A gel-less isolation of untagged plasmid DNA insert from vector backbone in homogeneous format.

Agarose gel electrophoresis with subsequent DNA extraction from gel is routinely used for DNA fragment isolation after plasmid DNA digestion. We describe a gel-less method for DNA fragment isolation after plasmid DNA digestion which is based on in-solution negative selection through depletion of vector backbone bearing LoxP sites by sorption on solid phase-immobilized mutated Cre recombinase. The method might be especially useful in preparation of DNA fragments for transgenic animal generation where residual agarose presence is a concern, and DNA fragments are frequently large in size and thus might be mechanically damaged during purification with conventional affinity-based gel extraction methods.

hTERT and BIRC5 gene promoters for cancer gene therapy: A comparative study.

Human telomerase reverse transcriptase (hTERT) and survivin (BIRC5) gene promoters are frequently used for transcriptional targeting of tumor cells, yet there is no comprehensive comparative analysis allowing rational choice of a promoter for a particular therapy. In the current study, the transcriptional activity of hTERT, human BIRC5 and mouse Birc5 promoters and their modifications were compared in 10 human cancer cell lines using the luciferase reporter gene activity assay. The results revealed that BIRC5- and hTERT-based promoters had strikingly different cell specificities with comparable activities in only 40% of cell lines. Importantly, relative hTERT and BIRC5 transcript abundance cannot be used to predict the most potent promoter. Among the hTERT-based promoters that were assessed, modification with the minimal cytomegalovirus promoter generally resulted in the most potent activity. Mouse Birc5 and modified human BIRC5 promoters were superior to the unmodified human survivin promoter; however, their tumor specificities must be investigated further. In summary, the present results emphasize the desirability for construction of more universal tumor-specific promoters to efficiently target a wide spectrum of tumor cells.

LY294002 enhances expression of proteins encoded by recombinant replication-defective adenoviruses via mTOR- and non-mTOR-dependent mechanisms.

Adenovirus-based drugs are efficient when combined with other anticancer treatments. Here we show that treatment with LY294002 and LY303511 upregulates expression of recombinant proteins encoded by replication-defective adenoviruses, including expression of therapeutically valuable combination of herpes simplex virus thymidine kinase controlled by human telomerase reverse transcriptase promoter (Ad-hTERT-HSVtk). In line with this, treatment with LY294002 synergized with Ad-hTERT-HSVtk infection in the presence of gancyclovir prodrug on Calu-I lung cancer cell death. The effect of LY294002 and LY303511 on adenovirus-delivered transgene expression was demonstrated in 4 human lung cancer cell lines. LY294002-induced upregulation of adenovirally delivered transgene is mediated in part by direct inhibition of mTOR protein kinase in mTORC2 signaling complex thus suggesting that anticancer drugs targeting mTOR will also enhance expression of transgenes delivered with adenoviral vectors. As both LY294002 and LY303511 are candidate prototypic anticancer drugs, and many mTOR inhibitors for cancer treatment are under development, our results have important implication for development of future therapeutic strategies with adenoviral gene delivery.

The RHOV gene is overexpressed in human non-small cell lung cancer.

Rho family GTPases act as molecular switches to regulate numerous cellular processes, including malignant transformation. Commonly, overexpression of Rho GTPases contributes to tumorigenesis. Elevated expression of several Rho GTPases has been reported in lung cancer and is associated with poor prognosis. The RHOV gene encodes the atypical Rho family GTPase Chp/RhoV, which is capable of transforming fibroblasts, although other functions of Chp remain largely elusive. RHOV is expressed in normal lung tissue in rats, but not in humans. RHOV expression was found in several human cancer cell lines, including non-small-cell lung cancer (NSCLC) cell line A549, but expression of RHOV in NSCLC tumors has never been investigated. Here we studied the expression of the RHOV gene in lung cancer cell lines and in 29 matched pairs of NSCLC tumors and adjacent nontumorous tissues. We found that RHOV is expressed in lung cancer cell lines and is upregulated in the majority of studied lung tumors. Analysis of the Oncomine database revealed correlation between elevated RHOV level and poor patient survival. We propose that the RHOV gene could be validated as a diagnostic or prognostic marker for NSCLC, and that observed overexpression of RHOV might contribute to tumorigenesis.

Rho family GTPase Chp/RhoV induces PC12 apoptotic cell death via JNK activation.

Rho GTPases regulate numerous cellular processes including apoptosis. Chp/RhoV is an atypical Rho GTPase which functions are poorly understood. Here we investigated the role of Chp in regulation of cell viability using PC12 cells with inducible expression of Chp as a model. We found that expression of Chp results in apoptosis in PC12 cells. Chp-induced apoptosis was accompanied by activation of JNK signaling and both death receptor-mediated and mitochondrial apoptotic pathways as justified by caspase-8 and caspase-9 activation, respectively. Moreover, inhibition of JNK by SP600125 rescued PC12 cells from Chp-triggered cell death and attenuated activation of caspases-9 and -3/7 suggesting that activation of JNK mediates pro-apoptotic effect of Chp. Expression of Chp resulted in increased phosphorylation of c-Jun in PC12 cells, and Chp expression in HE K293 cells upregulated AP-1-dependent transcription in a JNK-dependent manner. Together results of our study reveal the role of Chp GTPase as a putative regulator of JNK-dependent apoptotic death in PC12 cells, similarly to previously described pro-apoptotic activity of the related Cdc42 and Rac1 GTPases.