Hirotsugu Okuda

The copper-chelating agent, trientine, suppresses tumor development and angiogenesis in the murine hepatocellular carcinoma cells

Angiogenesis is now recognized as a crucial process in tumor development, including hepatocellular carcinoma (HCC). Since HCC is known as a hypervascular tumor, anti‐angiogenesis is a promising approach to inhibit the HCC development. Trientine dihydrochloride (trientine) is used in clinical practice as an alternative copper (Cu)‐chelating agent for patients with Wilsons disease of penicillamine intolerance. In our study, we examined the effect of Cu‐chelating agents on tumor development and angiogenesis in the murine HCC xenograft model. Although both trientine and penicillamine in the drinking water suppressed the tumor development, trientine exerted a more potent inhibitory effect than penicillamine. In combination with a Cu‐deficient diet, both trientine and penicillamine almost abolished the HCC development. Trientine treatment resulted in a marked suppression of neovascularization and increase of apoptosis in the tumor, whereas tumor cell proliferation itself was not altered. In vitro studies also exhibited that trientine is not cytotoxic for the tumor cells. On the other hand, it significantly suppressed the endothelial cell proliferation. These results suggested that Cu plays a pivotal role in tumor development and angiogenesis in the murine HCC cells, and Cu‐chelators, especially trientine, could inhibit angiogenesis and enhance apoptosis in the tumor with consequent suppression of the tumor growth in vivo. Since trientine is already used in clinical practice without any serious side effects as compared to penicillamine, it may be an effective new strategy for future HCC therapy.

Immunocytochemical localization of nicotinic acetylcholine receptor in rat cerebral cortex.

Localization of nicotinic acetylcholine receptor (nAChR) alpha 4 subunits was investigated in rat cerebral cortex using a monoclonal antibody against alpha 4 subunits. The antibody depleted more than 70% of the [3H]methylcarbamylcholine choline binding activity of the solubilized membrane fraction. By light microscopy alpha 4-like immunoreactivity (alpha 4-LI) was found through layers II to VI. The immunostaining was the most prominent in cell bodies and apical dendrites of pyramidal cells in layer V. By electron microscopy most immunoreaction products were observed in the rough endoplasmic reticulum, cytoplasmic matrix and synaptic membranes. Alpha 4-LI was detected in the postsynaptic membranes of neuronal cell bodies and apical dendrites. These findings suggest that alpha 4-containing subtypes serve as one possibly the postsynaptic nAChR in rat cerebral cortex.

The presence of corticotropin-releasing factor-like immunoreactive synaptic vesicles in axon terminals with nicotinic acetylcholine receptor-like immunoreactivity in the median eminence of the rat

Whether or not corticotropin-releasing factor (CRF) containing synaptic vesicles are located in axon terminals with nicotinic acetylcholine receptor (nAChR) in the median eminence (ME) of the rat was examined by electron microscopic double-labeling immunocytochemistry combining the pre-embedding avidin-biotin-peroxidase complex (ABC) method for nAChR with the post-embedding immunogold staining method for CRF. nAChR-like immunoreactivity (nAChR-LI) was found in the cell membranes of the axon terminals in the ME. CRF-like immunoreactivity (CRF-LI) was found in dense granular vesicles (about 100 nm in diameter) in the axon terminals. Double-labeling method revealed that some of nAChR-LI axon terminals were found to contain CRF-LI dense granular vesicles. The results indicate that nicotine may act on nAChR in axon terminals to release CRF.

Cytosine deaminase/5-fluorocytosine gene therapy can induce efficient anti-tumor effects and protective immunity in immunocompetent mice but not in athymic nude mice

Murine hepatocellular carcinoma cells were retrovirally transduced with the bacterial cytosine deaminase (CD) gene. CD‐transduced cells exhibited more than 120‐fold higher sensitivity to 5‐fluorocytosine (5‐FC) compared with parental cells. When syngeneic immunocompetent mice were inoculated s.c. with parental hepatocellular carcinoma cells containing as little as 5% CD‐transduced cells, significant inhibition of tumor formation was induced by 5‐FC treatment. Furthermore, established solid tumors in immunocompetent mice containing only 5% CD‐transduced cells were infiltrated markedly with CD4+ and CD8+ T lymphocytes and macrophages by 5‐FC treatment, such that significant reduction or even complete regression of tumors was observed. These tumor‐free mice resisted subsequent rechallenge with wild‐type tumor. Conversely, when athymic nude mice were inoculated with a cell mixture containing CD‐transduced cells and parental cells at a ratio of 40:60, all developed tumors despite 5‐FC treatment. Our results indicate that gene therapy using the CD/5‐FC system can induce efficient anti‐tumor effects and protective immunity in immunocompetent mice but not in athymic immunodeficient mice, suggesting that the hosts immunocompetence may be a critical factor for achieving successful gene therapy against cancer. Int. J. Cancer 81:592–597, 1999.

Immunocytochemical localization of nicotinic acetylcholine receptor in rat hypothalamus

Immunocytochemical localization of neuronal nicotinic acetylcholine receptor (nAChR) was examined in rat hypothalamus. Monoclonal antibody against alpha 4 ACh-binding subunits of nAChR was used in the avidin-biotin-peroxidase complex (ABC) immunocytochemical method at both the light and electron microscopic levels. By light microscopy nAChR-like immunoreactivity was found in many neuronal cell bodies and their fibers in the paraventricular nucleus (PVN) and in many axons and axon terminals in the median eminence (ME). The immunoreactivity of nAChR was the most intense in the ME. By electron microscopy immunoreaction products occurred on the rough endoplasmic reticulum, nuclear envelope, cytoplasmic matrices and postsynaptic densities of synaptic junctions in some neurons in the parvocellular part of the PVN. In the external layer of the ME, nAChR-like immunoreactivity was found over the entire plasma membranes of many axon terminals. Involvement of nAChRs in the release of neurotransmitters and neuropeptides both in the PVN and the ME is discussed.

Phosphorylation of rat brain nicotinic acetylcholine receptor by cAMP-dependent protein kinase in vitro

The participation of protein kinases in phosphorylation of nicotinic acetylcholine receptor (nAChR) in electric organ and muscle has been precisely investigated in vitro and in vivo whereas phosphorylation of neuronal nAChR is not yet fully characterized. Here, we first report the in vitro phosphorylation of brain nAChR. nAChR purified from rat brains was phosphorylated in vitro by cAMP-dependent protein kinase (PKA), immunoprecipitated with monoclonal antibody against the receptor, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. PKA specifically phosphorylated nAChR on the alpha 4 subunits, and H8, an inhibitor of PKA, inhibited completely the phosphorylation. Under the conditions used, a maximal stoichiometry of the phosphorylation by PKA was near to 1 mol of phosphate/mol of the alpha 4 subunits. The 32P-labeled subunits were digested with S. aureas V8 protease followed by SDS-PAGE autoradiography and the resultant phosphopeptide maps revealed three distinct phosphopeptide bands, one major band and two minor bands. Phosphoamino acid analysis of the 32P-labeled alpha 4 subunits showed that serine residues were exclusively phosphorylated. Based on these results, participation of PKA in the regulation of neuronal nAChR is discussed.

Transient cyclophosphamide treatment before intraportal readministration of an adenoviral vector can induce re-expression of the original gene construct in rat liver

Although adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenoviruses may be limited by their immunogenicity. Although immunosuppressive strategies around the time of initial exposure of adenoviruses have been shown to prevent the formation of neutralizing antibodies and permit the successful readministration of adenoviruses in animals, the practicality of the approaches remains questionable. Because the majority of prospective gene therapy patients have already been infected with wild-type adenoviruses, initial treatment with adenoviruses in humans may correspond to readministration of adenoviruses into animals. It is shown here that although intraportal infusion of adenoviruses carrying a reporter lacZ gene resulted in transient high levels of transgene expression in the rat liver, intraportal readministration of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with cyclophosphamide before the intraportal readministration of adenoviruses, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral readministration was significantly suppressed and successful re-expression of the transgene was achievable. These results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings.