ABSTRACT
Foxp3, a member of the forkhead transcription factor family, is a master gene that controls the development and function of CD4(+)CD25(+) regulatory T (Treg) cells. It is thought to contribute to pathogenesis of many different tumors, including ovarian carcinoma and pancreatic, breast and pancreatic ductal adenocarcinoma. Selectively depleted Foxp3-expressing cells with anit-CD25 antibodies or vaccination of Foxp3 mRNA-transfected dendritic cells engender protective immunity against tumor. This study targeted silencing Foxp3 gene expression using RNA interference (RNAi) delivered by a lentiviral vector to evaluate the therapeutic role of Foxp3 short-hairpin RNAs (shRNAs) in a murine model of leukemia. RLmale symbol1, a mouse CD4(+)CD25(+) leukemia cell with Foxp3 expression, was used as the leukemia animal model. By infecting RLmale symbol1 cells with Lenti-Foxp3-siRNA, we reduced Foxp3 gene expression and the suppressive function of CD4(+)CD25(-) effector cells stimulated with ConA. Moreover, lentiviral-mediated Foxp3 RNAi transduced into RLmale symbol1 cell or injected into the tumor showed suppressive effects on tumor growth and prolonged the survival of tumor-transplanted mice. However, this suppressive effect was abrogated in NOD-SCID mice transplanted with Lenti-Foxp3-siRNA-infected RLmale symbol1 cells. In conclusion, inhibiting Foxp3 gene expression by shRNAs effectively decreases tumor growth of Treg cell-like leukemia. The results may provide a novel strategy for future immunotherapy against cancers.
Subject(s)
Forkhead Transcription Factors/antagonists & inhibitors , Lentivirus/genetics , Leukemia, Experimental/therapy , Leukemia, T-Cell/therapy , RNA Interference , Animals , Cell Line, Tumor , Genetic Therapy , Leukemia, Experimental/genetics , Leukemia, T-Cell/genetics , Male , Mice , Mice, Inbred BALB C , RNA StabilityABSTRACT
A novel ligninolytic peroxidase gene (ACLnP) was cloned and characterized from a poroid brown-rot fungus, Antrodia cinnamomea. The genomic DNA of the fungus harboured two copies of ACLnP, with a length of 2111 bp, interlaced with 12 introns, while the full-length cDNA was 1183 bp, with a 66 bp signal peptide and an ORF of 990 bp. The three-dimensional molecular structure model was comparable to that of the versatile peroxidase of Pleurotus eryngii. ACLnP was cloned into vector pQE31, successfully expressed in Escherichia coli strain M15 under the control of the T5 promoter and produced a non-glycosylated protein of about 38 kDa, pI 5.42. The native and recombinant ACLnP was capable of oxidizing the redox mediator veratryl alcohol, and also decolorized bromophenol blue and 2,6-dimethoxyphenol dyes, implicating a functional extracellular peroxidase activity. The significance of discovering a functional ACLnP gene in A. cinnamomea in terms of wood degradation and colonization capacity in its unique niche is discussed.
Subject(s)
Antrodia/enzymology , Cloning, Molecular , Escherichia coli/genetics , Fungal Proteins/metabolism , Gene Expression , Lignin/metabolism , Peroxidase/metabolism , Escherichia coli/metabolism , Fungal Proteins/genetics , Fungi/classification , Fungi/enzymology , Molecular Sequence Data , Peroxidase/genetics , PhylogenyABSTRACT
Feeding of an "obligate" plant-parasitic nematode (nonfungal feeder), Pratylenchus scribneri, in the absence of plant tissue was demonstrated in an artificial system consisting of liquid media and indicator dyes including amaranth and various nontoxic food colors. Among the compounds tested, sucrose, dextrose, Gamborg's B5 medium, and DL-methionine stimulated a small percentage of feeding (12-36%). A high percentage of feeding (90-100%) occurred in a filtrate from excised corn roots cultured in Gamborg's B5 medium. This feeding system has the potential to develop an artificial medium for plant-parasitic nematodes and to screen novel nematicides that are stomach poisons.
