Neste trabalho foi transfectada uma linhagem de células embrionárias de rim humano (HEK293) com os genes das subunidades α e β da tireotrofina humana (hTSH), hormônio glicoproteico secretado pela hipófise. Após 5 dias de cultivo obteve-se uma concentração de hTSH no meio condicionado de 0,95μg/mL. O material foi concentrado e purificado utilizando uma estratégia envolvendo duas etapas, uma cromatografia de troca catiônica e uma cromatografia líquida de alta eficiência (HPLC) de fase reversa, que permitiu uma recuperação de 55% e uma pureza >90%. O produto purificado (hTSH-HEK) foi analisado e comparado a uma preparação comercial obtida em células CHO (hTSH-CHO) e a uma preparação hipofisária (hTSH-Pit). A identidade e a pureza do hTSH-HEK foram avaliadas por métodos físicoquímicos e imunológico (espectrometria de massa MALDI-TOF, HPLC de exclusão molecular e de fase reversa, SDS-PAGE e ensaio imunoradiométrico). A porção glicídica do hTSH-HEK foi avaliada pela análise do perfil dos N-glicanos e o comportamento biológico deste hormônio foi avaliado por bioensaio in vivo e estudo farmacocinético. As 3 preparações apresentaram pureza equivalente (97%) e a massa molecular relativa do hTSH-HEK foi 2,1% menor do que a do hTSH-CHO e 2,7% maior do que a do hTSH-Pit. A maior hidrofobicidade relativa, avaliada por RP-HPLC, foi a do hTSH-HEK. Os N-glicanos identificados no hTSH-HEK foram do tipo complexo, apresentando predominantemente estruturas tri-antenárias, enquanto no hTSH-CHO e no hTSH-Pit as estruturas bi-antenárias foram predominantes. Foram detectadas diferenças significativas relacionadas à composição dos carboidratos para estas preparações, um teor muito menor de ácido siálico e muito maior de fucose foram observados no hTSHHEK. Foi confirmada a atividade biológica das 3 preparações, sendo a bioatividade do hTSHHEK 39% e 16% inferior à do hTSH-CHO e hTSH-Pit, respectivamente. A meia-vida circulatória do hTSH-HEK foi menor (1,5 X) que a do hTSH-CHO e a do hTSH-Pit (1,2 X). De acordo com esses resultados o hTSH-HEK pode ser considerado uma alternativa viável para aplicações clínicas especialmente por sua origem humana e composição de carboidratos. / In this work a strain of embryonic human kidney cells (HEK293) was transfected with the genes of the α and β subunits of human thyrotropin (hTSH), a glycoproteic hormone secreted by the pituitary gland. After 5 days of culture, the concentration of hTSH in conditioned medium was 0.95μg/mL. The material was concentrated and purified utilizing a strategy involving two steps, a cation-exchange chromatography and a reversed phase high performance liquid chromatography (RP-HPLC), providing an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a recombinant commercial preparation obtained from CHO cells (hTSH-CHO) and to a human pituitary preparation (hTSH-Pit). Identity and purity of hTSH-HEK were evaluated through physicochemical and immunological methods (MALDI-TOF mass spectrometry, size exclusion HPLC, reversed-phase HPLC, SDS-PAGE, immunoradiometric assay). Glycidic portion of hTSHHEK was evaluated by N-glycoprofiling analysis and the biological behavior of this hormone was evaluated by an in vivo bioassay and via a pharmacokinetic study. The 3 preparations showed equivalent purity (97%) and hTSH-HEK molecular mass was 2.1% lower than hTSHCHO mass and 2.7% higher than hTSH-Pit mass. The highest relative hydrophobicity, evaluated by RP-HPLC, was shown by hTSH-HEK. Remarkable differences related to the carbohydrate moiety were found for these preparations, a much lower sialic acid content and a higher fucose content being observed in hTSH-HEK. Biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39% and 16% lower than hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t1/2) was lower than that of hTSHCHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK293-derived hTSH can be considered useful for clinical applications, also in view of its human origin and particular N-glycan composition.

Neste trabalho foi transfectada uma linhagem de células embrionárias de rim humano (HEK293) com os genes das subunidades α e β da tireotrofina humana (hTSH), hormônio glicoproteico secretado pela hipófise. Após 5 dias de cultivo obteve-se uma concentração de hTSH no meio condicionado de 0,95μg/mL. O material foi concentrado e purificado utilizando uma estratégia envolvendo duas etapas, uma cromatografia de troca catiônica e uma cromatografia líquida de alta eficiência (HPLC) de fase reversa, que permitiu uma recuperação de 55% e uma pureza >90%. O produto purificado (hTSH-HEK) foi analisado e comparado a uma preparação comercial obtida em células CHO (hTSH-CHO) e a uma preparação hipofisária (hTSH-Pit). A identidade e a pureza do hTSH-HEK foram avaliadas por métodos físicoquímicos e imunológico (espectrometria de massa MALDI-TOF, HPLC de exclusão molecular e de fase reversa, SDS-PAGE e ensaio imunoradiométrico). A porção glicídica do hTSH-HEK foi avaliada pela análise do perfil dos N-glicanos e o comportamento biológico deste hormônio foi avaliado por bioensaio in vivo e estudo farmacocinético. As 3 preparações apresentaram pureza equivalente (97%) e a massa molecular relativa do hTSH-HEK foi 2,1% menor do que a do hTSH-CHO e 2,7% maior do que a do hTSH-Pit. A maior hidrofobicidade relativa, avaliada por RP-HPLC, foi a do hTSH-HEK. Os N-glicanos identificados no hTSH-HEK foram do tipo complexo, apresentando predominantemente estruturas tri-antenárias, enquanto no hTSH-CHO e no hTSH-Pit as estruturas bi-antenárias foram predominantes. Foram detectadas diferenças significativas relacionadas à composição dos carboidratos para estas preparações, um teor muito menor de ácido siálico e muito maior de fucose foram observados no hTSHHEK. Foi confirmada a atividade biológica das 3 preparações, sendo a bioatividade do hTSHHEK 39% e 16% inferior à do hTSH-CHO e hTSH-Pit, respectivamente. A meia-vida circulatória do hTSH-HEK foi menor (1,5 X) que a do hTSH-CHO e a do hTSH-Pit (1,2 X). De acordo com esses resultados o hTSH-HEK pode ser considerado uma alternativa viável para aplicações clínicas especialmente por sua origem humana e composição de carboidratos. / In this work a strain of embryonic human kidney cells (HEK293) was transfected with the genes of the α and β subunits of human thyrotropin (hTSH), a glycoproteic hormone secreted by the pituitary gland. After 5 days of culture, the concentration of hTSH in conditioned medium was 0.95μg/mL. The material was concentrated and purified utilizing a strategy involving two steps, a cation-exchange chromatography and a reversed phase high performance liquid chromatography (RP-HPLC), providing an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a recombinant commercial preparation obtained from CHO cells (hTSH-CHO) and to a human pituitary preparation (hTSH-Pit). Identity and purity of hTSH-HEK were evaluated through physicochemical and immunological methods (MALDI-TOF mass spectrometry, size exclusion HPLC, reversed-phase HPLC, SDS-PAGE, immunoradiometric assay). Glycidic portion of hTSHHEK was evaluated by N-glycoprofiling analysis and the biological behavior of this hormone was evaluated by an in vivo bioassay and via a pharmacokinetic study. The 3 preparations showed equivalent purity (97%) and hTSH-HEK molecular mass was 2.1% lower than hTSHCHO mass and 2.7% higher than hTSH-Pit mass. The highest relative hydrophobicity, evaluated by RP-HPLC, was shown by hTSH-HEK. Remarkable differences related to the carbohydrate moiety were found for these preparations, a much lower sialic acid content and a higher fucose content being observed in hTSH-HEK. Biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39% and 16% lower than hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life (t1/2) was lower than that of hTSHCHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK293-derived hTSH can be considered useful for clinical applications, also in view of its human origin and particular N-glycan composition.

Le travail rapporté dans ce manuscrit s’inscrit dans la perspective de mieux comprendre les propriétés électriques des tissus biologiques dans la gamme de fréquence allant de 10 kHz à 100 MHz. La connaissance de ces propriétés est essentielle pour évaluer les effets du champ électromagnétique sur le corps humain en cas d’exposition involontaire mais aussi pour diagnostiquer un changement physiologique quand le tissu est exposé volontairement. De nombreux travaux ont été effectués pour mesurer les propriétés électriques des différents tissus mais le comportement électrique relevé n’est pas toujours bien compris en raison de la complexité de la structure du tissu. De plus, on constate une grande variabilité dans les données rapportées dans la littérature. Pour mieux appréhender le comportement électrique du tissu, la démarche originale proposée dans ce manuscrit consiste à partir de l’échelle de la cellule. Ainsi la première partie du manuscrit est consacrée à la caractérisation électrique de cellule humaines de type HEK (Human Embryonic Kidney) par électrorotation. La mise en oeuvre de cette technique a permis d’extraire les propriétés électrique de la membrane et du cytoplasme des cellules HEK. La deuxième partie du manuscrit se focalise sur la création d’agrégats de cellules à partir de cellules HEK isolées. Deux techniques ont été essayées. La première s’appuie sur la nature des jonctions intercellulaires. Même si elle donne des résultats, cette approche n’est pas satisfaisante dans la mesure où on ne peut contrôler ni la forme ni la taille des agrégats. L’autre technique consiste à utiliser un champ électrique pour manipuler les cellules par diélectrophorèse. Un microsystème a été conçu à cet effet avec un réseau d’électrodes quadripolaires. On a ainsi pu obtenir des agrégats permanents de cellules HEK ayant une taille et une forme contrôlée. La dernière partie du manuscrit porte sur l’étude du comportement électrique des agrégats de cellules par le biais de simulations numériques de spectres d’électrorotation. La comparaison du comportement électrique d’une cellule isolée et celui d’un agrégat de cellules montre que le milieu extra cellulaire influe fortement sur la réponse électrique de l’agrégat ; la différence de taille entre les cellules de même que la présence de jonctions intercellulaires ne semblent pas avoir un impact significatif. Ces résultats de simulation devront être appuyés par des études expérimentales. / The work reported in this manuscript aims at understanding the electrical properties of biological tissues in the frequency range between 10 kHz and 100 MHz. Knowing these properties is essential to assess the effects of involuntary exposure of human body to electromagnetic fields, but also to diagnose a physiological change when the tissue is exposed voluntarily. Many studies have been conducted to measure the electrical properties of different tissues but existing data are not well understood because of the complexity of tissues structure. Moreover, there is considerable variability of data among studies evaluating these properties. To better understand the electrical behavior of tissues, the original approach proposed in this manuscript consists in investigating their properties starting from the cell scale. The first part of the manuscript is dedicated to the electrical characterization of HEK 293 (HumanEmbryonic Kidney) cells by electrorotation. This technique has been used to extract the electrical properties of the membrane and the cytoplasm of HEK cells. The second part of the manuscript focuses on creating cell aggregates from isolated HEKcells. Two approaches have been proposed. The first one was based on the nature of intercellular junctions. This approach led to the formation of aggregates of irregularsize and shape. The other technique consisted in using an electric field to perform dielectrophoresis-assisted cell assembly. A microsystem comprising quadrupole electrodes was designed for this purpose. Permanent aggregates of HEK cells having a controlled size and shape could be obtained using this approach. The last part of the manuscript focuses on study of the electrical behavior of the cell aggregates through numerical simulations of electrorotation spectra. The confrontation between results obtained for a single cell and those obtained for an aggregate tend to show that the extracellular medium influences strongly the electrical response of the aggregate ; Conversely, the difference in size between the cells as well as the presence of intercellular junctions does not seem to have had a notable impact. These simulation results will have to be supported by experimental studies.

Calcium (Ca2+)/calmodulin-dependent kinase 2 (CaMK-II) is a multifunctional member of a family of Ca2+/calmodulin-dependent serine/threonine protein kinases that respond to transient intracellular calcium signaling. CaMK-II has been reported to be involved with transcription regulation, cell motility, neuronal development, cell cycle regulation, and more recently early development of vertebrates (Easley et al., 2008; Rothschild et al., 2009; Francescatto et al., 2010). Through previous work in the lab using tandem mass spectrometry and “substrate-trapping mutants”, tumor suppressor protein 53 (p53) was identified as a novel CaMK-II binding partner in tissue culture. In this study, I sought to provide characterization of the functional interaction of p53 and CaMK-II. First, a stable p53 knockdown human cell line (HEK) was established through lentiviral transduction of p53 shRNA and verified with immunoblots and immunostaining assays. Next, the localization of CaMK-II and the cell growth rate in these cells was determined. In wild type HEK cells, catalytically inactive CaMK-II inhibited cell growth, which is consistent with previous studies in mouse fibroblasts with pharmacological inhibition. p53-deficient cells were less sensitive to CaMK-II deficiencies using dominant negative CAMK-II, but not pharmacological disruption. The overall results of this study have provided significant clues to the mechanism between CaMK-II and p53 in the control of cell cycle progression.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

Serine proteases are a family of enzymes with a wide array of functions across both eukaryotes and prokaryotes. Here we have attempted to produce the serine proteases rat mast cell protease 2 and mouse mast cell protease 5 in a culture of HEK 293 cells; and mouse mast cell protease 4, platypus granzyme B-like protease and platypus hypothetical protease in a baculovirus expression system. Following production we wanted to analyse these serine proteases using a phage display assay and a battery of chromogenic substrates.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

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 &#948;; 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 PKC&#948;; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering.

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 &#948;; 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 PKC&#948;; b) involvement of high and sustained ROS levels correlated with late apoptosis triggering.

V této diplomové práci je popsána přechodná transfekce buněk 293 HEK adaptovaných na růst při suspenzní kultivaci bez přítomnosti séra za použití polyethyleniminů (PEI). Buňky byly transfekovány plasmidem pcDNA5/SEAP, který exprimuje sekretovanou formu lidské placentální alkalické fosfatázy. K porovnání účinnosti jednotlivých transfekcí byla měřena koncentrace exprimované fosfatázy v buněčném supernatantu. Cílem této práce bylo optimalizovat různé faktory ovlivňující účinnost transfekcí s důrazem na nalezení optimálního poměru DNA:PEI.