Efeito dual de FGF2 e PMA em células HEK 293 transformadas por H-rasV12 / Dual effects of FGF2 and PMA on H-rasV12 transformed HEK293 cell line

Sabe-se há décadas que mutações nos genes ras estão presentes em cerca de 20% dos cânceres humanos, mas o desenvolvimento de terapias eficazes para o tratamento de câncer dependente dos oncogenes ras permanece um desafio científico importante. Nesse contexto, o nosso grupo publicou recentemente resultados interessantes mostrando que FGF2 exógeno ou PMA, contrariamente à expectativa geral, inibem a proliferação de células de camundongo malignas dependentes dos oncogenes H- ou K-Ras. Para dar continuidade a estes estudos o projeto desta tese foi planejado para investigar os mecanismos subjacentes a possíveis efeitos citotóxicos de FGF2 e PMA em células humanas transformadas por ras. Para esse fim, a linhagem humana imortalizada HEK 293 foi condicionalmente transformada pela expressão ectópica da construção quimérica de DNA ER:H-rasV12, que codifica a oncoproteína de fusão ER:H-RasV12, cuja atividade é induzível por 4-hidroxi-tamoxifen (4OHT). Essa abordagem nos permitiu verificar os efeitos de FGF2 e PMA em sublinhagens HEK/ER:HrasV12 fenotipicamente "normais" ou transformadas por níveis crescentes da oncoproteína H-RasV12. Os principais resultados mostraram que tanto FGF2 como PMA tem efeito dual promovendo ou inibindo a proliferação das células transformadas em função da concentração intracelular crescente de H-RasV12. Ensaios de crescimento de colônias em suspensão de agarose mostraram que: a) as células parentais HEK293 não desenvolveram colônias mesmo quando tratadas com FGF2 ou PMA, resultados que estão de acordo com seu fenótipo não tumoral; b) mas, as sublinhagens HEK/ER:HrasV12 deram origem a colônias mesmo quando tratadas com concentrações pequenas de 4OHT, que condicionaram níveis intracelulares baixos de ER:HRasV12; nestas condições experimentais, FGF2 foi um forte promotor do crescimento de colônias, condizente com sua reconhecida atividade promotora do crescimento de células tumorais em suspensão; ainda nestas condições, PMA não teve efeito significante sobre o crescimento de colônias; c) coerentemente, concentrações elevadas de 4-OHT levaram aos níveis intracelulares mais altos de ER:HRasV12 e, por conseguinte, a desenvolvimento máximo de colônias de células HEK/ER:HrasV12, no entanto, nestas condições, ambos FGF2 e PMA inibiram completamente o crescimento de colônias. Por outro lado, transformação de HEK293 com um vetor de expressão constitutiva de HrasV12 levou à seleção e isolamento das sublinhagens tumorais HEK/HrasV12, cujo fenótipo se caracterizou por: a) nenhum efeito de FGF2 sobre a sua proliferação e b) forte inibição de sua proliferação por PMA. A ação citotóxica de PMA exclusivamente observada em células HEK 293 transformadas por H-rasV12 se caracterizou por: a) total dependência de PKC, provavelmente mediada pela ativação proteolítica específica de PKC δ; b) envolvimento de níveis elevados e sustentados de ROS com disparo tardio de apoptose

It is known for nearly 20 years that mutated ras oncogenes are found in 20% of human malignancies, however efficacious therapies are not yet available for Ras-driven cancer. Along of these lines, our group recently published provocative results showing, against common belief, that FGF2 and PMA inhibited proliferation of Ras-dependent malignant mouse cells. Aiming to gain insight into this intriguing phenomenon, the present thesis project was planned to investigate the possible cytotoxicity of FGF2 and PMA in human Ras-driven malignant cells. To this end an immortalized non-tumorigenic human cell line (HEK293) was stably transformed with the DNA construction ER:H-rasV12, which encodes the fusion protein ER:H-RasV12, whose activity requires activation by 4-hidroxitamoxifen (4-OHT). This approach allowed us to evaluate FGF2 and PMA effects on HEK/ER:HrasV12 sublines under switching from "normal" to transformed phenotypes upon 4-OHT induction. Our main results have shown that both FGF2 and PMA displayed dual effects promoting or inhibiting proliferation of HEK/ER:HrasV12 cells in function of ER:HRasV12 intracellular levels. Clonogenic assays in agarose suspension have shown: a) parental HEK293 line did not develop colonies under FGF2 and PMA treatment or not, in agreement with its non-tumorigenic nature; b) however, HEK/ER:HrasV12 sublines developed colonies even under low 4-OHT concentrations, which led to low ER:HRasV12 intracellular levels; under these conditions FGF2 strongly promoted colony growth and PMA had no effect; c) furthermore, in HEK/ER:HrasV12 sublines, elevated 4-OHT concentrations led to high ER:HRasV12 intracellular levels and maximal colony growth; but, under these experimental conditions both FGF2 and PMA abolished colony growth. On the other hand, HEK293 transformation with a vector that constitutively express HrasV12 yielded HEK/ER:HrasV12 sublines displaying the following phenotypic traits: a) non FGF2 effects on proliferation and b) severe proliferation inhibition by PMA. PMA toxicity, exclusively observed in HrasV12 -transformed HEK293 cells, was characterized by: a) total dependency on PKC, likely mediated by specific proteolytic activation of PKCδ; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector / Transferência de genes heterólogos para células corneanas epiteliais primárias utilizando vetor lentivírus

PURPOSE: To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP) reporter cassette to primary corneal epithelial cells ex vivo. METHODS: Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80 percent confluency) prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS). We evaluated the transduction efficiency (TE) over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells). RESULTS: Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5th post-transduction day (14.1 percent) and tended to stabilize after the 8th day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7 percent. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified. CONCLUSIONS: Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.

OBJETIVO: Avaliar a transferência de genes heterólogos expressando a proteí­na "Green Fluorescent Protein" (GFP) para células corneanas epiteliais primárias ex vivo utilizando vetor lentivírus. MÉTODOS: Tecido corneoescleral de coelhos foi usado para obtenção de suspensão de células corneanas epitelias. As células foram semeadas na densidade de 5×10³ células/cm² e expandidas por 5 dias até uma confluência de 70-80 por cento antes de serem transduzidas. A transferência genética foi monitorada por microscopia fluorescente e por um separador de células ativadas por fluorescência. Foram avaliadas a eficiência de transdução ao longo do tempo e o efeito dose-resposta de diferentes quantidades de partículas virais. Um grupo de células foi analisado pelo separador de células ativadas por fluorescência para avaliar a transdução de células com fenótipo de cé­lulas tronco do epitélio corneano (baseado na exclusão do corante "Hoechst dye"). RESULTADOS: Os vetores lentivírus foram efetivos na transdução de cé­lulas corneanas epiteliais primárias de coelhos ex vivo. Fotodocumentação das células vivas demonstrou células epiteliais de morfologia normal e expressando o gene fluorescente (GFP). A eficiência de transdução ao longo do tempo foi maior no quinto dia após a transdução (14,1 por cento) e demonstrou uma tendência à estabilidade a partir do oitavo dia após a transdução. O número de células transduzidas foi dose-dependente e atingiu 7 por cento com as maiores concentrações de partículas virais. Quando analisadas pelo separador de células ativadas por fluorescência para detecção de células transduzidas e também de células que excluíram o corante "Hoechst dye", foi detectado que células com fenótipo de células tronco do epitélio corneano ("side-population") também foram transduzidas. CONCLUSÕES: Os vetores lentivirais podem transferir genes heterolólogos para células corneanas epiteliais primárias expandidas ex vivo de forma eficiente. Os genes foram expressos de forma estável ao longo do tempo e puderam ser transferidos tanto para células epiteliais maduras como para presumíveis células tronco epiteliais. A eficiência de transdução foi obtida de forma dose-dependente.

Can painting human cells with exogenous maltoporin enable efficient therapeutic gene transfer by bacteriophage lambda vectors? / Las células humanas marcadas con maltoporinas exógenas, ¿permiten una terapia eficiente de tranferencia de genes por vectores bacteriofagos lambda?

Many gene therapy strategies require transfer of high-molecular weight DNA into human cells. To enable clinical trials, these vectors need to be produced on a large scale and at low cost. The production of effective high-capacity vectors like HSV-amplicons and helper-dependent adenoviral vectors is difficult to up-scale, so new inexpensive vectors are needed for the efficient delivery of high-molecular weight DNA to human cells. Bacteriophage lambda vectors can accommodate up to about 46 kb of therapeutic DNA and can be easily produced in an industrial setting. However, the lambda vectors transfer DNA into mammalian cells with only a low efficiency. It was shown that bacteriophage lambda virions ejected their DNA in the presence of the purified receptor for bacteriophage lambda, maltoporin (LamB protein), encoded by the malB gene of Shigella sonnei 3070. This property of S. sonnei maltoporin was exploited for the bacteriophage injection-driven DNA loading of liposomes and other polymer nanocontainers displaying maltoporin. Relying on the above evidence I hypothesize that the efficient gene transfer by industrially produced bacteriophage lambda vector virions, such as cosmid transducing particles, to human cells can be accomplished after incorporation (protein painting) of the purified S. sonnei maltoporin into the human plasma membrane.