Masahiro Higuchi

Regulation of apoptosis by respiration: cytochrome c release by respiratory substrates

Cytochrome c release from mitochondria is essential for apoptosis. Using human myelogenous leukemia ML‐1a, its respiration‐deficient and reconstituted cells, we demonstrated that respiratory function is essential for tumor necrosis factor‐induced cytochrome c release. In a cell free system using mitochondrial fraction from ML‐1a, initiation of respiration by substrates for complexes I, II, and III but not IV released cytochrome c, suggesting that reduction of coenzyme Q or complex III is essential for cytochrome c release. In the same system, disruption of mitochondrial outer membrane was neither enough nor the cause for cytochrome c release by succinate. These observations define an early pathway in which a change in respiration releases cytochrome c.

Inhibition of ligand binding and antiproliferative effects of tumor necrosis factor and lymphotoxin by soluble forms of recombinant P60 and P80 receptors

Tumor necrosis factor (TNF) is one of the mediators of inflammatory responses. Recently, the cDNA for two distinct receptors of TNF with predicted molecular masses of 60 kDa and 80 kDa, respectively, were isolated. In this report, we compare the inhibitory effects of these two forms of recombinant soluble TNF receptors (extracellular domains) on the ligand binding and on the antiproliferative effects of TNF and lymphotoxin (LT) in a human histiocytic lymphoma cell line (U-937). Our results show that the soluble form of the p60 receptor is approximately 100-fold more potent than the soluble form of the p80 receptor in inhibiting both the antiproliferative effects of TNF as well as in blocking TNF binding to U-937 cells. In contrast, the antiproliferative effects of LT and its binding to cells is inhibited equally by both the p60 and p80 forms of the soluble receptor. Thus, overall our results indicate that the two soluble receptors differ in their ability to inhibit TNF and LT. The impotance of these soluble receptors in blocking the harmful effects of TNF and LT is discussed.

Regulation of the Activation of Nuclear Factor κB by Mitochondrial Respiratory Function: Evidence for the Reactive Oxygen Species-Dependent and -Independent Pathways

Mitochondrial respiratory function regulates the redox status of cells, which, in turn, can control the activation of transcription factors. However, how mitochondria accomplish this modulation is not completely understood. Using the human myelogenous leukemia cells ML-1a, respiration-deficient clone 19 derived from ML-1a, and reconstituted clones, we demonstrated the role of respiratory function in the activation of nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1). Constitutive activation of NF-kappaB and AP-1 was observed in clone 19, but not in ML-1a, and the constitutive activation observed in clone 19 was completely inhibited in reconstituted clones that have functional mitochondria. Additionally, tumor necrosis factor (TNF)-induced activation of NF-kappaB and AP-1 observed in ML-1a was greatly reduced in clone 19. These results indicate that mitochondrial respiratory function regulates TNF-induced and constitutive activation of NF-kappaB and AP-1. We investigated the roles of reactive oxygen species in NF-kappaB activation. Generation of superoxide detected by hydroethidine, but not hydrogen peroxide detected by dehydrorhodamine 123, was transiently increased by TNF in both of the cells. The antioxidant, pyrrolidine dithiocarbamate, reduced TNF-induced, but not the constitutive, NF-kappaB activation. These results indicate that the increase in superoxide generation might be involved in TNF-induced, but not in constitutive, NF-kappaB activation. Our results thus demonstrate the involvement of mitochondrial respiratory function in the activation of reactive oxygen species-dependent and -independent pathways for NF-kappaB activation.

Microtiter plate radioreceptor assay for tumor necrosis factor and its receptors in large numbers of samples

We developed a rapid radioreceptor assay for tumor necrosis factor (TNF) and its receptors by utilizing serum-coated 96-well plates. This assay has an advantage over the traditional tube assay in speed, number of simultaneous samples, and amount of material needed for the assay. As many as 200 samples were assayed in less than 3 h. The usefulness of this assay to determine TNF and its cell surface receptors and to screen for TNF inhibitors in large numbers of biological fluids from patients is demonstrated. This assay may also prove useful for determining the activity of competitive antagonists of TNF.

Characterization of the apoptotic effects of human tumor necrosis factor: development of highly rapid and specific bioassay for human tumor necrosis factor and lymphotoxin using human target cells

Currently available bioassays for most cytokines require several days and therefore must be performed under sterile conditions. In this report we describe a bioassay for tumor necrosis factor (TNF) and lymphotoxin (LT) that is extremely rapid and specific and does not require sterile conditions. Using tritiated thymidine release, we could conveniently monitor degradation of DNA into small fragments following the incubation of human myelogenous leukemia ML-1a cells with TNF. The assay showed that TNF-dependent DNA fragmentation was potentiated by cycloheximide and occurred within 90 min. Treatment of cells to TNF lead to apoptosis as indicated by thymidine release, DNA laddering on agarose gels and morphological alterations. Under these conditions, plasma membrane were not damaged as indicated by lack of chromium release. This effect was linear with TNF concentration. This assay had high throughput, did not require sterile conditions, could be carried out in the absence of serum, and was sensitive only to TNF and LT and not to interferon (IFN)-alpha, IFN-beta, IFN-gamma, transforming growth factor beta, interleukin-4, leukemia inhibitory factor and granulocyte-monocyte colony stimulating factor; all cytokines known to inhibit different cell types. Besides detection of TNF in biological fluids, this assay may prove useful for the identification of novel inhibitors of TNF action.

P80 form of the human tumor necrosis factor receptor is involved in DNA fragmentation.

Two different types of TNF receptors with molecular masses of 60 kDa (p60) and 80 kDa (p80) have been identified. TNF is known to cause DNA fragmentation in certain tumor cell lines but the role of p60 and p80 in this action is not understood. In the present study, we examined the role of these receptors in TNF‐induced DNA fragmentation. Treatment of U‐937 cells with phorbol ester caused downregulation of both types of TNF receptors and this was accompanied by disappearance of the TNF‐induced DNA fragmentation. The removal of phorbol ester led to two time‐dependent events: (1) the rapid regeneration of the p80 form but not the p60 form of the TNF receptor; and (2) the reappearance of TNF‐induced DNA fragmentation. These results suggest that the p80 receptor could mediate the TNF‐induced DNA fragmentation.

Suramin inhibits tumor cell cytotoxicity mediated through natural killer cells, lymphokine-activated killer cells, monocytes, and tumor necrosis factor

Suramin, a polysulfonated naphthylurea, is an antitrypanosomal and antifilarial drug. Because of its anti-reverse transcriptase activity and antiproliferative activity, suramin is also used for the treatment of acquired immunodeficiency syndrome and cancer. In spite of these uses, very little is known about its effects on the immune system. In this report, we investigated the effects of suramin on peripheral blood mononuclear cells. We found that natural killer (NK) cell-mediated cytotoxicity against human erythroblastoid cell line K562 was completely inhibited by suramin in a dose-dependent manner. It also completely blocked lymphokine-activated killer (LAK) cell-mediated cytotoxicity against the human B lymphoblastoid cell lines Raji and Daudi. The cytotoxicity against the human melanoma tumor cell line A-375 mediated by unstimulated and stimulated monocytes was also suppressed by suramin. Maximum inhibition of monocyte-mediated cytotoxicity was observed when suramin was present during both the activation and the effector phases of cytotoxicity. Besides its effects on cell-mediated cytotoxicity, suramin also inhibited the cytotoxic effects of tumor necrosis factor (TNF) against different tumor cell lines. Furthermore, we found that suramin interferes with the binding of TNF with its receptor. Thus our results indicate that suramin overall downregulates the immune system by inhibiting cell-mediated and TNF-mediated cytotoxicity against different tumor cells.