Post-Transcriptional Control of LINE-1 Retrotransposition by Cellular Host Factors in Somatic Cells.

Long INterspersed Element-1 (LINE-1 or L1) retrotransposons form the only autonomously active family of transposable elements in humans. They are expressed and mobile in the germline, in embryonic stem cells and in the early embryo, but are silenced in most somatic tissues. Consistently, they play an important role in individual genome variations through insertional mutagenesis and sequence transduction, which occasionally lead to novel genetic diseases. In addition, they are reactivated in nearly half of the human epithelial cancers, contributing to tumor genome dynamics. The L1 element codes for two proteins, ORF1p and ORF2p, which are essential for its mobility. ORF1p is an RNA-binding protein with nucleic acid chaperone activity and ORF2p possesses endonuclease and reverse transcriptase activities. These proteins and the L1 RNA assemble into a ribonucleoprotein particle (L1 RNP), considered as the core of the retrotransposition machinery. The L1 RNP mediates the synthesis of new L1 copies upon cleavage of the target DNA and reverse transcription of the L1 RNA at the target site. The L1 element takes benefit of cellular host factors to complete its life cycle, however several cellular pathways also limit the cellular accumulation of L1 RNPs and their deleterious activities. Here, we review the known cellular host factors and pathways that regulate positively or negatively L1 retrotransposition at post-transcriptional level, in particular by interacting with the L1 machinery or L1 replication intermediates; and how they contribute to control L1 activity in somatic cells.

Role of cell cycle re-entry in neurons: a common apoptotic mechanism of neuronal cell death.

Currently, there is no effective treatment for neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Thus, a major focus of neuroscience research is to examine the mechanisms involved in neuronal loss in order to identify potential drug targets. Recent results indicate that DNA damage and re-entry into the cell cycle may constitute a common pathway in apoptosis in neurological diseases. The role of the cell cycle in such disorders is supported by data on the brain of patients who showed an increase in cell-cycle protein expression. Indeed, studies performed in neuronal cell preparations indicate that oxidative stress could be the main mechanism responsible for cell cycle re-entry. DNA damage and repair after oxidative stress may activate the enzyme ataxia telangiectasia mutated, which is a cell-cycle regulator. Once the cell cycle is activated, the increase in the expression of transcription factor E2F-1 could induce neuronal apoptosis. Furthermore, the potential routes involved in E2F-1 induced apoptosis could be p53-dependent or p53-independent. Under this E2F-1 hypothesis of cell death, multiple mitochondria-dependent pathways may be activated, including caspase and caspase-independent signaling cascades. Finally, given that cyclin-dependent kinase inhibitory drugs have neuroprotective and anti-apoptotic effects in experimental models, their potential application for the treatment of neurological disorders should be taken into account.

Antiapoptotic effects of roscovitine on camptothecin-induced DNA damage in neuroblastoma cells.

In the present study dopaminergic neuroblastoma B65 cells were exposed to Camptothecin (CPT) (0.5-10 µM), either alone or in the presence of roscovitine (ROSC). The results show that CPT induces apoptosis through the activation of ataxia telangiectasia mutated (ATM)-induced cell-cycle alteration in neuroblastoma B65 cells. The apoptotic process is mediated through the activation of cystein proteases, namely calpain/caspases. However, whereas a pan-caspase inhibitor, zVADfmk, inhibited CPT-mediated apoptosis, a calpain inhibitor, calpeptin, did not prevent cell death. Interestingly, CPT also induces CDK5 activation and ROSC (25 µM) blocked CDK5, ATM activation and apoptosis (as measured by caspase-3 activation). By contrast, selective inhibition of ATM, by KU55933, and non-selective inhibition, by caffeine, did not prevent CPT-mediated apoptosis. Thus, we conclude that CDK5 is activated in response to DNA damage and that CDK5 inhibition prevents ATM and p53ser15 activation. However, pharmacological inhibition of ATM using KU55933 and caffeine suggests that ATM inhibition by ROSC is not the only mechanism that might explain the anti-apoptotic effects of this drug in this apoptosis model. Our findings have a potential clinical implication, suggesting that combinatory drugs in the treatment of cancer activation should be administered with caution.

Resveratrol inhibits proliferation and promotes apoptosis of neuroblastoma cells: role of sirtuin 1.

Resveratrol prolongs lifespan and prevent cancer formation; however, the mechanisms are not understood. Here we evaluated the cell-cycle inhibition and apoptosis of resveratrol in B65 neuroblastoma cells, and we also studied the effects of resveratrol on the mammalian silent information regulator 2 (SIRT1). Results show that resveratrol reduces cell viability and causes apoptosis at 24 h of treatment. Resveratrol partially blocked cell proliferation, and significantly increased the fraction of cells arrested in the S phase. The role of SIRT1 in cell-cycle effects mediated by resveratrol was studied through changes in the expression of SIRT1 using western blot. Exposure to resveratrol decreased SIRT1 content, concomitant with an increase in the acetylated form of sirtuin substrates p53 and NFκ-ß. Treatment of B65 neuroblastoma cells with resveratrol also reduced the content of the phosphorylated form of AKT. Exposure to the SIRT1 inhibitors nicotinamide and sirtinol altered neither cell viability nor the fraction of apoptotic cells. Furthermore, when cells were exposed simultaneously to resveratrol and nicotinamide or sirtinol, no changes were observed in the fraction of apoptotic cells. Our results show that a decrease in SIRT1 content, caused by exposure to resveratrol, does not appear to be involved in cell-cycle arrest or activation of apoptosis.

Activation of ataxia telangiectasia muted under experimental models and human Parkinson's disease.

In the present study we demonstrated that neurotoxin MPP(+)-induced DNA damage is followed by ataxia telangiectasia muted (ATM) activation either in cerebellar granule cells (CGC) or in B65 cell line. In CGC, the selective ATM inhibitor KU-55933 showed neuroprotective effects against MPP(+)-induced neuronal cell loss and apoptosis, lending support to the key role of ATM in experimental models of Parkinson's disease. Likewise, we showed that knockdown of ATM levels in neuroblastoma B65 cells using an ATM-specific siRNA attenuates the phosphorylation of retinoblastoma protein without affecting other cell-cycle proteins involved in the G(0)/G(1) cell-cycle phase. Moreover, we demonstrated DNA damage, in human brain samples of PD patients. These findings support a model in which MPP(+) leads to ATM activation with a subsequent DNA damage response and activation of pRb. Therefore, this study demonstrates a new link between DNA damage by MPP(+) and cell-cycle re-entry through retinoblastoma protein phosphorylation.

ATM is involved in cell-cycle control through the regulation of retinoblastoma protein phosphorylation.

Ataxia telangiectasia mutated protein (ATM) is a member of the phosphatidylinositol-3 kinase (PI3K) family, which has a role in the cellular response to DNA double-strand breaks (DSBs). In the present study, we evaluated the role of ATM in cell-cycle control in dopaminergic rat neuroblastoma B65 cells. For this purpose, ATM activity was either inhibited pharmacologically with the specific inhibitor KU-55933, or the ATM gene was partially silenced by transfection with small interfering RNA (siRNA). Our data indicate that although ATM inhibition did not affect the cell cycle, both treatments specifically decreased the levels of cyclin A and retinoblastoma protein (pRb), phosphorylated at Ser780. Furthermore, ATM inhibition decreased the active form of p53, which is phosphorylated at Ser15, and also decreased Bax and p21 expression. Using H(2)O(2) as a positive control of DSBs, caused a rapid pRb phosphorylation, this was prevented by KU-55933 and siRNA treatment. Collectively, our data demonstrate how a new molecular network on ATM regulates the cell cycle through the control of pRb phosphorylation. These findings support a new target of ATM.

Effects of MPP+ on the molecular pathways involved in cell cycle control in B65 neuroblastoma cells.

The toxicity caused by cell exposure to 1-methyl-4-phenylpyridinium ion (MPP(+)) is a useful model in the study of Parkinson's disease (PD). However, the exact molecular mechanisms triggered by MPP(+) in cell death are currently unclear. In the present research, we show that exposure to MPP(+) induce the cell death of neuroblastoma-derived dopaminergic B65 cells, which is not reversed by the widely known caspase inhibitor Z-VAD fmk or by calpain inhibition. Likewise, when B65 cells were treated with MPP(+), the DNA damage pathway that involves p53 was activated, and cells were arrested in the G(2)/M phase of the cell cycle. Interestingly, MPP(+) has two effects on the expression of cell cycle-related proteins. It increases the content of cyclins A, E, cdk2 and the phosphorylated form of pRb (serine 780). However, MPP(+) 5mM decreased the expression of cyclin D1, B1 and cdk4. The decrease in the expression of cyclin B1 may be related to the arrest of cells observed in the G(2)/M phase of cell cycle. The increase in S phase cell cycle proteins and retinoblastoma protein phosphorylation was an unexpected result. As the antioxidant trolox attenuated the process of cell loss and changes in the cell cycle, as measured by flow cytometry, we concluded that oxidative stress was involved in the effects of MPP(+) in this cell line. In summary, the present work characterizes the molecular changes involved in damage caused by MPP(+) in B65 cells, and highlights the effects of MPP(+) on molecules involved in the control of cell cycle progression.

Oxidative stress-induced DNA damage and cell cycle regulation in B65 dopaminergic cell line.

Reactive oxygen species and oxidative stress are associated with neuronal cell death in many neurodegenerative conditions. However, the exact molecular mechanisms triggered by oxidative stress in neurodegeneration are still unclear. This study used the B65 rat neuroblastoma cell line as a model to study the molecular events that occur after H(2)O(2) treatment. Treatment of B65 cells with H(2)O(2) rapidly up-regulated the DNA damage pathway involved in double-strand breakage. Subsequently, proteins involved in p53 regulation, such as sirtuin 1 and STAT1, were modified. In addition, H(2)O(2) treatment altered the pattern of cell cycle protein expression. Specifically, a decrease was found in the expression of cyclin D1, cdk4 and surprisingly the levels of cyclin A and the retinoblastoma protein phosphorylated at ser780 were increased. Furthermore, this study shows that pre-treatment of B65 cells with 50 microM trolox confers almost total protection against apoptotic cell death and restores the cell cycle. Likewise, the increase in retinoblastoma phosphorylation was attenuated by KU-55993, a selective ATM inhibitor, and also by trolox. These observations indicate that DNA damage and oxidative stress are responsible for cell cycle regulation. In summary, this study describes the molecular mechanisms involved in cell cycle alterations induced by oxidative stress in B65 cells. These findings highlight the relevance of ATM in the regulation of cell cycle after oxidative stress.

A molecular study of pathways involved in the inhibition of cell proliferation in neuroblastoma B65 cells by the GSK-3 inhibitors lithium and SB-415286.

Pharmacological GSK-3 inhibitors are potential drugs for the treatment of neurodegenerative diseases, cancer and diabetes. We examined the antiproliferative effects of two GSK-3 inhibitors, lithium and SB-415286, on B65 neuroblastoma cell line. Treatment of B65 cells with either drug administered separately caused a decrease in cell proliferation that was associated with G(2)/M cell cycle arrest. Cell-cycle proteins such as cyclins D, E, A, cdk4 and cdk2 were up-regulated. Since lithium and SB-415286-induced G(2)/M arrest we studied changes in the expression of proteins involved in this phase, specifically cyclin B, cdc2 and the phosphorylated form of this protein (tyr15-cdc2). Both drugs increased the expression of tyr15-cdc2, thus inhibiting mitosis. On the other hand, SB-415286 increased the expression of SIRT2, involved in the regulation of proliferation. Moreover, cell-cycle arrest mediated by SB-415286 was accompanied by apoptosis that was not prevented by 100 microM of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), a pan-caspase inhibitor. Likewise, GSK-3 inhibitors did not affect the mitochondrial release of apoptosis inducing factor (AIF). We conclude that inhibitors of GSK-3 induced cell-cycle arrest, mediated by the phosphorylation of cdc2 and, in the case of SB-415286, SIRT2 expression, which induced apoptosis in a caspase-independent manner.

Neuroprotective effects of SB-415286 on hydrogen peroxide-induced cell death in B65 rat neuroblastoma cells and neurons.

Glycogen synthase kinase-3 (GSK-3) is involved in the pathogenesis of several neurodegenerative diseases. In addition, as oxidative stress has been implicated in all neurodegenerative disorders, the inhibition of both pathways offers a potential strategy for preventing or delaying neurodegeneration. We examined the cytoprotective effects of lithium and SB-415286, two inhibitors of GSK-3, using a rat B65 cell line and also in cerebellar granule cells (CGN). H(2)O(2) decreased the inactive form of GSK-3 (phospho-GSK-3 at Ser9), as measured by immunoblot experiments involving an antibody against the inactive form of the enzyme. Moreover, lithium inhibited this effect. While SB-415286 exerted a protective effect, lithium did not attenuate the toxic effects of H(2)O(2) (1mM). We then examined those mechanisms implicated in the protective effects of SB-415286. When we analyzed reactive oxygen species (ROS) production using the fluorescent probe 2,7-dichlorodihydrofluorescein diacetate in B65 cells, as well as in CGN, we found that SB-415286 strongly reduced DCF fluorescence. Lithium, however, did not exhibit any antioxidant properties. We conclude that the GSK-3 inhibitor SB-415286 has antioxidant properties, which may explain the cytoprotective effects against H(2)O(2) damage. Furthermore, inhibition of GSK-3 activity was not involved in this protective effect.

The antiproliferative activity of melatonin in B65 rat dopaminergic neuroblastoma cells is related to the downregulation of cell cycle-related genes.

A potential application of melatonin is its ability to rescue many cell types from cell death, because of its antioxidant properties. Likewise, recent studies suggest that melatonin may also be used as an anti-tumor drug, due to its anti-proliferative properties in tumor cells when administered at physiologic or pharmacologic doses. In the present study, we investigated the mechanisms involved in the apoptosis induced by acute exposure to melatonin and roscovitine in the rat dopaminergic neuroblastoma B65 cell line. Cell growth studies revealed that, at 24 hr of treatment, roscovitine blocked cell growth and induced apoptosis whereas melatonin delayed cell growth and induced a slight increase in the number of apoptotic nuclei. Melatonin also increased the percentage of cells in the G1-phase of the cell cycle, whereas roscovitine blocked cells in the G2/M-phase. Both compounds significantly downregulated the transcriptional activity of cdk4, while melatonin also downregulated cdk2 and cyclin D1. Taken together, our data show that melatonin at millimolar concentrations inhibits dopaminergic B65 proliferation, induces cell apoptosis, and modulates cell cycle progression by inhibiting the transcriptional activity of cyclins and cdks related to the progression of the G1-phase.