Dolores J. Takemoto

Oxidative Activation of Protein Kinase Cγ through the C1 Domain EFFECTS ON GAP JUNCTIONS

The accumulation of reactive oxygen species (ROS, for example H2O2) is linked to several chronic pathologies, including cancer and cardiovascular and neurodegenerative diseases (Gate, L., Paul, J., Ba, G. N., Tew, K. D., and Tapiero, H. (1999) Biomed. Pharmacother. 53, 169–180). Protein kinase C (PKC) γ is a unique isoform of PKC that is found in neuronal cells and eye tissues. This isoform is activated by ROS such as H2O2. Mutations (H101Y, G118D, S119P, and G128D) in the PKCγ Cys-rich C1B domain caused a form of dominant non-episodic cerebellar ataxia in humans (Chen, D.-H., Brkanac, Z., Verlinde, C. L. M. J., Tan, X.-J., Bylenok, L., Nochli, D., Matsushita, M., Lipe, H., Wolff, J., Fernandez, M., Cimino, P. J., Bird, T. D., and Raskind, W. H. (2003) Am. J. Hum. Genet. 72, 839–849; van de Warrenburg, B. P. C., Verbeek, D. S., Piersma, S. J., Hennekam, F. A. M., Pearson, P. L., Knoers, N. V. A. M., Kremer, H. P. H., and Sinke, R. J. (2003) Neurology 61, 1760–1765). This could be due to a failure of the mutant PKCγ proteins to be activated by ROS and to subsequently inhibit gap junctions. The purpose of this study was to demonstrate the cellular mechanism of activation of PKCγ by H2O2 and the resultant effects on gap junction activity. H2O2 stimulated PKCγ enzyme activity independently of elevations in cellular diacylglycerol, the natural PKC activator. Okadaic acid, a phosphatase inhibitor, did not affect H2O2-stimulated PKCγ activity, indicating that dephosphorylation was not involved. The reductant, dithiothreitol, abolished the effects of H2O2, suggesting a direct oxidation of PKCγ at the Cys-rich C1 domain. H2O2 induced the C1 domain of PKCγ to translocate to plasma membranes, whereas the C2 domain did not. Direct effects of H2O2 on PKCγ were demonstrated using two-dimensional SDS-PAGE. Results demonstrated that PKCγ formed disulfide bonds in response to H2O2. H2O2-activated PKCγ was targeted into caveolin-1- and connexin 43-containing lipid rafts, and the PKCγ phosphorylated the connexin 43 gap junction proteins on Ser-368. This resulted in disassembly of connexin 43 gap junction plaques and decreased gap junction activity. Results suggested that H2O2 caused oxidation of the C1 domain, activation of the PKCγ, and inhibition of gap junctions. This inhibition of gap junctions could provide a protection to cells against oxidative stress.

Synthesis and Anti-Breast Cancer Activities of Substituted Quinolines

Promising anti-breast cancer agents derived from substituted quinolines were discovered. The quinolines were readily synthesized in a large scale from a sequence of reactions starting from 4-acetamidoanisole. The Michael addition product was isolated as the reaction intermediate in the ring closing reaction of 4-amino-5-nitro-2-(3-trifluoromethylphenyloxy)anisole with methyl vinyl ketone leading to 6-methoxy-4-methyl-8-nitro-5-(3-trifluoromethylphenyloxy)quinoline (14). The amino function of 8-amino-6-methoxy-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline, prepared from 14, was connected to various side chains via alkylation with N-(3-iodopropyl)phthalimide, Michael addition with acrylonitrile, and reductive amination with various heterocycle carboxaldehydes, such as imidazole-4-carboxaldehyde, thiophene-2-carboxaldehyde, and 2-furaldehyde. Effects of the substituted quinolines on cell viability of T47D breast cancer cells using trypan blue exclusion assay were examined. The results showed that the IC(50) value of 6-methoxy-8-[(2-furanylmethyl)amino]-4-methyl-5-(3-trifluoromethylphenyloxy)quinoline is 16+/-3nM, the lowest IC(50) out of all the quinolines tested. IC(50) values of three other quinolines are in the nanomolar range, a desirable range for pharmacological testing.

Induction of tumor cytotoxic immune cells using a protein from the bitter melon (Momordica charantia)

The fruit and seeds of the bitter melon (Momordica charantia) have been reported to have anti-leukemic and antiviral activities. This anti-leukemic and antiviral action was associated with an activation of murine lymphocytes. A partially purified protein factor from the bitter melon caused an infiltration and activation of peritoneal exudate cells in C57B1/6J, C3H/HeJ, and C3H/HeN mice. When the extract was injected twice a week at 8 micrograms of protein per ip injection for 0-4 weeks, the peritoneal exudate cells from the treated mice were cytotoxic in a long-term (18-hr) 51Cr-release assay against a range of labeled targets: L1210, P388, and MOLT-4 tumor cells. Cytotoxicity was also observed against YAC-1 targets in a short-term (4-hr) assay. Fractionation of the cytotoxic immune cells implicated a nonadherent cell population which was capable of killing an NK-sensitive cell line in a 4-hr 51Cr-release assay. Unit gravity sedimentation studies indicated that the cytotoxicity was due to either a neutrophil or a large lymphocyte. Antibody depletion experiments using antibody to asialo GM1, an NK cell-specific antibody, depleted cytotoxicity observed in nonadherent, Ficoll/Hypaque-separated PEC. This suggests that at least part of the anti-leukemic activity of the bitter melon extract is due to the activation of NK cells in the host mouse.

Antitumor activity of wheats with high orthophenolic content

The purpose of the present study is to develop in vitro assays for rapid screening of a large number of food samples that contain components that prevent tumor formation in vivo and to identify the components that contribute to this antitumor effect. Wheat samples representing numerous strains and cultivars were screened for their in vitro ability to kill a human colon cancer cell, CaCo2, in culture by trypan blue dye exclusion assay. Wheat samples were assayed for orthophenolic acid content by use of a colorimetric assay using a bathochromatic shift at 350 nm. Blood levels of specific orthophenols were determined by high-performance liquid chromatography/mass spectrometry. Wheat samples, which contained low, mid-, and high in vitro protective ability, were used to formulate balanced diets fed to Min mice. Wheat samples with high ability to kill CaCo2 cells in culture had high levels of orthophenolic acids and produced elevated blood caffeic acid levels when used in diets. These factors correlated positively with their ability to prevent tumor formation in Min mice. When fiber content was equal in diets the content of orthophenolic acids in wheats predicted the antitumor activity in vivo.

Antibody Indications of Secondary and Superimposed Retinal Hypersensitivity in Retinitis Pigmentosa

Antibody reactions with recognized retinopathy-inducing retinal antigens may be interpreted to reflect ongoing autoimmune events responsible for some forms of vision loss. We sought evidence of secondary and superimposed retinal hypersensitivity indicated by such antibody reactivity in a random group of patients with retinitis pigmentosa. We identified patterns of immunologic reactivity within members of a group of 52 patients with retinitis pigmentosa, which suggests some patients with retinitis pigmentosa may experience consequential superimposed retinal hypersensitivity. Identifying subgroups of patients with retinitis pigmentosa who exhibit indications of retinal hypersensitivity to known uveitopathogenic retinal proteins may permit the reduction of their rate of retinal degradation by immunomodulation.

The cytotoxic and cytostatic effects of the bitter melon (Momordica charantia) on human lymphocytes.

We have previously reported that a crude extract from the bitter melon (Momordica charantia) killed human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocyte cells at these same doses (Takemoto et al., 1980). We now report that the crude preparation has both cytostatic and cytotoxic activities which are heat stable and trypsin-sensitive. Time and dose-response curves suggest that the factors act quickly, perhaps by entry into the cell. The effects of the crude extract are complete after only 2 hr of exposure. These activities are not due to the presence of the lectins from bitter melon seeds, as these purified proteins had no activity against human lymphocytic cells.

A novel role of gap junction connexin46 protein to protect breast tumors from hypoxia

Connexin proteins are the principle structural components of the gap junctions. Colocalization and tissue‐specific expression of diverse connexin molecules are reported to occur in a variety of organs. Impairment of gap junctional intercellular communication, caused by mutations, gain of function or loss of function of connexins, is involved in a number of diseases including the development of cancer. Here we show that human breast cancer cells, MCF‐7 and breast tumor tissues express a novel gap junction protein, connexin46 (Cx46) and it plays a critical role in hypoxia. Previous studies have shown that connexin46 is predominantly expressed in lens and our studies find that Cx46 protects human lens epithelial cells from hypoxia induced death. Interestingly, we find that Cx46 is upregulated in MCF‐7 breast cancer cells and human breast cancer tumors. Downregulation of Cx46 by siRNA promotes 40% MCF‐7 cell death at 24 hr under hypoxic conditions. Furthermore, direct injection of anti‐Cx46 siRNA into xenograft tumors prevents tumor growth in nude mice. This finding will provide an exciting new direction for drug development for breast cancer treatment and suggests that both normal hypoxic tissue (lens) and adaptive hypoxic tissue (breast tumor) utilize the same protein, Cx46, as a protective strategy from hypoxia.

Protection of Retinal Cells from Ischemia by a Novel Gap Junction Inhibitor

Retinal cells which become ischemic will pass apoptotic signal to adjacent cells, resulting in the spread of damage. This occurs through open gap junctions. A class of novel drugs, based on primaquine (PQ), was tested for binding to connexin 43 using simulated docking studies. A novel drug has been synthesized and tested for inhibition of gap junction activity using R28 neuro-retinal cells in culture. Four drugs were initially compared to mefloquine, a known gap junction inhibitor. The drug with optimal inhibitory activity, PQ1, was tested for inhibition and was found to inhibit dye transfer by 70% at 10 microM. Retinal ischemia was produced in R28 cells using cobalt chloride as a chemical agent. This resulted in activation of caspase-3 which was prevented by PQ1, the gap junction inhibitor. Results demonstrate that novel gap junction inhibitors may provide a means to prevent retinal damage during ischemia.

Cleavage from the N-terminal region of βBp crystallin during aging of the human lens

Polyclonal antisera have been made to synthetic peptides that correspond to the N-terminal (residues 1-12) and C-terminal (residues 195-204) sequences of bovine beta Bp crystallin. Both anti-beta Bp1-12 and anti-beta Bp195-204 recognize specifically the beta Bp component of bovine lens. In the young human lens, anti-beta Bp195-204 recognizes predominantly the 26,000 MW form of beta Bp, while in older lenses this same antiserum recognizes mainly the 22,000 MW in vivo proteolysis product. In contrast, during aging of the normal human lens anti-beta Bp1-12 recognizes only decreasing amounts of the 26,000 MW form of beta Bp, with no binding to the 22,000 MW form of this polypeptide. These results suggest that during aging of the normal human lens, the N-terminus of beta Bp is the preferred site of in vivo proteolysis.

Purification and Characterization of a Cytostatic Factor with Anti-Viral Activity from the Bitter Melon

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activities, and is a competitive inhibitor of guanylate cyclase activity. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses. In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity. The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons. The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse). As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated. The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h. The cytostatic component had no effect on cellular cyclic GMP metabolism.