Tanto hormônios glicocorticóides (GCs) como o gene supressor de tumor TP53, medeiam a resposta celular a uma diversidade de condições fisiológicas de estresse, sendo reguladores fundamentais do processo de vida/morte de diversos tipos celulares. A interação funcional entre estes fatores vem sendo explorada, recentemente, revelando que GCs exercem um efeito dual sobre p53. O modelo celular ST1/P7 de glioma de rato é particularmente interessante para investigar o papel de p53 na ação de GCs, já que estas linhagens apresentam respostas distintas a GCs. O tratamento com Hidrocortisona (Hy) leva as células ST1 a uma complexa reversão fenotípica tumoral→normal, enquanto as células P7 são altamente resistentes ao tratamento. Foi possível observar que a ativação de p53 por Hy ocorre apenas em células ST1, mas não em P7. Esta ativação é mediada pela indução de fosforilação da Ser15 de p53 e seu acúmulo nuclear, o que resulta no aumento de sua ligação a elementos responsivos a p53 no DNA e na sua capacidade de transativação de p53, levando a um aumento da expressão de alguns de seus genes-alvo. Contudo, o bloqueio de p53 através de siRNA não foi suficiente para alterar a resposta de células ST1 a GCs, indicando que a regulação positiva de p53 por GCs pode ser um evento secundário, mas não essencial, para a resposta anti-tumoral exercida por estes hormônios em células ST1. / Both glucocorticoid hormones (GCs) and the TP53 tumor suppressor gene mediate cellular responses to a diversity of physiological stress conditions, acting as crucial regulators of the life/death process in a wide variety of cell types. The ST1/P7 rat glioma model cell system is particularly interesting to investigate the role of p53 in the action of GCs, since these cell lines display opposite responses to GCs. Treatment with Hydrocortisone (Hy) leads ST1 cells to a complete tumoral→normal phenotypic reversion, while P7 cells are highly resistant to this treatment. It was possible to observe that activation of p53 by Hy occurs only in ST1 cells, but not in GC-resistant P7 cells. This activation is mediated by induction of phosphorylation of the Ser15 residue of p53 and its accumulation in the nucleus, resulting in increased binding of p53 to its responsive elements on the DNA and in activation of its transactivating potential, leading to increased expression of some of its target genes. However, blocking of p53 through siRNA was not sufficient to alter ST1 cells response to GCs, indicating that the positive regulation of p53 by GCs may be a secondary, non-essential, event for the anti-tumor response exerted by these hormones in ST1 cells.

Le mécanisme d’homéostasie de taille chez les cellules animales est très peu compris actuellement. Cette question est pourtant d’un intérêt majeur car le maintien de l’homéostasie de taille dans une population de cellules prolifératives doit se faire par une coordination entre la croissance et la division. Chez la levure S. pombe, il a ainsi été montré que la taille est une information cruciale pour déclencher l’entrée en mitose (Fantes, 1977). Chez plusieurs bactéries et les cellules filles de la levure S. cerevisiae au contraire, de récentes études ont au contraire montré que l’homéostasie de taille était le résultat d’une addition constante de volume, indépendamment de la taille initiale des cellules (Campos et al., 2014; Soifer et al., 2016; Taheri-Araghi et al., 2015). Ce mécanisme est appelé « adder » et génère une régression des tailles à la moyenne, génération après génération. Ces résultats ont été possibles grâce au développement de techniques permettant la mesure dynamique du volume à l’échelle de la cellule unique et sur plusieurs générations. Une telle mesure est cependant très difficile chez les cellules de mammifère dont le volume fluctue constamment et qui cyclent sur des temps plus longs (environ 20 heures). Pour cette raison, la plupart des approches proposées sont indirectes (Kafri et al., 2013; Sung et al., 2013; Tzur et al., 2009) ou reposent sur une mesure de la masse plutôt que du volume (Mir et al. 2014; Son et al., 2012). Ensemble, ces études ont montré que les cellules de mammifère croissaient de manière exponentielle. Elles ont aussi remis en cause le modèle traditionnel qui proposait que l’homéostasie de taille reposait sur l’adaptation de la durée du cycle et mis en avant un rôle de la régulation de la vitesse de croissance. Cependant, aucun modèle n’a réellement été proposé ou démontré. La nature et l’existence même d’un mécanisme maintenant l’homéostasie de taille des cellules de mammifère est en fait discutée (Lloyd, 2013).Pour caractériser l’homéostasie de taille des cellules de mammifères, nous avons développé une technique permettant pour la première fois la mesure du volume de ces cellules sur des cycles complets (Cadart et al., 2017; Zlotek-Zlotkiewicz et al. 2015). Nous montrons que plusieurs types cellulaires (HT29, MDCK et HeLa) se comportent d’une manière similaire à celle d’un « adder ». Pour tester davantage cette observation, nous induisons artificiellement des divisions asymétriques en confinant les cellules dans des micro-canaux. Nous observons que les asymétries de tailles sont réduites mais pas complètement corrigées au cours du cycle suivant, à la manière d’un « adder ». Pour comprendre comment la croissance et la progression dans le cycle sont coordonnées et génère cet « adder », nous combinons notre méthode de mesure de volume avec un suivi de la progression dans les différentes phases du cycle. Nous montrons que la durée de la phase G1 est inversement corrélée au volume initial des cellules. Cependant, cette corrélation semble contrainte par une durée minimale de G1 mise en évidence lors de l’étude de cellules artificiellement poussées à atteindre de grandes tailles. Néanmoins, même dans cette condition où la modulation de la durée du cycle est perdue, l’observation du « adder » est maintenue. Ceci suggère un rôle complémentaire de la régulation de la vitesse de croissance des cellules. Nous proposons donc une méthode pour estimer théoriquement la contribution relative de l’adaptation de la vitesse de croissance et de la durée du cycle dans le contrôle de la taille. Nous utilisons cette méthode pour proposer un cadre général où comparer le processus homéostatique des bactéries et de nos cellules. En conclusion, notre travail apporte pour la première fois la démonstration que les cellules de mammifères maintiennent l’homéostasie grâce à un mécanisme similaire au « adder ». Ce mécanisme semble impliquer à la fois une modulation de la durée du cycle et du taux de croissance. / The way proliferating mammalian cells maintain a constant size through generations is still unknown. This question is however central because size homeostasis is thought to occur through the coordination of growth and cell cycle progression. In the yeast S. pombe for example, the trigger for cell division is the reach of a target size (Fantes, 1977). This mechanism is referred to as ‘sizer’. The homeostatic behavior of bacteria and daughter cells of the yeast S. cerevisiae on the contrary was recently characterized as an ‘adder’ where all cells grow by the same absolute amount of volume at each cell cycle. This leads to a passive regression towards the mean generation after generation (Campos et al., 2014; Soifer et al., 2016; Taheri-Araghi et al., 2015). These findings were made possible by the development of new technologies enabling direct and dynamic measurement of volume over full cell cycle trajectories. Such measurement is extremely challenging in mammalian cells whose shape constantly fluctuate over time and cycle over 20 hours long periods. Studies therefore privileged indirect approaches (Kafri et al., 2013; Sung et al., 2013; Tzur et al., 2009) or indirect measurement of cell mass rather than cell volume (Mir et al. 2014; Son et al., 2012). These studies showed that cells overall grew exponentially and challenged the classical view that cell cycle duration was adapted to size and instead proposed a role for growth rate regulation. To date however, no clear model was reached. In fact, the nature and even the existence of the size homeostasis behavior of mammalian cells is still debated (Lloyd, 2013).In order to characterize the homeostatic process of mammalian cells, we developed a technique that enable measuring, for the first time, single cell volume over full cell cycle trajectories (Cadart et al., 2017; Zlotek-Zlotkiewicz et al. 2015). We found that several cell types, HT29, HeLa and MDCK cells behaved in an adder-like manner. To further test the existence of homeostasis, we artificially induced asymmetrical divisions through confinement in micro-channels. We observed that asymmetries of sizes were reduced within the following cell cycle through an ‘adder’-like behavior. To then understand how growth and cell cycle progression were coordinated in way that generates the ‘adder’, we combined our volume measurement method with cell cycle tracking. We showed that G1 phase duration is negatively correlated with initial size. This adaptation is however limited by a minimum duration of G1, unraveled by the study of artificially-induced very large cells. Nevertheless, the adder behavior is maintained even in the absence of time modulation, thus suggesting a complementary growth regulatory mechanism. Finally, we propose a method to estimate theoretically the relative contribution of growth and timing modulation in the overall size control and use this framework to compare our results with that of bacteria. Overall, our work provides the first evidence that proliferating mammalian cells behave in an adder-like manner and suggests that both growth and cell cycle duration are involved in size control.

Cell division of ggeneflesmus quadricauda (Turp.) Breb. (Scenedesmaceae) is enhanced by methylglyoxal, a general inhibitor of cell division, at threshold concentration in conjunction with treatment timing related to growth stage of batch cultures. At 0.5 mM methylglyoxal concentration, cell division was significantly enhanced in algae treated in the logarithmic phase. Specific growth rates of methylglyoxal-treated cultures were rapidly increased at the beginning of logarithmic phase. Cultures inoculated with high cell numbers were less sensitive, but still showed high specific growth rates in logarithmic phase. Cell division in cultures which had low cell numbers was inhibited by 0.5 mM methylglyoxal treatment. Both specific activity of Glyoxalase I and the ratio of Glyoxalase I to Glyoxalase II of methylgloxal-treated cultures were higher than those of controls (1.3 and 2.1- fold, respectively). Pyruvate concentration in treated cultures was increased after methylglyoxal treatment.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19930号 / 医博第4150号 / 新制||医||1017(附属図書館) / 33016 / 京都大学大学院医学研究科医学専攻 / (主査)教授 野田 亮, 教授 小川 誠司, 教授 高田 穣 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

This work examines the synthesis of poly(ADP-ribose) in normal and SV40-transformed monolayer cultures of 3T3 cells as a function of growth and DNA fragmentation. A review of the relevant literature is given in the introduction of this work. Poly(ADP-ribose) synthesis has been implicated in transcription, replication, repair, differentiation and regulation of cell growth. The results of this study suggest that poly(ADP-ribose) synthesis is involved in some aspect of cell-growth control and DNA repair.

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