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K Pfizenmaier

Publications and source records attributed to K Pfizenmaier.

At least 91 records · Page 5Linked to original sources
PubMed1989

PMA-responsive 5' flanking sequences of the human TNF gene.

In order to define functional domains involved in the control of TNF-gene transcription, 5'-flanking sequences of the TNF-gene were analysed by TNF-promoter deletion mutants linked to the CAT-gene and by gel retardation assays. To this, three TNF-promoter fragments of different length were fused to the CAT reporter gene and transiently transfected into several human cell lines. We found that a 315 bp long fragment encompassing positions -285 bp to +30 bp with respect to the TNF mRNA cap site is sufficient to function as a PMA inducible promoter/enhancer in all cell lines tested. By further deletion analysis of this clone we could narrow the PMA-inducible element within a 185 bp long sequence (-285 to -110 bp). In addition, we investigated specific interactions between nuclear proteins and TNF promoter sequences using gel-retardation assays. Besides several constitutive binding activities, we could demonstrate in several cell lines a nuclear protein that is induced by PMA and binds to the fragment of the TNF promoter containing the PMA-inducible element.

Cell Line↗
PubMed1988

Tumor necrosis factor and lymphotoxin gene expression in human tumor cell lines.

In this paper we show that eight of 17 tumor cell lines of various tissue origin constitutively express tumor necrosis factor (TNF) mRNA. Five of these eight cell lines concomitantly contained lymphotoxin (LT) mRNA. Of the remaining nine cell lines that lacked detectable TNF or LT gene expression, five could be induced by phorbol ester and/or cytokines to accumulate the respective mRNAs. While TNF mRNA was found not only in neoplastic hematopoietic cells, but also in cell lines derived from carcinomas, LT gene expression seemed to be restricted to lymphoid tumor cells. Tumor cells that expressed LT mRNA also secreted LT protein and proved to be resistant to the cytostatic/cytotoxic effects of their own protein product as well as to exogenous recombinant TNF and recombinant LT. In contrast, most of the cell lines containing TNF mRNA did not release TNF protein into the supernatants, indicating that TNF gene expression may be controlled predominantly at a post-transcriptional level. Thus, the presence of TNF mRNA does not necessarily reflect a TNF-resistant phenotype. Our findings demonstrate that TNF and/or LT mRNA expression is a rather common phenomenon in long-term cultured tumor cell lines and reveal that ectopic TNF and/or LT production by tumor cells may be involved in certain paraneoplastic syndromes as well as in tumorigenesis.

Cell Line↗
PubMed1988

Tumor necrosis factor-alpha affects LTR-controlled oncogene expression in transformed mouse fibroblasts at the post-transcriptional level.

Potential mechanisms of TNF-alpha action on tumor cells have been investigated in a model of mouse fibroblasts transformed by distinct retroviral vectors carrying the v-mos, c-myc, and v-Ha-ras oncogene, respectively. Treatment of v-mos and c-myc transformed cells with murine rTNF-alpha in non-cytotoxic concentrations caused a strong inhibition of both proliferative capacity in monolayer culture and colony formation in soft agar. In contrast, v-Ha-ras transformed cells showed little sensitivity to TNF-alpha treatment. These changes in cell growth characteristics of v-mos and c-myc transformants was preceded by a selective reduction of oncogene-specific steady RNA levels, whereas no RNA down-regulation was observed for v-Ha-ras transformants. An unchanged transcriptional activity of the LTR-controlled v-mos and c-myc genes, but a decreased half-life of oncogene-specific mRNA suggests that TNF-alpha primarily affects stability of v-mos and c-myc RNA without influencing the activity of retroviral promoters. This is confirmed by an unchanged chloramphenicol acetyl transferase activity in TNF-treated LTR-chloramphenicol acetyl transferase transformants. Removal of TNF-alpha from cultures of reverted cells provoked a rapid retransformation, with full recovery of neoplastic growth characteristics 10 days after abrogation of TNF-alpha treatment. These data suggest that in cases of non-cytocidal action of TNF-alpha persistent suppression of tumor cell growth requires continuous TNF-alpha treatment.

Animals↗
PubMed1988

IL-2 production in human T lymphotropic virus I-infected leukemic T lymphocytes analyzed by in situ hybridization.

In situ hybridization studies were performed with 35S-labeled anti-sense RNA probes to study IL-2 mRNA expression in three human T lymphotropic virus I-infected T cell lines at the single cell level. In HuT 102, MT-2, and MT-4 cells, IL-2 mRNA-expressing cells were identified, occurring at frequencies of 2 x 10(-2), 8 x 10(-3), and 5 x 10(-3), respectively. In these cell lines, IL-2 mRNA was not detectable in RNA extracted from whole adult T cell leukemia cell populations because of dilution by other RNA species from the vast majority of cells that do not contain IL-2 mRNA. The data indicate the possibility of paracrine growth stimulation via IL-2 and its receptor even in those human T lymphotropic virus I-infected T cell populations that apparently lack IL-2 activity when analyzed by conventional assay procedures.

Cell Line↗
PubMed1988

High affinity human IFN-gamma-binding capacity is encoded by a single receptor gene located in proximity to c-ros on human chromosome region 6q16 to 6q22.

We have used human-rodent somatic cell hybrids to investigate the regional localization of the IFN-gamma R gene on human chromosome 6 and studied functional and antigenic characteristics of the expressed IFN-gamma R by Scatchard analyses of 125I-IFN-gamma binding and binding of an anti-receptor mAb (A6C5). The data obtained revealed coordinate expression of IFN-gamma- and A6C5-binding capacity as well as competition in binding to chromosome 6-positive hybrids and normal cells, indicating that the A6C5-defined protein is by itself capable of high affinity IFN-gamma binding and, thus, is likely to constitute the major IFN-gamma R protein of distinct cell types. The receptor gene could be allocated to region 6q16 to 6q22, which also contains the c-ros oncogene. Genetic linkage of the IFN-gamma R gene to an oncogene located in a region of non-random chromosomal aberrations may have a causal relationship to the deregulated IFN-gamma R expression in several malignancies.

Antibodies, Monoclonal↗
PubMed1988

Tumor necrosis factor-induced changes of gene expression in U937 cells. Differentiation-dependent plasticity of the responsive state.

TNF-alpha alone or in combination with IFN-gamma differentially affects the proliferation and differentiation of the human leukemic cell line U937 and two derivatives C27 and G3. All three cell lines express similar numbers of functional, high affinity receptors for both TNF-alpha and IFN-gamma. In C27 and G3 cells, TNF-alpha as well as IFN-gamma induced changes in steady state levels of specific mRNA, which appear to be associated with TNF-alpha and IFN-gamma diverse effects on cell growth and differentiation. Constitutive differences in membrane phosphorylation patterns suggest that altered transduction of TNF-alpha signals may account for the differential response of these three cell lines. Several lines of evidence indicate that C27 and G3 cells, when compared with parental U937 cells represent discretely higher stages of monocytic differentiation, suggesting that cellular differentiation may contribute to the development of resistance to the action of TNF-alpha.

Antigens, Surface↗
PubMed1988

Distinct mechanisms of interferon-gamma and tumor necrosis factor-alpha action in oncogene-transformed mouse fibroblasts.

The potential mechanisms of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha action on tumor cells have been investigated in a model of mouse fibroblasts transformed by distinct retroviral vectors carrying the v-mos, c-myc, and v-Ha-ras oncogene, respectively. Treatment with both cytokines not only caused growth inhibition of v-mos- and c-myc-transformants, but also a reversion of transformation-induced suppression of major histocompatibility complex (MHC) class I antigen expression in all transformed cell lines. The phenotypical reversion of transformants was preceded by a selective modulation of LTR-controlled oncogene expression. TNF-alpha primarily affected stability of oncogene-specific RNAs without influencing the activity of retroviral promoters. In contrast, IFN-gamma was effective at the transcriptional level, apparently due to inhibition of LTR activity as revealed from reduced CAT activity in IFN-gamma-treated LTR-CAT transformants. This IFN-gamma-mediated down-regulation of retroviral promoter activity seemed to be selective for Moloney-virus-derived promoters, since the activity of other viral and cellular promoters was not suppressed by IFN-gamma.

Animals↗
PubMed1988

Gamma interferon regulates long terminal repeat-controlled oncogene expression in transformed mouse fibroblasts at the level of mRNA transcription.

In transformed NIH 3T3 cells, murine gamma interferon reduces the expression of the long terminal repeat-controlled oncogenes v-mos, c-myc, and v-Ha-ras by a direct effect on the activity of retroviral promoters, as revealed by analyses of RNA half-life and transcriptional activity of retroviral genes as well as by analyses of chloramphenicol acetyltransferase activity in cells transformed with the cat gene under the control of long terminal repeats.

Animals↗
PubMed1988

Mechanisms of TNF resistance: identification of membrane phosphoproteins associated with a dominant resistant phenotype in lymphoid-myeloid somatic cell hybrids.

Analysis of 53 somatic cell hybrids between TNF-sensitive myeloid cells (U937) and TNF-resistant T-cell lines HUT78 (UH-hybrids) and Jurkat (UJ-hybrids), respectively, revealed complete resistance to TNF-mediated cytostasis in all cases. Moreover, all hybrids remained insensitive to a combined treatment with TNF-alpha and IFN-gamma, which exert synergistic growth inhibition and cytotoxicity on parental U937 cells. Analyses of cell surface marker expression, membrane phosphoproteins, and expression of tissue-specific cytokine genes revealed differential conservation of myeloid and T-cell-specific properties in each of these hybrids, but invariant, dominant resistance to TNF-alpha-mediated growth inhibition. All TNF-resistant hybrids expressed a membrane phosphoprotein pattern, which closely resembled that of the respective parental T-cell lines. In particular, two membrane phosphoproteins of apparent molecular weight of 50,000 and 38,000 were common in the two parental T-cell lines and all UH- and UJ-hybrid clones, suggesting a possible role of these proteins in mediating TNF resistance.

Antigens, Surface↗
PubMed1988

Phase I study of recombinant human tumor necrosis factor-alpha in patients with advanced malignancies.

A clinical phase I trial with recombinant human tumor necrosis factor-alpha (rTNF-alpha) was performed in 30 patients with advanced malignancies. The maximal tolerated dose (MTD) by 3 times weekly intramuscular (i.m.) application was 150 micrograms m-2. Main subjective toxicities including chills, fever, hypotension, fatigue, and anorexia were dose-related. In addition, transient changes in hematologic parameters and lipid metabolism were noted. Two out of 25 evaluated patients showed a minor tumor response after eight weeks of therapy. There was evidence for an improvement of in vivo immuneresponsiveness as revealed from positive delayed type hypersensitivity (DTH) skin tests of 3 out of 6 pretherapeutically anergic patients. We conclude from this phase I trial that rTNF-alpha can be safely administered at doses up to 150 micrograms m-2 i.m., 3 times weekly, without evidence of cumulative toxicity in long-term treatment.

Adult↗
PubMed1987

Downregulation of tumor necrosis factor (TNF) sensitivity via modulation of TNF binding capacity by protein kinase C activators.

The regulatory action of activators for protein kinase C on the specific binding capacity for recombinant human tumor necrosis factor alpha (TNF-alpha) was studied on various human cell lines. Phorbol myristate acetate (PMA) and oleyl acetyl glycerol (OAG) both are able to rapidly downregulate TNF-binding capacity of normal and malignant cells derived from various tissues. As PMA treatment did not enhance internalization of TNF-alpha-receptor complexes at 37 degrees C, and since OAG was able to downregulate TNF-binding capacity under conditions where internalization and shedding of receptor protein are prevented, we conclude that protein kinase C controls ligand affinity of the TNF-receptor protein, possibly via direct phosphorylation. Protein kinase C triggered downregulation of TNF-alpha-binding capacity concomitantly resulted in reduction of TNF-alpha sensitivity, as revealed from decreased cytotoxic action of TNF-alpha on L 929 cells and from inhibition of TNF-alpha-mediated enhancement of HLA class II antigen expression in Colo 205 cells. Restoration of TNF-binding capacity upon abrogation of protein kinase C stimulation leads to full recovery of TNF responsiveness, further supporting the close linkage of TNF-receptor expression and TNF sensitivity. These data suggest that regulation of TNF-binding capacity by protein kinase C is one of the cellular control mechanisms of TNF responsiveness.

Cell Survival↗
PubMed1987

Immunoregulatory activity of recombinant human tumor necrosis factor (TNF)-alpha: induction of TNF receptors on human T cells and TNF-alpha-mediated enhancement of T cell responses.

The expression of specific tumor necrosis factor (TNF) membrane receptors and biological effects of recombinant TNF (rTNF)-alpha on normal human T lymphocytes were studied. Although resting T cells lacked specific binding capacity for rTNF-alpha, high affinity (Kd 70 pM) TNF receptors were de novo induced upon primary activation of T cells. Comparison of TNF receptor expression with that of high affinity interleukin 2 (IL-2) and interferon-gamma (IFN-gamma) receptors, respectively, revealed similarities to IL 2-receptor expression with respect to kinetics of induction. However, maximum expression of TNF receptors (approximately equal to 5000/cell at day 6) and subsequent decline occurred approximately 3 days after the peak of IL 2-receptor expression. In contrast, no change in the expression of IFN-gamma receptors (Kd 10 pM, 300 to 400 receptors/cell) was found in the course of T cell activation. On activated TNF receptor positive T cells, TNF-alpha exerted multiple stimulatory activities. Thus TNF increased the expression of HLA-DR antigens and high affinity IL 2 receptors. As a consequence, TNF-treated T cells showed an enhanced proliferative response to IL 2. Moreover, TNF-alpha was effective as a co-stimulator of IL 2-dependent IFN-gamma production. These data indicate that TNF-alpha may regulate growth and functional activities of normal T cells.

Glycoproteins↗
PubMed1987

Tumor necrosis factor enhances HLA-A,B,C and HLA-DR gene expression in human tumor cells.

Tumor necrosis factor-alpha (TNF-alpha) exerts multiple biologic activities, which include such diverse effects as direct tumoricidal activity and stimulation of metabolic activities as well as expression of cell surface antigens on nonmalignant cells. We report here that in various human tumor cells TNF-alpha upregulates constitutively expressed HLA genes. Further, in a priori HLA-negative tumor cells, TNF-alpha enhances IFN-gamma-induced class I and class II HLA gene expression. In vitro transcription, Northern blot, and immunofluorescence analysis performed with the leukemic cell line K 562 reveal that TNF-alpha reversibly enhances IFN-gamma-induced HLA gene expression at the level of mRNA transcription. In addition, a posttranscriptional regulation of HLA gene expression by TNF-alpha is suggested from enhancement of HLA class I membrane expression in Colo 205 cells without measurable changes in steady state mRNA levels. Therefore, it is conceivable that TNF-alpha, similar to IFN-gamma, might provoke tumor rejection not only via direct tumoricidal action but also via enhancement of HLA antigens and induction of tumor-specific immune responses.

Cell Line↗
PubMed1987

Stable expression of a selectable myeloproliferative sarcoma virus in murine T lymphocyte and monocyte cell lines.

We have investigated whether a retroviral vector based on the myeloproliferative sarcoma virus (MPSV) can be expressed in murine T cells and macrophages. This vector (neoR MPSV) carries the dominant selection marker for neomycin resistance (neoR) and the mos oncogene. The murine T cell line BW5147 and the monocytic cell line P388D1 were either transfected with neoR MPSV DNA or infected with neoR MPSV virus. From both lines, neoR cell clones could be established by retroviral infection, but not by calcium-phosphate precipitation-mediated DNA transfection. The efficiency of infection could be increased 60- to 200-fold upon cocultivation of target cells with irradiated neoR MPSV virus-producing cells. All neoR clones showed neoR MPSV specific sequences as revealed by dot blot and Southern blot analysis. The integration and expression of neoR MPSV was stable over a period of now more than 4 months, even in the absence of selection for neomycin resistance. Northern blot analysis showed that neoR clones express full length neoR MPSV. Further, clones of both T cell and monocyte origin were capable to produce infectious virus particles as revealed by focus formation on fibroblasts and conversion of neomycin sensitive fibroblasts to a neomycin resistant phenotype.

Animals↗
PubMed1987

Tumor necrosis factor inhibits MYC expression in HL-60 cells at the level of mRNA transcription.

Highly purified, recombinant human tumor necrosis factor type alpha (rTNF-alpha) exerts cytostatic activity on the human promyelocytic leukemic cell line HL-60. Between 4 and 10 hr after treatment with rTNF-alpha, HL-60 cells start to exhibit phagocytic activity, indicating the induction of a differentiation process, and show a marked decline in steady-state levels of MYC mRNA. In vitro transcription assays reveal that rTNF-alpha inhibits MYC gene expression at the level of transcription. rTNF-alpha has little or no effect on the half-life of MYC mRNA, indicating that the decreased MYC mRNA level is primarily due to reduced transcription, and the protein synthesis inhibitor cycloheximide does not prevent rTNF-alpha-mediated inhibition of MYC gene expression, suggesting that rTNF-alpha action is independent of de novo protein synthesis. In contrast to MYC, HLA-A, -B, -C mRNA levels are slightly increased in rTNF-alpha-treated HL-60 cells. Recombinant interferon-gamma, which also enhances HLA gene transcription in HL-60 cells, fails to reduce MYC mRNA levels and lacks cytostatic activity. Attenuation of HL-60 cell proliferation occurs as a late response to treatment with rTNF-alpha and thus may be secondary to both MYC inhibition and induction of cell differentiation.

Cell Line↗
PubMed1987

Murine gamma interferon inhibits v-mos-induced fibroblast transformation via down regulation of retroviral gene expression.

Expression of the retroviral vector Neor myeloproliferative sarcoma virus (MPSV), which contains the v-mos oncogene and the neomycin resistance gene, leads to neoplastic transformation of mouse fibroblasts. Murine recombinant gamma interferon (IFN-gamma) could revert the neoplastic properties of established Neor MPSV-transformed cell lines to an apparently untransformed phenotype. In the presence of IFN-gamma, the Neor MPSV transformants showed a greater than 97% reduction of cloning efficiency in soft agar, strongly reduced proliferative capacity, and morphological changes. The IFN-gamma-induced phenotypic reversion was preceded by a rapid and selective reduction of all retroviral RNA species, apparently due to IFN-gamma action on the long terminal repeat of Neor MPSV. The mRNA levels of cellular genes either remained unaffected (beta-actin) or were even enhanced (H-2) in IFN-gamma-treated Neor MPSV-transformed cells. Upon removal of IFN-gamma, retroviral gene expression was fully recovered and a gradual reappearance of the transformed phenotype of these cells within 3 weeks was noted. These data show that IFN-gamma can cause a virtually complete, but reversible, inhibition of v-mos-induced neoplastic properties in transformed fibroblasts by selective down regulation of retroviral RNA levels.

Cell Transformation, Viral↗
PubMed1987

Specific membrane receptors for human interferon-gamma (IFN-gamma).

IFN-gamma is a T-cell derived lymphokine which possesses antitumoral activity for a variety of malignant cells by virtue of its direct effect on cell growth and by its immunomodulatory activity. All IFN-gamma actions are initiated by binding to high affinity cell surface receptors, which are constitutively expressed in virtually all cell lines from various tissues. Although the detailed structure of the IFN-gamma receptor is still elusive, the available data suggest that the high affinity IFN-gamma binding site is a heterodimeric molecule of 128 kDa comprised of two subunits of 53 and 75 kDa, which is invariantly expressed in distinct tumor cells, differing in their response to IFN-gamma. Thus, the capability and type of cellular response to IFN-gamma appears to be largely determined at a post-receptor level. Nevertheless, in sensitive cell lines, the magnitude of response is proportional to the quantity of receptor ligand interactions. This could be important for the definition of effective doses in clinical applications of IFN-gamma, as distinct tumor cells are heterogeneous with respect to quantity of IFN-gamma receptors, with greater 20-fold differences of the number of receptors per cell.

Affinity Labels↗