REDUCTION IN VIABILITY OF HUMAN CERVIX CARCINOMA HeLa CELLS UNDER TRANSFER OF p21 GENE WITH BLOCK POLYMERIC DIMETHYLAMINOETHYL METHACRYLATE-BASED CARRIER
DOI: http://dx.doi.org/10.30970/sbi.1602.681
Abstract
Background. Various cationic polymers have been proposed for use as carriers of genetic materials in biotechnology and biomedicine. The poly(2-dimethylamino)ethyl-methacrylate (poly(DMAEMA)) demonstrated high transfection efficiency and low cytotoxicity when used as a gene delivery system. The aim of the present study was to evaluate the ability of poly(DMAEMA)-block-poly(N-vinylpyrrolidone)-co-(butyl-acrylate)-co-2-aminoethyl methacrylate carrier, BP83-1, to deliver p21 gene into human cervix carcinoma HeLa cells and to define its effects on the viability of tumor cells in vitro.
Methods. Transfection assay, Western-blot analysis, MTT test, DNA comet analysis in alkaline conditions, diphenylamine assay for DNA fragmentation (Barton’s assay), FACS analysis of cell cycling.
Results. The BP83-1 polymer effectively transferred pFlag-P21WT plasmid DNA containing p21 gene into human cervix carcinoma HeLa cells. The level of BP83-1-facilitated delivery of p21 into HeLa cells was significantly higher than the level achieved with linear polyethyleneimine (PEI). A significant (26.1 % and 40.1 %) reduction in the viability of HeLa cells transfected with pDNA/BP83-1 and pDNA/PEI polyplexes was detected compared to non-transfected cells. The reverse dependence between the elevated amount of p21 and the reduced amount of Cdk2 was observed in the transfected HeLa cells. The number of cells in G1 phase of the cell cycle in HeLa cells increased from 54.9 % to 65.8 % and to 64.9 % after their transfection with pFlag-P21WT/BP83-1 and pFlag-P21WT/PEI polyplexes, correspondingly. Besides, an increased number of single-strand breaks in DNA and content of the fragmented DNA was detected in HeLa cells transfected with pDNA/BP83-1 and pDNA/PEI polyplexes. The DNA damaging effects of the BP83-1 carrier and pDNA/BP83-1 polyplex were less pronounced in treated HeLa cells, compared with such effects of PEI and pDNA/PEI polyplex.
Conclusion. An effective transfer of p21 gene with BP83-1 carrier into human cervix carcinoma HeLa cells was demonstrated. The overexpression of p21 gene led to inhibition of viability of HeLa cells, DNA damage, and blocking of cell cycle progression from G1 phase to S phase via a reduction of the amount of cyclin-dependent kinase 2 (Cdk2) and accumulation of cells in G1 phase.
Keywords
poly(2-dimethylamino)ethyl-methacrylate, polymeric carrier, gene transfer, p21, cytotoxic action, cell cycle, DNA damage
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