Naofumi Yamauchi

Induction of Heat Shock Protein 47 Synthesis by TGF-β and IL-1β Via Enhancement of the Heat Shock Element Binding Activity of Heat Shock Transcription Factor 1

With most immunological reactions, tissue fibrosis, collagen overproduction caused by immune cytokines, is inevitably associated. Among the various immune cytokines, heat shock protein 47 (HSP47) is a procollagen-specific molecular chaperon and is essential for secretion of procollagen from cells. Induction of HSP47 by TGF-β has been previously reported in rat skeletal myoblasts and mouse osteoblasts, but not in human diploid fibroblasts. As for IL-1β, its effect on HSP47 has not been elucidated. In the present study, using human embryonic lung fibroblast cells, we first disclosed that both TGF-β and IL-1β induced HSP47 synthesis. We then revealed that the binding of the heat shock element (HSE) by heat shock transcription factor 1 (HSF1) was enhanced by both cytokines. We further demonstrated that trimer formation of HSF1, which is essential for its binding to HSE, was induced by these cytokines. The enhancement of HSP47 synthesis and their trimer formation of HSF1 were augmented by using a combination of both cytokines. Collectively, TGF- β and IL-1β were found to induce trimer formation of HSF1 which in turn bound to HSE of HSP47, resulting in the enhancement of HSP47 expression. Thus, HSP47 could well be a good candidate for molecular targeting in controlling tissue fibrosis, given that both principal fibrinogenetic cytokines (TGF-β, IL-1β) are commonly involved in its induction through HSF1 trimerization.

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate

To target disseminated tumors in vivo, transgenes [ β‐galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV‐TK)] were conjugated to transferrin (Tf) by a biotin‐streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf‐R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf‐β ‐galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf‐R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK‐1) whereas in normal diploid cells (HEL), which express low levels of Tf‐R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf‐HSV‐TK gene conjugate for massively metastasized k562 tumors in severe combined immunedeficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.—Sato, Y., Yamauchi, N., Takahashi, M., Sasaki, K., Fukaura, F., Neda, H., Fujii, S., Hirayama, M., Itoh, Y., Koshita, Y., Kogawa, K., Kato, J., Sakamaki, S., Niitsu, Y. In vivo gene delivery to tumor cells by transferrin‐streptavidin‐DNA conjugate. FASEB J. 14, 2108–2118 (2000)

Serum tumor necrosis factor activity in inflammatory bowel disease

Serum tumor necrosis factor (TNF) levels in 33 patients with inflammatory bowel disease (IBD) were measured by using a sensitive enzyme immunoassay. Four of five Crohns diseases (CD) and nine of twenty eight ulcerative colitis (UC) had elevated levels of serum TNF. In active CD or UC, a greater fraction of patients studied had significantly increased serum TNF levels (3/3 for CD and 8/11 for UC). Production of TNF by peripheral blood monocytes when stimulated by lipopolysaccharide was also increased in these patients and correlated with their serum TNF levels. These results suggest that TNF may have some pathoetiological meaning in IBD.

Treatment of murine collagen-induced arthritis by ex vivo extracellular superoxide dismutase gene transfer

UNLABELLED OBJECTIVE; Superoxide dismutase (SOD) is a potent antiinflammatory enzyme that has received growing attention for its therapeutic potential. This study was undertaken to examine the efficacy of extracellular SOD (EC-SOD) gene therapy in murine collagen-induced arthritis. METHODS Embryonic DBA/1 mouse fibroblasts were infected with a recombinant retrovirus expressing human EC-SOD. DBA/1 mice that had been treated with type II collagen were administered subcutaneous injections of 2 x 10(7) EC-SOD-expressing fibroblasts on day 29, when symptoms of arthritis were already present. The severity of arthritis in individual mice was evaluated in a double-blind manner; each paw was assigned a separate clinical score, and hind paw thickness was measured with a caliper. Mice were killed on day 50 for histologic examination of the joints. RESULTS High serum concentrations of EC-SOD were maintained for at least 7 days. Mice treated with the transgene exhibited significant suppression of clinical symptoms such as disabling joint swelling, deformity, and hind paw thickness, compared with the untreated group (mean +/- SD maximum clinical score in the untreated and the transgene-treated groups 2.71 +/- 1.08 and 1.35 +/- 1.22, respectively; P < 0.01, and hind paw thickness 3.04 +/- 0.18 mm and 2.56 +/- 0.12 mm, respectively; P < 0.05). Histologic abnormalities, including destruction of cartilage and bone, infiltration of mononuclear cells, and proliferation of synovial cells, were also markedly improved in the EC-SOD-treated mice compared with the control group (histopathologic score 7.50 +/- 1.13 and 4.13 +/- 1.88 in the untreated and transgene-treated groups, respectively; P < 0.05). CONCLUSION These results indicate that EC-SOD gene transfer may be an effective form of therapy for rheumatoid arthritis.

Mechanism of Synergistic Cytotoxic Effect between Tumor Necrosis Factor and Hyperthermia

We previously reported that recombinant human tumor necrosis factor (rhTNF) and hyperthermia had a synergistic effect against tumors, in vitro and in vivo. We have now investigated the mechanism of this synergy by measuring the lysosomal enzyme activity and hydroxyl radical production of L‐M cells treated with rhTNF and/or hyperthermia. A synergistic activation of lysosomal enzyme and the induction of hydroxyl radical production in L‐M cells treated with both rhTNF and hyperthermia was observed. A synergistic cytotoxic effect was observed when rhTNF and hyperthermia were combined, and was inhibited by the addition of a reactive oxygen scavenger, dimethyl sulfoxide or bipyridine. The results show that the augmenting effect of hyperthermia on lysosomal enzyme activation and induction of hydroxyl radical production by rhTNF plays an important role in the synergistic cytotoxic effect.

Cytotocidal Mechanism of TNF: Effects of Lysosomal Enzyme and Hydroxyl Radical Inhibitors on Cytotoxicity

The participation of lysosomal enzymes, hydroxyl radicals, and mitochondrial respiration in the cytocidal effect of TNF on tumor cells was investigated. The cytotoxicity of TNF on L-M cells was clearly reduced by lysosomotropic agents, DMSO (hydroxyl radical scavenger), NDGA (lipoxygenase inhibitor), and sodium azide (mitochondrial respiration inhibitor). The results suggest that lysosomal enzyme and hydroxyl radicals play an important triggering role in the destruction of tumor cells by TNF, and that the process of destruction might require ATP.

Synergistic Cytotoxicity of Recombinant Human TNF and Various Anti-Cancer Drugs

A synergistic increase in the cytotoxic effects of recombinant human tumor necrosis factor (rH-TNF) and anti-cancer drugs was demonstrated in vitro. The cytotoxicity of rH-TNF against L-M cells in combination with Mitomycin C (MMC), Adriamycin (ADM), Cytosine arabinoside (Ara-C), Actinomycin D (ACD), Daunomycin (DM), Cisplatin (CDDP), Vincristine (VCR), and 5-Fluorouracil (5FU), based on the concentration necessary for 50% inhibition of cell growth (IC50), was 4 to 347 times as high as that of rH-TNF alone. The results suggest that combination therapy including rH-TNF and anti-cancer drugs may be of value in the treatment of malignancy in human patients.

Endogenous Tumor Necrosis Factor Inhibits the Cytotoxicity of Exogenous Tumor Necrosis Factor and Adriamycin in Pancreatic Carcinoma Cells

Pancreatic carcinoma is one of the most devastating neoplasms with regard to its resistance to conventional therapy. In a previous report, we found that endogenous tumor necrosis factor (enTNF) exerts an intracellular protective effect against exogenous TNF- and Adriamycin (ADM)-induced cytotoxicity by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD). We also know that glutathione S-transferase pi (GST-pi) and glutathione (GSH) also scavenge OFR. It remains unclear to what extent enTNF and MnSOD induced by enTNF regulate the sensitivity to ADM and exogenous TNF among different carcinoma cells. In this study, we examined the relationship between ADM and exogenous TNF sensitivity and en-TNF expression and MnSOD activity in four pancreatic carcinoma lines. We determined whether ADM and exogenous TNF sensitivity could be predicted by measuring enTNF expression and MnSOD activity in the carcinoma cells. The sensitivity to TNF and ADM varied with the cell lines, and TNF sensitivity correlated well with Adriamycin sensitivity. Moreover, enTNF expression and Mn-SOD activity correlated positively with resistance to ADM and exogenous TNF. When MIAPaCa-2 cells, which had the lowest enTNF expression and the highest sensitivity to exogenous TNF and ADM, were transfected with the nonsecretory-type human TNF gene (pTNF delta pro) to increase enTNF synthesis, their intracellular MnSOD activity and exogenous TNF and ADM resistance were increased. These findings suggest that MnSOD plays a critical role in scavenging OFR induced by ADM and exogenous TNF. enTNF is the most important factor that regulates the production of MnSOD. Therefore, it is plausible that inhibition of enTNF expression or MnSOD activity in pancreatic carcinoma would improve the efficacy of therapies for pancreatic carcinoma.

Recombinant Human Tumor Necrosis Factor Causes Regression in Patients with Advanced Malignancies

Fifteen patients with advanced solid tumors of various types were treated by the intratumoral administration of recombinant human tumor necrosis factor (rH-TNF). The treatment appeared to benefit the 4 cases of superficial tumors: there were 1 complete response, 1 partial response and 2 minor responses. In all 11 patients with deep-seated tumors, including 6 cases of pancreatic cancer, 4 of liver cell cancer and 1 of metastatic liver tumor, no tumor regression was observed, but progression stopped in all these tumors. Seven of the 11 with deep-seated tumors showed a decrease in tumor markers and/or the development of tumor necrosis. Fever, hypotension and fatigue were the main clinical side effects. No significant changes were found in hematologic, renal or liver parameters. These results suggest that administration of rH-TNF to the tumor site has the potential for controlling local tumor growth.

Endogenous Tumor Necrosis Factor Functions as a Resistant Factor against Hyperthermic Cytotoxicity in Pancreatic Carcinoma Cells via Enhancement of the Heat Shock Element-Binding Activity of Heat Shock Factor 1

To elucidate the relationship between two distinct resistant factors, endogenous tumor necrosis factor (enTNF) and heat shock proteins (HSPs), against hyperthermia, we assessed whether enTNF enhances HSP72 expression. Although there was a variability in the sensitivity of pancreatic carcinoma cell lines to heat, enTNF and HSP72 expression as well as MnSOD activity correlated positively with heat resistance. When MIAPaCa-2 pancreatic carcinoma cells, which had the lowest enTNF expression and highest heat sensitivity, were transfected with a nonsecretory-human TNF gene (pTNF delta pro), intracellular manganous superoxide dismutase (MnSOD) activity, HSP72 expression, and heat resistance were significantly increased. Furthermore, in these cells, enTNF expression correlated with the binding activity of heat shock factor 1 (HSF 1) to an oligonucleotide containing the human heat shock element. These results indicate that enTNF participates in the intrinsic resistance against heat via induction of MnSOD, enhances HSF1-binding activity, and augments of HSP72 expression. Therefore, inhibition of enTNF expression in pancreatic carcinoma cells would improve the efficacy of hyperthermia for pancreatic carcinoma.