Search

The efficiency of cell reprogramming in two-dimensional (2D) cultures is limited. Given that cellular stemness is intimately related to microenvironmental changes, 3D cell cultures have the potential of overcoming this limited capacity by allowing cells to self-organize by aggregation. In 3D space, cells interact more efficiently, modify their cellular topology, gene expression, signaling, and metabolism. It is yet not clear as how 3D culture environments modify the reprogramming potential of fibroblasts. We demonstrate that 3D spheroids from dermal fibroblasts formed under ultra-low attachment conditions showed increased lactate production. This is a requisite for cell reprogramming, increase their expression of pluripotency genes, such as OCT4, NANOG and SOX2, and display upregulated cystathionine-β-synthase (CBS) and hydrogen sulfide (H2S) production. Knockdown of CBS by RNAi suppresses lactic acid and H2S production and concomitantly decreases the expression of OCT4 and NANOG. On the contrary, H2S donors, NaHS and garlic-derived diallyl trisulfide (DATS), promote the expression of OCT4, and support osteogenic trans-differentiation of fibroblasts. These results demonstrate that CBS mediated release of H2S regulates the reprogramming of dermal fibroblasts grown in 3D cultures and supports their trans-differentiation. [ABSTRACT FROM AUTHOR]

Recently, we reported that cancer cells that recover from a potentially lethal damage gain new phenotypic features comprised of mitochondrial structural remodeling associated with increased glycolytic dependency and drug resistance. Here, we demonstrate that a subset of cancer cells, upon recovery from a potentially lethal damage, undergo dedifferentiation and express genes, which are characteristic of undifferentiated stem cells. While these cells are competent in maintaining differentiated progeny of tumor, they also exhibit transdifferentiation potential. Dedifferentiation is characterized by accumulation of hydrogen sulfide (H 2 S), which triggers up-regulation of nicotinamide phosphoribosyltransferase (Nampt) accompanied by changes in the redox state. The molecular events triggered by Nampt include elevated production of NAD + and up-regulation of H 2 S producing enzymes, cystathionine beta synthase (CBS) and cystathionase (CTH) with 3-mercaptopyruvate sulfurtransferase (MST) being detectable only in 3D spheroids. Suppression of Nampt, or inactivation of H 2 S producing enzymes, all reduce H 2 S production and reverse the ability of cells to dedifferentiate. Moreover, H 2 S induced stem cell markers in parental cancer cells in a manner similar to that observed in damage recovered cells. These data suggest of existence of a positive feedback loop between H 2 S and Nampt that controls dedifferentiation in cancer cells that recover from a potentially lethal damage. [ABSTRACT FROM AUTHOR]

We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer. [ABSTRACT FROM AUTHOR]

Highlights: [•] Some cancer cells recover from severe damage that causes cell death in majority of cells. [•] Damage-Recovered (DR) cancer cells show reduced mitochondria, mDNA and mitochondrial enzymes. [•] DR cells show increased aerobic glycolysis, ATP, cell proliferation, and resistance to damage. [•] DR cells recovered from in vivo damage also show increased glycolysis and proliferation rate. [Copyright &y& Elsevier]

[Display omitted] • Levels of α-synuclein in plasma and cerebral CSF distinguish PD from ET. • Metabolic pathway profiles in plasma and CSF distinguish PD from ET. • Increase in plasma NADH/NAMPT support pentose phosphate pathway dysregulation in PD. Parkinson's disease (PD) and essential tremor (ET) are two common adult-onset tremor disorders in which prevalence increases with age. PD is a neurodegenerative condition with progressive disability. In ET, neurodegeneration is not an established etiology. We sought to determine whether an underlying metabolic pattern may differentiate ET from PD. Circulating metabolites in plasma and cerebrospinal fluid (CSF) were analyzed using gas chromatography-mass spectroscopy. There were several disrupted pathways in PD compared to ET plasma including glycolysis, tyrosine, phenylalanine, tyrosine biosynthesis, purine and glutathione metabolism. Elevated α-synuclein levels in plasma and CSF distinguished PD from ET. The perturbed metabolic state in PD was associated with imbalance in the pentose phosphate pathway, deficits in energy production, and change in NADPH, NADH and nicotinamide phosphoribosyltransferase levels. This work demonstrates significant metabolic differences in plasma and CSF of PD and ET patients. [ABSTRACT FROM AUTHOR]

Abstract: The p53 transcription factor is involved in cell cycle, apoptosis and differentiation. However, the mechanism of p53 mediated differentiation is not fully understood. Here, we show that recently discovered dual oxidase maturation factor 1 (DUOXA1), which was implicated in neuronal differentiation, is regulated by p53 and may be an important factor in neuronal differentiation. We show that DUOXA1 is highly expressed in mouse neuronal stem cells with intensive nuclear localization. A strong interaction between DUOXA1 and p53 is observed in undifferentiated cells and declines in terminally differentiated neurons. Overexpressed p53 induces marked DUOXA1 expression in P19 cells and intensifies neuronal differentiation in the presence of retinoic acid, which suggests that p53 and DUOXA1 possess a neural differentiation potential. At day 3 of retinoic acid induced differentiation when cells showed a typical morphology of neuronal progenies, CD133 expression was down-regulated. The expression level of CD133 was significantly decreased in p53 over-expressing cells and was accompanied by a substantial increase in the expression level of neurofilament. In conclusion, DUOXA1 is a novel p53-regulated neurogenic factor involved in p53 dependent neuronal differentiation. [Copyright &y& Elsevier]

In this study, we describe a role for the mammalian Numb-interacting protein 1 (Nip1) in regulation of neuronal differentiation in stem cells. The expression of Nip1 was detected in the developing mouse brain, embryonic stem cells, primary neuronal stem cells, and retinoic acid-treated P19 embryonal carcinoma cells. The highest expression of Nip1 was observed in undifferentiated neuronal stem cells and was associated with Duox1-mediated reactive oxygen species ROS production. Ectopic nip1 expression in P19 embryonal carcinoma cells induced neuronal differentiation, and this phenotype was also linked to elevated ROS production. The neuronal differentiation in nip1-overexpressing P19 cells was achieved in a retinoic acid-independent manner and was corroborated by an increase in the expression of the neuronal basic helix-loop-helix transcription factors and neural-lineage cell markers. Furthermore, depletion of nip1 by short hairpin RNA led to a decrease in the expression of neuronal basic helix-loop-helix transcription factors and ROS. However, inhibition of ROS production in nip1-overexpressing P19 cells restricted but did not extinguish neuronal differentiation. Microarray and mass spectrometry analysis identified intermediate filaments as the principal cytoskeletal elements affected by up-regulation of nip1. We show here the first evidence for a functional interaction between Nip1 and a component of the nuclear lamina, lamin A/C. associated with a neuronal-specific phenotype. Taken together, our data reveal an important role for Nip1 in the guidance of neuronal differentiation through ROS generation and modulation of intermediate filaments and implicate Nip1 as a novel intrinsic regulator of neuronal cell fate. [ABSTRACT FROM AUTHOR]

Abstract: The signaling lymphocyte activation molecule (SLAM)-associated protein (SAP or SH2D1A) is an important regulator of immune function which, when mutated or deleted, causes the X-linked lymphoproliferative syndrome (XLP). Because B cell lymphoma is a major phenotype of XLP, it is important to understand the function of SAP in B cells. Here we report that SAP is expressed endogenously in mouse splenic B cells, is inducibly expressed in the human BJAB cells, and co-localizes and interacts with CD22. We also show that SAP binding to the inhibitory immunoreceptor CD22 regulates calcium mobilization in B cells. Moreover, forced expression of SAP leads to constitutive CD22 tyrosine phosphorylation and decreased Ca2+ response in B cells. Biochemical analysis reveals that, in response to IgM cross-linking, the phosphorylation of Syk, Blnk, or PLCγ2 and their interactions with one another were either diminished or completely abolished in SAP-expressing cells compared to cells that lack SAP. Collectively our work identifies a novel role for SAP in B cells and extends its function to inhibitory immunoreceptor signaling and calcium mobilization. [Copyright &y& Elsevier]

Abstract: The phosphoinositide 3′-kinase (PI3K)/Akt signaling cascade controls cellular processes such as apoptosis and proliferation. Moreover, it is a mediator of insulin effects on target cells and as such is a major regulator of fuel metabolism. The PI3K/Akt cascade was demonstrated to be activated by stressful stimuli, including heat shock and reactive oxygen species (ROS). This minireview focuses on activation of the pathway by exposure of cells to heavy metal ions, Cu2+ and Zn2+. It is hypothesized that stimulation of PI3K/Akt is the molecular mechanism underlying the known insulin-mimetic effects of copper and zinc ions. Following a brief summary of PI3K/Akt signaling and of activation of the cascade by Cu2+ and Zn2+, mechanisms of metal-induced PI3K/Akt activation are discussed with a focus on the role of ROS and of cellular thiols (glutathione, thioredoxin) and protein tyrosine phosphatases in Cu2+ and Zn2+ signaling. Finally, consequences of metal-induced PI3K/Akt activation are discussed, focusing on the modulation of FoxO-family transcription factors by Cu2+ and Zn2+. [Copyright &y& Elsevier]

The signaling lymphocyte-activating molecule (SLAM) family immunoreceptors are expressed in a wide array of immune cells, including both T and B lymphocytes. By virtue of their ability to transduce tyrosine phosphorylation signals through the so-called ITSM (immunoreceptor tyrosine-based switch motif) sequences, they play an important part in regulating both innate and adaptive immune responses. The critical role of the SLAM immunoreceptors in mediating normal immune reactions was highlighted in recent findings that SAP, a SLAM-associated protein, modulates the activities of various immune cells through interactions with different members of the SLAM family expressed in these cells. Importantly, mutations or deletions of the sap gene in humans result in the X-linked lymphoproliferative syndrome. In this review, we summarize current knowledge and survey the latest developments in signal transduction events triggered by the activation of SLAM family receptors in different cell types. [ABSTRACT FROM AUTHOR]

Abstract: Previous reports have shown that metallothionein (MT) may modulate p53 activity through zinc exchange. However, little is known on a direct interaction between MT and p53 in cells. The results demonstrate an interaction between MT and p53 can occur in vitro. The complex between MT and p53 was observed in breast cancer epithelial cells with both wild and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially associated with Apo-MT. Our data suggest that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells. [Copyright &y& Elsevier]

Copper is implicated in metabolic disorders, such as Wilson''s disease or Alzheimer''s disease. Analysis of signaling pathways regulating cellular survival and function in response to a copper stress is crucial for understanding the pathogenesis of such diseases. Exposure of human skin fibroblasts or HeLa cells to Cu2+ resulted in a dose- and time-dependent activation of the antiapoptotic kinase Akt/protein kinase B, starting at concentrations as low as 3 μM. Only Cu(II), but not Cu(I), had this effect. Activation of Akt was accompanied by phosphorylation of a downstream target of Akt, glycogen synthase kinase-3. Inhibitors of phosphoinositide-3-kinase (PI3K) completely blocked activation of Akt by Cu2+, indicating a requirement of PI3K for Cu2+-induced activation of Akt. Indeed, cellular PI3K activity was strongly enhanced after exposure to Cu2+. Copper ions may lead to the formation of reactive oxygen species, such as hydrogen peroxide. Activation of Akt by hydrogen peroxide or growth factors is known to proceed via the activation growth factor receptors. In line with this, pretreatment with inhibitors of growth factor receptor tyrosine kinases blocked activation of Akt by hydrogen peroxide and growth factors, as did a src-family tyrosine kinase inhibitor or the broad-spectrum tyrosine kinase inhibitor genistein. Activation of Akt by Cu2+, however, remained unimpaired, implying (i) that tyrosine kinase activation is not involved in Cu2+ activation of Akt and (ii) that activation of the PI3K/Akt pathway by Cu2+ is initiated independently of that induced by reactive oxygen species. Comparison of the time course of the oxidation of 2′,7′-dichlorodihydrofluorescein in copper-treated cells with that of Akt activation led to the conclusion that production of hydroperoxides cannot be an upstream event in copper-induced Akt activation. Rather, both activation of Akt and generation of ROS are proposed to occur in parallel, regulating cell survival after a copper stress. [Copyright &y& Elsevier]

Transitioning from a differentiated state to a higher-order of plasticity, by partial rather than full reactivation of pluripotency genes, might be a better approach in regenerative medicine. Hydrogen sulfide plays a crucial role in the maintenance and differentiation of mesenchymal stem cells (MSC) that have the potential to differentiate to a diverse group of mesenchymally derived cells. It was shown that these cells show a heavy reliance on cystathionine-β-synthase (CBS)-derived hydrogen sulfide (H 2 S) during differentiation. We have found that expression and activity of 3-mercaptopyruvate sulfurtransferase (MPST), one of three enzymes that hat regulates H 2 S biosynthesis, is significantly lower in MSC as compared with lineage-restricted dermal fibroblasts. Here, we tested the hypothesis that suppression of MPST in dermal fibroblasts might induce plasticity-related changes and broaden the transdifferentiation potency. Inactivation of MPST with phenylpyruvate (PP) or by siRNA silencing led to diminished H 2 S production associated with increased production of reactive oxygen species (ROS) and lactic acid. Accumulation of α-ketoglutarate (α-KG), a key metabolite required for the expression of ten-eleven translocation hydroxylase (TET), was associated with stimulated transcription of pluripotency related genes including OCT4 , KLF4 , SOX2 , and NANOG. Suppression of TET1 gene and inhibition of glycolysis with glucose analog, 2-desoxy- d -glucose, or hexokinase II inhibitor significantly reduced expression of pluripotency genes following MPST inactivation or knockdown. MPST disruption promoted the conversion of fibroblasts into adipocytes as evidenced by a significant increase in expression of adipocyte-specific genes, PPARγ, and UCP1 , and intracellular accumulation of oil Red-O positive fat droplets. Inhibition of glycolysis inhibited these changes. Under induced differentiation conditions, fibroblasts with disrupted MPST show the potency to differentiate to white adipogenic lineage. Thus, MPST inactivation or silencing enhances the plasticity of dermal fibroblasts in a TET1 and glycolysis dependent manner and promotes adipogenic transdifferentiation. [ABSTRACT FROM AUTHOR]