Marcos Seoane

Interferon beta promotes survival in primary astrocytes through phosphatidylinositol 3-kinase

Although interferon-beta (IFN-beta) has been demonstrated to be effective in the treatment of multiple sclerosis (MS) patients, the mechanism(s) underlying its beneficial effects has not been uncovered yet. Until now, most of the effort in the study of the relevant mechanisms of IFN-beta has dealt with its ability to modulate the immune response. Only recently, it has been proposed that the beneficial effects of IFN-beta in MS patients could depend on its ability to modulate astrocyte function. In the present work, we have found that IFN-beta treatment promotes the survival of astrocytes through stimulation of the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. We propose that the beneficial effects of IFN-beta in MS therapy may depend, at least in part, on its capacity to protect astrocytes against the apoptotic cell death that occurs in the early steps of the pathogenesis of MS.

The Mitochondrial Genome Is a “Genetic Sanctuary” during the Oncogenic Process

Since Otto Warburg linked mitochondrial physiology and oncogenesis in the 1930s, a number of studies have focused on the analysis of the genetic basis for the presence of aerobic glycolysis in cancer cells. However, little or no evidence exists today to indicate that mtDNA mutations are directly responsible for the initiation of tumor onset. Based on a model of gliomagenesis in the mouse, we aimed to explore whether or not mtDNA mutations are associated with the initiation of tumor formation, maintenance and aggressiveness. We reproduced the different molecular events that lead from tumor initiation to progression in the mouse glioma. In human gliomas, most of the genetic alterations that have been previously identified result in the aberrant activation of different signaling pathways and deregulation of the cell cycle. Our data indicates that mitochondrial dysfunction is associated with reactive oxygen species (ROS) generation, leading to increased nuclear DNA (nDNA) mutagenesis, but maintaining the integrity of the mitochondrial genome. In addition, mutational stability has been observed in entire mtDNA of human gliomas; this is in full agreement with the results obtained in the cancer mouse model. We use this model as a paradigm of oncogenic transformation due to the fact that mutations commonly found in gliomas appear to be the most common molecular alterations leading to tumor development in most types of human cancer. Our results indicate that the mtDNA genome is kept by the cell as a “genetic sanctuary” during tumor development in the mouse and humans. This is compatible with the hypothesis that the mtDNA molecule plays an essential role in the control of the cellular adaptive survival response to tumor-induced oxidative stress. The integrity of mtDNA seems to be a necessary element for responding to the increased ROS production associated with the oncogenic process.

Retinoblastoma Loss Modulates DNA Damage Response Favoring Tumor Progression

Senescence is one of the main barriers against tumor progression. Oncogenic signals in primary cells result in oncogene-induced senescence (OIS), crucial for protection against cancer development. It has been described in premalignant lesions that OIS requires DNA damage response (DDR) activation, safeguard of the integrity of the genome. Here we demonstrate how the cellular mechanisms involved in oncogenic transformation in a model of glioma uncouple OIS and DDR. We use this tumor type as a paradigm of oncogenic transformation. In human gliomas most of the genetic alterations that have been previously identified result in abnormal activation of cell growth signaling pathways and deregulation of cell cycle, features recapitulated in our model by oncogenic Ras expression and retinoblastoma (Rb) inactivation respectively. In this scenario, the absence of pRb confers a proliferative advantage and activates DDR to a greater extent in a DNA lesion-independent fashion than cells that express only HRasV12. Moreover, Rb loss inactivates the stress kinase DDR-associated p38MAPK by specific Wip1-dependent dephosphorylation. Thus, Rb loss acts as a switch mediating the transition between premalignant lesions and cancer through DDR modulation. These findings may have important implications for the understanding the biology of gliomas and anticipate a new target, Wip1 phosphatase, for novel therapeutic strategies.

Mechanisms of Interferon-β-Induced Survival in Fetal and Neonatal Primary Astrocytes

Background: We have previously shown that interferon-β (IFN-β) is a potent promoter of astrocyte survival. Although the mechanism(s) by which IFN-β promotes astrocyte survival have not been completely elucidated, it has been shown that IFN-β directly stimulates survival signaling pathways. In the present report, we took advantage of the differences in the susceptibility of fetal and neonatal astrocytes to apoptosis to further investigate the mechanism(s) underlying the antiapoptotic effect of IFN-β. Methods: Primary monolayer cultures of cortical astrocytes were established from neonatal (3- to 6-day-old) or fetal (embryonic days: E15 or E17) Sprague-Dawley rat cerebral cortices. Apoptotic cell death was determined by fluorescent-microscopic analysis of staining patterns of cell DNA with Hoechst 33258, and determination of annexin V binding.Akt phosphorylation was detected by Western blottingusing a commercial kit that allows specific recognition of both non-phosphorylated and serine-phosphorylated Akt. Results: In the present work, we have found that primary astrocytes obtained from neonatal rats are resistant to apoptosis induced by serum starvation, though cell death may be induced by combining serum starvation with sodium butyrate treatment. This effect is counteracted by IFN-β treatment through a mechanism that involves phosphatidylinositol 3-kinase stimulation. Conclusions: IFN-β can be considered as a neuroprotective agent and, therefore, part of its beneficial effects in multiple sclerosis (MS) treatment may depend on its capacity to protect astrocytes against the apoptotic cell death that occurs in the course of the MS lesions.

Fas/CD95 Ligation Induces Proliferation of Primary Fetal Astrocytes Through a Mechanism Involving Caspase 8-Mediated ERK Activation

Background: Fas/CD95 is the best-studied member of the death receptor (DR) superfamily in the central nervous system where it can trigger cellular responses other than apoptosis, including the promotion of neurogenesis and neuritogenesis, stimulation of the progression of gliomas, and regulation of the immune response of astrocytes. Methods: We have investigated the role of Fas/CD95 in the regulation of the proliferation of fetal astrocytes in vitro, as well as the signalling pathways involved. Results: Fas/CD95 ligation stimulated the proliferation of primary fetal astrocytes, through a mechanism that depends on the activation of caspase 8 and subsequent phosphorylation of extracellular signal regulated kinase (ERK). Interestingly this proliferative effect is only observed with a low dose of the Fas/CD95 agonist. In contrast, when primary astrocytes are challenged with a high dose of the Fas/CD95 agonist significant cell death occurs. Conclusions: Our findings support that, besides its effects on cell survival, Fas/CD95 may play a complex and prominent role in the regulation of astrocyte proliferation during development.

Bimodal effect of interferon-β on astrocyte proliferation and survival: Importance of nuclear factor-κB

Accumulating evidence indicates that interferon-β (IFN-β) can modify the complex immunopathogenic scenario causing clinical relapse activity and disease progression in MS. However, the beneficial effects of IFN-β in MS patients may also depend on non-immune mechanisms, including the modulation of astrocyte function. In the present report, we have shown that, depending on the dose, IFN-β treatment can either promote astrocyte proliferation and survival, or result astrocyte death. These actions depend, at least in part, on the regulation of nuclear factor-kappa B (NF-κB), an inducible transcription factor present in neurons and glia. This bimodal effect of IFN-β adds a new layer of complexity in the actions of IFN-β within the CNS.

Comparison of holding power of three different pin designs for external skeletal fixation in avian bone: a study in common buzzard (Buteo buteo).

OBJECTIVE (1) To evaluate resistance to axial extraction of 3 pin designs in avian humerus and tibiotarsus; (2) to assess the effect of pin location within the bone on holding power; and (3) to assess the influence of thread pitch on holding power. STUDY DESIGN Resistance of pins to axial extraction was measured immediately after insertion. ANIMALS Adult common buzzards (Buteo buteo; n=9). METHODS Different pin designs (1 smooth; 2 threaded pins, differing in pitch) were inserted into the proximal and distal metaphysis and the proximal, middle, and distal diaphysis of the humerus and tibiotarsus. Maximum force required for axial extraction of pins was recorded. RESULTS Smooth pins had the lowest extraction force (P<.05). Pins inserted into the diaphysis (proximal, middle and distal) of the humerus and the distal metaphysis of the tibiotarsus had a greater pullout strength than pins in other locations. Pins with a smaller pitch inserted into the proximal diaphysis and distal metaphysis of the humerus, and the proximal metaphysis of the tibiotarsus had significantly greater holding power than pins with a larger pitch (P<.05). CONCLUSIONS Pins inserted into the diaphysis of humerus and the distal metaphysis of the tibiotarsus are better at resisting extraction. Pins with a smaller pitch possess greater holding power than pins with a larger pitch in avian humerus and tibiotarsus. CLINICAL RELEVANCE Consideration should be given to pin location and thread pitch, when choosing external skeletal fixation to repair an avian humeral or tibiotarsal fracture.Objective— (1) To evaluate resistance to axial extraction of 3 pin designs in avian humerus and tibiotarsus; (2) to assess the effect of pin location within the bone on holding power; and (3) to assess the influence of thread pitch on holding power. Study Design— Resistance of pins to axial extraction was measured immediately after insertion. Animals— Adult common buzzards (Buteo buteo; n=9). Methods— Different pin designs (1 smooth; 2 threaded pins, differing in pitch) were inserted into the proximal and distal metaphysis and the proximal, middle, and distal diaphysis of the humerus and tibiotarsus. Maximum force required for axial extraction of pins was recorded. Results— Smooth pins had the lowest extraction force (P<.05). Pins inserted into the diaphysis (proximal, middle and distal) of the humerus and the distal metaphysis of the tibiotarsus had a greater pullout strength than pins in other locations. Pins with a smaller pitch inserted into the proximal diaphysis and distal metaphysis of the humerus, and the proximal metaphysis of the tibiotarsus had significantly greater holding power than pins with a larger pitch (P<.05). Conclusions— Pins inserted into the diaphysis of humerus and the distal metaphysis of the tibiotarsus are better at resisting extraction. Pins with a smaller pitch possess greater holding power than pins with a larger pitch in avian humerus and tibiotarsus. Clinical Relevance— Consideration should be given to pin location and thread pitch, when choosing external skeletal fixation to repair an avian humeral or tibiotarsal fracture.

Uncoupling Oncogene-Induced Senescence (OIS) and DNA Damage Response (DDR) triggered by DNA hyper-replication: lessons from primary mouse embryo astrocytes (MEA)

Oncogene-induced senescence (OIS) is a complex process, in which activation of oncogenic signals during early tumorigenesis results in a high degree of DNA replication stress. The ensuing response to the DNA damage produces a permanent G1 arrest that prevents unlimited cell proliferation and lessens the development of tumours. However, despite the role of OIS in the proliferative arrest resulting from an activating oncogenic-lesion has obtained wide support, there is also evidence indicating that cells may overcome oncogene-induced senescence under some circumstances. In this study, we have investigated the possibility that some of the assumptions on the role of DNA damage response (DDR) in triggering OIS may depend on the fact that most of the available data were obtained in mouse embryo fibroblast. By comparing the degree of OIS observed in mouse embryo fibroblasts (MEF) and mouse embryo astrocytes (MEA) obtained from the same individuals we have demonstrated that, despite truthful activation of DDR in both cell types, significant levels of OIS were only detected in MEF. Therefore, this uncoupling between OIS and DDR observed in astrocytes supports the intriguingly possibility that OIS is not a widespread response mechanism to DDR.

Abstract 2893: PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo

Although PARP-1 has been traditionally related to DNA repair, in the last years an increasing number of studies have linked this polymerase to other cellular processes such as metabolism and cell mitosis. In this study we wanted to investigate the biological importance of the interaction between PARP-1 and the transcription factor E2F-1, and more specifically in scenarios where the activity, or hyperactivity, of E2F-1 is of critical importance such as embryonic development or oncogenesis. In this regard, we have found that the treatment either with enzymatic inhibitors of PARP-1 has an effect on the transcriptional activity of E2F-1 as well as the proliferative rate of treated cells. This effect is significantly increased with the treatment with gossypol, a specific inhibitor of PARP-1 protein-protein interactions occurring through the BRCT domain, as in the case of E2F-1. This effect is also observed in vivo since the severity of histological malformations in Rb-/- embryos is significantly reduced in Parp-1-/- Rb-/- embryos, which phenotype closely resembles that of E2f-1-/- Rb-/- mice. Finally, we also found that the deletion or inhibition of PARP-1 in an animal model of gliomagenesis helps the cell to block oncogenic stimuli derived from E2F-1 hyperactivity, by reactivating critical signalling pathways involved in oncogene-induced senescence. In summary, the importance of the relationship between PARP-1 and E2F-1 in different biological contexts leads us to believe that the search for ways of altering or disrupting this interaction could be a novel strategy for the development of molecules of therapeutic potential. Citation Format: Pablo Iglesias, Marcos Seoane, Isabel Castro-Piedras, Victor M. Arce, Jose A. Costoya. PARP-1 regulates cell cycle by downregulating E2F-1 transcriptional activity in vitro and in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2893.

Abstract 2950: Regulation of the functional interface between nucleotide excision repair and transcription by MITF modulates melanoma growth

Nucleotide excision repair (NER) is an essential instrument to maintain genomic stability in eukaryotes especially towards ultraviolet radiation (UVR). There is profound knowledge of the mechanistic execution of NER, however its regulation is not well understood. Here we provide evidence that the lineage-dependent oncogene MITF regulates the functional interface between NER and transcription through transactivation of XPG and GTF2H1, which constitute core elements of the NER and general transcription factor machineries, respectively. For this purpose, flow cytometry and microscopy fluorescence assays were applied to evaluate the temporal repair kinetics of DNA lesions after UVR in the presence and upon depletion of MITF in human primary melanocytes and several melanoma cell lines. Moreover, ChIP and luciferase reporter assays were performed to show the in vivo occupancy and a MITF dependent transactivation of the XPG and GTF2H1 promoters. Transcriptome as well as tissue microarrays (TMA) analysis were used to demonstrate the relationship between MITF and TFIIH. Finally, in vivo experiments were performed utilizing a subcutaneous xenograft tumor model in SCID mice to confirm our hypothesis. Our results showed that the NER/TFIIH complex is controlled by MITF in the presence and absence of UVR mediated genotoxic attack resulting in nucleotide repair deficiency and breakdown of global transcription upon MITF depletion. Importantly, in primary human melanoma the TFIIH subunit GTF2H1, which is involved in NER and RNA transcription, is linked to MITF abundance and prognostic melanoma stage. Moreover, RNA-interference mediated repression of GTF2H1 led to a significant reduction in tumor formation in a melanoma xenograft model. Collectively, these results describe an unanticipated role of MITF in the regulation of intimately linked NER and transcription machineries in the melanocytic lineage, which is preserved upon transformation into melanoma. Through its control of the NER/TFIIH complex MITF might coordinately regulate repair and transcription processes, thereby optimizing the rapid resumption of transcriptional activity after completion of strand repair, which is vitally important for cellular survival. The very same mechanism may drive the genesis of melanoma and its progression in the context of aberrant transcriptional activity of MITF due to SUMOylation defective germline mutation or genomic amplification. Citation Format: Marcos Seoane, Laia Pagerols Raluy, Karoline Kaufmann, Julia Strauss, Kevin Dierck, Juergen Thomale, Johanna M. Brandner, Udo Schumacher, Peter Nollau, Peter J. Wild, Martin Zimmermann, Jinyan Du, David E. Fisher, Martin A. Horstmann. Regulation of the functional interface between nucleotide excision repair and transcription by MITF modulates melanoma growth. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2950. doi:10.1158/1538-7445.AM2014-2950