi fibroblast growth factor genes induce resistance to and receptor modulation of tumor necrosis factor

Tumor necrosis factor (TNF) has been shown to inhibit the growth of some cell types and stimulate the proliferation of others by a mechanism that is not understood. In the present study, we investigated the effect of transfection of NIH‐3T3 cells with either the basic fibroblast growth factor gene (bFGF) or the kaposi FGF gene (K‐fgf) on the growth‐modulatory effects of TNF. Our results show that transformation of cells with either gene leads to resistance to the growth‐inhibitory effects of TNF. The K‐fgf gene was found to be a more potent inducer of cellular resistance than the bFGF gene. The cellular resistance correlated with the inhibition of TNF‐induced activation of phospholipase A2 and downmodulation of TNF receptors. Overall, our results indicate that both K‐fgf and bFGF play an important role in suppression of antiproliferative effects of TNF.

Temporary Tolerance or Suppressive Regulation Induced By Non MHC Alloantigens in Transplantation and Pregnancy

A special type of tolerance or suppressive regulation will be reported, which can be characterized by the complementary participation of two alloantigen systems; the major histocompatibility complex, and either a minor histocompatibility or certain type of differentiation antigen (secondary, subordinated) alloantigen system of functional importance. There are representative experimental observations on this phenomena, from which one characteristic model, reported by Hutchinson and Morris (Hutchinson and Morris 1987) is explained. Prior to kidney transplantation between RTL incompatible rats recipients were transfused with blood obtained from various strains characterized by either matching or mismatching with the transfusion and organ donor or recipient strain in the minor histocompatibility system. The matching between transfusion and kidney donor and a mismatching between transfusion donor and organ recipient as regards minor histocompatibility alloantigen system has to be emphasized as a new requirement. No similar situation has been reported in human beings. However, the induction of tolerance by transfusion in certain models is a well established phenomena. The importance of class II antigen matching between transfusion donor and recipient were outlined and proved in series of clinical observations based on in vivo and in vitro parameters including cytotoxic antibody production, MLC, cytotoxic precursor cell function and kidney survival (Lagaay et al. 1989, Claas et al 1991, van Rood and Claas 1990, de Waal and van Twuyer 1991).