No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.     

Mutations in chemosensory cilia cause resistance to paraquat in nematode Caenorhabditis elegans

The relationship between oxidative stress and longevity is a matter of concern in various organisms. We isolated mutants resistant to paraquat from nematode Caenorhabditis elegans. One mutant named mev-4 was long-lived and showed cross-resistance to heat and Dyf phenotype (defective in dye filling). Genetic and sequence analysis revealed that mev-4 had a nonsense mutation on the che-11 gene, homologues of which are involved in formation of cilia and flagella in other organisms. The paraquat resistance was commonly observed in various Dyf mutants and did not depend on the daf-16 gene, whereas the extension of life span did depend on it. Expression of antioxidant enzyme genes seemed normal. These results suggest that chemosensory neurons are a target of oxidative stress and influence longevity dependent on the daf-16 signaling in C. elegans.     

Establishment of a detection system for demethylating agents using an endogenous promoter CpG island

Disturbances of epigenetic information that result in changes in DNA methylation patterns are involved in carcinogenesis and other human disorders. Detection of agents that can cause epigenetic alterations--i.e. epimutagens--is therefore an important objective. We have developed and now describe the first detection system for demethylating agents that involves an endogenous promoter CpG island (CGI). After screening 10 promoter CGIs of genes silenced in human cancers, a CGI of the FLJ32130 gene was found to respond sensitively to a known demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC), by abundantly re-expressing its mRNA. After introducing the Hyg(r)-EGFP fusion gene into exon 3 of the FLJ32130 gene by homologous recombination, we isolated one clone that had the expected recombination outcomes and designated it F117. Two subclones (F117-47 and F117-123) of this original clone that did not share its propensity for leaky expression of the Hyg(r)-EGFP mRNA were then isolated, and methylation of their 5' CGI was confirmed. The addition of 5-aza-dC at doses of 0.1 microM or higher led to their 5' CGI being demethylated, and to Hyg(r)-EGFP being expressed; the anticipated fluorescence was readily confirmed by fluorescence microscopy. We believe that this is the first assay system that detects agents that disturb the methylated status of a CGI that regulates an endogenous promoter.     

Persistence of gene expression changes in stomach mucosae induced by short-term N-methyl-N'-nitro-N-nitrosoguanidine treatment and their presence in stomach cancers

Cancers induced by different carcinogens show distinct expression profiles. In addition to the specific alterations of tumor-related genes induced by specific carcinogens, it is possible that some initial responses induced by a carcinogen could persist for long periods and are consistently present in the cancers induced. We have analyzed the initial responses in the rat pyloric mucosae after treatment for 2 weeks with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Gene expression was monitored 1 day, 2 weeks and 4 weeks after MNNG treatment by oligonucleotide microarray analysis. Of the differentially expressed genes showing greater than three-fold difference 1 day after MNNG treatment, 143 and 26 genes were up- and down-regulated, respectively, in MNNG-induced stomach cancers. Among these genes, 25 and 6 genes were up- and down-regulated, respectively, in the histologically normal pyloric mucosae, even 4 weeks after cessation of MNNG treatment. Among the up-regulated genes, many genes involved in tissue remodeling (Spi15, Serpine1 and Fst) and cellular growth (Bdnf, Ros1 and Fgf10) were present. The six down-regulated genes included TGF-beta-inducible early growth response gene. These findings demonstrate that some expression changes induced by MNNG persist for a prolonged period and are present in cancers. Persistent expression changes are considered to be important for prediction of past carcinogen exposure, and could provide a molecular environment favorable for malignant transformation.     

Comparative analysis of DNA alkylation by conjugates between pyrrole–imidazole hairpin polyamides and chlorambucil or seco-CBI

We investigated sequence-specific DNA alkylation using conjugates between the N-methylpyrrole (Py)-N-methylimidazole (Im) polyamide and the DNA alkylating agent, chlorambucil, or 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI). Polyamide–chlorambucil conjugates 1–4 differed in the position at which the DNA alkylating chlorambucil moiety was bound to the Py–Im polyamide. High-resolution denaturing polyacrylamide gel electrophoresis (PAGE) revealed that chlorambucil conjugates 1–4 alkylated DNA at the sequences recognized by the Py–Im polyamide core moiety. Reactivity and sequence specificity were greatly affected by the conjugation position, which reflects the geometry of the alkylating agent in the DNA minor groove. Polyamide–seco-CBI conjugate 5 was synthesized to compare the efficacy of chlorambucil with that of seco-CBI as an alkylating moiety for Py–Im polyamides. Denaturing PAGE analysis revealed that DNA alkylation activity of polyamide–seco-CBI conjugate 5 was similar to that of polyamide–chlorambucil conjugates 1 and 2. In contrast, the cytotoxicity of conjugate 5 was superior to that of conjugates 1–4. These results suggest that the seco-CBI conjugate was distinctly active in cells compared to the chlorambucil conjugates. These results may contribute to the development of more specific and active DNA alkylating agents.     

Differential regulation of intracellular redox state by extracellular matrix proteins in glomerular mesangial cells: potential role in diabetic nephropathy

Advanced diabetic nephropathy is characterized by abnormal synthesis of extracellular matrix (ECM) proteins, such as collagen I (COL I). The present experiments were designed to test the hypothesis that the presence of abnormal ECM proteins may be responsible for increased generation of reactive oxygen species (ROS) that are thought to have an important role in the pathogenesis of diabetic nephropathy. SV40 MES 13 murine mesangial cells were plated on COL I or collagen IV (COL IV) for 3 h at 5.5 or 25 mM D-glucose concentration. Increased intracellular ROS generation and reduced intracellular nitric oxide (NO) production was measured in cells attached to COL I compared with cells attached to COL IV. Treatment with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NO synthase, reduced this difference in ROS generation between cells attached to either COL I or IV. The results using antibodies against integrins also indicated that an alpha(2) integrin-mediated pathway was involved in the different response in ROS generation caused by ECM proteins. These results suggest that contact between altered ECM proteins that are present in advanced diabetic nephropathy and mesangial cells has the potential to increase intracellular oxidative stress, leading to progressive glomerular damage.     

Metabolic engineering to produce phytochromes with phytochromobilin, phycocyanobilin, or phycoerythrobilin chromophore in Escherichia coli

By co-expression of heme oxygenase and various bilin reductase(s) in a single operon in conjunction with apophytochrome using two compatible plasmids, we developed a system to produce phytochromes with various chromophores in Escherichia coli. Through the selection of different bilin reductases, apophytochromes were assembled with phytochromobilin, phycocyanobilin, and phycoerythrobilin. The blue-shifted difference spectra of truncated phytochromes were observed with a phycocyanobilin chromophore compared to a phytochromobilin chromophore. When the phycoerythrobilin biosynthetic enzymes were co-expressed, E. coli cells accumulated orange-fluorescent phytochrome. The metabolic engineering of bacteria for the production of various bilins for assembly into phytochromes will facilitate the molecular analysis of photoreceptors.