A New Opportunity for "Old" Molecules: Targeting PARP1 Activity through a Non-Enzymatic Mechanism.

In recent years, new therapies have been developed based on molecules that target molecular mechanisms involved in both the initiation and maintenance of the oncogenic process. Among these molecules are the poly(ADP-ribose) polymerase 1 (PARP1) inhibitors. PARP1 has emerged as a target with great therapeutic potential for some tumor types, drawing attention to this enzyme and resulting in many small molecule inhibitors of its enzymatic activity. Therefore, many PARP inhibitors are currently in clinical trials for the treatment of homologous recombination (HR)-deficient tumors, BRCA-related cancers, taking advantage of synthetic lethality. In addition, several novel cellular functions unrelated to its role in DNA repair have been described, including post-translational modification of transcription factors, or acting through protein-protein interactions as a co-activator or co-repressor of transcription. Previously, we reported that this enzyme may play a key role as a transcriptional co-activator of an important component of cell cycle regulation, the transcription factor E2F1. Here, we show that PARP inhibitors, which interfere with its activity in cell cycle regulation, perform this without affecting its enzymatic function.

From neural stem cells to glioblastoma: A natural history of GBM recapitulated in vitro.

Due to its aggressive and invasive nature glioblastoma (GBM), the most common and aggressive primary brain tumour in adults, remains almost invariably lethal. Significant advances in the last several years have elucidated much of the molecular and genetic complexities of GBM. However, GBM exhibits a vast genetic variation and a wide diversity of phenotypes that have complicated the development of effective therapeutic strategies. This complex pathogenesis makes necessary the development of experimental models that could be used to further understand the disease, and also to provide a more realistic testing ground for potential therapies. In this report, we describe the process of transformation of primary mouse embryo astrocytes into immortalized cultures with neural stem cell characteristics, that are able to generate GBM when injected into the brain of C57BL/6 mice, or heterotopic tumours when injected IV. Overall, our results show that oncogenic transformation is the fate of NSC if cultured for long periods in vitro. In addition, as no additional hit is necessary to induce the oncogenic transformation, our model may be used to investigate the pathogenesis of gliomagenesis and to test the effectiveness of different drugs throughout the natural history of GBM.

Immortalization of a cell line with neural stem cell characteristics derived from mouse embryo brain.

BACKGROUND: Neural stem cells (NSC) have been extensively used as a tool to investigate the mechanisms responsible for neural repair, and they have been also considered as the source for a series of promising replacement therapies in various neurodegenerative diseases. However, their use is limited by their relative rarity and anatomical localization, and also because, the methods for isolation and characterization are usually time consuming and have some technical limitations. RESULTS: In this study, we describe a resource and method for obtaining immortalized cells with NSC characteristics obtained from mouse brain embryo. CONCLUSIONS: Because these cells can be maintained indefinitely in culture, they may constitute a permanent source of NSC that can be used for research studies on neural development and regeneration.

Expression of myostatin in human hematopoietic cells unveils novel autocrine/paracrine actions for the hormone.

Myostatin is a member of the transforming growth factor ß (TGFß) superfamily that has a well-established role as a mediator of muscle growth and development. However, myostatin is now emerging as a pleiotropic hormone with multiple actions in the regulation of the metabolism as well as several aspects of both cardiac and smooth muscle cells physiology. In addition, myostatin is also expressed in several nonmuscular cells where its physiological role remains to be elucidated in most cases. In this report, we have shown that both myostatin and its receptor system are expressed in blood cells and in hematopoietic cell lines. Furthermore, myostatin treatment promotes differentiation of both HL60 and K562 cells through a mechanism that involves activation of extracellular signal-regulated kinases 1/2 and p38-mitogen-activated protein kinase, thus leading to the possibility that myostatin may be a paracrine/autocrine factor involved in the control of haematopoiesis. In addition, the presence of myostatin expression in immune cells could envisage a novel role for the hormone in the pathogenesis of inflammatory diseases.

GPE Promotes the Proliferation and Migration of Mouse Embryonic Neural Stem Cells and Their Progeny In Vitro.

This study was designed to investigate a possible role of the N-terminal tripeptide of insulin-like growth factor-1 (IGF-I), Gly-Pro-Glu (GPE), physiologically generated in neurons following IGF-I-specific cleavage, in promoting neural regeneration after an injury. Primary cultures of mouse neural stem cells (NSCs), obtained from 13.5 Days post-conception (dpc) mouse embryos, were challenged with either GPE, growth hormone (GH), or GPE + GH and the effects on cell proliferation, migration, and survival were evaluated both under basal conditions and in response to a wound healing assay. The cellular pathways activated by GPE were also investigated by using specific chemical inhibitors. The results of the study indicate that GPE treatment promotes the proliferation and the migration of neural stem cells in vitro through a mechanism that involves the activation of extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase PI3K-Akt pathways. Intriguingly, both GPE effects and the signaling pathways activated were similar to those observed after GH treatment. Based upon the results obtained from this study, GPE, as well as GH, may be useful in promoting neural protection and/or regeneration after an injury.

Growth hormone pathways signaling for cell proliferation and survival in hippocampal neural precursors from postnatal mice.

BACKGROUND: Accumulating evidence suggests that growth hormone (GH) may play a major role in the regulation of postnatal neurogenesis, thus supporting the possibility that it may be also involved in promoting brain repair after brain injury. In order to gain further insight on this possibility, in this study we have investigated the pathways signaling the effect of GH treatment on the proliferation and survival of hippocampal subgranular zone (SGZ)-derived neurospheres. RESULTS: Our results demonstrate that GH treatment promotes both proliferation and survival of SGZ neurospheres. By using specific chemical inhibitors we have been also able to demonstrate that GH treatment promotes the activation of both Akt-mTOR and JNK signaling pathways, while blockade of these pathways either reduces or abolishes the GH effects. In contrast, no effect of GH on the activation of the Ras-ERK pathway was observed after GH treatment, despite blockade of this signaling path also resulted in a significant reduction of GH effects. Interestingly, SGZ cells were also capable of producing GH, and blockade of endogenous GH also resulted in a decrease in the proliferation and survival of SGZ neurospheres. CONCLUSIONS: Altogether, our findings suggest that GH treatment may promote the proliferation and survival of neural progenitors. This effect may be elicited by cooperating with locally-produced GH in order to increase the response of neural progenitors to adequate stimuli. On this view, the possibility of using GH treatment to promote neurogenesis and cell survival in some acquired neural injuries may be envisaged.

AB067. New drug development for the use of PARP1-E2F1 transcriptional inhibitors in the treatment of glioblastoma.

BACKGROUND: Glioblastoma (GBM) is the most malignant brain tumor and ranks among the most lethal of all human cancers, without improvements in survival over the last 30 years. Data obtained in our group suggest that PARP1, a well-known DNA-repairing protein, could also play a key role in the regulation of cell cycle through its interaction with the transcription factor E2F1. Therefore, considering that most oncogenic processes are associated with cell cycle deregulation, we hypothesized that disruption of PARP1-E2F1 interaction would provide a novel therapeutic approach to different types of cancer. METHODS: The identification of novel compounds disrupting PARP1-E2F1 interaction was carried out by combining in silico and in vitro screening, using a rational drug design. The virtual screen was performed using a molecular library of several million compounds at the selected target site, using AtomNet® (Atomwise, San Francisco, CA, USA), the first deep learning neural network for structure-based drug design and discovery. Since there is no complete structural information of the PARP1-E2F1 protein-protein interaction, a homologous structure of the BRCT domain of BRCA1 complex with the phospho-peptide (PDBID: 1T2V) was used to identify the potential binding interface of BRCT domain of PARP-1 (PDBID: 2COK) and the E2F1 protein. Top scoring compounds were clustered and filtered to obtain a final subset of 83 compounds that were incorporated to our in vitro screening, which included both transcriptional E2F1 activity and survival studies. Complete culture medium supplemented with the compounds selected in the in silico screening (10 µM) were added and incubated for 24 hours. E2F1 activity was observed by measuring luminescence. For the viability assay, the fluorescence reading was performed (excitation 544 nm and emission 590 nm). RESULTS: The in silico and in vitro screening resulted in 12 compounds that inhibited E2F1 transcriptional activity and significantly reduced cell number. The highest inhibition of both E2F1 transcriptional activity and cell growth was observed with compound 3797, which was selected for further studies. CONCLUSIONS: Both in silico and in vitro results indicate that inhibition of PARP1-E2F1 transcriptional activity may provide a new rationale for designing novel therapeutic approaches for the treatment of GBM.

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.

Sex differences in thermoregulation in mammals: Implications for energy homeostasis.

Thermal homeostasis is a fundamental process in mammals, which allows the maintenance of a constant internal body temperature to ensure an efficient function of cells despite changes in ambient temperature. Increasing evidence has revealed the great impact of thermoregulation on energy homeostasis. Homeothermy requires a fine regulation of food intake, heat production, conservation and dissipation and energy expenditure. A great interest on this field of research has re-emerged following the discovery of thermogenic brown adipose tissue and browning of white fat in adult humans, with a potential clinical relevance on obesity and metabolic comorbidities. However, most of our knowledge comes from male animal models or men, which introduces unwanted biases on the findings. In this review, we discuss how differences in sex-dependent characteristics (anthropometry, body composition, hormonal regulation, and other sexual factors) influence numerous aspects of thermal regulation, which impact on energy homeostasis. Individuals of both sexes should be used in the experimental paradigms, considering the ovarian cycles and sexual hormonal regulation as influential factors in these studies. Only by collecting data in both sexes on molecular, functional, and clinical aspects, we will be able to establish in a rigorous way the real impact of thermoregulation on energy homeostasis, opening new avenues in the understanding and treatment of obesity and metabolic associated diseases.

Growth hormone treatment enhances the functional recovery of sciatic nerves after transection and repair.

INTRODUCTION: Although nerves can spontaneously regenerate in the peripheral nervous system without treatment, functional recovery is generally poor, and thus there is a need for strategies to improve nerve regeneration. METHODS: The left sciatic nerve of adult rats was transected and immediately repaired by epineurial sutures. Rats were then assigned to one of two experimental groups treated with either growth hormone (GH) or saline for 8 weeks. Sciatic nerve regeneration was estimated by histological evaluation, nerve conduction tests, and rotarod and treadmill performance. RESULTS: GH-treated rats showed increased cellularity at the lesion site together with more abundant immunoreactive axons and Schwann cells. Compound muscle action potential (CMAP) amplitude was also higher in these animals, and CMAP latency was significantly lower. Treadmill performance increased in rats receiving GH. CONCLUSION: GH enhanced the functional recovery of the damaged nerves, thus supporting the use of GH treatment, alone or combined with other therapeutic approaches, in promoting nerve repair.

Growth hormone (GH) treatment may cooperate with locally-produced GH in increasing the proliferative response of hippocampal progenitors to kainate-induced injury.

PRIMARY OBJECTIVE: This study was designed to investigate the effect of growth hormone treatment on the proliferation of endogenous neural progenitor cells in the dentate gyrus (DG) of the brain stimulated by kainic acid (KA)-induced neurotoxicity. RESEARCH DESIGN: Neurotoxicity was induced by intraperitoneal injection of KA. GH treatment lasted 4 days, starting either immediately or after 10 days of administration of the neurotoxic insult. METHODS AND PROCEDURE: Proliferating cells were immunodetected after labelling by in vivo administration of 5-bromodeoxyuridine (BrdU). GH expression was detected by in situ hybridization and immunofluorescence. MAIN OUTCOMES AND RESULTS: KA administration stimulated the proliferation of hippocampal precursors and this effect was significantly enhanced by GH treatment. Hippocampal GH expression was also up-regulated in response to KA administration. CONCLUSIONS: The findings support the possibility that the proliferative response observed in the hippocampus of rats treated with KA and GH is a consequence of cooperation between the exogenous and the locally-produced hormone and their synergism with other mitogenic factors generated in response to the neurotoxic damage. Therefore, GH treatment could be used to cooperate with other physiological or pathological stimuli in order to promote cell proliferation.

In-Silico Detection of Oral Prokaryotic Species With Highly Similar 16S rRNA Sequence Segments Using Different Primer Pairs.

Although clustering by operational taxonomic units (OTUs) is widely used in the oral microbial literature, no research has specifically evaluated the extent of the limitations of this sequence clustering-based method in the oral microbiome. Consequently, our objectives were to: 1) evaluate in-silico the coverage of a set of previously selected primer pairs to detect oral species having 16S rRNA sequence segments with ≥97% similarity; 2) describe oral species with highly similar sequence segments and determine whether they belong to distinct genera or other higher taxonomic ranks. Thirty-nine primer pairs were employed to obtain the in-silico amplicons from the complete genomes of 186 bacterial and 135 archaeal species. Each fasta file for the same primer pair was inserted as subject and query in BLASTN for obtaining the similarity percentage between amplicons belonging to different oral species. Amplicons with 100% alignment coverage of the query sequences and with an amplicon similarity value ≥97% (ASI97) were selected. For each primer, the species coverage with no ASI97 (SC-NASI97) was calculated. Based on the SC-NASI97 parameter, the best primer pairs were OP_F053-KP_R020 for bacteria (region V1-V3; primer pair position for Escherichia coli J01859.1: 9-356); KP_F018-KP_R002 for archaea (V4; undefined-532); and OP_F114-KP_R031 for both (V3-V5; 340-801). Around 80% of the oral-bacteria and oral-archaea species analyzed had an ASI97 with at least one other species. These very similar species play different roles in the oral microbiota and belong to bacterial genera such as Campylobacter, Rothia, Streptococcus and Tannerella, and archaeal genera such as Halovivax, Methanosarcina and Methanosalsum. Moreover, ~20% and ~30% of these two-by-two similarity relationships were established between species from different bacterial and archaeal genera, respectively. Even taxa from distinct families, orders, and classes could be grouped in the same possible OTU. Consequently, regardless of the primer pair used, sequence clustering with a 97% similarity provides an inaccurate description of oral-bacterial and oral-archaeal species, which can greatly affect microbial diversity parameters. As a result, OTU clustering conditions the credibility of associations between some oral species and certain health and disease conditions. This significantly limits the comparability of the microbial diversity findings reported in oral microbiome literature.

PARP1 Deficiency Reduces Tumour Growth by Decreasing E2F1 Hyperactivation: A Novel Mechanism in the Treatment of Cancer.

In recent years, poly (ADP-ribose) polymerase (PARP) inhibitors have been evaluated for treating homologous recombination-deficient tumours, taking advantage of synthetic lethality. However, increasing evidence indicates that PARP1 exert several cellular functions unrelated with their role on DNA repair, including function as a co-activator of transcription through protein-protein interaction with E2F1. Since the RB/E2F1 pathway is among the most frequently mutated in many tumour types, we investigated whether the absence of PARP activity could counteract the consequences of E2F1 hyperactivation. Our results demonstrate that genetic ablation of Parp1 extends the survival of Rb-null embryos, while genetic inactivation of Parp1 results in reduced development of pRb-dependent tumours. Our results demonstrate that PARP1 plays a key role as a transcriptional co-activator of the transcription factor E2F1, an important component of the cell cycle regulation. Considering that most oncogenic processes are associated with cell cycle deregulation, the disruption of this PARP1-E2F1 interaction could provide a new therapeutic target of great interest and a wide spectrum of indications.

Interferon-beta protects astrocytes against tumour necrosis factor-induced apoptosis via activation of p38 mitogen-activated protein kinase.

Several large clinical trials have demonstrated that interferon-beta (IFN-beta) therapy is effective in the treatment of multiple sclerosis (MS) patients. However, the mechanisms underlying the beneficial effects of IFN-beta are not fully understood. Most of the effort in the study of the relevant mechanisms of IFN-beta has dealt with its immunomodulatory actions. However, the beneficial effects of IFN-beta in MS patients may also depend on non-immune mechanisms, including the modulation of astrocyte function. In the present work, we have found that IFN-beta treatment protects astrocytes against tumour necrosis factor-induced apoptosis via activation of p38 mitogen-activated protein kinase. We propose that this effect may be of importance to protect astrocytes against apoptosis within the demyelinated plaques of the MS.

Chronic limb-threatening ischemia could benefit from growth hormone therapy for wound healing and limb salvage.

Revascularization for chronic limb-threatening ischemia (CLTI) is necessary to alleviate symptoms and wound healing. When it fails or is not possible, there are few alternatives to avoid limb amputation in these patients. Although experimental studies with stem cells and growth factors have shown promise, clinical trials have demonstrated inconsistent results because CLTI patients generally need arteriogenesis rather than angiogenesis. Moreover, in addition to the perfusion of the limb, there is the need to improve the neuropathic response for wound healing, especially in diabetic patients. Growth hormone (GH) is a pleiotropic hormone capable of boosting the aforementioned processes and adds special benefits for the redox balance. This hormone has the potential to mitigate symptoms in ischemic patients with no other options and improves the cardiovascular complications associated with the disease. Here, we discuss the pros and cons of using GH in such patients, focus on its effects on peripheral arteries, and analyze the possible benefits of treating CLTI with this hormone.

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.

Myostatin expression in muscular dystrophies and mitochondrial encephalomyopathies.

The demonstration that myostatin may negatively regulate muscle mass in adult individuals has raised the possibility of targeting the myostatin pathway in order to increase muscle growth in a variety of muscle degenerative and wasting conditions. In this regard, blockade of endogenous myostatin results in anatomic, biochemical, and physiologic improvement in the dystrophic phenotype in the mdx mouse. Moreover, myostatin messenger ribonucleic acid levels are decreased in the regenerated muscle of these mice, suggesting that myostatin may also be involved in the pathogenesis of the disease. To gain further insight into the possible role of myostatin in muscle degenerative diseases, the present work investigates the expression of muscle myostatin in children with muscular dystrophies and mitochondrial encephalomyopathies. No differences in the pattern of myostatin expression were evident in any case, even in those patients with prominent muscular atrophy. These findings suggest that muscle loss that can be observed in muscle degenerative diseases does not depend on changes in myostatin expression.