RESUMO
BACKGROUND: It is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse. RESULTS: Using an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal. CONCLUSIONS: We conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor.
Assuntos
Melanoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Cutâneas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fibroblastos , Humanos , Queratinócitos , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pele/metabolismoRESUMO
We have previously demonstrated that in renal cortical collecting duct cells (RCCD(1)) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT-RCCD(1) (not expressing aquaporins) and AQP2-RCCD(1) (transfected with AQP2). Our results showed that when most RCCD(1) cells are in the G(1)-phase (unsynchronized), the blockage of barium-sensitive K(+) channels implicated in rapid RVD inhibits cell proliferation only in AQP2-RCCD(1) cells. Though cells in the S-phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down-regulation in the rapid RVD response only in AQP2-RCCD(1) cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2-besides increasing water permeability-would play some other role. These observations together with evidence implying a cell-sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G(1), volume tends to increase but it may be efficiently regulated by an AQP2-dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down-regulated when volume needs to be increased in order to proceed into the S-phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2.
Assuntos
Aquaporina 2/metabolismo , Proliferação de Células/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Rim/citologia , Animais , Aquaporina 2/genética , Compostos de Bário/farmacologia , Sinalização do Cálcio , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Cloretos/farmacologia , Citometria de Fluxo , Pontos de Checagem da Fase G1 do Ciclo Celular , Microscopia de Vídeo , Pressão Osmótica , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Transporte Proteico , Ratos , Rutênio Vermelho/farmacologia , Fase S , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , TransfecçãoRESUMO
Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid-base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD(1) (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes.
Assuntos
Adaptação Fisiológica , Alcalose/patologia , Alcalose/fisiopatologia , Apoptose , Aquaporina 2/metabolismo , Ciclo Celular , Túbulos Renais Coletores/patologia , Animais , Proliferação de Células , Córtex Renal/patologia , Ratos , Fatores de TempoRESUMO
BACKGROUND INFORMATION: A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin-2 during apoptosis in renal-collecting duct cells. We evaluated the putative relationship between aquaporin-2 expression and the activation of the ionic pathways involved in the regulatory volume response. RESULTS: Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin-2. Typical features of apoptosis were evaluated with different approaches and the water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin-2 cells and it is linked to the rapid activation of volume-regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin-2 was strongly reduced during the apoptotic process and it occurs before DNA degradation. CONCLUSIONS: These results let us propose that under apoptotic stimulation aquaporin-2 would act as a sensor leading to a co-ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to activate the enzymatic apoptotic cascade.
Assuntos
Apoptose/fisiologia , Aquaporina 2/metabolismo , Túbulos Renais Coletores , Animais , Aquaporina 2/genética , Compostos de Bário/metabolismo , Linhagem Celular , Permeabilidade da Membrana Celular , Tamanho Celular , Cloretos/metabolismo , Cicloeximida/metabolismo , Fragmentação do DNA , Glibureto/metabolismo , Humanos , Canais Iônicos/metabolismo , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/metabolismo , Concentração Osmolar , Ratos , Água/metabolismoRESUMO
Metastatic cancer relapses following the reactivation of dormant, disseminated tumour cells; however, the cells and factors involved in this reactivation are just beginning to be identified. Using an immunotherapy-based syngeneic model of melanoma dormancy and GFP-labelled dormant cell-derived cell lines, we determined that vaccination against melanoma prevented tumour growth but did not prevent tumour cell dissemination or eliminate all tumour cells. The persistent disseminated melanoma tumour cells were quiescent and asymptomatic for one year. The quiescence/activation of these cells in vitro and the dormancy of melanoma in vivo appeared to be regulated by glucocorticoid-induced leucine zipper (GILZ)-mediated immunosuppression. GILZ expression was low in dormant cell-derived cultures, and re-expression of GILZ inactivated FOXO3A and its downstream target, p21CIP1. The ability of dormancy-competent cells to re-enter the cell cycle increased after a second round of cellular dormancy in vivo in association with shortened tumour dormancy period and faster and more aggressive melanoma relapse. Our data indicate that future cancer treatments should be adjusted according to the stage of disease progression.
Assuntos
Proteína Forkhead Box O3/genética , Melanoma/genética , Células-Tronco Neoplásicas/metabolismo , Fase de Repouso do Ciclo Celular/genética , Fatores de Transcrição/genética , Animais , Biomarcadores Tumorais , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Genes Reporter , Humanos , Melanoma/imunologia , Melanoma/mortalidade , Melanoma/patologia , Melanoma Experimental , Camundongos , Metástase Neoplásica , Células-Tronco Neoplásicas/patologia , Prognóstico , Transdução de SinaisRESUMO
Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration.