RESUMO
A synthetic genetic chain was created-a two-promoter integrator, the activity of the output signal of which is high only with the high activity of two promoters at the input of the chain. The integrator is shown to work by the example of cancer-specific promoters of the human survivin and telomerase genes that can be used for specific transcription of target genes in tumor cells. To create the integrator, we used the well-known TET-ON 3G transactivator, regulated by doxycycline.
Assuntos
Doxiciclina/farmacologia , Regiões Promotoras Genéticas , Transativadores , Humanos , Survivina/genética , Telomerase/genética , TetraciclinaRESUMO
Targeted toxins are promising anticancer agents that allow selectively destroying cancer cells due to the increased content of onco-specific markers on their surface. The use of such anti-cancer toxins in medicine is mainly hampered by their high non-specific toxicity, in particular, hepatotoxicity. In our work on human cell line, we have shown that the removal of the DARPin-PE40 translocation toxin domain leads to a decrease in hepatotoxicity. The same effect is also observed when inactivation of the furin cleavage site in the DARPin-PE40 molecule was done. Simultaneous removal of both the translocation domain and the furin cleavage site showed the best results. This toxin modification can be used to create more selective anti-cancer toxins.
Assuntos
Antineoplásicos/efeitos adversos , Furina/química , Furina/metabolismo , Fígado/efeitos dos fármacos , Terapia de Alvo Molecular , Antineoplásicos/uso terapêutico , Sítios de Ligação , Linhagem Celular Tumoral , Células Hep G2 , Humanos , Domínios Proteicos , Transporte Proteico/efeitos dos fármacosRESUMO
Stable red fluorescing line of human ovarian epithelial cancer cells SK-OV-3ip-red was generated expressing gene coding for protein TurboFP635 (Katushka) fluorescing in the far-red spectrum region with excitation and emission peaks at 588 and 635 nm, respectively. Fluorescence of SK-OV-3ip-red line remained high during long-term cell culturing and after cryogenic freezing. The obtained cell line SK-OV-3ip-red can serve a basis for a model of a scattered tumor with numerous/extended metastases and used both for testing anticancer drugs inhibiting metastasis growth and for non-invasive monitoring of the growth dynamics with high precision.
Assuntos
Proteínas Luminescentes/biossíntese , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/patologia , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Clonagem Molecular , Feminino , Expressão Gênica , Humanos , Proteínas Luminescentes/genética , Transfecção , Proteína Vermelha FluorescenteRESUMO
The HER2/neu receptor is over-expressed on the surface of many types of cancer cells, and is widely used for tar- geted delivery of anticancer drugs. We have created geneti- cally engineered construct expressing the PE40 fragment of Pseudomonas toxin bound with the DARPin molecule which recognizes the HER2/neu receptor with high specificity. The construct destroyed transfected tumor cells in vitro. Intra-tumor injections of the construct complexed with polyethyleneimine led to growth retardation of D2F2/E2 tumors in mice. These results suggest a possibility of using this approach to develop new anticancer drugs.
Assuntos
Toxinas Bacterianas , Terapia Genética/métodos , Neoplasias/terapia , Pseudomonas/genética , Receptor ErbB-2/agonistas , Proteínas Recombinantes de Fusão , Células 3T3 , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Células CHO , Cricetulus , Células HEK293 , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genéticaRESUMO
Postnatal development of mammalian oocytes is accompanied by functional and structural remodeling of the nucleolar apparatus: the final stage of this process is the formation of large objects (up to 10 µm in diameter) termed nucleolus-like bodies (NLBs) in preovulatory GV oocytes. N LB material was shown to be essential for early embryonic development, but its composition is still uncharacterized. In the present study, the protein-binding dye fluorescein-5-isothiocyanate (FITC) was used to show that proteins characterized by a high local concentration are essential NLB components in mouse GV oocytes. One of these proteins was able to be identified for the first time using a mild treatment of oocytes with proteinase K; the protein identified was fibrillarin, a factor of early pre-rRNA processing. Fibrillarin is present in the inner NLB mass of all oocytes capable of synthesizing rRNA; however, it is not colocalized with BrUTP microinjected into oocytes in order to identify transcribed ribosomal genes, in contrast to the "surface" fibrillarin. These observations imply the accumulation of nucleolar proteins not involved in ribosome biogenesis inside the NLB. All NLBs present in an individual nucleus of an NSN-type GV oocyte contain fibrillarin and are associated with active ribosomal genes. The results obtained in the present work demonstrate that proteinase K treatment of GV mouse oocytes allows for: (1) identification of "cryptic" proteins inside the densely packed NLB material and (2) the enhancement of oocyte image quality during BrUTP-based identification of rRNA synthesis sites but (3) not for the detection of active ribosomal genes in the inner mass of the NLB. The fluorescent dye FITC can be recommended for assessment of intracellular protein localization in the oocytes of all mammalian species.
Assuntos
Nucléolo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Endopeptidase K/química , Oócitos/metabolismo , Precursores de RNA/biossíntese , Transcrição Gênica/fisiologia , Animais , Feminino , Camundongos , Oócitos/citologiaRESUMO
In this study, we investigated the possibility of phototoxic flavoprotein miniSOG (photosensitizer) excitation in cancer cells by bioluminescence occurring when luciferase NanoLuc oxidizes its substrate, furimazine. We have shown that the phototoxic flavoprotein miniSOG expressed in eukaryotic cells in fusion with NanoLuc luciferase is activated in the presence of its substrate, furimazine. Upon such condition, miniSOG possesses photoinduced cytotoxicity and causes a 48% cell death level in a stably transfected cell line.
RESUMO
During the past two decades, there have been numerous attempts at using animals in order to produce recombinant human proteins and monoclonal antibodies. However, it is only recently that the first two therapeutic agents isolated from the milk of transgenic animals, C1 inhibitor (Ruconest) and antithrombin (ATryn), appeared on the market. This inspires hope that a considerable number of new recombinant proteins created using such technology could become available for practical use in the near future. In this review, the methods applied to produce transgenic animals are described and the advantages and drawbacks related to their use for producing recombinant human proteins and monoclonal antibodies are discussed.