At present, the etiology of idiopathic pulmonary fibrosis (IPF) remains elusive. Over the past two decades, however, researchers have identified and described the underlying processes that result in metabolic dysregulation, metabolic reprogramming, and mitochondrial dysfunction observed in the cells of IPF lungs. Metabolic changes and mitochondrial dysfunction in IPF include decreased efficiency of electron transport chain function with increasing production of reactive oxygen species, decreased mitochondrial biogenesis, and impaired mitochondrial macroautophagy, a key pathway for the removal of dysfunctional mitochondria. Metabolic changes in IPF have potential impact on lung cell function, differentiation, and activation of fibrotic responses. These alterations result in activation of TGF-β and predispose to the development of pulmonary fibrosis. IPF is a disease of the aged, and many of these same bioenergetic changes are present to a lesser extent with normal aging, raising the possibility that these anticipated alterations in metabolic processes play important roles in creating susceptibility to the development of IPF. This review explores what is known regarding the cellular metabolic and mitochondrial changes that are found in IPF, and examines this body of literature to identify future research direction and potential points of intervention in the pathogenesis of IPF. Aging is life’s natural destiny. It is biologically defined by a progressive impairment in vital functions coupled with diminished fitness to adapt to environmental stimuli and respond to stress (homeostenosis) . Among tOver the past few decades, our understanding of the function and role of mitochondria has moved beyond the initial view of mitochondria as nothing more than a cellular energy generator producing ATP through oxidative phosphorylation (OXPHOS). Mitochondria are now understood to have their own life cycle consisting of biogenesis, fission/fusion dynamics, and recycling through macroautophagy (mitophagy), and to act as an important bidirectional signaling platform, communicating with the nucleus as well as other organelles. Multiple signaling pathways converge and interact to regulate the linked processes of mitochondrial energetics, biogenesis, production of reactive oxygen species (ROS), mitochondrial DNA (mtDNA) preservation and repair, and mitophagy. Dysregulation of many of these regulatory mechanisms that control mitochondrial function have recently been identified in the epithelial cells, fibroblasts, and macrophages in IPF lungs . MitochoMitochondrial biogenesis, the process of producing additional mitochondria and by extension cellular energy production capacity, is under the control of master regulators PPARγ coactivator-1α (PGC-1α) and PGC-1β, which are nutritional sensors able to induce expression of nuclear respiration factors 1 and 2 (NRF). NRFs upregulate expression of the nuclear and mitochondrial mechanisms necessary for mitochondrial biogenesis . With agIdiopathic pulmonary fibrosis and experimental models of pulmonary fibrosis are associated with telomere shortening and DNA damage, which activates the DNA damage sensor poly[ADP-ribose] polymerase 1 (PARP-1) and the checkpoint inhibitor p53. These signaling mechanisms feedback to reduce activation of PPARγ coactivators and reduce mitochondrial biogenesis , 16. ImpMitophagy is a selective and adaptive response that targets mitochondria for turnover, regulates the number of mitochondria to match cellular energy needs, and removes damaged and dysfunctional mitochondria that can cause cellular stress. Mitophagy and mitochondrial turnover are important processes in maintaining cellular integrity and health. Mitochondrial damage, whether from metabolic alterations, increased ROS production, or an accumulation of mtDNA damage and mutations affecting transcription, results in a reduction of mitochondrial transmembrane potential, reduced ATP production, further increases in ROS, and leakage of mitochondrial contents into the cytosol that, if left unchecked, would eventually lead to cellular injury and apoptosis –23.Selective mitophagy of damaged mitochondria occurs through PTEN-induced putative kinase 1 (PINK1)-Parkin signaling with PINK1 acting as a sensor of mitochondrial membrane depolarization and subsequently activating Parkin, which labels the dysfunctional mitochondrion for trafficking to the autophagosome , 24. WitDeficient mitophagy has been associated with IPF and development of pulmonary fibrosis in response to injury , 25–27. As adaptive responses, autophagy and mitophagy are essential to maintaining normal fibroblast function and preventing apoptosis. Impairment of mitophagy, mediated by Parkin deficiency, in IPF lung fibroblasts has been associated with increased deposition of extracellular matrix under profibrotic conditions such as exposure to TGF-β. Defects in mitophagy and autophagy result in increased production of ROS and activation of platelet-derived growth factor receptor (PDGFR)/mammalian target of rapamycin (mTOR) signaling pathways that enhance fibroblast to myofibroblast transformation . SupportTGF-β has been shown to variably affect key mediators of mitophagy and promote fibrogenesis. Treatment of alveolar epithelial cells with TGF-β results in initial stabilization of PINK1 on the surface of mitochondria and induction of mitophagy, which appears to be an adaptive response to increased ROS production due to TGF-β’s effect on the ETC . Longer 2O2 through activation of the pro-survival kinase, protein kinase B (Akt1). Increased Akt1 activation and ROS production induce mitophagy as a protective measure and prevent macrophage apoptosis, which stabilizes macrophages to release additional TGF-β and promote local fibroblast activation and proliferation. Blocking mitophagy in alveolar macrophages is protective against bleomycin-induced fibrosis , an enzyme that increases levels of fructose-2,6-bisphosphate and drives glycolysis . Glycolyin vivo.By contrast, a recent study comparing IPF lung tissue to normal human lung using a combined metabolomic and microarray analysis of key metabolic enzymes showed a decrease in the late-stage glycolytic products, fructose 1,6-bisphosphate and phosphoenolpyruvate, and a decrease in PFK and PFKFB3, suggesting an overall reduction in glycolysis in IPF whole lung tissue . These fvia canonical Smad and PDGFR signaling resulting in increased cellular glucose uptake. Inhibiting GLUT1 upregulation is protective against bleomycin-induced fibrosis , fibrosis , and GLUfibrosis . Since min vivo  and senolytic agents (dasatinib)  , 74. Thiin vitro and in animal models of pulmonary fibrosis. These early investigations of the role of altered bioenergetics in the development of fibrotic lung disease pave the way for translational research to implicate these pathways in human IPF. Other pathways, specifically those involved in maintaining normal mitophagy and turnover of dysfunctional mitochondria and mitochondrial-targeted antioxidants, present potential avenues for altering the natural history of pulmonary fibrosis but require further investigation to identify a point of intervention. Few potential interventions appear ready for human trials to restore mitochondrial biogenesis and mitophagy in alveolar epithelial cells and use of combination senolytic and antioxidant agents to remove senescent cells from injured lung and prevent propagation of SASP signaling  involved in the development of IPF. Additionally, cellular perturbations seen with normal aging such as decreased macroautophagy and cellular proteostasis have the potential to incite mitochondrial dysfunction and changes in cellular bioenergetics. Development of animal models capable of isolating defects in mitochondrial homeostasis to a particular cell type may be revelatory of the role each cell type plays and shed light on the optimal targets for intervention. Particular attention to the effects of mitochondrial dysfunction on telomere length in the resident progenitor cells of the lungs responsible for regenerating the alveolar epithelium may present novel approaches for preventing stem cell exhaustion in IPF.While many of these pathways have shown potential as therapeutic targets in isolation, an integrated approach to the bioenergetic and homeostatic changes that are seen with aging and IPF, such as telomere shortening, reduced mitochondrial biogenesis, increased ROS production, and increased nuclear and mtDNA damage, is essential to determining the pathways and therapeutic targets that are most likely to affect the natural history of IPF.DZ wrote the manuscript along with MB. The manuscript was conceptualized, supervised and modified by ALM and MR. All authors offered intellectual contribution and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Expansion of the CGG repeats beyond 200 copies results in protein deficiency by leading to aberrant methylation of the FMR1 promoter and the switch from active to repressive histone modifications. Additionally, the CGGs become increasingly unstable, resulting in high degree of variation in expansion size between and within tissues of affected individuals. It is still unclear how the FMR1 protein (FMRP) deficiency leads to disease pathology in neurons. Nor do we know the mechanisms by which the CGG expansion results in aberrant DNA methylation, or becomes unstable in somatic cells of patients, at least in part due to the lack of appropriate animal or cellular models. This review summarizes the current contribution of pluripotent stem cells, mutant human embryonic stem cells, and patient-derived induced pluripotent stem cells to disease modeling of FXS for basic and applied research, including the development of new therapeutic approaches.Fragile X syndrome (FXS) is the most common heritable form of cognitive impairment. It results from a loss-of-function mutation by a CGG repeat expansion at the 5′ untranslated region of the X-linked  Fragile X syndrome  is the most common heritable form of cognitive impairment , and is the leading known genetic cause of autism. It is inherited as an X-linked trait and results from a deficiency in the fragile X mental retardation protein (FMRP) [fragile X mental retardation 1 (FMR1) gene [FMR1 [FMR1 in FXS patients, and how the loss-of-function of FMRP leads to neural deficits in FXS fetuses. In addition, the underlying mechanisms for germ line and somatic CGG instabilities in FXS are unknown. In terms of treatment, it would be enormously beneficial to find how/whether FMR1 gene inactivation can be reversed in patients. As is the case for other pathologies resulting from untranslated repeat expansions, modeling FXS can be particularly challenging since it is difficult to clone lengthy repetitive elements. Nevertheless, numerous transgenic and knockin animal models of FXS have been created [Nearly all FXS patients lack FMRP due to an unusual loss-of-function mutation; namely, a CGG tri-nucleotide microsatellite repeat expansion in the 5′ untranslated region (5′-UTR) of the X-linked R1) gene ,4,5. WhiR1) gene . When thne [FMR1 ,10,11,12ne [FMR1 ,13. It i created ,20,21,22 created ,24,25. THuman embryonic stem cells (hESCs) are undifferentiated pluripotent cell lines with unrestricted self-renewal and the ability to differentiate into a wide range of cell types . They arThus far, over a dozen of FXS hESC lines have been established worldwide see  and utilThis review summarizes the current contribution of mutant PSCs, hESCs and patient-derived iPSCs to research of FXS. For each we summarize the currently available stem cell based models, highlight how they have contributed to better understanding of the related mechanism, their limitations and the ways in which they can be utilized in future investigations.FMR1 is not fully understood. In addition, it remains unclear when FMR1 gene inactivation takes place during embryo development. Previous reports related to the timing of FMR1 silencing showed that the FMR1 locus is still active at 8–10 weeks of gestation. It begins to be repressed in fetal tissues by the end of the first trimester, and frequently remains partly active in extra embryonic tissues [FMR1 inactivation occurs through a developmentally regulated process. These studies also provided evidence that the CGG expansion is a necessary, but not sufficient condition for gene inactivation and that additional differentiation-dependent factors are required to achieve FMR1 silencing. When the first male FXS hESC line was established (HEFX), it was found to express FMR1 mRNA at levels comparable to wild type (WT) hESCs and to be completely unmethylated at FMR1 [FMR1 epigenetic silencing is triggered by differentiation and encouraged investigators to posit that the cellular phenotype of FXS neurons could be corrected simply by removing the repressive epigenetic marks that are incorrectly acquired by cell de-differentiation. However, when patients’ skin fibroblasts were reprogrammed using standard reprogramming procedures, FMR1 remained inactive and hypermethylated in iPSCs derived from FXS fibroblasts [FMR1 gene, it recurrently resulted in hypermethylation and complete inactivation of FMR1 in the newly established iPSC clones [FMR1.Despite the tight correlation between transcriptional inactivation and chromatin compaction, the precise mechanism governing the silencing of  tissues ,48,49,50 at FMR1 . This varoblasts ,36,38,39C clones . This leFMR1 hypermethylation is not restricted to somatic cells but can also be acquired in the undifferentiated state, since studies have shown that FXS hESC lines are fairly heterogeneous for methylation levels despite the presence of a full mutation [FMR1 hypermethylation , more closely resemble epiblast cells (primed ESCs) than inner cell mass (ICM) cells (naïve ESCs) [FMR1 gene in FXS iPSCs [FMR1 in FXS hESC/iPSCs clones, respectively [FMR1 methylated, and if so whether FMR1 silencing can be achieved before the blastocyst stage. If methylation is found, it would be valuable to assess the extent of its variation within affected embryos. In any case and regardless of the exact time at which hypermethylation is first established, the failure to methylate expansions greater than 200 CGGs may point to a “window of opportunity” within which aberrant methylation can occur such that expansions that have coincidentally escaped de novo methylation persist. A model that relates to the timing and nature of FMR1 hypermethylation suggests that abnormal methylation is first acquired on full expansions in FXS at a restricted time point before/during embryo implantation. Once established, it is irreversible and is clonally maintained. Expansions that escape abnormal methylation during this limited time frame remain unmethylated or become inactivated later during differentiation  ,53,54. IXS iPSCs . Howeverectively . Hence ition see . The relMR1 mRNA .FMR1 by taking advantage of FXS hESCs which, unlike the somatic cells of patients and patient-derived iPSCs, provide an unusual opportunity to uncouple CGG expansion and heterochromatin induction in a developmentally regulated context. Given the accumulated data related to epigenetic gene silencing in other loci, particularly at repetitive elements, there is a common view that FMR1 inactivation is elicited in a way that is RNA-directed [FMR1 silencing is achieved, although none have yet been validated. One model put forward by Ladd et al. argued that the insulator-binding protein CTCF differentially binds to the flanking regions of the repeats in normal but not expanded alleles [ASFMR1) [FMR1. To find the potential mechanistic link between FMR1 inactivation and CTCF binding, Avitzour et al. took advantage of FXS hESCs and examined whether methylation is coupled with the binding loss of CTCF adjacent to the repeats [FMR1 unmethylated hESCs. This excluded the possibility of CTCF binding loss as a trigger for heterochromatin by CGG expansion. However it does not rule out the potential role of bidirectional transcription as a mediator of silencing, given the capacity of long dsRNA to be processed into small RNA molecules by the RNA interference (RNAi) machinery [Dicer activity [Dicer, Ago1 or Ago2 did not prevent FMR1 epigenetic gene silencing in FXS hESCs [Mechanistically, researchers are attempting to address the question of how CGG expansion leads to de novo methylation of the 5′-UTR of directed ,57,58. O alleles ,60. Furt[ASFMR1) ,60. Cons[ASFMR1) ,62,63,64 repeats , as sugg repeats ,60. Howeachinery . Anotheractivity ,67. Regaactivity ,69). By XS hESCs , which iXS hESCs .FMR1 gene silencing involving FXS hESCs have pointed to the role of cell differentiation in this process. For instance, Colak and colleagues, who employed two FXS hESC lines (WCMC-37 and SI-214) reported they had uncovered an mRNA-mediated mechanism that drives epigenetic gene silencing in a way that relies on neuronal differentiation [FMR1 mRNA hybridize to the complementary CGG-repeat portion of the FMR1 gene to form an RNA:DNA duplex. They demonstrated that upon directed differentiation into nerve cells, the destruction of the naturally formed RNA:DNA hybrids at the 5′-UTR of the FMR1 locus is consistent with the repression of FMR1 transcription, and is coupled with a switch from active to repressive histone marks . This could suggest that RNA:DNA hybrid formation at a critical time point during cell differentiation prevents neural-induced FMR1 silencing. However, a careful examination of the methylation status of FMR1 in both FXS hESC lines indicated that they were partly methylated to begin with. It remains to be determined whether FMR1 inactivation by neuronal differentiation reflects a second wave of gene inactivation. In addition, it is unclear whether silencing in FXS undifferentiated cells is achieved by the same mechanism as in differentiating cells, and whether the block in FMR1 transcription in FXS HESC derived neurons coincides with DNA hypermethylation. It would be of great interest to explore the potential role of R-loops (three-stranded nucleic acid structures formed between an RNA strand to one of the DNA strands) in maintaining the FMR1 in a transcriptionally active configuration, in light of recent findings on their potential role as a mechanism protecting against epigenetic gene silencing [FXN gene [Other studies exploring the mechanism of ntiation . They clilencing . ConversFXN gene .FMR1 inactivation raises the issue of whether the inactive state is maintained once it is achieved. For years, the question was whether the maintenance of the inactive state depends on the presence of the CGG expansion, or whether once established it is endlessly preserved (similar to the effect of X-inactive specific transcript (XIST), which is essential for initiation and spread but is not needed for the maintenance of X inactivation) [FMR1 gene in male FXS iPSCs [FMR1 gene expression and FMRP protein levels in a pair of clones by targeting a deletion at the CGG repeats and immediate 5′-flanking region that led to nearly complete reversion of hypermethylation (≤10%) concomitant to the gain of H3K4me3 and the partial loss of H3K9me2 marks. Demethylation and re-activation of the FMR1 led to sustained expression through long-term differentiation into mature neurons. These findings suggest that the mechanism responsible for maintaining the inactive state does not depend on epigenetic memory, but rather on persistent de novo acquisition of abnormal epigenetic marks conditioned by the presence of the expansion at each cell division. If indeed the expanded repeats are crucial for the maintenance of aberrant DNA hypermethylation at each cell cycle, it would be stimulating to investigate which molecular mechanism enables this process and how is it mediated.A different feature related to epigenetic ivation) . HoweverXS iPSCs . Using tFMR1, when the CGGs increase beyond the normal range (>55 CGGs) they tend to further expand when transmitted through the female germ line (germ line instability). Once the CGGs expand over 200 repeats, they lead to FXS and become increasingly unstable. This results in a high degree of variation in expansion size between and within tissues of affected individuals, a phenomenon termed repeat somatic instability [Repeat instability describes a mutational event that refers to the addition or deletion of DNA repeats arranged in tandem. It accounts for over 20 neuromuscular, neurodevelopmental and neurodegeneration pathologies, including FXS (for comprehensive review see ). In FMRtability ,76. In Ftability ,78. Whiltability ,80. TherFMR1 is hypomethylated. This supports, but still does not prove, that somatic instability may be inhibited by the gain of aberrant methylation. It would be interesting to explore whether methylation and repeat somatic instability are mechanistically associated, or whether they are two unrelated molecular events that have regulators in common. The mechanism for instability, both germ line and somatic, is unknown. Evidence for the involvement of DNA replication, DNA repair and recombination has been obtained using different model systems (for a comprehensive review see ). RegardEvidence emerging from diverse systems, including bacterial plasmids and various replication defective strains of yeast, support the hypothesis that somatic instability in FXS is promoted by DNA replication ,84,85,86Finally, while all of the aforementioned studies deal with the use of PSCs to address fundamental issues concerning somatic instability in FXS, it would be worthwhile investigating the mechanisms underlying germ line instability; i.e., to determine how a PM converts into a FM when transmitted through the mother’s germ line. This can be accomplished once efficient protocols for inducing differentiation of hESC/iPSC into functional gametes are developed. In fact, ESCs have already been differentiated into fully matured oocytes and viable offspring produced from in vitro derived gametes in mice , thus paFMRP is ubiquitously expressed in the body, but is most abundant in the brain and testes. It interacts with approximately 4% of all mRNA species in the brain and is responsible for transporting them out of the cell nucleus to the synapses of neurons . Most ofFMR1 epigenetic gene silencing occurs and FMRP is turned off. The absence of FMRP from that point on during development eventually leads to intellectual impairment, the clinical phenotype of FXS [FMR1 knockout mice have been extremely informative for studying the clinical pathology of FMRP deficiency, they fail to recapitulate the course of the disease in a developmentally regulated context because they are FMRP protein deficient from day 1 [FMR1 by homologous recombination, the PM rarely expanded into the full mutation range in the offspring, and when it did, it often contracted and manifested almost no aberrant methylation, leaving the FMR1 gene active [FMR1 expressing (PM) and un-expressing (FM) isogenic iPSC clones, and compared their neural differentiation properties. Whereas FM iPSCs (methylated and inactive FMR1) exhibited fewer Tuj1-positive cells and shorter branch processes, in glial cells no clear difference could be detected between PM and affected clones. However, since FMR1 is already inactive from the start in the FXS iPSCs, it may not properly reflect the molecular cascade that leads to the pathology as it occurs in vivo. On the other hand, using three different male FXS hESC lines as a substitute for patient-derived iPSCs, Telias et al. were able to generate electrophysiologically active cells using a differentiation protocol that enhanced the formation of fully developed neurons [FMR1 may be crucial for investigating the neural phenotype of the disease, since FXS iPSCs neurons are likely to present a more drastic phenotype than their FXS hESCs counterparts. On the other hand, as disease severity is fairly heterogeneous among patients, the expected phenotype may be highly variable between the different cell lines. This should be taken into account when trying to assess pathogenesis, and could be addressed by defining standard criteria for differentiation, and by verifying loss-of-function of the FMRP protein at equivalent differentiation stages.Based on numerous studies on FXS aborted fetuses ,102, FMRe of FXS ,104,105.e of FXS ,107. Thee of FXS ,18,20,22om day 1 . On the e active . These ce active , who est neurons . Althoug neurons ,109. By  neurons . This maRE1 Silencing Transcription Factor (REST), which functions to repress neural genes in non-neuronal tissues. It is thought to be a master regulator in neurogenesis. By comparing gene expression in WT and affected FXS iPSCs, Halevy et al. provided a list the downregulated genes responsible for axon guidance that are typically controlled by REST [REST itself is upregulated in FXS iPSC-derived neurons, most likely by the increase in mir-382 mRNA levels. Rescue experiments by overexpression of mir-382 in FXS iPSCs-derived neurons reduced elevated levels of REST and restored axon guidance related gene expression. A full characterization of the in vitro derived neurons in terms of subtype and degree of maturation were not reported. Another candidate is the SOX family of transcription factors, and in particular SOX9 and SOX2, which are known for their key role in neurogenesis and are both highly expressed in NPCs [SOX9 expression levels concurrent with a rise in SOX2 mRNA levels [SOX2 and SOX9 expression directly or indirectly, and to better understand the overall involvement of the SOX superfamily in the neuron defects associated with FXS. Finally, in a subsequent study the researchers used expression analysis for the GABA-A receptor subunits and reported increased levels of α2 subunit mRNA levels, which could possibly explain the lack of mature response to GABA signaling [Regarding the molecular mechanisms that are involved with impaired neurogenesis of FXS, it is hard to point to major downstream targets/specific pathways that play a central role in disease pathology since FMRP, as mentioned earlier, has many (hundreds to thousands ) downstr by REST . Interes in NPCs ,113. By A levels . These rignaling . This laignaling ,119,120,Neuronal differentiation protocols are improving and thus becoming more available to research labs worldwide. Apart from shortening the protocols considerably, it facilitates our ability to manipulate the cells to differentiate into specific lineages and particular subtypes of neurons in a more controlled and efficient fashion which will enable better evaluation. In addition, large scale gene expression profiles at various time points during nerve cell differentiation are expected to provide new insights and perhaps the identification of new key players in FXS pathogenesis. Hopefully, this will eventually lead to the development of new therapeutic approaches to cure, or more likely to relieve disease symptoms.FMR1 gene silenced by the FM. In fact, the existence of rare unmethylated full mutation individuals [FMR1 transcription may be possible. Given the large number of mRNAs targeted by FMRP and the various known dysregulated pathways, including the GABAergic pathway (reviewed in ), clearlividuals , suggestFMR1 silencing is produced by epigenetic modifications and the coding sequence for FMR1 remains intact in patients, scientists are aiming to develop alternative therapeutic approaches to treat patients by developing strategies to re-activate the gene and restore its function. Mutant PSCs provide a powerful platform for drug screening and development since they can potentially differentiate into all cell types in large numbers so that targeted tissues are available for large-scale drug screening  . Howeveriewed in ). This iiewed in  and therFMR1 gene in iPSCs and in vitro differentiated neurons [FMR1 mRNA levels, and was validated by immunostaining for FMRP. However, only 5-azaC was able to robustly reactivate gene expression in FXS neurons, although its association with TSA treatment led to a slight increase in mRNA levels. At high concentrations (10 µM) the effect of 5-azaC on DNA methylation reached 57% and was coupled with enrichments in H3K4me3 and H3-acetylation (active histone modification marks) in the neurons, and to a lesser extent in undifferentiated FX iPSCs. However, FMR1 mRNA and protein levels were only partially restored (15%–45% mRNA levels of WT control). This may have resulted from the inability of 5-azaC to remove the repressive methylation mark of H3K9me3 from the locus, and is consistent with previously established data in patients’ somatic cells where FMR1 re-activation only persisted for a limited time (5–7 weeks) after drug withdrawal. It is worth noting that following drug treatment massive cell death was observed in the undifferentiated FXS and WT iPSCs, most likely since the drug concentrations were beyond physiological levels found in the plasma of cancer patients that were treated with this agent [Over the last few years there have been several attempts to use in vitro NPCs and fully matured in vitro differentiated neurons from FXS PSCs (mainly iPSCs) to pinpoint the chemical reagents that can efficiently remove the repressive epigenetic marks that are wrongly acquired by the mutation. At first, researchers aimed to explore the effect of a few Food and Drug Administration (FDA)- approved chromatin remodeling compounds including the general histone deacetylase inhibitor trichostatin-A (TSA) and the de-methylating agents 5-azacytidine (5-azaC) and its deoxy derivative, 5-aza-2′-deoxycytidine (5-aza-dC), which at low doses lead to the loss of DNA methylation by inhibition of DNA methyltransferases . Taking  neurons . The effis agent . An addiis agent ,129,130.FMR1 re-activation. Using NPCs derived from the iPSCs of a single patient, Kumari and colleagues screened a collection of approximately 5000 known FDA approved drugs by a highly sensitive fluorescent detection assay for FMRP expression [FMR1 mRNA levels. Nevertheless, this study provided a proof-of-principle for how FXS iPSCs could be exploited as a platform for high-throughput screening to identify top compounds for FXS therapy. In a different screen, Kaufmann et al. tested a set of 50,000 compounds, including modulators of several epigenetic targets, with a high-content imaging system that measures FMRP, the most significant readout for the expected phenotype [FMR1 locus in FXS iPSCs [FMR1 gene. Overall, although identifying the mode of action of leading compounds were beyond the scope of these studies, they provided a proof-of-concept for how patient derived NPCs may pave the way for the discovery of new drugs and/or approaches to correct the FXS phenotype. That said, it is uncertain whether any molecule will ever be gene-specific and safe. Therefore, the ultimate therapy for FXS will be to specifically target the FMR1 gene rather than treating patients with agents that act globally on the genome. A more general and promising approach would be to screen for a large number of compounds in a way that does not rely on the best candidate approach and/or educated speculation. This could be done by high-throughput screenings to identify compounds that increase FMRP expression in FXS iPSCs-derived neurons. To date, three high-throughput screens based on iPSCs have been reported for pression . They idhenotype . This enXS iPSCs . By carrFMR1 by 5-azaC fails to remove the repressive mark H3K9me3, along with its inability to fully restore FMR1 mRNA expression levels, it may be impossible to re-establish the transcriptional activity of the gene permanently as long as the extra CGGs remain. To explore whether re-activation can be achieved through the elimination of the repeats, Park et al. managed to delete the entire region by gene editing [FMR1 mRNA to normal levels. This was accompanied by a gain in H3K4me3 and a significant decrease in H3K9me3. Interestingly, when comparing gene expression profiles between the isogenic edited and non-edited FXS neurons, differential expression of glutamate receptor genes were found, which is consistent with the deregulation of glutamate signaling pathway in FXS [FMR1 locus, and thus consistently remove the epigenetic modifications that are repeatedly deposited due to the expansion. A different approach to consider would be to target the locus by gene manipulation. Given that demethylation of  editing . Using ty in FXS . Altogety in FXS . Moreovey in FXS  specificFMR1 gene, how the loss-of-function of FMRP leads to neural deficits in FXS fetuses, or how the CGGs become extensively unstable in patients. This is, in part, because currently available animal models do not accurately reproduce the molecular and cellular phenotypes of FXS in humans, and since the diseased organ—the developing brain—is rarely available for research. Hence, there is a need to complement human-based model systems in a way that can overcome the difficulties associated with obtaining a sufficient supply of disease relevant cells for basic and applied research. These systems should also bypass the need for gene targeting since lengthy repeat expansions are particularly difficult to artificially induce and maintain. For all of these reasons researchers are currently relying on mutant pluripotent stem cells, hESCs and iPSCs, as a way to address some of the gaps in our knowledge on the molecular mechanisms that go wrong in FXS developing fetuses. Thus far, over a dozen FXS hESC lines and many patient-derived iPSC clones have been established. These exclusive cell lines are expected to be particularly useful because they may provide an exceptional opportunity to look at affected neurons while they differentiate and become functionally mature. In addition, they provide a valuable way to genetically intervene and carry out functional assays and rescue experiments while they propagate and differentiate in vitro. Finally, they are expected to be a powerful tool in the development of new therapeutic approaches to correct, or more likely reduce, disease symptoms by searching for drug targets or by genetic engineering. However, we still need to better define how well these cell lines mimic the molecular and cellular pathways that are disturbed in vivo, and better evaluate the differences between FXS hESCs and patient-derived iPSCs, to be able to best exploit them to respond to the remaining unanswered questions regarding FXS.Despite the identification of the CGG expansion as the cause of FXS over two decades ago, little is known about the mechanisms underlying FXS. For example, it is still unclear how CGG expansion leads to epigenetic gene silencing of the


Saccharomyces cerevisiae cells, which constitute an adequate model to study such toxicity at the molecular level and the functions participating in protection against Se compounds. Those Se forms enter the yeast cell through other oxyanion transporters. Once inside the cell, inorganic Se forms may be converted into selenide through a reductive pathway that in physiological conditions involves reduced glutathione with its consequent oxidation into diglutathione and alteration of the cellular redox buffering capacity. Selenide can subsequently be converted by molecular oxygen into elemental Se, with production of superoxide anions and other reactive oxygen species. Overall, these events result in DNA damage and dose-dependent reversible or irreversible protein oxidation, although additional oxidation of other cellular macromolecules cannot be discarded. Stress-adaptation pathways are essential for efficient Se detoxification, while activation of DNA damage checkpoint and repair pathways protects against Se-mediated genotoxicity. We propose that yeast may be used to improve our knowledge on the impact of Se on metal homeostasis, the identification of Se-targets at the DNA and protein levels, and to gain more insights into the mechanism of Se-mediated apoptosis.Inorganic Se forms such as selenate or selenite (the two more abundant forms in nature) can be toxic in  Selenium (Se) is an element that shares characteristics of both metals and nonmetals, being therefore considered as a metaloid. Similarly to sulfur, Se has different oxidation states ranging from +VI to -II. In the external environments found on earth it is present in low amounts, generally as selenate Se(+VI) or selenite Se(+IV), although these amounts vary considerably depending on the geographical areas Saccharomyces cerevisiae has no metabolic need for Se, it is an adequate organism to study the toxic effects of Se forms on cell functions In addition to their known role in combating various forms of degenerative diseases, the impact of organoselenium compounds in cancer chemoprevention has been studied, and thus, doses up to 200-300 µg per day are proposed to protect against diverse types of cancer  on the basis of the Se antioxidant role 56781011121315Thauera selenatis can respire toxic oxyanions of Se, namely selenate and selenite 2119In the environment, Se is found in four oxidation states, elemental selenium Se(0) and soluble selenate Se(+VI), selenite Se(+IV), and selenide Se(-II) . Se(-II) is found as volatile, methylated species or as organoselenium typically in the form of proteins containing the amino acids selenocysteine and selenomethionine Inorganic Se compounds can also be metabolized inside the cell through a reductive pathway from selenate to selenide . This process involves reduced glutathione (GSH) with the consequent formation of oxidized glutathione (GSSG) and reactive oxygen species (ROS) 25262829S. cerevisiae, selenate probably enters through the high affinity sulfate permeases Sul1 and Sul2. Thus, a double mutant lacking both transporters is hyperresistant to selenate as well as to chromate S. cerevisiaesul1 mutant cells allows increased uptake of molybdate, while increasing molybdate concentration in the medium interferes with sulfate entry S. cerevisiae.Specific transporters for uptake of inorganic Se compounds still need to be characterized in eukaryotic cells. In S. cerevisiae or other eukaryotic organisms.Initial work on the kinetics of selenite uptake indicated the existence of both a high affinity and a low affinity transport system operating at different selenite concentrations et al. S. cerevisiae38PHO84 and PHO89 genes is upregulated at low phosphate concentration depending on the PHO signal transduction pathway, with the Pho4 transcription factor as effector of the pathway PHO regulon 38S. cerevisiae cells through the Pho84 and phosphate low affinity transporters 42A detailed study by Lazard JEN1 gene codes for a plasma membrane high affinity transporter of monocarboxylic acids such as lactate, pyruvate or acetate JEN149Glucose was the only carbon/energy source used in the studies described above but apparently, the Jen1 transporter acts as an alternative selenite and arsenite transporter in the presence of other carbon sources SSU1 gene  formation and show increased mutation rates 5259S. cerevisiae cells has revealed that HR mutants are selenite-hypersensitive 526163In yeast cells DNA DSBs are mainly repaired by the homologous recombination (HR) pathway, taking advantage of sister chromatids as DNA repair templates et al. revealed that selenide treatment causes oxygen-dependent DNA phosphodiester-bond breaks in vitro, and chromosome fragmentation in vivoA recent study by Peyroche Despite the advance in our knowledge on the mechanisms that contribute to the formation and repair of Se-mediated DNA damage, little is known on the possible impact of Se on proteins involved in DNA metabolism. It will be interesting to see if Se compounds interfere with disulfide bridge formation and protein folding, or the function of metalloproteins such as DNA polymerases 2+, thiol oxidants, ROS, and members of the Bcl-2 family of proteins 757677S. cerevisiae has been very useful to dissect the underlying mechanisms that contribute to apoptosis but little is known on the impact of Se on yeast mitochondrial function. In human cells, apoptotic events that are mediated by selenite, selenocystine, and selenodioxide are related to oxidation of protein thiol groups and ROS generation 8182S. cerevisae is lacking mitochondrial selenoproteins, toxic Se may as well induce apoptotic events in yeast S. cerevisae growth Mitochondria play an important role in cellular energy supply and apoptosis in yeast and higher eukaryotes 71S. cerevisiae mutants displaying selenite tolerance revealed that the mutants in genes involved in the conversion of sulfate into sulfur were tolerant to selenite and also to tellurite, supporting an opportunistic common assimilation pathway for both toxic oxyanions gsh2Δ mutant defective in GSH biosynthesis is also unable to accumulate elemental selenium 632890GLR1 partially rescues the inhibitory effect of selenite on yeast cell growth glr1∆ mutant is hypersensitive to selenite, as well as a gsh1∆ mutant involved in the first step of GSH biosynthesis glr1∆ and gsh1∆ mutants have also been described as hypersensitive to sodium selenide Once inside the cell selenite would employ the sulfate assimilation pathway for its conversion into the more toxic form selenide ycf1∆ mutant is hypersensitive to heavy metals. On the contrary, the ycf1∆ mutant is resistant to selenite, while overexpression of Ycf1 exacerbates selenite toxicity The observed drop in the intracellular glutathione pool upon selenite treatment cannot be simply explained by the reduction of the cytosolic GSH pool due to reduction of selenite to selenide and ROS. Detoxification of heavy atoms such as cadmium, arsenic or mercury in yeast involves the participation of the vacuolar membrane located ABC transporter Ycf1 44S. cerevisiae cells revealed the induction of genes participating in the oxidative stress response 5463TTR1) and glutathione reductase (GLR1), both coding for activities required for functional (NADPH-dependent) thioredoxin and (glutathione-dependent) glutaredoxin systems, respectively. These two systems control the redox state of protein thiol groups through their thiol oxidoreductase activity and consequently, are important for repairing oxidation of these thiol groups and for protein redox modulation 94SOD1Transcriptome analysis of selenide- and selenite-treated S. cerevisiaegrx1∆ grx2∆ mutant is hypersensitive to selenite 49grx1∆ mutant has also been described as selenide-hypersensitive The alteration of the GSH/GSSG ratio by selenite may result in general oxidation of protein thiol groups. This would explain the reported protection of dithiol glutaredoxins Grx1 and Grx2 against selenite toxicity 49Diverse stress response pathways sense the lesions caused by Se compounds in yeast cells and induce protective responses. Given the diversity of the toxic effects triggered by selenite or selenide, it is not surprising that several response transducers and effectors may be acting in parallel. Disruption of the pathways or effector loss of function results in hypersensitivity to Se molecules.RAD9 and other genes of the pathway are hypersensitive to DNA damage mediated by UV light, DNA alkylating agents or selenite 5452RAD9 and other genes coding for central components of the DNA damage response are not hypersensitive to selenomethionine Upon DNA damage, cell cycle arrest by G1/S, S and G2/M cell cycle checkpoints is essential to avoid unscheduled repair of DNA lesions 100101et al. Yeast Snf1 was identified as having a general role in the oxidative stress response and selenite tolerance 2H2 zinc finger transcriptional regulators that was initially characterized as modulator of the response of yeast cells to alkaline pH. Later studies showed its implication in processes such as sporulation and invasive growth, protection against Na+ and Li+ toxicity, cell wall assembly, protection against weak organic acids and regulation of calcium homeostasis 109110111114114110+-ATPase (V-ATPase) complex becomes downregulated, providing a rationale for the selenite hypersensitivity of the rim101∆ mutant Rim101 is a member of the fungal PacC family of CS. cerevisiae cells and interfere with cellular processes are summarized in Fig. 2, which also depicts the activation of pathways required for Se tolerance and detoxification. Entrance of selenate and selenite (the two more abundant free forms of Se in nature) occurs through oxyanions transporters, and once in the cell they are transformed into selenide through a reductive pathway that may involve GSH. In the presence of oxygen, selenide can promote the formation of superoxide and other ROS species, which may damage DNA, proteins and probably also other cellular macromolecules. Thus, changes in the redox buffering of the cell and ROS overproduction, which themselves are two interrelated processes, would be an origin of Se toxicity.The mechanisms by which Se compounds enter into in vitro with different metal anions, confirming in fact its interaction with ferrous iron and that the selenide- iron complexes become biologically inactive in vivo interference can be extended to other anions is open. In addition, yeast as model organism offers the possibility to address the identification of hot spots of Se-mediated DNA damage, or to extend our knowledge on factors involved in signaling and repair of Se-mediated damage. It will be interesting to determine which proteins are prone to selenite or selenide-dependent modifications, including irreversible (i.e. carbonylation) or reversible  protein modifications. Finally, yeast may serve as an excellent tool for the characterization of events related to Se-mediated aging and apoptosis. Such studies are important for a better understanding of the molecular mechanism underlying Se-mediated pathologies in multicellular organisms, including humans.However, many questions remain. Selenite has been shown to activate expression of genes under transcriptional control of the Aft1 regulon 117


Osteoarthritis (OA), a degenerative disease of diarthrodial joints, is influenced by mechanical and inflammatory factors with aging, obesity, chronic injuries, and secondary diseases thought to be major factors driving the process of articular cartilage degeneration. Chondrocytes, the cellular component of cartilage, reside in an avascular environment and normally have limited potential to replicate. However, extrinsic factors such as injury to the joint or intrinsic alterations to the chondrocytes themselves can lead to an altered phenotype and development of OA. Synovial inflammation is also a pivotal element of the osteoarthritic, degenerative process: influx of pro-inflammatory cytokines and production of matrix metalloproteinases accelerate advanced cellular processes such as synovitis and cartilage damage. As well as a genetic input, recent data have highlighted epigenetic factors as contributing to disease. Studies conducted over the last decade have focused on three key aspects in OA; inflammation and the immune response, genome-wide association studies that have identified important genes undergoing epigenetic modifications, and finally how chondrocytes transform in their function during development and disease. Data highlighted here have identified critical inflammatory genes involved in OA and how these factors impact chondrocyte hypertrophy in the disease. This review also addresses key inflammatory factors in synovial inflammation, epigenetics, and chondrocyte fate, and how agents that inhibit epigenetic mechanisms like DNA methylation and histone modifications could aid in development of long-term treatment strategies for the disease. Osteoarthritis (OA) is the most common musculoskeletal disorder impacting life quality of patients with about 35–40 million Europeans and ~355 million affected worldwide  secrete synovial fluid for joint lubrication with hyaluronic acid the principal component that provides the required viscosity; (2) supplement nutrition to chondrocytes and draining waste metabolites, and (3) act as the source of synoviocytes, macrophages, and fibroblasts that express a wide range of cellular markers required for specialized functions relating to innate and adaptive immunity  angiogenesis with increased levels of vascular endothelial growth factor (VEGF), blood vessel formation (factor VIII), and intercellular adhesion molecule-1 and the pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α), and IL1ß in both early and late OA, with significantly higher expression in early OA  for many years . The process of acetylation is orchestrated by histone acetyltransferases, located on specific lysine residues on the histone N-terminal tails, resulting in the unraveling of the histone and providing access to the DNA structure and transcriptional machinery  (de Andrés et al., in vitro DNA methylation reduced basal activity of the IL-8 promoter (Table Interleukin-8, also called CXCL-8, is a more recent inflammatory chemokine to be studied in the context of OA. Alongside IL-1β and leukemia inhibitory factor, IL-8 induces chondrocyte hypertrophy and differentiation (Borzì et al., er Table . IL-6 iser Table . These sTranscription factors, also known to regulate chondrocyte differentiation, act by controlling the transcriptional rate of target genes and aberrant expression is known to influence OA pathogenesis as a consequence of chondrocyte hypertrophy (van der Kraan and van den Berg, NFAT1 is a well-known regulator of expression of cytokine genes during the immune response, and interestingly, exhibits an age-dependent expression in mouse articular cartilage (Zhang and Wang, A key transcription factor for chondrogenesis during the development of skeletal system, SOX9 is one of the earliest markers expressed by mesenchymal stem/stromal cells and is essential for expression of cartilage-specific matrix proteins (Han and Lefebvre, Among many genes subjected to candidate gene association analysis (Ryder et al., Maintaining optimal amounts of ECM components, such as collagen and proteoglycan, is also crucial for preserving normal articular cartilage architecture and function. Gene mutations of collagen proteins are typically associated with early-onset of OA (Chan et al., Interactions of HDACi with chondrocytes have conflicting effects on COL2A1, COL9A1, and ACAN gene expression. Short-term HDACi treatment of chondrocytes enhances the expression of these genes, whereas long-term treatment represses their expression. This switch in effects could be attributed to overexpression of HDAC1 and 2 Table . HypermeNormal human articular cartilage expresses very low levels of matrix proteinases such as aggrecanases, collagenases, and MMPs with elevated levels in OA associated with ECM degradation and altered chondrocyte phenotype (Burrage et al., MicroRNAs have a strong role to play in the regulation of MMPs and aggrecanases. ADAMTS-5 expression is highly regulated by miRNA in human OA (Ukai et al., Osteoarthritis is indeed a multifactorial disease underpinned by a complex interplay of inflammation and epigenetic modifications. Genome-wide analyses and studies focused on the identification of candidate OA-risk associated genes conducted in the last decade have thrown light on the mechanism of how chondrocytes undergo hypertrophy and cause progression of the disease. Future studies targeting markers involved in both chondrocyte and synovial hypertrophy could perhaps provide further insight into the use of epigenetic knockdown models and inhibitors to attenuate chondrocyte terminal differentiation and in turn restore cartilage homeostasis.SR contributed to the development of the concept underpinning the review, researched the relevant literature, and wrote the body of the review. UF also contributed to the concept of the submission and provided guidance on the scientific writing. MM participated in conception and review design, manuscript preparation, on-going and final review of the submission.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


GNAS gene, other genetic alterations and affected pathways are, to a large degree, poorly known. In this study, we sequenced whole coding genome of nine PMP tumors and paired normal tissues in order to identify additional, commonly mutated genes and signaling pathways affected in PMP. These exome sequencing results were validated with an ultra-deep amplicon sequencing method, leading to 14 validated variants. The validated results contain seven genes that contribute to the protein kinase A (PKA) pathway. PKA pathway, which also contains GNAS, is a major player of overproduction of mucin, which is the characteristic feature of PMP. In addition to PKA pathway, we identified mutations in six genes that belong to the transforming growth factor beta (TGF-β) pathway, which is a key regulator of cell proliferation. Since either GNAS mutation or an alternative mutation in the PKA pathway was identified in 8/9 patients, inhibition of the PKA pathway might reduce mucin production in most of the PMP patients and potentially suppress disease progression.Pseudomyxoma peritonei (PMP) is a subtype of mucinous adenocarcinoma mainly restricted to the peritoneal cavity and most commonly originating from the appendix. The genetic background of PMP is poorly understood and no targeted treatments are currently available for this fatal disease. While RAS signaling pathway is affected in most if not all PMP cases and over half of them also have a mutation in the  Pseudomyxoma peritonei (PMP) is a rare subtype of intestinal-derived mucinous adenocarcinoma, with prevalence of approximately 1–2 persons per million per year . CurrentKRAS and GNAS alterations in PMP, together with a lower frequency of e.g. SMAD4, SMAD2, SMAD3, TP53, PIK3CA, and AKT1 mutations. Exome sequencing enables identification of variants in the whole coding region of the human genome and allows to expand the analysis of the mutations to pathway level.The genetic background of PMP is poorly understood due to its low incidence and challenges related to collection of representative tissue material of this remarkably mucinous tumor type. The published next-generation sequencing (NGS) studies –11 have KRAS mutation and 12/19 (63%) of the patients harbored a mutation in the GNAS gene. Here, we have sequenced the whole coding genome of nine of these PMP tumors and paired normal tissues in order to identify common mutated genes and pathways outside the scope of the targeted hotspot panels. Mutations in the protein kinase A (PKA) pathway were under special focus, since PKA pathway activation is involved in the overproduction of mucin ,9–117,9–diseases , it indi pathway . Intereseoplasms . In addin of PMP . Both LT cancers . ACVR1B d in CRC . Interested KRAS . AltogetTP53 has been shown to be mutated in HG PMPs [PIK3CA and AKT1 mutations [MCL1 amplification [CTNNB1 mutation [JUN [Most likely also other dysregulated pathways than RAS, PKA and TGF-β have a role in the development and progression of PMP, and further investigation of these pathways is needed  Tables.  HG PMPs –11. The utations –11, and fication , and themutation ,11 and aion [JUN . FurtherDue to the low proportion of neoplastic cells in the PMP samples, sensitivity and functional prioritizing was emphasized in the exome sequencing data analysis. Ultra-deep targeted sequencing was then used to eliminate false positives resulting from the reduced specificity. Transition substitutions, C→T or G→A, are the most abundant mutations in PMP , which wIn conclusion, we have used FFPE samples with deep sequencing to characterize the mutational landscape in PMP. FFPE samples are more abundant and easier to access than fresh samples but their computational analysis is challenging. Thus, the successful use of the freely available computational pipeline developed herein paves way to use FFPE samples in future PMP studies. Our major finding include novel mutated genes in the PKA pathway possibly contributing to the characteristic mucin production of the PMP disease. Inhibition of the PKA pathway could potentially be a universal target in reducing mucin production in the PMP patients, which could prolong survival of these patients. Our results also highlight the importance of TGF-β pathway in the development and progression of PMP and therapies targeting this pathway could also suppress progression of the disease.KRAS and GNAS mutations, somatic mutations in the PKA and TGF-β pathways possibly account for the increased mucus secretion and cell proliferation, respectively.Genetic background of pseudomyxoma peritonei (PMP) is not well characterized and there are no targeted therapies for the treatment of this disease. In this study, we used exome sequencing to expand the mutational analysis of PMP tumors beyond the mutational hotspot areas of known cancer genes and validated the results with an ultra-deep amplicon sequencing method. Validated results indicate that in addition to the fundamental S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


Reelin is an extracellular signaling protein synthesized by Cajal-Retius cells in utero and early after birth, its presence being signaled in adult life too. Reelin acts on its receptors, VLDLR and ApoER2, acting on cytoskeleton, controlling migration and subsequently positioning and stabilizing the cortical neurons. We investigated the reelin presence and its receptors, VLDLR and ApoER2, in melanocytic nevi considering the neural crest origin of the nevus cells and their migration into skin during embrionary period.Melanocytic nevi present a strict cellular architecture and an increased malignant transforming capacity. We investigated reelin presence in 32 melanocytic nevi . The assessment of reelin presence was performed by histological semiquantitative criteria.Results showed the presence of reelin in 29 cases (29/ 32). The presence of reelin was elevated in junctional areas as in dysplastic nevi. VLDLR presented positive values in 16 cases (16/ 32) and ApoER2 was weak positive in 7 cases. Reelin or its receptors was peritumorally absent. Our study showed the presence of reelin in nevus cells from cutaneous melanocytic nevi and, in these cells, only the VLDLR receptor was present in half of the cases.The significance of the reelin presence in cutaneous nevus cells may be hypothetically considered correlated with the position maintenance of the nevus cells or migration of these cells in malignant transforming situation.Abbreviations: ApoER2 = apolipoprotein receptor 2, VLDLR = very low density lipoprotein receptor, DAB-1 = DIABLO protein, HMB45 = gene HMB45 Reelin acts by connecting to its receptors, VLDLR (very low-density lipoprotein receptor) and ApoER2 (apolipoprotein receptor 2), associated to the DIABLO protein (DAB-1). This results in the phosphorylation of DAB-1 tyrosine by the kinases of the src family [2]. The reelin-induced signal regulates the intracellular DAB-1 levels, with effect on the reorganization of the neuronal cytoskeleton, which is fundamental for the migration, positioning, and stability of the cortical neurons [3]. Reelin-deficient mutant mice show massive disorganization of the neurons from several cortical areas [4]. Beside the cortex, reelin can be temporarily seen in adult mice, in the peripheral nerves [5]. In adult mammals, reelin is also present in the liver; this organ may be considered a reelin source, the pituitary gland, and blood [6]. Reelin plasma levels  are increased both in experimental cirrhosis in mice and human cirrhotic patients [7]. Reelin is an extracellular signaling protein synthesized by the Cajal Retius cells in utero or early after birth, which controls the migration and the positioning of the cortical neurons [8]. Reelin was detected in the brain and CSF in adult humans [9]. Reelin is weakly expressed in gastric cancer, due to the aberrant hyper-methylation of the reelin-encoding gene promoter [10]. The increase in cellular motility and induced invasivity were obtained in pancreatic cancer by blocking ApoER2, VLDLR, and DAB-1 [11]. In breast cancer, the loss of reelin expression in tumor cells is associated with tumor proliferation. The increase in reelin expression in these cells is inversely correlated with the methylation of the reelin-encoding gene promoter. In vitro incubation of MDA-MD231 cells with recombinant reelin suppresses cellular migration [12]. Reelin was shown in several tumor cell types. Thus, epithelial prostate cancer cells show a massive expression of reelin. The reelin expression in these cells is significantly correlated with the Gleason score. Reelin was not expressed in benign prostatic proliferations .The increased expression of the VLDLR receptor vs. the ApoER2 receptor, seen in our study, supports this hypothesis. The opposing effects of the two reelin receptors are well known, since the coupling of reelin to its VLDLR receptor is followed by the phosphorylation of DAB1, resulting in migration arrest [14].We may also hypothetically presume that, in certain normal or pathological circumstances of the adult life, reelin could promote cellular migration. Evidence supporting this hypothesis includes the presence of reelin in normal retinogenesis and its increased expression after retinal and corneal injuries. The authors suggested that reelin controls the migration of stem cells in neuronal and non-neuronal tissues during the adult life, in the same manner as during normal ontogeny .An example is the presence of reelin as a matrix protein at collector blood vessels level. Reelin is involved in the cooperation between the endothelial cells and the smooth muscle cells without the implication of VLDLR and APOR2 receptors. Mice deficient in RLin gene present massive alterations of morphology and function of lymphatic vessels, meanwhile VLDLR and APOR2 deficient mice present a normal development of collector lymphatic vessels. On the other hand, reelin is involved in morphology and physiology of dermal collector lymphatic vessels and not at the nervous system level [The association between reelin and cellular migration, both in the embryonic and adult life, led to the assumption that reelin could be involved in the proliferation and migration of tumor cells. Several studies associated the presence or absence of reelin in tumor cells with the malignant proliferation of these cells.Thus, reelin was increased in prostate carcinoma, but not in benign prostatic hyperplasia. In gastric, pancreatic, and breast cancer, the decrease of reelin was associated with tumoral proliferation and increased invasivity.Reelin was well expressed in our study involving nevocellular nevi – i.e., benign tumor with risk of malignant transformation. In one of our studies carried out in malignant melanoma (grade Clark IV invasive tumors), reelin was inconstantly expressed and unequally located within the tumoral mass. In some cases, reelin was absent or weakly expressed, but there were also other cases in which reelin stained moderately positive (unpublished data).In conclusion, our data showed the significant presence of reelin and partially of its VLDLR receptor in the nevus cells, cells originating from the neural crest but situated outside the central nervous system. Reelin was not expressed in the peritumoral tissue. In the absence of more detailed data, it may be assumed that reelin could play the same role in nevus cells as it does in the central nervous system. In addition, reelin may be involved in maintaining and stability of nerves architecture by VLDLR receptor coupling and in some circumstances, it facilitates normal or tumor melanocyte migration. The demonstration of the presence of reelin in nevus cells, as well as the investigation of its role in neuronal migration in adult life, opened new study prospects in tumoral proliferation and invasivity.The authors state no conflict of interest.


Brief periods of ischaemia followed by reperfusion of one tissue such as skeletalmuscle can confer subsequent protection against ischaemia-induced injury inother organs such as the heart. Substantial evidence of this effect has beenaccrued in experimental animal models. However, the translation of thisphenomenon to its use as a therapy in ischaemic disease has been largelydisappointing without clear evidence of benefit in humans. Recently, innovativeexperimental observations have suggested that remote ischaemic preconditioning(RIPC) may be largely mediated through hypoxic inhibition of the oxygen-sensingenzyme PHD2, leading to enhanced levels of alpha-ketoglutarate and subsequentincreases in circulating kynurenic acid (KYNA). These observations provide vitalinsights into the likely mechanisms of RIPC and a route to manipulating thismechanism towards therapeutic benefit by direct alteration of KYNA,alpha-ketoglutarate levels, PHD inhibition, or pharmacological targeting of theincompletely understood cardioprotective mechanism activated by KYNA. Furthermore, experimental evidence for a large number ofpotential mediators and mechanisms has been obtained, but a clear understanding ofthe mechanisms is lacking. This commentary focuses on recent work examining a novelmechanism that may underlie remote ischaemic preconditioning (RIPC) etal., demonstrated that brief cycles of ischaemia and reperfusion of thecoronary arteries protect the myocardium from subsequent prolonged ischaemia andreperfusion, leading to a reduction in infarct size 2.Ischaemic preconditioning is the phenomenon whereby brief periods of ischaemiafollowed by tissue reperfusion confer subsequent protection againstischaemia-induced injury. The concept, proposed 30 years ago by Murry 3. Birnbaum et al. went on to demonstrate that“remote” transient ischaemia of non-myocardial tissues could also beassociated with reductions in the extent of myocardial infarction. They combinedpartial reduction of blood flow to the hindlimb with increased oxygen demand byrapid electrical stimulation of the gastrocnemius muscle and showed reducedmyocardial infarct size in rabbits 4. Subsequently, Kharbanda et al. showed similar beneficialeffects in a porcine model of myocardial infarction and applied the concept of RIPCto healthy human volunteers by inducing transient non-invasive ischaemia with theuse of a blood pressure cuff applied to one arm and demonstrated improvedendothelial function in the contralateral arm 5.The concept was developed further with the observation that ischaemia in one coronaryterritory could protect cardiac tissue supplied by other epicardial arteries 9. Hu et al. undertook a systematic review of 30 randomisedcontrolled trials to investigate the effects of RIPC on the incidence and outcomesof acute kidney injury (AKI) and found evidence of benefit in preventingcontrast-induced AKI 10. However, there was not benefit in ischaemia reperfusion-induced AKI 10, and more recent trials have also failed to see clear benefit in that setting 11. The REmote preconditioning for Protection Against Ischaemia-Reperfusion inrenal transplantation (REPAIR) trial found some evidence that RIPC using transientarm ischaemia-reperfusion improved renal transplant function 12.The important clinical question has emerged of whether RIPC can be usedtherapeutically in the wide range of medical conditions in which ischaemic injuryoccurs. RIPC has been applied in elective cardiac surgery, vascular surgery,percutaneous coronary intervention, and organ transplantation in attempts to improvecardiac, renal, and other outcomes. Individual, small randomised controlled trialshave been reported to show potential benefit 13 nor have more recent larger-scale studies. The Effect of RIPC on ClinicalOutcomes in Coronary Artery Bypass Graft (CABG) Surgery (ERICCA) study, a randomisedcontrolled clinical trial in 1,612 patients, showed no effect of RIPC on clinicaloutcomes 14. RIPC consisting of four 5-minute cycles of ischaemia-reperfusion of theupper arm did not improve clinical outcomes in patients undergoing elective CABG. Nodifferences were seen in mortality, stroke, myocardial infarction, or AKI. The RIPCfor Heart Surgery (RIPHeart) trial of 1,385 patients used a similar upper limbischaemia protocol but also failed to see benefit 15.In the setting of cardiac surgery, meta-analyses have not confirmed any therapeuticbenefit from RIPC Overall, these results are disappointing but convincing in their failure to see atherapeutic benefit of RIPC in most patients. The optimum type, duration, and timingof the ischaemic intervention is uncertain; skeletal muscle mass, hepatic function,concurrent medications, choice of anaesthetic, and the effect on different targetorgans may also vary and influence the effect of the intervention. How can thebenefits seen in experimental studies be translated to a useful therapy, and doesRIPC operate in humans? Understanding the mechanism of effect might enableoptimisation of the clinical use of RIPC.17. Recent work has implicated the hypoxia response and the generation ofcirculating molecular mediators. Hypoxia is a central component of ischaemia, andthe hypoxia-inducible factor (HIF) transcription factors play a dominant role inco-ordinating the transcriptional response to hypoxia. The abundance of theHIF-α factors is controlled by oxygen-dependent prolyl hydroxylation by thePHD family of 2-oxoglutarate dioxygenases 20  and theirtranscriptional potency by the FIH-1 asparaginyl hydroxylase 22. Several studies have implicated the HIF–PHD system in the mechanismof RIPC. These include impaired RIPC in mice heterozygous for a knockout alleleencoding HIF-1α 23, activation of HIF-1α by ischaemic preconditioning, and enhancement ofcardiac protection by pharmacological and genetic enhancement of HIF-1α 24. Mice with genetically reduced levels of PHD2 (and hence enhancedHIF-1α levels) showed greater resistance to cardiac ischaemia 26, as did animals with activation of HIF by pharmacological PHD inhibition orVHL deficiency 27, though other studies have suggested that HIF-1α upregulation isunnecessary in acute RIPC 28.Whilst definitive evidence for therapeutic benefit in humans is lacking, evidencethat experimental manipulations can have a protective benefit is strong  31, that might act as circulating mediators of RIPC. Whilst these studies dosuggest a role for the HIF–PHD system in RIPC, they have not fullydisentangled the requirement for HIF activation in the remote ischaemic tissueversus that in the target protected organ nor the relative contributions of neuralor hormonal mediators.There are a broad array of HIF-mediated responses to hypoxia that might help mediateischaemic preconditioning, including the promotion of anaerobic metabolism,vascularity, and vasodilatation, reactive oxygen species protection, and alterationsin cell survival and cell cycle. Some of these HIF-dependent hypoxic responsesinclude the release of circulating mediators by ischaemic tissue, such as itscanonical target erythropoietin 1. They initially provided further evidence for the protective effects of HIFactivation by showing that genetic and chronic PHD2 inactivation in mice heartsconferred protective benefit against permanent and transient cardiac ischemia.Similar beneficial effects were also seen with acute systemic PHD2 geneticinactivation and with systemic administration of a pharmacological PHD inhibitor. Todetermine whether manipulations of the HIF–PHD system in the remote ischaemictissue (but not the target heart) affected RIPC, they studied mice with PHD2inactivated only in skeletal muscle. Such mice again showed enhanced myocardialprotection following ischaemia. They then undertook parabiotic experiments toprovide important evidence that this protective effect was mediated by a circulatingfactor. To determine the nature of this circulating factor, they tested for cytokineand metabolite differences in the blood of mice with and without PHD2 skeletalmuscle inactivation. No significant changes were seen in cytokines or molecules suchas erythropoietin, which has previously been suggested to act as a circulatingmediator of RIPC. Similarly, no plausible secreted candidates were identified fromgenetic expression analyses between mice with and without PHD2 skeletal muscleinactivation. However, when blood was compared by analysis with liquidchromatography and mass spectroscopy, significant differences in tryptophanmetabolites were observed. Similar alterations were also seen in blood shortly afterpharmacological PHD inhibition with significant elevations in the level of thetryptophan metabolite kynurenic acid (KYNA).A major insight into the mechanism of RIPC and the role of the HIF–PHD systemand circulating mediators has come from the recent work of Kaelin and colleagues 32.Further evidence implicating KYNA as a mediator of ischaemic preconditioning wereobtained by abrogating RIPC with inhibitors of the tryptophan pathway and from thebeneficial effects of administration of KYNA itself. Studies were then undertaken toexplore the mechanism by which PHD2 inactivation in muscle resulted in increases incirculating KYNA and mediation via an increase in levels of the obligatory PHDco-substrate alpha-ketoglutarate with subsequent hepatic generation of KYNA  . Systemi33, or by the known influence of KYNA on the aryl hydrocarbon receptor (AHR)response 34 (which shares with the HIF pathway the heterodimeric transcription factor AHRnuclear translocator [ARNT])? Whilst protective effects of AHR activation have beensuggested in some models of ischaemia, in others activation of the AHR response bytryptophan metabolites can have deleterious effects 35.These findings provide vital insights into a potential mechanism of RIPC and generateintriguing questions  . NotablyBox 1. What is the mechanism of the cardioprotective effect, and does it operatein other tissues?Can the manipulation of KYNA or alpha-ketoglutarate levels or directpharmacological targeting of the cardioprotective mechanism activated byKYNA produce therapeutic benefit in patients with ischaemicdiseases?What mass of tissue ischaemia is necessary to achieve sufficientperturbations in the levels of circulating KYNA?46 have protective benefits?Will the emerging PHD inhibitors currently being trialled for theirerythropoietic effect in vivo?Do the other known influences on PHD function, such as oxygenavailability, iron and ascorbate, or perturbations ofalpha-ketoglutarate metabolism, influence protective mechanisms via thiseffect What effect is produced by acute versus chronic elevations in the levelsof such molecules, and to what extent do metabolic compensations or thecomplex feedback loops operating in the PHD–hypoxia-induciblefactor (HIF) system affect the operation of RIPC?Does ischaemia operate locally to mediate protective effects through thismechanism?Does this mechanism operate in other situations, such as hypoxictumours?Are there associations between levels of KYNA and outcomes in ischaemicdiseases?Is KYNA the dominant mediator of RIPC in humans, or are othermediators/mechanisms more important?1 and other animal studies, does the failure of RIPC to achieve improvedclinical outcomes reflect inadequate suppression of PHD2 activity and/orinsufficient increases in levels of KYNA? Improved understanding of the transductionof the RIPC signal from remote tissue to protected target may now allow improvementsin clinical strategies to deliver the enormous potential benefits of RIPC and thedevelopment of new pharmacological approaches that directly activate the protectivepathway.What is the relative importance of this newly defined mechanism of RIPC to otherpathways, how is it related to neurally mediated effects, and how do they interact?Can manipulation of KYNA or alpha-ketoglutarate levels or direct pharmacologicaltargeting of the cardioprotective mechanism activated by KYNA produce therapeuticbenefit in patients with ischaemic diseases? In contrast to the impressiveprotective benefits seen in the work of Olenchock and colleagues


An emerging vision for toxicity testing in the 21st century foresees in vitro assays assuming the leading role in testing for chemical hazards, including testing for carcinogenicity. Toxicity will be determined by monitoring key steps in functionally validated molecular pathways, using tests designed to reveal chemically-induced perturbations that lead to adverse phenotypic endpoints in cultured human cells. Risk assessments would subsequently be derived from the causal in vitro endpoints and concentration vs. effect data extrapolated to human in vivo concentrations. Much direct experimental evidence now shows that disruption of epigenetic processes by chemicals is a carcinogenic mode of action that leads to altered gene functions playing causal roles in cancer initiation and progression. In assessing chemical safety, it would therefore be advantageous to consider an emerging class of carcinogens, the epigenotoxicants, with the ability to change chromatin and/or DNA marks by direct or indirect effects on the activities of enzymes  that convey the epigenetic information. Evidence is reviewed supporting a strategy for in vitro hazard identification of carcinogens that induce toxicity through disturbance of functional epigenetic pathways in human somatic cells, leading to inactivated tumour suppressor genes and carcinogenesis. In the context of human cell transformation models, these in vitro pathway measurements ensure high biological relevance to the apical endpoint of cancer. Four causal mechanisms participating in pathways to persistent epigenetic gene silencing were considered: covalent histone modification, nucleosome remodeling, non-coding RNA interaction and DNA methylation. Within these four interacting mechanisms, 25 epigenetic toxicity pathway components  were found to have experimental evidence showing that functional perturbations played “driver” roles in human cellular transformation. Measurement of epigenotoxicants presents challenges for short-term carcinogenicity testing, especially in the high-throughput modes emphasized in the Tox21 chemicals testing approach. There is need to develop and validate in vitro tests to detect both, locus-specific, and genome-wide, epigenetic alterations with causal links to oncogenic cellular phenotypes. Some recent examples of cell-based high throughput chemical screening assays are presented that have been applied or have shown potential for application to epigenetic endpoints. Somatic cells can develop long-lasting or multi-generational cellular phenotypes in response to a host of input signals originating from homeostatic physiological pathways, conditions of physiological stress, or abnormal cellular environments . InducedEpigenetic controls on gene expression mediated by cytosine methylation in DNA, covalent histone modifications, histone remodelling and long non-coding RNAs have often been studied for their independent or joint contributions to tumour initiation, promotion and progression. Epigenetic alterations have frequently been shown to accompany in vitro and/or in vivo experimental chemical exposure, but the series of essential, cause-and-effect molecular steps that lead from such exposure to the observed molecular-level alterations and beyond, to stable phenotypic changes, have seldom been explored in detail. At the outset of undertaking this review, there appeared to be an open question around whether or not there was sufficient understanding of epigenetic pathways controlling gene expression driving consequent cellular effects related to carcinogenesis and whether that knowledge might be usefully applied to furthering the development and implementation of chemical toxicity testing and risk assessment.Currently, carcinogenic hazard characterization is dependent on two year rodent bioassays supported by adjunct in vivo or in vitro bioassays, such as the genotoxicity testing battery which assesses various damages and alterations to DNA sequence or copy number as processes causally linked to carcinogenicity. Additional assays may provide molecular biomarkers of effect related to increased cell proliferation, inhibition of programmed cell death, and receptor activation. The adjunct assays typically provide information on the mode or mechanism of action of the chemical agent in question, and help to establish whether the apical events in the animal model would be relevant to human cells and tissue responses. There is now much recognition that carcinogens may act through a large and diverse variety of mechanisms, including through induced epigenetic effects. Numerous investigations have shown that environmental influences  elicit epigenetic changes associated with adverse cell and tissue responses and a large number of these studies are related to cancer as an outcome ,4,5,6,7.In addition to further understanding mechanisms of action for carcinogens, there are unique advantages in considering epigenetic measurements ,15. EpigThe present review focuses on what is known about epigenetic mechanisms of gene expression control that are causal contributors to the oncogenic transformation of human cells by environmental chemicals. The review highlights some notable experimental studies in human somatic cell cultures, combined with overviews from detailed reviews in order to summarize the basic molecular components forming in vitro epigenetic pathways leading to tumour suppressor gene silencing and to oncogenic cellular phenotypes. An emphasis has been put on knowledge obtained from human in vitro cellular systems, drawing on studies from over about the last 10 years that have included functional assessments of oncogenic phenotypic endpoints. Especially informative in this regard are studies with direct, experimental perturbations to epigenetic pathway components created by genetically engineered manipulations or by using chemical agents that are known activators or inhibitors of the pathway components. It is expected that when similar in direction and magnitude, perturbations caused by exposure to chemicals would function similarly to the engineered manipulations that created toxicity pathways leading to transformed cell variants. The review finally provides a summary of how the measurements and tools that have provided underlying mechanistic knowledge are beginning to be harnessed to provide high-throughput chemical screening assays based on key steps in the pathways.Despite the increased insight that may become available by incorporating epigenetic information into carcinogenicity testing strategies, the costs incurred, in terms of financial resources, time, and issues regarding animal welfare, that are involved to produce traditional apical endpoint data combine to form a significant impediment to obtaining information for the large number of chemicals that await evaluation. These inherent difficulties accompanying animal testing have encouraged development of the Tox21 strategies for health risk identification and assessment that are summarized in the following sections.p-dioxin (TCDD)]. In addition, maladaptive responses in the homeostatic controls on cellular physiology may cause changes to critical metabolites involved in epigenetic functions. Important metabolic co-factors in epigenetic functions include: glutathione which is important for DNA and histone methylation reactions; ATP for histone phosphorylations; acetyl-coenzyme A for histone acetylations; flavin adenine dinucleotide (FAD) as a cofactor for the lysine-specific histone demethylase 1 (LSD1) histone demethylase; NAD+ as cofactor for the SIRTUIN family of histone deacetylases; α-ketoglutarate, as a co-factor for histone demethylases (HDMs) and for the Ten-eleven translocation (TET) protein dioxygenase family members that oxidize the methyl group of 5-methylcytosine and facilitate DNA demethylation pyrene- or 3-methycholanthrene-transformed colonies (identified as transformed upon reaching approximately 200 to 500 cells) had fully lost its expression. The loss of expression continued in animal tumours derived from the transformed cells, many generations after the chemical exposures, therefore demonstrating a mitotically stable change had occurred. Loss of H19 expression was correlated with hypermethylation within the gene as revealed by a loss of methylation-sensitive restriction enzyme cutting sites within the H19 locus in DNA obtained from both the transformed cells or subsequently from animal- derived tumours. Re-introduction of a functional H19 RNA into the transformed cells was accomplished by transfection of a plasmid-based expression vector which resulted in a high level of H19 RNA in those cells. Re-expression of H19 RNA in this way caused reduced tumour size and increased tumour latency relative to H19 non-expressing controls when transfected cells were injected subcutaneously into athymic nude mice. It is important to note, in support of the epigenetic reprogramming theory of carcinogenesis, that expression of H19 and expression of other differentiation-associated genes, is consistently lost when mouse embryonic fibroblasts are experimentally reprogrammed with transcription factors to become pluripotent stem cells (iPSC) or when the fibroblasts are oncogenically transformed by transfection with oncogenes pyrene or nickel chloride (a non-mutagenic carcinogen) pyrene induced morphological transformants were picked and serially cultured up to the point of culture senescence at 100 population doublings pyrene, or the stress of serum-free medium, causes some cells within to overcome the culture-induced barrier termed “stasis”  and the cells often become visible as a subpopulation of morphological variants (vHMEC) within cell monolayers. Stasis is defined as a proliferative halt mediated by p16/INK4A inhibition of CYCLIN D-mediated phosphorylation of retinoblastoma protein thereby allowing unphosphorylated pRB to sequester E2F transcription factors and down-regulate the proliferation-related target genes  causes centrosome dysfunction and related aneuploidy pyrene, benzo[a]pyrene diol epoxide, methylnitrosourea, cigarette smoke condensate, cadmium chloride, nickel chloride) or non-genotoxic  properties. The phenotypic endpoint most often measured in these studies centered on selectable malignant transformations producing variant cells that have the capacity to grow without anchorage within a soft agar culture medium. The transformed cells arising from these experiments are thought to correspond to cells that have undergone steps 1 and 2 in the outline multi-step tumourigenesis model. The transformation experiments, in this case, would therefore most likely mimic the clonal outgrowth of preneoplastic cells in a tissue setting (preneoplastic tissue field), wherein epigenetic alterations contribute to a more advanced tumour pathogenesis. Examples of changes in gene expression caused by chemically-induced hypermethylated and hypomethylated DNA at gene promoters, as well as increases in binding of polycomb repressive complex are given. Reversal of the epigenetic effects produced by chemical inhibitors of DNA methylation and chromatin remodeling enzymes or engineered re-establishment of gene expression states affected by the epigenetic perturbations had significant effects on reducing the malignant properties of the transformed cells in those studies.A collection of (twenty) related studies examining causal connections between chemically-induced epigenetic perturbations and aspects of more progressed transformed cellular phenotypes in human cells are listed in RARβ2 gene  and the genes for cellular retinol binding protein 1 (CRBP1) and CYP26A1 (a P450 retinoic acid-specific hydrolase), that are direct transcriptional targets of RARβ2. Silencing at these loci involved both hypoacetylation of histone 4 and DNA hypermethylation. Highly diminished RARβ2 transcriptional activity due to establishment of an homozygous repressive epigenotype at both alleles of this gene and throughout the network of its target genes, particularly CRBP1, lead to several phenotypic changes involved in breast epithelial cell transformation, such as loss of epithelial polarization, inability to form a hollow lumen in 3D basement membrane cultures, and anchorage-independent growth. The specific mechanism(s) involved in targeting epigenetic modifiers to these gene loci remain unknown. It is interesting to note, with respect the epigenetic effects cascade controlled by retinoic acid, that others have more recently found that 24 h exposure of human oesophageal cancer cells to benzo[a]pyrene diol epoxide (BPDE) suppressed expression of RARβ2 gene expression by an induced methylation of its promoter [RARβ2 and consequently its target gene network, in the immortalized breast epithelial cell line discussed above, as a model for a chemically-perturbed toxicity pathway leading to heritable oncogenic phenotypic changes.A developing idea, not exemplified in promoter . The proTSG silencing, and chemically-induced human cell transformation, it would be most helpful to conduct screening projects in cell systems, like those discussed throughout the earlier sections of this review, that could subsequently be used to confirm the abilities of chemicals or their metabolized derivatives to transform cells into oncogenic variants.During the last decade, there has been growing effort directed toward the design and implementation of human in vitro phenotypic assays for screening chemical libraries, in intact, viable cells, in order to identify and predict potential safety risks as well as to identify candidate chemicals with potential drug activities ,322,323.Capability to be performed in 96- or 1536-well plate formats10 steps or lessFast assay times (24–48 h)Minimal assay steps, 4 or lessRobust signals, greater than 3-foldSeveral published studies have presented results from practicable phenotypic, cell-based, high- or medium-throughput chemical screening assays for epigenetic effects targeting epigenetic pathway components , or their effects on target genes, in a variety of human and rodent cells . Also liIn TSG loci. Finally, lower-throughput phenotypic measures related to oncogenic transformation , confirmed by follow-up measures using medium-throughput assays of specific epigenetic pathway components  or effects at epigenetic reporter genes or at individual endogenous 2 assays ) could bIt will be important, from a toxicological perspective, to begin to develop protocols that would allow such assays to be performed with untransformed cell lines and perhaps primary human cell cultures in which causal connections to increases in oncogenic phenotypes can be made in the higher tier assays. It has also been suggested that existing, validated toxicological tests could be adapted to include epigenomic studies . In thisTSGs) playing roles in transformed phenotypes of primary or immortalized human cells.Testing for chemical hazards in vitro, as envisioned by the Tox21 strategy, requires cellular- and molecular-level insights into the key, causal steps along pathways of toxicity in human cells. A principal goal for this review was, therefore, to survey published studies containing direct experimental evidence demonstrating that epigenetic disruptions are a carcinogenic mode of action, producing altered gene expression patterns that influence human oncogenic transformation in vitro with relevance to in vivo tumours. A current overview of epigenetic pathway components, marks, writers, editors/erasers and readers, of DNA methylation and HPTM was provided, with an emphasis on those players capable of creating stable epigenetic changes in gene expression known to participate in carcinogenesis, in particular, among known tumour suppressor genes. Examples of engineered biological systems or specific molecular tools that were used to generate perturbations of twenty five epigenetic toxicity pathway components showed that conditions or activities outside of the ranges measured in model human cell cultures were “drivers” of cellular transformation events in those cultures , representing the four tissues characterized by the highest cancer incidences, were the most frequently applied models in the functional characterizations of molecular- or xenobiotic-induced perturbations of epigenetic pathways leading to oncogenic transformation in vitro. Human cellular models of oncogenic transformation provide highly relevant contexts for further development of in vitro chemical testing of epigenetic toxicity pathways and, under Tox21 assessment strategies, should provide useful information for human health risk assessment and decision-making processes by chemical regulatory agencies. TSG expression as a mode of carcinogenic action with potential for application within the TT21C testing and assessment paradigm and it will be particularly important to develop existing knowledge around TSG epigenetic silencing into informative screening assays. In this regard, there is need for information in several areas: (1) knowledge of the contributions to cell transformation resulting from silencing events among the wide range of known TSGs in the cell models chosen for study; (2) calibrations of changes in the toxicity pathways that belong to differentiation or physiological processes considered as normal in those cell models; (3) determinations of the magnitudes of changes forming “tipping points” that begin to contribute to disease and carcinogenic processes; (4) measures of persistence of the epigenetic changes; and (5) reproducible concentration-response and exposure-duration curves that would allow extrapolation of the adverse concentrations to tissue concentrations expected to occur in exposed humans. One specific obstacle that may need to be overcome in this endeavor is that cell transformation as a phenotypic endpoint and its underlying epigenetic changes that act to effectively silence TSGs may be longer term events resulting from chronic chemical exposures. The relatively short time-frames currently employed in high throughput Tier 1 TT21C testing strategies will necessitate validations of reporter-gene constructs based upon TSG targets, or any other epigenetic assays based on cell-by-cell measures, to determine if they will be sufficiently sensitive in short term assays. Application of multiple, confirmatory approaches to identifying further targets of epigenetic toxicity and to querying how perturbations of these targets lead to the phenotypic endpoint of cell transformation will benefit the uptake of this mode of action into cancer risk assessments. Some general considerations on applicable technologies and biological issues in the traditional weight-of-evidence-based assessment of epigenetic carcinogens have previously been offered [Causal epigenetic events have been identified among the human cell transformation models presented in this review, it should be a near-term priority to develop and employ aspects of these particular events as functional biomarker targets for quantitative high through-put screens of chemical libraries. The review has emphasized the epigenetic repression of  offered ,339. Goi