Kinetics of DNA methylation inheritance by the Dnmt1-including complexes during the cell cycle

<p>Abstract</p> <p>Background</p> <p>The clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during the cellular division is a crucial biological process controlled by the DNA methyltransferase Dnmt1, mainly. To investigate possible dynamic mechanisms of DNA methylation inheritance during the cell cycle, we used a Proximity Ligation <it>In Situ </it>Assay (P-LISA) to analyze the kinetic of formation and DNA recruitment of Dnmt1-including complexes.</p> <p>Results</p> <p>P-LISA, sequential chromatin immunoprecipitation and quantitative methylation specific PCR revealed that the Dnmt1/PCNA/UHRF1-including complexes are mainly formed and recruited on DNA during the S-phase of cell cycle, while the formation and the DNA recruitment of several Dnmt1/transcription factors-including complexes are not S-phase dependent but are G0/G1 and/or G2/M phases dependent.</p> <p>Conclusion</p> <p>Our data confirm that DNA methylation inheritance occurs in S-phase, and demonstrate that DNA methylation inheritance can also occur in G0/G1 and G2/M phases of the cell cycle.</p

Impact of pH on Bax α conformation, oligomerisation and mitochondrial integration

AbstractThe change in the conformation of Bax at the onset of apoptosis is a determinant for the execution of this cell death programme. However, very few models can account for this modification and the factors involved in this process remain elusive. We have analysed the modifications in the conformation induced by a variation in pH using a cell-free assay. We show that a moderate basic or acidic pH can induce apoptotic-like changes in the conformation of Bax, such as the exposure of the N-terminal or the BH3 domain. These changes in the conformation are associated with the binding of Bax to mitochondria and an enhanced Bax homo- and oligomerisation. Our results suggest that variations in the pH, in a range consistent with that often observed during apoptosis, are sufficient to trigger Bax translocation to mitochondria and the subsequent release of apoptogenic factors from this organelle

Differential Dependence on Beclin 1 for the Regulation of Pro-Survival Autophagy by Bcl-2 and Bcl-xL in HCT116 Colorectal Cancer Cells

Autophagy is described to be involved in homeostasis, development and disease, both as a survival and a death process. Its involvement in cell death proceeds from interrelationships with the apoptotic pathway. We focused on survival autophagy and investigated its interplays with the apoptotic machinery. We found that while Mcl-1 remained ineffective, Bcl-2 and Bcl-xL were required for starved cells to display a fully functional autophagic pathway as shown by proteolysis activity and detection of autophagic vesicles. Such pro-autophagic functions of Bcl-2 and Bcl-xL were independent of Bax. However they appeared to operate through non redundant mechanisms as Bcl-xL wielded a tighter control than Bcl-2 over the regulation of autophagy: unlike Bcl-2, Bcl-xL and Atg7 manipulation yielded identical phenotypes suggesting they could be components of the same signalling pathway; Bcl-xL subcellular localisation was modified upon starvation, and importantly Bcl-xL acted independently of Beclin 1. Still an intact BH3-binding site was required for Bcl-xL to stimulate a fully functional autophagic pathway. This study highlights that, in addition to their well-established anti-death function during apoptosis, Bcl-2 and Bcl-xL have a broader role in cell survival. Should Bcl-2 and Bcl-xL stand at the cross-roads between pro-survival and pro-death autophagy, this study introduces the new concept that the regulation of autophagy by Bcl-2 and Bcl-xL is adjusted according to its survival or death outcome

Endothelial Secreted Factors Suppress Mitogen Deprivation-Induced Autophagy and Apoptosis in Glioblastoma Stem-Like Cells

International audienceRapidly growing and highly vascularized tumors, such as glioblastoma multiforme, contain heterogeneous areas within the tumor mass, some of which are inefficiently supplied with nutrients and oxygen. While the cell death rate is elevated in such zones, tumor cells are still suspected to grow and survive independently of extracellular growth factors. In line with this, glioblastoma stem-like cells (GSCs) are found closely associated with brain vasculature in situ, and as such are most likely in a protected microenvironment. However, the behavior of GSCs under deprived conditions has not been explored in detail. Using a panel of 14 patient-derived GSCs, we report that ex vivo mitogen deprivation impaired self-renewal capability, abolished constitutive activation of the mTor pathway, and impinged on GSC survival via the engagement of autophagic and apoptotic cascades. Moreover, pharmacological inhibition of the mTor pathway recapitulated the mitogen deprivation scenario. In contrast, blocking either apoptosis or autophagy, or culturing GSCs with endothelial-secreted factors partly restored mTor pathway activation and rescued GSC survival. Overall, our data suggest that GSCs are addicted to mTor, as their survival and self-renewal are profoundly dependent on this signaling axis. Thus, as mTor governs the fate of GSCs under both deprivation conditions and in the presence of endothelial factors, it could be a key target for therapeutic purposes

Cellular Heterogeneity and Cooperativity in Glioma Persister Cells Under Temozolomide Treatment

Factor de impacto: 6,684 Q1.We have observed a drug-tolerant/persister state in a human glioblastoma (GBM) cell line after exposure to temozolomide, the standard-of-care chemotherapeutic agent for GBM. We used a multicolor lentiviral genetic barcode labeling to follow cell population evolution during temozolomide treatment. We observed no change in the distribution of the different colored populations of cells in persister or resistant cells suggesting that pre-existing minor subpopulations, which would be expected to be restricted to a single color, were not amplified/selected during the response to the drug. We have previously identified four genes (CHI3L1, FAT2, KLK5, and HB-EGF) that were over-expressed during the persister stage. Single-cell analysis of these four genes indicated that they were expressed in different individual cells ruling out the existence of a single persister-specific clone but suggesting rather a global answer. Even so, the transitory silencing of CHI3L1, FAT2, or KLK5 influenced the expression of the other three genes and the survival of U251 cells in absence of temozolomide. Since proteins encoded by the four genes are all localized in the extracellular matrix or interact within the extracellular compartment, we propose that cellular interactions and communications are important during the persister stage before the acquisition of chemo-resistance. Thus, persisters might be a new therapeutically relevant target in GBM.This research was founded by a grant from the “Ligue contre le Cancer-Grand Ouest” and a Région Pays de la Loire special fund (ERRATA program).S

Evidence for a mitochondrial localization of the retinoblastoma protein

<p>Abstract</p> <p>Background</p> <p>The retinoblastoma protein (Rb) plays a central role in the regulation of cell cycle, differentiation and apoptosis. In cancer cells, ablation of Rb function or its pathway is a consequence of genetic inactivation, viral oncoprotein binding or deregulated hyperphosphorylation. Some recent data suggest that Rb relocation could also account for the regulation of its tumor suppressor activity, as is the case for other tumor suppressor proteins, such as p53.</p> <p>Results</p> <p>In this reported study, we present evidence that a fraction of the total amount of Rb protein can localize to the mitochondria in proliferative cells taken from both rodent and human cells. This result is also supported by the use of Rb siRNAs, which substantially reduced the amount of mitochondrial Rb, and by acellular assays, in which [³⁵S]-Methionine-labeled Rb proteins bind strongly to mitochondria isolated from rat liver. Moreover, endogenous Rb is found in an internal compartment of the mitochondria, within the inner-membrane. This is consistent with the protection of Rb from alkaline treatment, which destroys any interaction of proteins that are weakly bound to mitochondria.</p> <p>Conclusion</p> <p>Although a few data regarding an unspecific cytosolic localization of Rb protein have been reported for some tumor cells, our results are the first evidence of a mitochondrial localization of Rb. The mitochondrial localization of Rb is observed in parallel with its classic nuclear location and paves the way for the study of potential as-yet-unknown roles of Rb at this site.</p

Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes Tumorigenesis from Human and Mice Glial Cells

Global DNA hypomethylation is a hallmark of cancer cells, but its molecular mechanisms have not been elucidated. Here, we show that the disruption of Dnmt1/PCNA/UHRF1 interactions promotes a global DNA hypomethylation in human gliomas. We then demonstrate that the Dnmt1 phosphorylations by Akt and/or PKC abrogate the interactions of Dnmt1 with PCNA and UHRF1 in cellular and acelluar studies including mass spectrometric analyses and the use of primary cultured patient-derived glioma. By using methylated DNA immunoprecipitation, methylation and CGH arrays, we show that global DNA hypomethylation is associated with genes hypomethylation, hypomethylation of DNA repeat element and chromosomal instability. Our results reveal that the disruption of Dnmt1/PCNA/UHRF1 interactions acts as an oncogenic event and that one of its signatures (i.e. the low level of mMTase activity) is a molecular biomarker associated with a poor prognosis in GBM patients. We identify the genetic and epigenetic alterations which collectively promote the acquisition of tumor/glioma traits by human astrocytes and glial progenitor cells as that promoting high proliferation and apoptosis evasion

Opposite role of Bax and BCL-2 in the anti-tumoral responses of the immune system

BACKGROUND: The relative role of anti apoptotic (i.e. Bcl-2) or pro-apoptotic (e.g. Bax) proteins in tumor progression is still not completely understood. METHODS: The rat glioma cell line A15A5 was stably transfected with human Bcl-2 and Bax transgenes and the viability of theses cell lines was analyzed in vitro and in vivo. RESULTS: In vitro, the transfected cell lines (huBax A15A5 and huBcl-2 A15A5) exhibited different sensitivities toward apoptotic stimuli. huBax A15A5 cells were more sensitive and huBcl-2 A15A5 cells more resistant to apoptosis than mock-transfected A15A5 cells (pCMV A15A5). However, in vivo, in syngenic rat BDIX, these cell lines behaved differently, as no tumor growth was observed with huBax A15A5 cells while huBcl-2 A15A5 cells formed large tumors. The immune system appeared to be involved in the rejection of huBax A15A5 cells since i) huBax A15A5 cells were tumorogenic in nude mice, ii) an accumulation of CD8+ T-lymphocytes was observed at the site of injection of huBax A15A5 cells and iii) BDIX rats, which had received huBax A15A5 cells developed an immune protection against pCMV A15A5 and huBcl-2 A15A5 cells. CONCLUSIONS: We show that the expression of Bax and Bcl-2 controls the sensitivity of the cancer cells toward the immune system. This sensitization is most likely to be due to an increase in immune induced cell death and/or the amplification of an anti tumour immune respons

Distinct Roles of Bcl-2 and Bcl-Xl in the Apoptosis of Human Bone Marrow Mesenchymal Stem Cells during Differentiation

Background: Adult mesenchymal stem cells (MSCs) can be maintained over extended periods of time before activation and differentiation. Little is known about the programs that sustain the survival of these cells. Principal Findings: Undifferentiated adult human MSCs (hMSCs) did not undergo apoptosis in response to different cell death inducers. Conversely, the same inducers can readily induce apoptosis when hMSCs are engaged in the early stages of differentiation. The survival of undifferentiated cells is linked to the expression of Bcl-Xl and Bcl-2 in completely opposite ways. Bcl-Xl is expressed at similar levels in undifferentiated and differentiated hMSCs while Bcl-2 is expressed only in differentiated cells. In undifferentiated hMSCs, the down-regulation of Bcl-Xl is associated with an increased sensitivity to apoptosis while the ectopic expression of Bcl-2 induced apoptosis. This apoptosis is linked to the presence of cytoplasmic Nur 77 in undifferentiated hMSCs. Significance: In hMSCs, the expression of Bcl-2 depends on cellular differentiation and can be either pro- or anti-apoptotic. Bcl-Xl, on the other hand, exhibits an anti-apoptotic activity under all conditions

Changes in liver mitochondrial plasticity induced by brain tumor

BACKGROUND: Accumulating data suggest that liver is a major target organ of systemic effects observed in the presence of a cancer. In this study, we investigated the consequences of the presence of chemically induced brain tumors in rats on biophysical parameters accounting for the dynamics of water in liver mitochondria. METHODS: Tumors of the central nervous system were induced by intraveinous administration of ethylnitrosourea (ENU) to pregnant females on the 19th day of gestation. The mitochondrial crude fraction was isolated from the liver of each animal and the dynamic parameters of total water and its macromolecule-associated fraction (structured water, H(2)Ost) were calculated from Nuclear Magnetic Resonance (NMR) measurements. RESULTS: The presence of a malignant brain tumor induced a loss of water structural order that implicated changes in the physical properties of the hydration shells of liver mitochondria macromolecules. This feature was linked to an increase in the membrane cholesterol content, a way to limit water penetration into the bilayer and then to reduce membrane permeability. As expected, these alterations in mitochondrial plasticity affected ionic exchanges and led to abnormal features of mitochondrial biogenesis and caspase activation. CONCLUSION: This study enlightens the sensitivity of the structured water phase in the liver mitochondria machinery to external conditions such as tumor development at a distant site. The profound metabolic and functional changes led to abnormal features of ion transport, mitochondrial biogenesis and caspase activation