Killing of cancer cells through the use of eukaryotic expression vectors harbouring genes encoding nucleases and ribonuclease inhibitor.

Cancer gene therapy vectors are promising tools for killing cancer cells with the purpose of eradicating malignant tumours entirely. Different delivery methods of vectors into the cancer cells, including both non-viral and viral, as well as promoters for the targeted expression of genes encoding anticancer proteins were developed for effective and selective killing of cancer cells without harming healthy cells. Many vectors have been created to kill cancer cells, and some vectors suppress malignant tumours with high efficiency. This review is focused on vectors bearing genes for nucleases such as deoxyribonucleases (caspase-activated DNase, deoxyribonuclease I-like 3, endonuclease G) and ribonucleases (human polynucleotide phosphorylase, ribonuclease L, α-sarcin, barnase), as well as vectors harbouring gene encoding ribonuclease inhibitor. The data concerning the functionality and the efficacy of such vectors are presented.

Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells.

BACKGROUND: Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. RESULTS: Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. CONCLUSION: Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins.

Eukaryotic expression vectors bearing genes encoding cytotoxic proteins for cancer gene therapy.

Cancer gene therapy is a promising direction for the treatment of cancer patients. A primary goal of all cancer therapies is to selectively target and kill tumour cells. Such therapies are administered via different approaches, including both viral and non-viral delivery; however, both methods have advantages and disadvantages. Transcriptional targeting enables genes encoding toxic proteins to be expressed directly in cancer cells. Numerous vectors have been created with the purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Data concerning the function of vectors bearing genes that encode cytotoxic proteins under the control of different promoters, including tissue/tumour specific and constitutive promoters, is summarised here. This review focuses on vectors that bear genes encoding diphtheria toxin, Pseudomonas exotoxin A, caspases, gef, streptolysin, and melittin. Data describing the efficacy of such vectors have been summarised. Notably, there are vectors that killed cancer cell lines originating from the same type of cancer with differential efficiency. Thus, there is differential inhibition of cancer cell growth dependent on the cell line. In this review, the constructs employing genes whose expression induces cell death and the efficiency with which they suppress cancer cell growth will be summarised.

Purification and partial characterisation of pectin methylesterase produced by Fusarium asiaticum.

Fusarium asiaticum is the predominant causal agent of Fusarium head blight (FHB) in China. When grown in liquid cultures containing potato tuber extract as the sole carbon source, F. asiaticum (strain 7071) from wheat (China) produced pectin methylesterase (PME), polygalacturonase (PG), and pectin lyase (PNL). The activity of these pectolytic enzymes was detected by a gel diffusion assay. Three forms of PME were identified in a culture filtrate of F. asiaticum. Two forms of PME with molecular weights of 31 kDa and 42.5 kDa, as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), were purified using a combination of chromatographic techniques. These PMEs did not bind to Concanavalin A (Con A), which was confirmed by rechromatography using a Con A agarose column. The 31 kDa purified PME was thermostable in a temperature range of 25-55 °C. The optimal pH for the PME of F. asiaticum was 6.5. This research provides the basis for future investigations of pectolytic enzymes from F. asiaticum.

Construction of the plasmid for expression of ETA-EGFP fusion protein under control of the cytomegalovirus promoter and its effects in HeLa cells.

Tumor-targeted vectors encoding toxic protein genes are promising tools for treating malignant tumors. We used the pEGFP-N1 vector to construct a novel plasmid (pCMV-ETA-EGFP) for eukaryotic expression of a truncated Pseudomonas aeruginosa exotoxin A (ETA) that is known to inhibit protein synthesis, and subsequently induce cell death, by inactivation of elongation factor-2. ETA was linked to the enhanced green fluorescent protein (EGFP) gene, and ETA-EGFP gene expression was driven by the cytomegalovirus (CMV) promoter. The time-lapse effects of pCMV-ETA-EGFP expression were examined in transiently transfected HeLa cells. HeLa cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and small er, Cyrilliccapital IE, CyrillicGFP-N1 showed lower fluorescence intensity than cells transfected with pEGFP-N1 alone. Analysis of the number of dead cells further confirmed the highly toxic effect of the ETA-EGFP fusion protein on cells transfected with pCMV-ETA-EGFP or cotransfected with pCMV-ETA-EGFP and small er, Cyrilliccapital IE, CyrillicGFP-N1. ETA-EGFP fusion protein induced apoptotic cell death through the caspase-3 activation. By using the antibody against a marker nucleolar antigen A3 [Grigoryev, A.A., Bulycheva, T.I., Sheval, E.V., Kalinina, I.A., Zatsepina, O.V., 2008. Cytological indicators of the overall suppression of protein synthesis revealed by staining with new monoclonal antibody. Cell Tissue Biol. 2, 191-199], the distribution of which changes when HeLa cells are treated with known translation inhibitors, we obtained evidence to support the idea that protein synthesis is inhibited in transfected cells in situ. ETA-EGFP fusion protein was identified in lysates of transfected cells using anti-GFP-BL antibodies. Collectively, our results indicate that HeLa cells transfected with pCMV-ETA-EGFP synthesize the ETA-EGFP fusion protein that efficiently inhibits protein synthesis, leading to massive cell death by an apoptosis-mediated pathway with a participation of caspase-3. The constructed vector can be used in suicidal gene therapy of cancer and may also be useful for investigating the general effects of translational downregulation in human cancer cells. We also suggest a novel approach for detecting the activity of new vectors in transfected cells, which is based on the redistribution of nucleolar proteins in transfected cells.

A new vector for controllable expression of an anti-HER2/neu mini-antibody-barnase fusion protein in HEK 293T cells.

Tumor-targeted vectors with controllable expression of therapeutic genes and specific antitumor antibodies are promising tools for the reduction of malignant tumors. Here we describe a new plasmid for the eukaryotic expression of an anti-HER2/neu mini-antibody-barnase fusion protein (4D5 scFv-barnase-His(5)) with an NH(2)-terminal leader peptide. The 4D5 scFv-barnase-His(5) gene was placed downstream of the tetracycline responsive-element minimal promoter in the vector using the Tet-Off gene-expression system. The Bacillus amyloliquefaciens ribonuclease barnase is toxic for the host cells. To overcome this problem, barstar gene under its own minimal cytomegalovirus promoter was used in designed vector. Barstar inhibits the background level of barnase in the cells in the presence of tetracycline in culture medium. The HEK 293T cells were transfected with the designed vector, and the 4D5 scFv-barnase-His(5) fusion protein was identified by anti-barnase antibodies in cell culture medium and after purification from cell lysates using metal-affinity chromatography. The overexpression of the anti-HER2/neu mini-antibody-barnase fusion protein decreased the intensity of fluorescence of HEK 293T cells co-transfected with the generated plasmid and a plasmid containing the gene of enhanced green fluorescent protein (pEGFP-N1), in comparison with the intensity of fluorescence of HEK 293T cells transfected with pEGFP-N1, in the absence of tetracycline in the medium. The effect of the 4D5 scFv-barnase-His(5) on EGFP fluorescence indicates that the introduced barnase functions as a ribonuclease inside the cells. The anti-HER2/neu mini-antibody could be used to deliver barnase to HER2/neu-positive cells and provide its penetration into the target cells, as HER2/neu is a ligand-internalizing receptor. This expression vector has potential applications to both gene and antibody therapies of cancer.