[Tumor necrosis factor alpha (TNF alpha)].
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Humans↗
Biomedical subjects
N Watanabe
Publications and source records attributed to N Watanabe.
[CEA (carcinoembryonic antigen) in nipple discharge].
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Biomarkers, Tumor↗
[A combination of low-dose carboplatin (CBDCA) and radiation therapy in head and neck cancer patients--response and hematologenic toxicity].
A combination of carboplatin (CBDCA) and radiation therapy was performed in patients with head and neck cancer. The intravenous administration of CBDCA at a weekly dose of 100 mg/body was combined with external irradiation at a dose of 1.8 Gy/day x 5/week during the same therapy period. We evaluated the effects of this method not only on survival and neoadjuvant chemotherapy in patients with advanced cancer but also on local control and prevention of distant metastases in patients with early cancer. The subjects consisted of 31 patients with head and neck cancer who visited the Department of Otolaryngology, Showa University Hospital, Kanto Rosai Hospital and Yokohama Rosai Hospital between March 1993 and March 1995. Squamous cell carcinoma was found in all but one patient who had adenocarcinoma in the parotid gland. The patients were 27 males and 4 females ranging between 40 and 81 years of age (mean, 59 years). Twenty-six patients had previously untreated tumor, while 5 patients had recurrent tumor. The clinical stage was Stage I in 6, Stage II in 10, Stage III in 5 and Stage IV in 10 patients. The original site of cancer was the larynx in 9, hypopharynx in 6, nasopharynx in 6 and elsewhere in 10 patients. The combined therapy was repeated for 2-9 courses (mean, about 5 courses). The total dose of CBDCA was 500 mg on average with a maximum of 900 mg. The total effective rate of the combined therapy was 93.3% with 12 cases of complete response (CR) and 16 cases of partial response (PR). The effective rate in patients with previously untreated tumor was 92% because one patient showed a minor response (MR) and one patient showed no change (NC). The effective rate in patients with recurrent tumor was 100%. Concerning the clinical stage, all patients with Stage I-III disease showed CR or PR, while MR was found in 1, NC in 1 and progressive disease (PD) in 1 of 10 patients with Stage IV disease, resulting in an effective rate of 71.4%. There were no CRs in Stage IV patients. Therapy was terminated due to side effects in 2 patients. Excellent safety was confirmed by laboratory data.
Adult↗
UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway that is associated with inactivation of the Wee1Hu kinase and activation of the Cdc25C phosphatase.
We have previously demonstrated that UCN-01, a potent protein kinase inhibitor currently in phase I clinical trials for cancer treatment, abrogates G2 arrest following DNA damage. Here we used murine FT210 cells, which contain temperature-sensitive Cdc2 mutations, to determine if UCN-01 abrogates G2 arrest through a Cdc2-dependent pathway. We report that UCN-01 cannot induce mitosis in DNA-damaged FT210 cells at the non-permissive temperature for Cdc2 function. Failure to abrogate G2 arrest was not due to UCN-01-inactivation at the elevated temperature because parental FM3A cells, which have wild-type Cdc2, were sensitive to UCN-01-induced G2 checkpoint abrogation. Having established that UCN-01 acted through Cdc2, we next assessed UCN-01's effect on the Cdc2-inhibitory kinase, Wee1Hu, and the Cdc2-activating phosphatase, Cdc25C. We found that Wee1Hu was indeed inactivated in UCN-01-treated cells, possibly just prior to Cdc2 activation and entry of DNA-damaged cells into mitosis. This inhibition appeared, however, to be a consequence of a further upstream action since in vitro studies revealed purified Wee1Hu was relatively resistant to UCN-01-inhibition. Consistent with such an upstream action, UCN-01 also promoted the hyperphosphorylation (activation) of Cdc25C in DNA-damaged cells. Our results suggest that UCN-01 abrogates G2 checkpoint function through inhibition of a kinase residing upstream of Cdc2, Wee1Hu, and Cdc25C, and that changes observed in these mitotic regulators are downstream consequences of UCN-01's actions.
Alkaloids↗
Production of proinflammatory cytokines by phorbol myristate acetate-treated THP-1 cells and monocyte-derived macrophages after phagocytosis of apoptotic CTLL-2 cells.
Because it is generally believed that apoptosis is not associated with inflammation, we hypothesized that the interaction of phagocytes with apoptotic cells provides a negative or null signal for inflammation. However, we recently found that the interaction led to the production of proinflammatory cytokines but not antiinflammatory cytokines, although the apoptotic cell membranes appeared to be intact. In this study, we examined in detail the relationship among the kinetics of apoptosis, phagocytosis and production of cytokines by macrophages. Among the time points examined, murine CTLL-2 cells became apoptotic in terms of cell size and exposure of phosphatidylserine after 12 h of culture in the absence of IL-2, and at the same time they began to be phagocytosed and lead to proinflammatory cytokine production by PMA-treated THP-1 cells (human macrophages). The phagocytosis of apoptotic cells by macrophages was also confirmed by confocal laser microscopy. The coculturing of human macrophages with murine apoptotic cells led to the production of human proinflammatory cytokines, notably IL-8, at both the mRNA level and the protein level. The coculturing of monocyte-derived macrophages with the apoptotic cells also led to the production of IL-8 protein. Both the phagocytosis and production of the cytokines were suppressed by either phospho-L-serine or RGDS (Arg-Gly-Asp-Ser), but not by RGES (Arg-Gly-Glu-Ser). Thus, the production of proinflammatory cytokines and phagocytosis of apoptotic CTLL-2 cells appear to be closely interrelated.
Animals↗
Differentiation induction by a tumor-necrosis-factor mutant 471 in human myelogenous leukemic cells via tumor-necrosis-factor receptor-p55.
The present study examined differentiation-inducing activity by various tumor-necrosis-factor(TNF) mutants against the human leukemic cell lines HL-60 and U-937. Mutant TNF 471, from which 7 N-terminal amino acids of native TNF were deleted and Pro8, Ser9 and Asp10 were replaced by Arg, Lys and Arg, possessed the highest activity among the TNF mutants, and its activity was 120-fold that of native TNF. The various biological activities of TNF were signaled through 2 distinct receptors, p55 and p75. Although cytotoxicity was reported to involve mainly p55, this differentiation-inducing activity was not well understood. The fact that the affinity of TNF 471 was higher to p55 and lower to p75 than that of native TNF by a binding competition assay suggested that the differentiation-inducing activity was also signaled through p55. To verify this hypothesis, the human myelogenous leukemic cell line, KG-1, which scarcely expresses either receptor and does not differentiate with TNF, was transduced with the p55 or p75 gene. Subsequently p55 transfectants manifested a greater ability to differentiate; however, p75 transfectants did not differ from parental cells or from mock-transfectants. Further, the differentiation of p55 transfectants induced by TNF was reduced by the inhibitor of protein-kinase-C (PKC), staurosporine. These results indicate that the differentiation-inducing activity was signaled through the TNF receptor, p55, via PKC and that the excellent ability of TNF 471 to induce differentiation was related to its high affinity for p55.
Antigens, CD↗
4-Benzylamino-1-chloro-6-substituted phthalazines: synthesis and inhibitory activity toward phosphodiesterase 5.
We synthesized various 4-benzylamino-1-chloro-6-substituted phthalazines (15) and 4-benzylamino-1-chloro-7-substituted phthalazines (16) and evaluated their inhibitory activity toward phosphodiesterase 5 (PDE5) purified from porcine platelets. The PDE5-inhibitory activities of 15 were greater than those of the isomers (16). The preferred substituent at the 4-position of phthalazine was a (3-chloro-4-methoxybenzyl)amino group, and those at the 6-position were cyano, nitro, and trifluoromethyl groups. Compounds 15a (IC50 = 4.8 nM), 15f (3.5 nM), and 15i (5.3 nM) were more potent inhibitors than E4021 (8.6 nM). Compounds 15a and 15f also showed vasorelaxant activity in isolated porcine coronary arteries precontracted with prostaglandin F2alpha (10(-5) M). The EC50 values for vasorelaxant action of 15a, 15f, and E4021 were 150, 160, and 980 nM, respectively. These results show that novel PDE5 inhibitors possessing a potent vasorelaxant effect may exist among phthalazine derivatives.
3',5'-Cyclic-GMP Phosphodiesterases↗
Promoter/repressor system of Lactobacillus plantarum phage og1e: characterization of the promoters pR49-pR-pL and overproduction of the cro-like protein cng in Escherichia coli.
The Lactobacillus plantarum phage og1e (42 259 bp) has two repressor-like genes cng and cpg oriented oppositely, accompanied by three potential promoters pR, pL and pR49, and seven operator-like sequences (GATAC-boxes) (Kodaira et al., 1997). In this study, the og1e putative promoters were introduced into the Escherichia coli promoter-detecting plasmid pKK232-8. In E. coli CK111, pR (pKPR1), pL (pKPL1) and pR49 (pKPR49) exhibited distinct CAT activities. When pKPR1 or pKPL1 was coexistent with a compatible plasmid pACYC184 carrying pR-cng (pA4PRCN1), the CAT activity was decreased significantly. On the other hand, cng directed a protein (Cng) of 10.1 kDa in E. coli under the control of T7 promoter. Gel mobility-shift assays demonstrated that Cng binds specifically to a DNA region containing the GATAC-boxes. In addition, primer extension analyses demonstrated that the two sequences pR and pL act as a promoter in L. plantarum as well as in E. coli. These results suggested that the potential promoters pR and pL probably function for the lytic and lysogenic pathways, respectively, and Cng may act as a repressor presumably through the GATAC-boxes as operators.
Bacteriophages↗
Role of citron kinase as a target of the small GTPase Rho in cytokinesis.
During mitosis, a ring containing actin and myosin appears beneath the equatorial surface of animal cells. This ring then contracts, forms a cleavage furrow and divides the cell, a step known as cytokinesis. The two daughter cells often remain connected by an intercellular bridge which contains a refringent structure known as the midbody. How the appearance of this ring is regulated is unclear, although the small GTPase Rho, which controls the formation of actin structures, is known to be essential. Protein kinases are also thought to participate in cytokinesis. We now show that a splice variant of a Rho target protein, named citron, contains a protein kinase domain that is related to the Rho-associated kinases ROCK14 and ROK, which regulate myosin-based contractility. Citron kinase localizes to the cleavage furrow and midbody of HeLa cells; Rho is also localized in the midbody. We find that overexpression of citron mutants results in the production of multinucleate cells and that a kinase-active mutant causes abnormal contraction during cytokinesis. We propose that citron kinase regulates cytokinesis at a step after Rho in the contractile process.
Alternative Splicing↗
Molecular dissection of the Rho-associated protein kinase (p160ROCK)-regulated neurite remodeling in neuroblastoma N1E-115 cells.
A critical role for the small GTPase Rho and one of its targets, p160ROCK (a Rho-associated coiled coil-forming protein kinase), in neurite remodeling was examined in neuroblastoma N1E-115 cells. Using wild-type and a dominant-negative form of p160ROCK and a p160ROCK-specific inhibitor, Y-27632, we show here that p160ROCK activation is necessary and sufficient for the agonist-induced neurite retraction and cell rounding. The neurite retraction was accompanied by elevated phosphorylation of myosin light chain and the disassembly of the intermediate filaments and microtubules. Y-27632 blocked both neurite retraction and the elevation of myosin light chain phosphorylation in a similar concentration-dependent manner. On the other hand, suppression of p160ROCK activity by expression of a dominant-negative form of p160ROCK induced neurites in the presence of serum by inducing the reassembly of the intermediate filaments and microtubules. The neurite outgrowth by the p160ROCK inhibition was blocked by coexpression of dominant-negative forms of Cdc42 and Rac, indicating that p160ROCK constitutively and negatively regulates neurite formation at least in part by inhibiting activation of Cdc42 and Rac. The assembly of microtubules and intermediate filaments to form extended processes by inhibitors of the Rho-ROCK pathway was also observed in Swiss 3T3 cells. These results indicate that Rho/ROCK-dependent tonic inhibition of cell process extension is exerted via activation of the actomysin-based contractility, in conjunction with a suppression of assembly of intermediate filaments and microtubules in many cell types including, but not exclusive to, neuronal cells.
3T3 Cells↗
Suppression of intracellular resistance factors by adriamycin augments heat-induced apoptosis via interleukin-1beta-converting enzyme activation in pancreatic carcinoma cells.
Combination of heat and various anticancer drugs can exert a synergistic antitumor effect in vitro and in vivo, though the mechanism is not clear. We have previously shown that endogenous tumor necrosis factor (enTNF) acts as an intracellular resistance factor to inhibit the cytotoxic effect of heat by scavenging oxygen-free radicals via the induction of manganous superoxide dismutase (MnSOD). Consequently, we examined whether the suppression of these resistance factors by combining anticancer drugs and heat causes an augmentation of heat-induced cytotoxicity. The human pancreatic carcinoma cell line, PANC-1, constitutively expresses appreciable amounts of enTNF and also demonstrates heat resistance. After treatment of these cells for 15 hr with adriamycin (ADM), the expression of enTNF was decreased by 43%, and MnSOD activity was suppressed by 55%. The cytotoxic effects of the treatment of PANC-1 cells with ADM followed by heat were greater than the sum of those observed with the agents administrated individually. Heat-induced apoptosis was also augmented by pretreatment with ADM. Furthermore, the interleukin-1beta-converting enzyme inhibitor, Ac-YVAD-CMK, reversed the augmented cytotoxicity. Our results indicate that suppression of intracellular resistance factors such as enTNF and MnSOD plays an important role in apoptosis seen after heat and ADM combined therapy.
Apoptosis↗
Requirement of expression of P-glycoprotein on human natural killer leukemia cells for cell-mediated cytotoxicity.
The requirement of P-glycoprotein, a product of the multidrug resistance (MDR)1 gene, for natural killer (NK) cell-mediated cytotoxicity was examined by using a human NK-like cell line, YTN, which is cytotoxic toward JY cells. YTN cells express P-glycoprotein, a judged by flow cytometry and polymerase chain reaction of reverse-transcribed mRNA. YTN cell-mediated cytotoxicity was inhibited by MDR-reversing reagents as well as the F(ab')2 fragment of a monoclonal antibody against P-glycoprotein. Furthermore, antisense oligonucleotides for MDR1 mRNA inhibited expression of P-glycoprotein as well as YTN cell-mediated cytotoxicity. Thus, this study provides firm evidence that P-glycoprotein plays an essential role in cell-mediated cytotoxicity.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Processing and release of tumor necrosis factor alpha.
Tumor necrosis factor alpha (TNFalpha) is synthesized as a transmembrane precursor form that is proteolytically processed and released as the soluble mature form. In human monocytes and monocytic cell lines, the production, processing, and release of TNFalpha are co-induced by certain activators, such as lipopolysaccharide. To investigate the mechanism of TNFalpha processing and release, we established a cell line which constitutively produced TNFalpha, by transfecting the TNFalpha precursor form cDNA into NIH/3T3 cells. In these cells, the processing and release of TNFalpha were augmented by phorbol 12-myristate 13-acetate (PMA), mediated through a protein kinase C (PKC) signalling pathway. Various protease inhibitors were tested and it was found that matrix metalloproteinase (MMP) inhibitors blocked the processing and release of TNFalpha both in the absence and presence of PMA. This result is compatible with the recent reports that MMP are involved in the processing and release of TNFalpha. In contrast, 3,4-dichloroisocoumarin, N alpha-p-tosyl-L-phenylalanine chloromethane, iodoacetamide, and o-phenanthroline inhibited the processing and release of TNFalpha only in the presence of PMA, suggesting that serine proteases requiring SH for their activity, a combination of serine proteases and cysteine proteases, or MMP, may be involved in the PKC-mediated induction of TNFalpha processing and release.
3T3 Cells↗
Inelastic X-ray scattering in molecular liquids and electron correlation effects.
Inelastic X-ray scattering spectra of liquid water and cyclohexane have been measured with 2 eV resolution for a momentum transfer range between 0.69 and 2.77 au at BL16A of the Photon Factory, KEK, Tsukuba. Observed spectra are transformed to the dynamic structure factor which is normalized by using the Bethe sum rule, and the static structure factor is obtained. From a comparison with extended CI calculations by the use of various basis sets, correlation effects are proved to be of vital importance in inelastic X-ray scattering.
Journal Article↗
Imaging soft X-ray microscope at Rits Synchrotron Radiation Center.
An imaging soft X-ray microscope with zone plates has been installed at Rits SR Center (Ritsumeikan University, Kusatsu, Japan). With this microscope, specimens were set in air, which made it possible to investigate the specimens without breaking the vacuum of the microscope. The specimens can be prefocused with an optical microscope. Dry and wet biospecimens in air were observed. A new optical system was designed to improve the resolution.
Journal Article↗
Three-dimensional tomography using a soft X-ray holographic microscope and CCD camera.
The depth resolution of a soft X-ray hologram is much worse than its transverse resolution because a single soft X-ray hologram has a small numerical aperture. To obtain a three-dimensional image, in-line holograms of a specimen were recorded from various directions and reconstructed to obtain two-dimensional projection data. Then, a three-dimensional reconstruction was performed by back-projection of these reconstructed holograms. Three-dimensional images of a tungsten wire of diameter 10 micro m and a fossil of a diatom were obtained.
Journal Article↗
An apoptosis-inducing gene therapy for pancreatic cancer with a combination of 55-kDa tumor necrosis factor (TNF) receptor gene transfection and mutein TNF administration.
Intratumoral injection of recombinant human tumor necrosis factor (TNF) for inoperable pancreatic cancer has shown some efficacy in suppressing tumor growth or decreasing tumor markers. However, complete regression has not yet been achieved, possibly due to a lack of TNF receptors on tumor cells or an abundance of intracellular resistance factors. Recently, two distinct types of TNF receptors, R55 and R75, were identified, which are responsible for signaling of cytotoxicity and of proinflammation, respectively. In this study, a novel type of suicide gene therapy is proposed that is based on transfection of the R55 gene into human pancreatic cancer cells (AsPC-1 and PANC-1) and subsequent administration of TNF. The transfectants from both cell lines showed higher TNF susceptibility than their parental cells. In vivo tumor formation of an AsPC-1 clone (clone 10) inoculated in nude mice was substantially suppressed by administration of TNF. For practical use of this strategy, however, the adverse effects of TNF may become an obstacle. We previously produced mutein TNF 471, which had a higher affinity for R55, superior antitumor activity, and fewer adverse effects. This mutein TNF 471 manifested greater antitumor activity against clone 10. Because the R55 receptor is known to be involved in augmentation of cellular immunity by TNF, mutein TNF 471 is also expected to be highly potent in this function. In fact, the mutein TNF 471 induced higher splenic natural killer cell activity in nude mice inoculated with clone 10 than did native TNF. This property of augumenting cellular responses may be advantageous in the eradication of viable tumor cells left untransfected in practical gene therapy regimens in which 100% transfection of the R55 gene into tumors is not feasible. Thus, gene therapy combining transfection of the TNF-R55 gene with administration of mutein TNF 471 may provide a new modality for the treatment of pancreatic cancer.
Adenocarcinoma↗
Administration of recombinant human interleukin 12 to chronically SIVmac-infected rhesus monkeys.
With the demonstration that interleukin 12 can enhance natural killer (NK) cell activity and drive CD4+ lymphocytes toward T helper type 1 (Thl) responses, there is a strong rationale for exploring the use of this cytokine as an immunomodulatory therapy in HIV-1-infected individuals. To assess its potential safety and effects on both immune and virologic aspects of HIV-1 infection, recombinant human IL-12 (rhIL-12) was assessed in rhesus monkeys chronically infected with the simian immunodeficiency virus of macaques (SIVmac). The activity of rhIL-12 on rhesus monkey lymphocytes was confirmed with the demonstration that peripheral blood lymphocyte lysis of the NK-sensitive cell line Colo was enhanced by this recombinant cytokine. Further, rhIL-12 was shown to induce interferon-gamma production by rhesus monkey lymphocytes in vitro. Then, in separate studies, two treatment regimens of rhIL-12 were assessed in SIVmac-infected monkeys: a low-dose regimen (0.1 microg/kg, daily for 4 weeks) and a high-dose regimen (2.5 microg/kg, every 3-4 days, for 3 weeks). Both rhIL-12 treatment regimens were well tolerated by these virus-infected animals. The high-dose regimen of rhIL-12 induced transient decreases in circulating lymphocytes in the SIVmac-infected monkeys. Furthermore, no changes in lymphocyte-associated SIVmac DNA or SIVmac plasma RNA levels were seen in the treated monkeys. These studies indicate that short-term treatment with rhIL-12 is well tolerated and causes no measurable changes in virus load in chronically SIVmac-infected rhesus monkeys.
Animals↗