Hypoxia inducible factor activates the transforming growth factor-alpha/epidermal growth factor receptor growth stimulatory pathway in VHL(-/-) renal cell carcinoma cells

Bi-allelic-inactivating mutations of the VHL tumor suppressor gene are found in the majority of clear cell renal cell carcinomas (VHL(-/-) RCC). VHL(-/-) RCC cells overproduce hypoxia-inducible genes as a consequence of constitutive, oxygen-independent activation of hypoxia inducible factor (HIF). While HIF activation explains the highly vascularized nature of VHL loss lesions, the relative role of HIF in oncogenesis and loss of growth control remains unknown. Here, we report that HIF plays a central role in promoting unregulated growth of VHL(-/-) RCC cells by activating the transforming growth factor-alpha (TGF-alpha)/epidermal growth factor receptor (EGF-R) pathway. Dominant-negative HIF and enzymatic inhibition of EGF-R were equally efficient at abolishing EGF-R activation and serum-independent growth of VHL(-/-) RCC cells. TGF-alpha is the only known EGF-R ligand that has a VHL-dependent expression profile and its overexpression by VHL(-/-) RCC cells is a direct consequence of HIF activation. In contrast to TGF-alpha, other HIF targets, including vascular endothelial growth factor (VEGF), were unable to stimulate serum-independent growth of VHL(-/-) RCC cells. VHL(-/-) RCC cells expressing reintroduced type 2C mutants of VHL, and which retain the ability to degrade HIF, fail to overproduce TGF-alpha and proliferate in serum-free media. These data link HIF with the overproduction of a bona fide renal cell mitogen leading to activation of a pathway involved in growth of renal cancer cells. Moreover, our results suggest that HIF might be involved in oncogenesis to a much higher extent than previously appreciated.     

<Emphasis Type="Italic">Rol</Emphasis> (root loci) gene as a positive selection marker to produce marker-free <Emphasis Type="Italic">Petunia hybrida</Emphasis>

The presence of marker genes conferring antibiotic or herbicide resistance in transgenic plants has been a serious problem for their public acceptance and commercialization. MAT (multi-auto-transformation) vector system has been one of the strategies to excise the selection marker gene from transgenic plants. Agrobacterium tumefaciens strain EHA105 harboring a rol-type MAT vector, pMAT101, was used to produce morphologically normal transgenic Petunia hybrida ‘Dainty Lady’ employing rol gene as the selection marker gene. LacZ gene was used as a model gene of interest. Infected explants were cultured on plant growth regulator (PGR)- and antibiotic-free half-strength MS medium. Sixty-five percent of the infected explants produced hairy roots. The hairy roots were separated and proliferated on 1/2 MS hormone-free medium. Shoots produced from the hairy roots on 1/2 MS medium supplemented with benzylaminopurine (BA) and naphthalene acetic acid (NAA) exhibited hairy root syndrome (Ri syndrome) such as dwarfed, reduced apical dominance, short internodes and increased rooting, but subsequently produced normal-looking marker-free shoots. Molecular analysis of DNA from the hairy roots, shoots with Ri syndrome and morphologically normal shoots revealed that the normal shoots had only LacZ gene, and the removable cassette consisting of rol, R (recombinase) and GUS genes was excised. From this study it can be concluded that the chimeric rol genes can be used as a selection marker for Agrobacterinum-mediated transformation of Petunia hybrida and that the production of marker-free normal transgenic plants is possible without using selective chemical agents employing rol-type MAT vector.     

Screening and Characterization of Some Lactobacillaceae for Detection of Cholesterol-Lowering Activities

Probiotics and Antimicrobial Proteins - Dyslipidemia, specifically abnormal levels of low-density lipoprotein cholesterol (LDL-C), is an important risk factor of cardiovascular disease. Evidence...     

Caveolin-1-dependent and -independent membrane domains

Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1^−/−) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1^−/−-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1^−/− compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.     

Stable integration and expression of wasabi defensin gene in “Egusi” melon (Colocynthis citrullus L.) confers resistance to Fusarium wilt and Alternaria leaf spot

Production of “Egusi” melon (Colocynthis citrullus L.) in West Africa is limited by fungal diseases, such as Alternaria leaf spot and Fusarium wilt. In order to engineer “Egusi” resistant to these diseases, cotyledonary explants of two “Egusi” genotypes, ‘Ejagham’ and NHC1-130, were transformed with Agrobacterium tumefaciens strain EHA101 harbouring wasabi defensin gene (isolated from Wasabia japonica L.) in a binary vector pEKH1. After co-cultivation for 3 days, infected explants were transferred to MS medium containing 100 mgl^−l kanamycin to select transformed tissues. After 3 weeks of culture, adventitious shoots appeared directly along the edges of the explants. As much as 19 out of 52 (36.5%) and 25 out of 71 (35.2%) of the explants in genotype NHC1-130 and ‘Ejagham’, respectively, formed shoots after 6 weeks of culture. As much as 74% (14 out of 19) of the shoots regenerated in genotype NHC1-130 and 72% (18 out of 25) of those produced in genotype ‘Ejagham’ were transgenic. A DNA fragment corresponding to the wasabi defensin gene or the selection marker nptII was amplified by PCR from the genomic DNA of all regenerated plant clones rooted on hormone-free MS medium under the same selection pressure, suggesting their transgenic nature. Southern blot analysis confirmed successful integration of 1–5 copies of the transgene. RT-PCR, northern and western blot analyses revealed that wasabi defensin gene was expressed in transgenic lines. Transgenic lines showed increased levels of resistance to Alternaria solani, which causes Alternaria leaf spot and Fusarium oxysporum, which causes Fusarium wilt, as compared to that of untransformed plants.     

Tetrasubstituted naphthalene diimide ligands with selectivity for telomeric G-quadruplexes and cancer cells

A series of tetrasubstituted naphthalene diimide compounds with N-methylpiperazine end groups has been synthesized and evaluated as G-quadruplex ligands. They have high affinity and selectivity for telomeric G-quadruplex DNA over duplex DNA. CD studies show that they induce formation of a parallel G-quadruplex topology. They inhibit the binding of hPOT1 and topoisomerase IIIα to telomeric DNA and inhibit telomerase activity in MCF7 cells. The compounds have potent activity in a panel of cancer cell lines, with typical IC(50) values of ～0.1 μM, and up to 100-fold lower toxicity in a normal human fibroblast cell line.     

The role of glutathione reductase in the cytotoxicity of chromium (VI) in isolated rat hepatocytes

Chromium (VI) is an environmental and occupational carcinogen, and it is accepted that intracellular reduction is necessary for DNA damage and cytotoxicity. We have investigated the interaction of Cr(VI) with hepatocytes in vitro to determine the contribution of various hepatic enzymes to the reduction of Cr(VI). Cr(VI) caused a dose-dependent decrease in cell viability and intracellular reduced glutathione (GSH) levels between 100 and 500 microM within 3 h exposure of hepatocytes. Both DT-diaphorase and cytochrome P450 play only a minor role in detoxifying Cr(VI) and/or its metabolites. (GSH) appears to act as a non-enzymatic reductant, reducing Cr(VI) to a toxic form. The evidence for this is two-fold. Firstly, GSH was depleted during the metabolism of Cr(VI) and, secondly, pretreatment of the cells with diethylmaleate to deplete GSH levels, partially protected the cells from Cr(VI) toxicity. Glutathione reductase appears to play an important role in the enzymatic reduction of Cr(VI) as inhibition of this enzyme by carmustine (BCNU) markedly protected the cells from cytotoxicity.     

Targeting pancreatic cancer with a G-quadruplex ligand

The integrity of telomeres in most cancer cells is maintained by the action of the telomerase enzyme complex, which catalyzes the synthesis of telomeric DNA repeats in order to replace those lost during replication. Telomerase is especially up-regulated in metastatic cancer and is thus emerging as a major therapeutic target. One approach to telomerase inhibition involves the sequestration of the single-stranded 3' ends of telomeric DNA into higher-order quadruplex structures. We have recently shown that tetra-substituted naphthalene diimide compounds are potent quadruplex-stabilizing molecules with telomerase inhibitory activity in cells. We show here that one such compound, BMSG-SH-3, which has been optimized by computer modeling, has significant in vivo antitumor activity against a model for pancreatic cancer, a cancer that is especially resistant to current therapies. A large reduction in telomerase activity in treated tumors was observed and the naphthalene diimide compound was found to be selectively localized in the treated tumors. We find that the expression of the therapeutically important chaperone protein HSP90, a regulator of telomerase is also reduced in vivo by BMSG-SH-3 treatment. The compound is a potent stabilizer of two G-quadruplex sequences found in the promoter region of the HSP90 gene, as well as a G-quadruplex from human telomeric DNA. It is proposed that the simultaneous targeting of these quadruplexes may be an effective anti-tumor strategy.     

Structure-based design of benzylamino-acridine compounds as G-quadruplex DNA telomere targeting agents

The design, synthesis, biophysical and biochemical evaluation is presented of a new series of benzylamino-substituted acridines as G-quadruplex binding telomerase inhibitors. Replacement of the previously reported anilino substituents by benzylamino groups results in enhanced quadruplex interaction, and for one compound, superior telomerase inhibitory activity.