Regulation of HSP70 on activating macrophages using PDT‐induced apoptotic cells

Although anti‐tumor immunological responses have been mainly associated with necrosis, apoptosis‐associated immune responses have been recently suggested as well. In this study, we investigated anti‐tumor immune responses and regulatory mechanisms of HSP70 using apoptotic cells induced by photodynamic therapy (PDT). The relationships between HSP70 release, HSP70 translocation and macrophage responses were studied using confocal fluorescence microscopy, FACS and ELISA. Macrophages incubated with apoptotic cells as well as necrotic tumor cells showed a high level of TNFα secretion. Apoptotic cells but not the apoptotic cell supernatants induced TNFα secretion. During both necrosis and apoptosis processes, the TNFα production was diminished drastically when HSP70 or TLR‐2 was inhibited. After the PDT treatment, cytoplasmic HSP70 was released from the necrotic cells, while HSP70 rapidly translocated to the surface of the apoptotic cells. Furthermore, the TNFα secretion and the tumor cytotoxicity of splenocytes from mice immunized with apoptotic cells appeared similar to that of splenocytes immunized with necrotic cells. Our in vitro and in vivo results show that apoptosis can potentially have higher impact in inducing immunological responses, hence clarifying the immunological regulatory mechanisms of HSP70 under cell apoptosis and necrosis induced by PDT treatment. These findings could lead to an optimal PDT treatment based on immunological responses. © 2009 UICC     

EFFECTS OF PHYTOLACCA ACINOSA POLYSACCHARIDES I ON CYTOTOXICITY OF MACROPHAGES AND ITS PRODUCTION OF TUMOR NECROSIS FACTOR AND INTERLEUKIN 1

The in vivo effects of Phytolacca acinosa poly-saccharides I (PEP-I) on immunologic cytotoxicity of mouse peritoneal macrophages and its production of tumor necrosis factor (TNF) and interleukin 1 (IL-1) were studied. PEP-I 80 or 160 mg kg was given ip twice every 4 day. Both doses were found to have significant enhancing activity on macrophages cytotoxicity against S180 sarcoma cells and malignant transformed fibroblast L929 cells. Peritoneal activated macrophages were incubated with LPS for 2 and 24 hrs to induce TNF and IL-1, respectively. The TNF and IL-1 activities were tested from cytotoxicity against L929 cells in an absorbence assay of enzymatic reaction and proliferation of thymocytes co-stimulated assay separately. The optimal time for TNF production was found on day 8. Significant increases in TNF and IL-1 were observed. In comparison of the effect of PEP-I on TNF with that of known priming agent BCG, there was no difference between them, but PEP-I had a high effect on IL-1. These results sug     

Recombinant human tumor necrosis factor alpha does not potentiate cell killing after photodynamic therapy with a silicon phthalocyanine in A431 human epidermoid carcinoma cells

Photodynamic therapy (PDT) is a novel cancer treatment utilizing a photosensitizer, visible light and oxygen. PDT with the silicon phthalocyanine Pc 4, a new photosensitizer, is highly effective in cancer cell destruction and tumor ablation. The mechanisms underlying cancer cell killing by PDT are not fully understood. Tumor necrosis factor alpha (TNF) is a multifunctional cytokine that has been implicated in photocytotoxicity. We asked whether recombinant human TNF (rhTNF) affects Pc 4-PDT cytotoxicity in A431 human epidermoid carcinoma cells. Co-treatment of A431 cells with various doses of Pc 4-PDT and a sub-lethal rhTNF dose led to a sub-additive reduction in cell survival. In addition, in the presence of Pc 4-PDT or rhTNF, caspase-3 activity and apoptosis were induced. The combined treatment, however, did not potentiate either caspase-3 activity or apoptosis. Similar to previous findings we observed that Pc 4-PDT initiated a time-dependent extracellular TNF accumulation. The data suggest that: a) PDT and rhTNF induce cancer cell killing through different mechanisms; and b) Pc 4-PDT-induced TNF production is a stress response that may not directly affect photocytotoxicity.     

Celecoxib and NS-398 enhance photodynamic therapy by increasing in vitro apoptosis and decreasing in vivo inflammatory and angiogenic factors

Photodynamic therapy (PDT) elicits both apoptotic and necrotic responses within treated tumors and produces microvascular injury leading to inflammation and hypoxia. PDT also induces expression of angiogenic and survival molecules including vascular endothelial growth factor, cyclooxygenase-2 (COX-2), and matrix metalloproteinases. Adjunctive administration of inhibitors to these molecules improves PDT responsiveness. In the current study, we examined how the combination of PDT and COX-2 inhibitors improve treatment responsiveness. Photofrin-mediated PDT combined with either celecoxib or NS-398 increased cytotoxicity and apoptosis in mouse BA mammary carcinoma cells. Immunoblot analysis of protein extracts from PDT-treated cells also showed poly(ADP-ribose) polymerase cleavage and Bcl-2 degradation, which were further enhanced following combined therapy. Tumor-bearing mice treated with PDT and either celecoxib or NS-398 exhibited significant improvement in long-term tumor-free survival when compared with PDT or COX-2 inhibitor treatments alone. The combined procedures did not increase in vivo tumor-associated apoptosis. Administration of celecoxib or NS-398 attenuated tissue levels of prostaglandin E2 and vascular endothelial growth factor induced by PDT in treated tumors and also decreased the expression of proinflammatory mediators interleukin-1beta and tumor necrosis factor-alpha. Increased tumor levels of the antiinflammatory cytokine, interleukin 10, were also observed following combined treatment. This study documents for the first time that adjunctive use of celecoxib enhances PDT-mediated tumoricidal action in an in vivo tumor model. Our results also show that administration of COX-2 inhibitors enhance in vitro photosensitization by increasing apoptosis and improve in vivo PDT responsiveness by decreasing expression of angiogenic and inflammatory molecules.     

Suppressive effects of intracellular glutathione on hydroxyl radical production induced by tumor necrosis factor

Protective effects of intracellular glutathione (GSH) against the cytotoxicity of human recombinant tumor necrosis factor (TNF) were investigated. Three tumor cell lines (L-M, B-16, HeLa) were used as target cells. Exposure of these cells to buthionine sulfoximine (BSO) or diethyl maleate (DEM) resulted in the depletion of intracellular GSH content to 5.2-43.0% of control values and enhancement of their susceptibility to TNF cytotoxicity. The hydroxyl radical production in L-M cells stimulated by TNF was increased by treatment with BSO or DEM. These results are consistent with the suggestion that intracellular GSH exerts its protective function against the cytocidal effect of TNF by inhibiting the hydroxyl radical production stimulated by TNF.     

Endogenous tumor necrosis factor functions as a resistant factor against hyperthermic cytotoxicity.

One of the mechanisms of cytotoxicity by tumor necrosis factor (TNF) is the induction of reactive oxygen molecules. Cells producing endogenous tumor necrosis factor (enTNF) show resistance to the cytotoxicity of exogenous TNF by scavenging the reactive oxygen molecules. The intracellular hydroxyl radical production is also known to be involved in the heat-induced cytotoxicity. In the present study, we therefore examined the possibility that enTNF may act as a protective protein against the heat-induced cytotoxicity in a manner similar to that of exogenous TNF. Heat-sensitive L-M (mouse tumorigenic fibroblast) cells, originally expressing no enTNF, were transfected with a human TNF expression vector to produce enTNF. The stable transfectants showed apparent resistance to heat treatment. Conversely, when HeLa (human uterine cervical cancer) cells, originally producing an appreciable amount of enTNF, were transfected with an antisense TNF mRNA expression vector to inhibit enTNF synthesis, their heat sensitivity was enhanced. Furthermore, L-M cells which were transfected with nonsecretory human TNF expression vector also acquired resistance to heat treatment. In these cells, heat resistance correlated well with expression of enTNF and intracellular levels of manganous superoxide dismutase. These results indicate that enTNF exerts its intracellular protective effect against the heat-induced cytotoxicity by scavenging reactive oxygen with induced manganous superoxide dismutase in a manner similar to that found in cells treated with exogenous TNF.     

Antitumoral effects of lipopolysaccharides, tumor necrosis factor, interferon and activated macrophages: synergism and tissue distribution

Treatment of C57BL/6 mice bearing Lewis lung carcinoma or of BALB/c mice bearing EMT6 sarcoma with tumor necrosis factor (TNF), lipopolysaccharides (LPS) or interferon caused necrosis of the solid tumors and regression. Toxicity was observed in tumor-bearing animals when TNF or LPS were used at effective antitumoral doses. Similar antitumoral effects could be achieved using less than 1 million macrophages from C57BL/6, lung of from BALB/c peritoneal cavity expanded in vitro, and spontaneously fully activated to cytotoxicity during culture. This effect, observed after transfer twice a week by intravenous or peritumoral route, was not dependent on histocompatibility. Additive effects were observed after combined treatment with activated macrophages and a low dose of LPS or TNF. The biodistribution of labelled LPS and of labelled cytotoxic macrophages was studied in tumor-bearing mice. Although, as expected, LPS was concentrated essentially in the liver, a slow accumulation in the center of the tumor was observed. Macrophages injected intravenously accumulated in the lung and were then redistributed towards liver, kidney and the tumor periphery. Macrophages injected locally remained essentially in the tumor periphery with a slow redistribution in the body. The complementary localization of LPS and of cytotoxic macrophages respectively in the center and periphery of solid tumors might explain their synergism.     

TNF expressed by tumor-associated macrophages, but not microglia, can eliminate glioma

It is well known that tumor necrosis factor (TNF) can have both contrary and pleiotropic effects in anti-tumor immune response. In the present study, we prepared two different tumor cell-based immunotherapy models: MCA38 adenocarcinoma and GL261 glioma intracranial interleukin-2 (IL-2)-based. Each tumor was transfected to express IL-2 with or without expression of the soluble form of tumor necrosis factor receptor type II (sTNFRII). Although mice in which TNF is blocked survive longer than IL-2 alone (35.2 versus 26 days), the reverse was observed for GL261 glioma. The differential effect on tumor growth implies enhanced TNF sensitivity of GL261 compared to MCA38. This notion is supported by the observation that TNF induces apoptosis in GL261 but not MCA38 tumors. We further examined tumor infiltrating CD11b+F4/80+ macrophages (or tumor-associated macrophages: TAM) for TNF production in vivo and found that TAM express cell surface TNF implying a role in eliminating glioma cells mediated by the cell surface form of TNF.     

Prolonged exposure to IL-1beta and IFNgamma induces necrosis of L929 tumor cells via a p38MAPK/NF-kappaB/NO-dependent mechanism

Interleukin-1beta (IL-1beta) is a cytokine that shares with tumor necrosis factor (TNF) the ability to initiate largely similar signaling pathways, leading to proinflammatory gene expression. In contrast to TNF, however, IL-1beta is not believed to induce tumor cell death. Here we demonstrate that prolonged treatment with IL-1beta, in combination with interferon-gamma (IFNgamma), is cytotoxic for L929 tumor cells. IL-1beta/IFNgamma-induced cytotoxicity requires only minimal amounts of IL-1beta and shows morphological features of necrosis. Although TNF induces a similar response, we could exclude a contribution of endogenous TNF production in the effect of IL-1beta/IFNgamma. Cell death in response to IL-1beta/IFNgamma is independent of caspases, but requires the IL-1beta/IFNgamma-induced production of inducible nitric oxide synthase (iNOS) and NO. Moreover, necrosis and iNOS/NO production could be prevented by treatment of the cells with a p38 mitogen activated protein kinase (p38MAPK) or IkappaB kinase beta inhibitor. Altogether, these findings demonstrate that prolonged exposure to IL-1beta plus IFNgamma induces L929 tumor cell necrosis, via a p38MAPK and nuclear factor-kappaB (NF-kappaB)-dependent signaling pathway, leading to the expression of iNOS and the production of toxic NO levels.     

Augmentation by tumor necrosis factor alpha of the systemic therapeutic effect of lymphokine-activated killer cells in adoptive immunotherapy of murine tumor

The therapeutic effect of a combined modality of lymphokine-activated killer (LAK) cells and tumor necrosis factor alpha (TNF alpha) on MBL-2 tumor in C57BL/6 mice was studied. Murine LAK cells induced from splenocytes by interleukin 2 (IL2) could lyse MBL-2 target cells in vitro. but no enhancement of the LAK activity was found by the treatment of LAK cells with TNF alpha in vitro. However, the treatment of MBL-2 with TNF alpha enhanced the sensitivity to LAK cells. Moreover, administration of TNF alpha to mice bearing solid MBL-2 tumor led to increased tumor vascular permeability within 1 h, and resulted in the enhanced accumulation of systemically transferred LAK cells in tumor tissue. Based on these results, we treated MBL-2-bearing mice with TNF alpha and then with LAK cells 1 h later. No therapeutic effect was observed when tumor-bearing mice were treated with TNF alpha alone or LAK cells plus IL2. However, adoptive immunotherapy using LAK cells and TNF alpha had therapeutic effects, i.e., growth inhibition of tumor nodules and prolongation of survival. These results indicated that appropriately timed pretreatment of tumor-bearing mice with TNF alpha augmented the anti-tumor efficacy of LAK cells.     

Synergistic cytotoxic effects of tumor necrosis factor, interferon-gamma and tamoxifen on breast cancer cell lines.

The combined effects of recombinant human tumor necrosis factor (TNF), interferon-gamma (IFN) and tamoxifen (TAM) on the proliferation of human breast cancer cell lines were investigated. In estrogen receptor positive MCF-7 cells, relatively resistant to TAM or TNF, cytotoxicity significantly increased in combinations of TNF and IFN, and of a cytokine and TAM. The cytotoxicity of TNF increased when cells were pretreated with IFN, but not vice versa. Sequential treatment with IFN following TNF and TAM also exhibited significant antiproliferative effect on both cell lines. The combined or sequential cytokines and TAM treatments are possible modalities to overcome breast cancers unresponsive to endocrine treatment.     

Impaired production of tumor necrosis factor in breast cancer

Spontaneous and lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF) by peripheral blood macrophages was investigated in breast cancer. Whereas spontaneous TNF production by macrophages derived from patients with breast cancer was comparable with the one found in healthy controls (P greater than 0.1), LPS-stimulated macrophages derived from patients in the disease-free interval as well as with metastatic breast cancer were found to produce significantly lower amounts of TNF, as compared with macrophages derived from healthy control individuals (P less than 0.0005). However, the production of TNF did not significantly differ between the two patient populations (P greater than 0.05). The impairment of LPS-induced TNF production did not depend upon such characteristics of the primary tumor as size, axillary lymph node and estrogen receptor status, or upon the fact of administration of adjuvant chemotherapy and, in patients with metastatic disease, hormone treatment. To further investigate cytokine production by macrophages, spontaneous and LPS-induced interleukin-1 (IL-1) production was investigated also. However, no difference was found between patients and controls concerning IL-1 generation. The authors thus conclude that LPS-induced TNF production was impaired in breast cancer independent of the presence of detectable metastatic disease, whereas IL-1 production remained unimpaired.     

Production of Tumor Necrosis Factor-α by Alveolar Macrophages of Lung Cancer Patients

The abilities of human alveolar macrophages (AM) obtained from healthy donors and patients with lung cancer to produce tumor necrosis factor (TNF) were compared with those of their blood monocytes after activation with lipopolysaccharide (LPS). TNF activity was assayed by measuring cytotoxicity against actinomycin D-treated L929 cells and TNF was determined quantitatively by sandwich enzyme-linked immnnosorbent assay (ELISA) with polyclonal and monoclonal antibodies against TNF-α. Unstimulated AM from healthy donors released variable amounts of TNF spontaneously, whereas blood monocytes did not. When treated with LPS for 24 h, AM and monocytes produced TNF dose-dependently, but TNF production by AM was significantly more than that by blood monocytes. This TNF activity was inhibited completely by monoclonal anti-TNF-α antibody. Macrophages generated by in vitro maturation of monocytes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) produced more TNF than freshly isolated monocytes. No difference was found in the abilities of AM from healthy donors and patients with lung cancer to produce TNF after activation stimuli. These observations suggest that human AM may be important in in vivo antitumor defense of the lung through TNF-α production.     

Production of tumor necrosis factor-alpha by alveolar macrophages of lung cancer patients

The abilities of human alveolar macrophages (AM) obtained from healthy donors and patients with lung cancer to produce tumor necrosis factor (TNF) were compared with those of their blood monocytes after activation with lipopolysaccharide (LPS). TNF activity was assayed by measuring cytotoxicity against actinomycin D-treated L929 cells and TNF was determined quantitatively by sandwich enzyme-linked immunosorbent assay (ELISA) with polyclonal and monoclonal antibodies against TNF-alpha. Unstimulated AM from healthy donors released variable amounts of TNF spontaneously, whereas blood monocytes did not. When treated with LPS for 24 h, AM and monocytes produced TNF dose-dependently, but TNF production by AM was significantly more than that by blood monocytes. This TNF activity was inhibited completely by monoclonal anti-TNF-alpha antibody. Macrophages generated by in vitro maturation of monocytes induced by granulocyte-macrophage colony-stimulating factor (GM-CSF) produced more TNF than freshly isolated monocytes. No difference was found in the abilities of AM from healthy donors and patients with lung cancer to produce TNF after activation stimuli. These observations suggest that human AM may be important in in vivo antitumor defense of the lung through TNF-alpha production.     

Differential stimulation of macrophages for tumor cytostasis and monokine production.

We have investigated whether antitumor activity could be expressed independently of cytokine production. Resident macrophages treated with interferon-gamma (IFN-gamma), lipopolysaccharide (LPS) plus muramyldipeptide (MDP) expressed a cytostatic activity against P815 tumor cells and released interleukin 6 (IL-6) and nitrite but produced neither IL-1 nor tumor necrosis factor (TNF). Thioglycollate-elicited macrophages required only LPS plus IFN-gamma for cytostatic activity which was expressed concomitantly with the release of high levels of TNF, IL-1 and IL-6, whereas C3H/HeJ macrophages produced low levels of monokines and were not cytostatic. LPS, alone, was sufficient for triggering Concanavalin A-primed macrophages leading to a full cytostatic activity, even in C3H/HeJ macrophages that was expressed, for these latter, in the absence of monokine production. TNF did not appear to play a role either in autocrine stimulation of macrophages or in the cytostatic process because anti-TNF antiserum affected neither the cytostatic activity nor the nitrite production.     

Photodynamic therapy with hypericin in a mouse P388 tumor model: vascular effects determine the efficacy

Hypericin, a polycyclic quinone obtained from plants of the Hypericum genus, exhibits strong photodynamic antitumor effects. In the present study, PDT efficacy of hypericin under different conditions was compared in a P388 mouse tumor model. Plasma and tumor drug measurements and assessment of vascular damage by fluorescein dye exclusion were performed to determine the relative contributions of vascular effects and direct tumor cytotoxicity. Furthermore, the influence of modifying tumor oxygenation on PDT effect was also evaluated. Study of PDT efficacy and tissue distribution revealed that PDT efficacy was more dependent on plasma concentration than tumor drug level. Fluorescein dye exclusion indicated the complete microvascular occlusion in the tumor and surrounding skin immediately after effective PDT treatments, while only a limited vascular occulation was observed after non-effective PDT treatment. It was found that neither tumor hypoxia induced by hydralazine nor increasing tumor oxygenation achieved by nicotinamide could significantly affect the effectiveness of various PDT protocols. These results suggest that tumor vasculature damage might be the primary mechanism of hypericin-mediated PDT effect. The existence of this potent secondary vascular effect is likely to account for the inability of tumor oxygenation modifiers to affect tumor response after PDT with hypericin.     

Sensitivity of resistant human tumor cell lines to tumor necrosis factor and adriamycin used in combination: correlation between down-regulation of tumor necrosis factor-messenger RNA induction and overcoming resistance.

A number of human tumor cell lines of various histological origin were examined for their sensitivity and resistance to tumor necrosis factor-alpha (TNF) and Adriamycin (ADR). Six ovarian lines, and one each of a renal, lung, and B-cell line, were tested for putative mechanisms of resistance to these agents. Cytotoxicity resulting from TNF or ADR showed no overall correlation in these lines. The combination of TNF and ADR produced enhanced cytotoxicity against these tumor lines and furthermore resulted in overcoming the resistance of TNF or ADR alone or in combination. A proposed mechanism of TNF resistance in tumor cells is the endogenous production of TNF mRNA and protein. There was a positive correlation between resistance to TNF and the constitutive production of TNF mRNA and protein. The TNF-resistant lines that did not constitutively produce TNF mRNA and protein and the three TNF-sensitive tumor lines exhibited up-regulation of their TNF mRNA in the presence of TNF or phorbol myristate acetate/ionophore, but did not secrete any detectable protein. Due to the enhanced cytotoxicity seen with the combination of TNF and ADR, the effect of this combination on the level of TNF mRNA was examined. ADR alone reduced the constitutive level of TNF mRNA and in combination with TNF reduced the level of induction produced by TNF. This down-regulation of TNF mRNA by ADR may play a role in the enhanced cytotoxicity seen with the combination of these 2 agents.     

Influences of interleukins 2 and 4 on tumor necrosis factor production by murine mononuclear phagocytes

Administration of recombinant human interleukin 2 (IL-2) to mice gave rise to peritoneal macrophages and blood monocytes that were primed to produce large amounts of tumor necrosis factor (TNF). Macrophages from IL-2-treated athymic mice responded less well than those from euthymic mice. In addition to its in vivo priming effect, IL-2 was able to directly stimulate TNF production in vitro by purified monocytes. Macrophages responded to IL-2 generally less well than monocytes both in vitro and in vivo. In contrast to IL-2, recombinant murine interleukin 4 (IL-4) down-regulated TNF synthesis by macrophages. In vitro pretreatment of macrophages with IL-4 largely abolished their ability to synthesize TNF in response to IL-2 or lipopolysaccharide. Also, administration of IL-4 to mice blocked the ability of IL-2 and lipopolysaccharide to prime macrophages in vivo for TNF production. Overall, these results demonstrate that IL-2 and IL-4 can act antagonistically to regulate TNF production by macrophages. In spite of its down-regulatory action on TNF production, IL-4 was unable to protect mice against the lethal toxic effects of lipopolysaccharide or IL-2.     

Role of tumor necrosis factor and interleukin 1 in gamma-interferon-promoted activation of mouse tumoricidal macrophages

The purpose of this study was to determine if recombinant murine interleukin 1 beta (rMu-IL-1 beta) alone or in combination with recombinant murine gamma-interferon (rMu-IFN-gamma) could activate murine macrophages to be tumoricidal against tumor necrosis factor (TNF)-insensitive target cells and to evaluate the possible role of interleukin 1 (IL-1) in murine macrophage activation by recombinant murine tumor necrosis factor (rMu-TNF) plus rMu-IFN-gamma. rMu-IL-1 beta and rMu-TNF alone or in combination could neither directly lyse the TNF-insensitive P815 mastocytoma nor activate resident peritoneal macrophages to be tumoricidal for this target. A synergistic induction of tumoricidal macrophage activity against P815 occurred, however, when either of these monokines was combined with rMu-IFN-gamma. The tumoricidal activity obtained was transitory, and the level of activity was dependent upon the monokine concentration and the length of induction period. Murine macrophages stimulated under the same conditions used to induce tumoricidal activity with rMu-TNF plus rMu-IFN-gamma or with rMu-IL-1 plus rMu-IFN-gamma were shown to produce low concentrations of IL-1 or TNF, respectively. Thus, a bidirectional cross-induction of the production of the two monokines occurred. The monokine production was also quite transitory, and the time of peak production of the monokines (12 h) was found to precede the time of peak tumoricidal activation (24 h). Using neutralizing antisera specific for rMu-IL-1s and rMu-TNF, the cross-induced production of TNF was shown to be required for macrophage tumoricidal activation by rMu-IL-1 beta alone (TNF-sensitive targets) or in combination with rMu-IFN-gamma (TNF-insensitive targets). There was no evidence, however, that the production of IL-1 was required for macrophage activation by rMu-TNF in combination with rMu-IFN-gamma.     

Endogenous tumor necrosis factor functions as a resistant factor against adriamycin

One of the mechanisms of cytotoxicity by tumor necrosis factor (TNF) and heat is the induction of reactive oxygen molecules. Cells producing endogenous tumor necrosis factor (enTNF) show resistance to the cytotoxicity of exogenous TNF and heat by inducing manganous superoxide dismutase (MnSOD) to scavenge the reactive oxygen molecules. Intracellular hydroxyl radical production is also involved in adriamycin-induced cytotoxicity. In this study, we therefore examined the possibility that enTNF may act as a protective protein against adriamycin-induced cytotoxicity in a manner similar to that in which it protects against exogenous TNF and heat. Adriamycin-sensitive L-M (mouse tumorigenic fibroblast) cells, originally expressing no enTNF, were transfected with an expression vector which directs the synthesis of non-secretory-type human TNF (enTNF). The stable transformants became resistant to adriamycin with increased levels of MnSOD. Conversely, when HeLa (human uterine cervical cancer) cells, which originally produce an appreciable amount of enTNF, were transfected with an anti-sense TNF mRNA expression vector to inhibit enTNF synthesis, their intracellular MnSOD activity was suppressed and adriamycin sensitivity was enhanced. However, no alterations in expression of multidrug-resistant gene products—P-170 glycoprotein, glutathione S-transferase π (GST-π) and the intracellular concentrations of glutathione (GSH)-were observed in these transfectants as compared to their parent cells. These results indicate that enTNF exerts its intracellular protective effect against adriamycin-induced cytotoxicity by the same mechanism as that against exogenous TNF and heat, namely scavenging reactive oxygen with induced MnSOD.