RESUMO
The alkylating agent platinum is first-line chemotherapy treatment for high-grade serous carcinomas (HGSC) of tubal-ovarian origin. Platinum compounds cause DNA damage and induce apoptotic cell death in the bulk tumor population. However, subpopulations of tumor cells may exhibit diverging behaviors from the bulk tumor due to an alternate stress response that diverts tumor cells from apoptotic death. In this study, we identified a salvage survival pathway in which G2-arrested tumor cells bypassed apoptosis and progressed through aberrant mitotic events to then emerge as a distinct subpopulation of viable large hyperploid cells but with uncertain long-term propagation potential. Platinum-induced large hyperploid cells were flow sorted and showed rare regrowth capacity as compared to their more proficiently regenerating non-hyperploid counterparts. However, detailed time-lapse microscopy provided direct evidence that these hyperploid cells were mitotically active and could divide successfully to produce viable daughter cells. The hyperploid survival response was observed across different cell lines and utilization of this survival pathway was dependent on the strength of the G2-M checkpoint. Conceivably, this salvage survival strategy may contribute to increased genomic diversity of the regenerating tumor cell line through a coupled hyperploidization and de-polyploidization process that may be relevant for drug resistance.
RESUMO
The evolutionarily-conserved protein phosphatase 1 (PP1) plays a central role in dephosphorylation of phosphoproteins during the M phase of the cell cycle. We demonstrate here that the PP1 inhibitor inhibitor-2 protein (Inh-2) induces an M-phase arrest in Xenopus cycling egg extracts. Interestingly, the characteristics of this M-phase arrest are similar to those of mitogen-activated protein kinase (p42MAPK)-induced M-phase arrest. This prompted us to investigate whether Inh-2-induced M-phase arrest was dependent on activation of the p42MAPK pathway. We demonstrate here that MAPK activity is required for Inh-2-induced M-phase arrest, as inhibition of MAPK by PD98059 allowed cycling extracts to exit M phase, despite the presence of Inh-2. We next investigated whether Inh-2 phosphorylation by the MAPK pathway was required to induce an M-phase arrest. We discovered that while p90Rsk (a MAPK protein required for M-phase arrest) is able to phosphorylate Inh-2, this phosphorylation is not required for Inh-2 function. Overall, our results suggest a novel mechanism linking p42MAPK and PP1 pathways during M phase of the cell cycle.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Divisão Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Oócitos/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Divisão Celular/fisiologia , Extratos Celulares/química , Ativação Enzimática/efeitos dos fármacos , Escherichia coli , Flavonoides/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Oócitos/citologia , Fosforilação/efeitos dos fármacos , Plasmídeos , Proteína Fosfatase 1/genética , Proteína Fosfatase 1/metabolismo , Proteínas/genética , Proteínas/metabolismo , Coelhos , Proteínas Recombinantes , Proteínas Quinases S6 Ribossômicas 90-kDa/genética , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Transformação Bacteriana , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMO
Exposure to microgravity has been associated with several physiological changes in astronauts, including an osteoporosis-like loss in bone mass. Despite many in vivo and in vitro studies in both microgravity and simulated microgravity conditions, the mechanism for bone loss is still not clear. The lack of weight-bearing forces makes microgravity an ideal physical stimulus to assess bone cell responses. In this work, we conduct a unique investigation of the effects of microgravity on bone-producing osteoblasts and, in parallel, on bone-resorbing osteoclasts. An increase in total number of discrete resorption pits is observed in osteoclasts that experienced microgravity versus ground controls. We further show that osteoblasts exposed to 5 days of microgravity have shorter and wavier microtubules (MTs), smaller and fewer focal adhesions, and thinner cortical actin and stress fibers. Space-flown osteoblasts present extended cell shapes as well as significantly more disrupted and often fragmented or condensed nuclei. The absence of gravitational forces therefore causes both an increase in bone resorption by osteoclasts, and a decrease in osteoblast cellular integrity. The observed effects on both major bone cell types likely accelerate bone loss in microgravity environments, and additionally offer a potential explanation to the development of disuse osteoporosis on Earth.
Assuntos
Reabsorção Óssea , Adesão Celular , Citoesqueleto/metabolismo , Osteoblastos/citologia , Osteoclastos/citologia , Ausência de Peso , Animais , Proliferação de Células , Células Cultivadas , Camundongos , Microscopia Eletrônica de VarreduraRESUMO
Phagocytosis is a complex sequence of events involving coordinated remodeling of the plasma membrane with the underlying cytoskeleton. Although the role of the actin cytoskeleton is becoming increasingly elucidated, the role of microtubules (MTs) remains poorly understood. Here, we examine the role of MTs during FcgammaR-mediated phagocytosis in RAW264.7 mouse macrophages. We observe that MTs extend into the phagosomal cups. The MT-depolymerizing agents, colchicine and nocodazole, cause a sizeable reduction in phagocytosis of large particles in RAW264.7 cells. Phagocytosis in primed macrophages is unaffected by MT-depolymerizing agents. However, activation of macrophages coincides with an increased population of drug-stable MTs, which persist in functional phagocytic cups. Scanning electron microscopy analysis of unprimed macrophages reveals that pseudopod formation is reduced markedly following colchicine treatment, which is not a consequence of cell rounding. MT depolymerization in these cells does not affect particle binding, Syk, or Grb2-associated binder 2 recruitment or phosphotyrosine accumulation at the site of phagocytosis. Ras activation also proceeds normally in macrophages treated with colchicine. However, MT disruption causes a decrease in accumulation of AKT-pleckstrin homology-green fluorescent protein, a probe that binds to PI-3K products at the sites of particle binding. A corresponding decline in activated AKT is observed in colchicine-treated cells using immunoblotting with a phospho-specific-AKT (ser473) antibody. Furthermore, the translocation of the p85alpha regulatory subunit of PI-3K is reduced at the phagocytic cup in colchicine-treated cells. These findings suggest that MTs regulate the recruitment and localized activity of PI-3K during pseudopod formation.
Assuntos
Macrófagos Peritoneais/imunologia , Microtúbulos/fisiologia , Fagocitose , Fosfatidilinositol 3-Quinases/metabolismo , Receptores de IgG/imunologia , Animais , Linhagem Celular , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/fisiologia , Macrófagos Peritoneais/ultraestrutura , Camundongos , Camundongos Endogâmicos BALB C , Receptores de IgG/metabolismoRESUMO
After invasion of epithelial cells, Salmonella enterica Typhimurium resides within membrane-bound vacuoles where it survives and replicates. Like endocytic vesicles, the Salmonella-containing vacuoles (SCVs) undergo a maturation process that involves sequential acquisition of Rab5 and Rab7 and displacement toward the microtubule-organizing center. However, SCVs fail to merge with lysosomes and instead develop subsequently into a filamentous network that extends toward the cell periphery. We found that the initial centripetal displacement of the SCV is due to recruitment by Rab7 of Rab7-interacting lysosomal protein (RILP), an effector protein that can simultaneously associate with the dynein motor complex. Unlike the early SCVs, the Salmonella-induced filaments (Sifs) formed later are devoid of RILP and dynein, despite the presence of active Rab7 on their membranes. Kinesin seems to be involved in the elongation of Sifs. SifA, a secreted effector of Salmonella, was found to be at least partly responsible for uncoupling Rab7 from RILP in Sifs and in vitro experiments suggest that SifA may exert this effect by interacting with Rab7. We propose that, by disengaging RILP from Rab7, SifA enables the centrifugal extension of tubules from the Salmonella-containing vacuoles, thereby providing additional protected space for bacterial replication.