Tumor necrosis factor induced DNA fragmentation of HL-60 cells

Tumor necrosis factor (TNF) induces differentiation of HL-60 cells, with only slight effects upon proliferation and little or no cytotoxicity. TNF induced cytotoxicity of other target cell lines has been associated with DNA fragmentation. To assess whether TNF-induced DNA fragmentation might also contribute to HL-60 differentiation, studies were performed using a [3H]-dThd release assay. Between 1 and 2 hours of culture, significant [3H]-dThd release was induced by TNF at concentrations of 10 U/ml and greater. This response was blocked by inhibiting energy metabolism, but not by several inhibitors of cell surface signal transduction, protein or RNA synthesis, or free radical scavengers. DNA electrophoresis of the released DNA disclosed a wide range of low molecular weight fragments. It is possible that TNF-induced DNA fragmentation contributes to HL-60 differentiation.     

Electrophoretic analysis of the internucleosome fragmentation of DNA in irradiated thymocytes of rats

Total DNA and DNA of chromatin degradation products obtained from rat thymocytes 6 h after irradiation with a dose of 10 Gy were separated electrophoretically. Relative shares of mononucleosomes and their oligomers were determined. Experimental distributions of DNA fragments differ from those calculated on the basis of the assumption of a random breakage of bonds between the nucleosomes.     

Internucleosomal fragmentation of nuclear DNA in the mucosa of the small intestine and in bone marrow cells of irradiated rats

DNA was isolated from thymus, bone marrow and small intestine epithelium of gamma-irradiated rats and analyzed by electrophoresis in 3% polyacrylamide gel - 0.5% agarose. The internucleosomal chromatin fragmentation was observed in all tissues investigated. In the thymus and bone marrow, DNA fragmentation increased to a maximum by the 6th hour, and in the small intestine, by the 4th hour following irradiation. No low molecular weight fragments were found in the organs under study by the 16th hour after irradiation.     

Epidermal growth factor and tumor promoters prevent DNA fragmentation by different mechanisms

Serum deprivation of C3H 10T 1/2 fibroblasts resulted in DNA fragmentation which was prevented by growth factors such as Epidermal Growth Factor or the tumor promoters, 12-0-tetradecanoyl-13-0-phorbol acetate and Dihydroteleocidin B. Palmityl carnitine, an inhibitor of Ca2+-phospholipid-dependent protein kinase C, reversed the effects of the tumor promoters, but not the effect of Epidermal Growth Factor.     

The action of the tumor necrosis factor (TNF) on the recovery processes of hemopoietic colony-forming cells in irradiated mice

The influence of a tumor-necrotic factor (TNF) on the CFUs population has been studied normally and after irradiation. An inhibitory effect on the pool of the seven-day and doubling of the yield of the eleven-day colonies have been observed in mice received TNF 20 h before bone marrow removal as compared with the controls. The kinetics of restoration of bone marrow cellularity and CFUs number in mouse donors treated with TNF 20 h before irradiation (5.0 Gy) has demonstrated the stimulatory effect of the agent on both indices.     

Signaling mechanisms in tumor necrosis factor alpha-induced death of microvascular endothelial cells of the corpus luteum

The microvasculature of the corpus luteum (CL), which comprises greater than 50% of the total number of cells in the CL, is thought to be the first structure to undergo degeneration via apoptosis during luteolysis. These studies compared the apoptotic potential of various cytokines (tumor necrosis factor α, TNFα; interferon gamma, IFNγ; soluble Fas ligand, sFasL), a FAS activating antibody (FasAb), and the luteolytic hormone prostaglandin F_2α (PGF_2α) on CL-derived endothelial (CLENDO) cells. Neither sFasL, FasAb nor PGF_2α had any effect on CLENDO cell viability. Utilizing morphological and biochemical parameters it was evident that TNFα and IFNγ initiated apoptosis in long-term cultures. However, TNFα was the most potent stimulus for CLENDO cell apoptosis at early time points. Unlike many other studies described in non-reproductive cell types, TNFα induced apoptosis of CLENDO cells occurs in the absence of inhibitors of protein synthesis. TNFα-induced death is typically associated with acute activation of distinct intracellular signaling pathways (e.g. MAPK and sphingomyelin pathways). Treatment with TNFα for 5–30 min activated MAPKs (ERK, p38, and JNK), and increased ceramide accumulation. Ceramide, a product of sphingomyelin hydrolysis, can serve as an upstream activator of members of the MAPK family independently in numerous cell types, and is a well-established pro-apoptotic second messenger. Like TNFα, treatment of CLENDO cells with exogenous ceramide significantly induced endothelial apoptosis. Ceramide also activated the JNK pathway, but had no effect on ERK and p38 MAPKs. Pretreatment of CLENDO cells with glutathione (GSH), an intracellular reducing agent and known inhibitor of reactive oxygen species (ROS) or TNFα-induced apoptosis, significantly attenuated TNFα-induced apoptosis. It is hypothesized that TNFα kills CLENDO cells through elevation of reactive oxygen species, and intracellular signals that promote apoptosis.     

Signalling pathway of tumor necrosis factor in normal and tumor cells

Summary Several aspects of the activity and effects of tumor necrosis factor (TNF) were investigated to gain further insight into its cytotoxic mechanism. The relation between number of TNF receptors and TNF susceptibility of both tumor cells and normal cells was studied, utilizing a specific binding assay. Among the tumor cells, a fairly close correlation (r=0.855) was observed between receptor number and sensitivity to TNF. No cytotoxic effect by TNF was observed on any of the normal cells tested, even though TNF receptors were shown to be present, and cell proliferation was apparently stimulated by TNF in some cases. TNF internalization and intracellular distribution were studied by pulse-labelling and Percoll density gradient centrifugation. In L-M (murine tumorigenic fibroblasts, highly sensitive to TNF cytotoxicity) cells and HEL (human embryonic lung cells, non-sensitive to TNF cytotoxicity) cells, receptor-bound ¹²⁵I-labelled recombinant human TNF was rapidly internalized and delivered to lysosomes within 15–30 min, and this was followed by degradation and release into the culture medium. The presence of either a cytoskeletal disrupting agent or a lysosomotropic agent was observed to inhibit the cytotoxic effect of TNF, thus also indicating that TNF internalization, followed by delivery to lysosomes, is essential in the cytolytic mechanism of TNF.As observed by [³H]uridine incorporation, TNF did not affect RNA synthesis in L-R cells (TNF-resistant cell lines derived from L-M cells) and HEL cells, but markedly stimulated (by 3.5 times) RNA synthesis in L-M cells.     

Genomic instability in human lymphocytes irradiated with individual charged particles: involvement of tumor necrosis factor alpha in irradiated cells but not bystander cells

Exposure to ionizing radiation can increase the risk of cancer, which is often characterized by genomic instability. In environmental exposures to high-LET radiation (e.g. 222Ra), it is unlikely that many cells will be traversed or that any cell will be traversed by more than one alpha particle, resulting in an in vivo bystander situation, potentially involving inflammation. Here primary human lymphocytes were irradiated with precise numbers of 3He2+ ions delivered to defined cell population fractions, to as low as a single cell being traversed, resembling in vivo conditions. Also, we assessed the contribution to genomic instability of the pro-inflammatory cytokine tumor necrosis factor alpha (TNFA). Genomic instability was significantly elevated in irradiated groups (> or = two-fold over controls) and was comparable whether cells were traversed by one or two 3He2+ ions. Interestingly, substantial heterogeneity in genomic instability between experiments was observed when only one cell was traversed. Genomic instability was significantly reduced (60%) in cultures in which all cells were irradiated in the presence of TNFA antibody, but not when fractions were irradiated under the same conditions, suggesting that TNFA may have a role in the initiation of genomic instability in irradiated cells but not bystander cells. These results have implications for low-dose exposure risks and cancer.     

Mechanism of the hypertriglyceridemia induced by tumor necrosis factor administration to rats

4 h after intravenous injection of recombinant HuTNF-alpha to fed rats, an increase in heart, diaphragm, and plasma lipoprotein lipase activity was observed. At the same time, a 40-60% decrease in enzymic activity in epididymal fat pad and kidney and 40% decrease in hepatic lipase activity in liver had occurred. Similar results were obtained 20 h after injection of recombinant HuTNF-alpha into fasted rats. Pretreatment with Indomethacin did not affect the changes in tissue lipoprotein lipase activity observed following recombinant HuTNF-alpha administration. Serum triacylglycerol concentration increased by 2- and 6-fold; 4 and 20 h after recombinant HuTNF-alpha administration. Disappearance of 14C-labeled triacylglycerol from the circulation after injection of small chylomicrons, biosynthetically labeled in their triacylglycerol and cholesterol moieties, was lower in TNF-treated than in control rats. However, the clearance rate of triacylglycerol was the same or even higher in recombinant HuTNF-alpha treated rats (assuming that 14C-labeled chylomicron triacylglycerol represents the serum triacylglycerol pool). The livers of recombinant HuTNF-alpha-treated rats and controls contained similar amounts of 14C-labeled lipids, but less [3H]cholesterol, suggesting that in recombinant HuTNF-alpha-treated rats, the liver took up chylomicron remnant particles enriched with triacylglycerol. Separation of the d less than 1.04 g/ml fraction of serum obtained from control and recombinant HuTNF-alpha treated rats by zonal ultracentrifugation revealed that in recombinant HuTNF-alpha-treated rats the lipoprotein particles were less lipolyzed than in controls. The secretion rate of [3H]triacylglycerol into the serum was determined 90 min after injection of [3H]palmitate albumin complex and Triton WR 1339. In recombinant HuTNF-alpha-treated rats, the secretion of [3H]triacylglycerol into plasma was 48% higher than in controls. It is suggested that the increase in lipoprotein lipase activity of heart and diaphragm resulted from an indirect effect of TNF. It is concluded that the increase in serum triacylglycerol in the recombinant HuTNF-alpha-treated rats is due mainly to an increased secretion of triacylglycerol by the liver. Impaired lipolysis, probably due to a fall in hepatic lipase could also contribute to the rise in plasma triacylglycerol.     

Canarypox virus-induced maturation of dendritic cells is mediated by apoptotic cell death and tumor necrosis factor alpha secretion

Recombinant avipox viruses are being widely evaluated as vaccines. To address how these viruses, which replicate poorly in mammalian cells, might be immunogenic, we studied how canarypox virus (ALVAC) interacts with primate antigen-presenting dendritic cells (DCs). When human and rhesus macaque monocyte-derived DCs were exposed to recombinant ALVAC, immature DCs were most susceptible to infection. However, many of the infected cells underwent apoptotic cell death, and dying infected cells were engulfed by uninfected DCs. Furthermore, a subset of DCs matured in the ALVAC-exposed DC cultures. DC maturation coincided with tumor necrosis factor alpha (TNF-alpha) secretion and was significantly blocked in the presence of anti-TNF-alpha antibodies. Interestingly, inhibition of apoptosis with a caspase 3 inhibitor also reduced some of the maturation induced by exposure to ALVAC. This indicates that both TNF-alpha and the presence of primarily apoptotic cells contributed to DC maturation. Therefore, infection of immature primate DCs with ALVAC results in apoptotic death of infected cells, which can be internalized by noninfected DCs driving DC maturation in the presence of the TNF-alpha secreted concomitantly by exposed cells. This suggests an important mechanism that may influence the immunogenicity of avipox virus vectors.     

Modulation of life and death by the tumor necrosis factor receptor-associated factors (TRAFs)

The TNF receptor-associated factor (TRAF) family is a group of adapter proteins that link a wide variety of cell surface receptors. Including the TNF and IL-1 receptor superfamily to diverse signaling cascades, which lead to the activation of NF-kappaB and mitogen-activated protein kinases. In addition, TRAFs interact with a variety of proteins that regulate receptor-induced cell death or survival. Thus, TRAF-mediated signals may directly induce cell survival or interfere with the death receptor-induced apoptosis.     

Targeting of tumor necrosis factor to tumor cells: secretion by myeloma cells of a genetically engineered antibody-tumor necrosis factor hybrid molecule

The construction, synthesis and secretion of a genetically engineered antibody-cytokine fusion molecule is described. To target tumor necrosis factor (TNF) to tumor cells, recombinant antibody techniques were used to produce a Fab-like antibody-TNF conjugate. At the gene level, the heavy chain gene of an antitransferrin receptor antibody was linked to a synthetic TNF gene encoding human TNF. Transfection of the heavy chain-TNF gene into a myeloma derived cell line which was producing the light chain of the same antibody, allowed the isolation of a cell line secreting a fusion protein of the expected molecular weight and composition. The culture supernatant of the cell line contained TNF cytotoxic activity towards murine L929 cells and human MCF-7 cells. Cytotoxicity towards the human cancer cells was inhibited by an excess of the original antitransferrin receptor antibody, indicating that the antibody-TNF molecules are targeted to the transferrin receptor rich tumor cells. Since the antibody genes used are chimeric (i.e. composed of mouse variable and human constant regions) and since DNA encoding human TNF was used, the hybrid protein is an example of a humanized immunotoxin-like molecule. These results illustrate the possibilities of antibody engineering technology to create and produce improved agents for cancer therapy. Furthermore, they demonstrate for the first time the ability of myeloma cells to secrete an antibody-cytokine chimeric molecule.     

Ceramide generation by two distinct pathways in tumor necrosis factor alpha-induced cell death

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor alpha. When cells were pre-treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near-complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.     

Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis

L929 cells were growth-inhibited after 1 to 2 days of treatment with human recombinant tumor necrosis factor (rTNF). This effect of rTNF was largely reversible, and L929 cells resumed normal growth when rTNF was removed. The rTNF showed growth inhibitory and cytotoxic activity when L929 cells approached a high cell density and grew slowly. This was shown in experiments in which L929 cells approached confluency at different times after being seeded at increasing initial densities. The rTNF had little effect on the growth of cells seeded at the lowest density tested. L929 cells cultured to high density synthesized RNA at a reduced rate. This suggested that a reduced rate of RNA synthesis may be at least in part responsible for the growth inhibitory and cytotoxic activities of rTNF on cells grown to high density. Treatment with inhibitors of RNA synthesis potentiated the cytotoxic activity of rTNF. Inhibition of mRNA synthesis was apparently responsible for the enhanced sensitivity to rTNF, as shown by experiments with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, an inhibitor of the synthesis of poly(A)-containing RNA.     

Caspase 3 inhibition attenuates hydrogen peroxide-induced DNA fragmentation but not cell death in neuronal PC12 cells

Exposure of neurons to H(2)O(2) results in both necrosis and apoptosis. Caspases play a pivotal role in apoptosis, but exactly how they are involved in H(2)O(2)-mediated cell death is unknown. We examined H(2)O(2)-induced toxicity in neuronal PC12 cells and the effects of inducible overexpression of the H(2)O(2)-scavenging enzyme catalase on this process. H(2)O(2) caused cell death in a time- and concentration-dependent manner. Cell death induced by H(2)O(2) was found to be mediated in part through an apoptotic pathway as H(2)O(2)-treated cells exhibited cell shrinkage, nuclear condensation and marked DNA fragmentation. H(2)O(2) also triggered activation of caspase 3. Genetic up-regulation of catalase not only significantly reduced cell death but also suppressed caspase 3 activity and DNA fragmentation. While the caspase 3 inhibitor DEVD inhibited both caspase 3 activity and DNA fragmentation induced by H(2)O(2) it did not prevent cell death. Treatment with the general caspase inhibitor ZVAD, however, resulted in complete attenuation of H(2)O(2)-mediated cellular toxicity. These results suggest that DNA fragmentation induced by H(2)O(2) is attributable to caspase 3 activation and that H(2)O(2) may be critical for signaling leading to apoptosis. However, unlike inducibly increased catalase expression and general caspase inhibition both of which protect cells from cytotoxicity, caspase 3 inhibition alone did not improve cell survival suggesting that prevention of DNA fragmentation is insufficient to prevent H(2)O(2)-mediated cell death.     

Apoptosis of L929 cells induced by tumor necrosis factor

We investigated the mode of TNF-dependent death of L929 murine fibroblasts and the influence of overexpression of bcl-2 family genes on this process. Based on morphological and biochemical data it has been shown that L929 cells died after TNF treatment by apoptosis irrespective of TNF dose and protein synthesis inhibition. Analysis of bcl-2 family gene transfectants revealed a down-regulation of TNF-induced apoptosis by bcl-2 and bclX overexpression, and an up-regulation by bax gene.     

Regulation of death complexes formation in tumor necrosis factor receptor signaling

TNFα stimulation triggers both cell death and survival programs. Since dysregulated apoptosis or cell growth can cause inflammatory diseases, cancer, or autoimmune disorders, it is important to understand the molecular mechanism of controlling cell death and survival by TNFR downstream signaling molecules. In this study, we used normal diploid cells, mouse embryonic fibroblasts (MEFs), to mimic the general TNFα-resistant phenomenon seen under physiological conditions. We elucidated the TNFα-induced death signaling complexes in TNF α-resistant WT MEFs and TNFα-sensitive MEFs that were cFLIP-, RelA-, TRAF2- or RIP1-deficient. Consistent with TNFα-mediated killing, we detected TNFα-induced high molecular weight complexes containing caspase-8 and FADD by gel filtration in the deficient MEFs, especially in those devoid of cFLIP. In addition to the presence of caspase-8-FADD in the TNFα-induced-death complex in the deficient MEFs, we also detected an intermediate protein complex containing RIP1, TRAF2 and caspase-8. Moreover, we demonstrated a correlation between TNFα-sensitivity and death-inducing complex ability in two transformed cell lines, E1A- and Ras- transformed MEFs and PDGF-B-transformed NIH-3T3 cells with PDGF-B signaling inhibited by the tyrosine kinase inhibitor STI571. Taken together, our results suggest the involvement of cFLIP-, RelA-, RIP1-, or TRAF2-related mechanisms for preventing FADD-caspase-8 interaction in wild-type MEFs.     

Nonspecific inhibition of tumor growth in vivo by admixed allogeneic tumor-sensitized lymphoid cells and identical inactivated allogeneic tumor cells.

With the in vivo tumor neutralization test (Winn test), growth of a transplanted (KMT-17) from Wistar-King-Aptekman rats was inhibited by allogeneic tumor (AH-66 from Donryu rats)-sensitized syngeneic lymphoid cells admixed with mitomycin C (MMC)-treated AH-66 cells. The observed tumor inhibition may be immunologically nonspecific, since no cross-antigens were detected by membrane immunofluorescence on the surfaces of KMT-17 and AH-66 cells. Close contact among KMT-17, AH-66-sensitized lymphoid cells and MMC-treated AH-66 cells was required for the inhibition of KMT-17 growth. AH-66 cells pretreated with formalin or ultrasonication lost tumor inhibitory activity when they were admixed with AH-66-sensitized lymphoid cells, and only MMC-treatment effectively preserved the tumor inhibitory activity of AH-66 cells. The sensitized spleen cells, draining lymph node, or peripheral blood cells inhibited tumor growth when they were admixed with MMC-treated AH-66 cells, whereas nucleated cells from bone marrow, thymus, or distal lymph node did not. Growths of KMT-17 were inhibited by admixed sensitized spleen cells and MMC-treated AH-66 even when pre-irradiated rats were used as recipients.