Protein Carbonylation in Patients with Myelodysplastic Syndrome: An Opportunity for Deferasirox Therapy

Control of oxidative stress in the bone marrow (BM) is key for maintaining the interplay between self-renewal, proliferation, and differentiation of hematopoietic cells. Breakdown of this regulation can lead to diseases characterized by BM failure such as the myelodysplastic syndromes (MDS). To better understand the role of oxidative stress in MDS development, we compared protein carbonylation as an indicator of oxidative stress in the BM of patients with MDS and control subjects, and also patients with MDS under treatment with the iron chelator deferasirox (DFX). As expected, differences in the pattern of protein carbonylation were observed in BM samples between MDS patients and controls, with an increase in protein carbonylation in the former. Strikingly, patients under DFX treatment had lower levels of protein carbonylation in BM with respect to untreated patients. Proteomic analysis identified four proteins with high carbonylation levels in MDS BM cells. Finally, as oxidative stress-related signaling pathways can modulate the cell cycle through p53, we analyzed the expression of the p53 target gene p21 in BM cells, finding that it was significantly upregulated in patients with MDS and was significantly downregulated after DFX treatment. Overall, our results suggest that the fine-tuning of oxidative stress levels in the BM of patients with MDS might control malignant progression.

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The Role of MicroRNAs in Diabetes-Related Oxidative Stress

International Journal of Molecular Sciences ◽

10.3390/ijms20215423 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5423 ◽

Cited By ~ 3

Author(s):

Mirza Muhammad Fahd Qadir ◽

Dagmar Klein ◽

Silvia Álvarez-Cubela ◽

Juan Domínguez-Bendala ◽

Ricardo Luis Pastori

Keyword(s):

Oxidative Stress ◽

Target Gene ◽

Cellular Stress ◽

Cellular Damage ◽

Oxygen Species ◽

Non Coding Rnas ◽

And Oxidative Stress

Cellular stress, combined with dysfunctional, inadequate mitochondrial phosphorylation, produces an excessive amount of reactive oxygen species (ROS) and an increased level of ROS in cells, which leads to oxidation and subsequent cellular damage. Because of its cell damaging action, an association between anomalous ROS production and disease such as Type 1 (T1D) and Type 2 (T2D) diabetes, as well as their complications, has been well established. However, there is a lack of understanding about genome-driven responses to ROS-mediated cellular stress. Over the last decade, multiple studies have suggested a link between oxidative stress and microRNAs (miRNAs). The miRNAs are small non-coding RNAs that mostly suppress expression of the target gene by interaction with its 3’untranslated region (3′UTR). In this paper, we review the recent progress in the field, focusing on the association between miRNAs and oxidative stress during the progression of diabetes.

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The Autophagy Status of Cancer Stem Cells in Gliobastoma Multiforme: From Cancer Promotion to Therapeutic Strategies

International Journal of Molecular Sciences ◽

10.3390/ijms20153824 ◽

2019 ◽

Vol 20 (15) ◽

pp. 3824 ◽

Cited By ~ 14

Author(s):

Larisa Ryskalin ◽

Anderson Gaglione ◽

Fiona Limanaqi ◽

Francesca Biagioni ◽

Pietro Familiari ◽

...

Keyword(s):

Stem Cells ◽

Treatment Resistance ◽

Primary Brain Tumor ◽

Small Subset ◽

Self Renewal ◽

Neuronal Stem Cells ◽

Proliferation And Differentiation ◽

The Common ◽

Pluripotency Maintenance

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor featuring rapid cell proliferation, treatment resistance, and tumor relapse. This is largely due to the coexistence of heterogeneous tumor cell populations with different grades of differentiation, and in particular, to a small subset of tumor cells displaying stem cell-like properties. This is the case of glioma stem cells (GSCs), which possess a powerful self-renewal capacity, low differentiation, along with radio- and chemo-resistance. Molecular pathways that contribute to GBM stemness of GSCs include mTOR, Notch, Hedgehog, and Wnt/β-catenin. Remarkably, among the common biochemical effects that arise from alterations in these pathways, autophagy suppression may be key in promoting GSCs self-renewal, proliferation, and pluripotency maintenance. In fact, besides being a well-known downstream event of mTOR hyper-activation, autophagy downregulation is also bound to the effects of aberrantly activated Notch, Hedgehog, and Wnt/β-catenin pathways in GBM. As a major orchestrator of protein degradation and turnover, autophagy modulates proliferation and differentiation of normal neuronal stem cells (NSCs) as well as NSCs niche maintenance, while its failure may contribute to GSCs expansion and maintenance. Thus, in the present review we discuss the role of autophagy in GSCs metabolism and phenotype in relationship with dysregulations of a variety of NSCs controlling pathways, which may provide novel insights into GBM neurobiology.

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Absence of the Rho GTPase Activating Protein p190-B Enhances Long Term Hematopoietic Stem Cell Engraftment

Blood ◽

10.1182/blood.v112.11.614.614 ◽

2008 ◽

Vol 112 (11) ◽

pp. 614-614 ◽

Cited By ~ 1

Author(s):

Haiming Xu ◽

Hartmut Geiger ◽

Kathleen Szczur ◽

Deidra Deira ◽

Yi Zheng ◽

...

Keyword(s):

Bone Marrow ◽

Ex Vivo ◽

Gtpase Activating Protein ◽

Self Renewal ◽

Hematopoietic Stem ◽

Proliferation And Differentiation ◽

Serial Transplantation

Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. While their role in HSC functions has begun to be understood, the role of their regulator in vivo has been understudied. P190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, has been implicated in regulating cell size and adipogenesis-myogenesis cell fate determination during fetal development (Sordella, Dev Cell, 2002; Cell 2003). Here, we investigated the role of p190-B in HSC/P engraftment. Since mice lacking p190-B die before birth, serial competitive repopulation assay was performed using fetal liver (FL) tissues from day E14.5 WT and p190-B−/− embryos. WT and p190-B−/− FL cells exhibited similar levels of engraftment in primary recipients. However, the level of contribution of p190-B−/− cells to peripheral blood and bone marrow was maintained between the primary and secondary recipients and still easily detectable in tertiary recipients, while the level of contribution of FL WT cells dramatically decreased with successive serial transplantion and was barely detectable in tertiary recipients. The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. A pool of HSC was maintained in serially transplanted p190-B−/− animals, since LinnegScaposKitpos (LSK) cells were still present in the BM of p190-B−/− secondary engrafted mice while this population disappeared in WT controls. Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of p190-B−/− HSC compared to WT HSC since highly enriched p190-B−/− HSC (LSK) demonstrated similar enhanced serial transplantation potential. Because previous studies have suggested that the loss of long term function of HSC during serial transplantation can depend, at least in part, on the upregulation of the cyclin dependent kinase inhibitor p16Ink4a (Ito et al, Nat Med 2006), the expression of p16Ink4a was examined during serial transplantation. While expression of p16Ink4a increased in WT HSC in primary and secondary recipients, p16Ink4a remained low in p190-B−/− HSC, which indicated that p190-B-deficiency represses the upregulation of p16Ink4a in HSC in primary and secondary transplant recipients. This provides a possible mechanism of p190-B-mediated HSC functions. We next examined whether p190-B-deficiency may preserve the repopulating capacity of HSC/P during ex vivo cytokine-induced culture. While freshly isolated LSK cells from WT and p190-B−/− mice exhibited comparable intrinsic clonogenic capacity, the frequency of colony-forming unit after 7 days in culture was 2 fold-higher in p190-B−/− compared with WT cultures, resulting in a net CFU expansion. Furthermore, competitive repopulation assays showed significantly higher repopulating activity in mice that received p190-B−/− cultured cells compared with WT cells equivalent to a 4.4-fold increase in the estimated frequency of repopulating units. Interestingly, p190-deficiency did not alter cell cycling rate or survival both in vivo and in vitro. Therefore, p190-B-deficiency maintains key HSC functions either in vivo or in ex vivo culture without altering cycling rate and survival of these cells. These findings define p190-B as a critical regulator of HSC functions regulating self renewal activity while maintaining a balance between proliferation and differentiation.

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CDK6 kinase activity is required for thymocyte development

Blood ◽

10.1182/blood-2010-08-300517 ◽

2011 ◽

Vol 117 (23) ◽

pp. 6120-6131 ◽

Cited By ~ 31

Author(s):

Miaofen G. Hu ◽

Amit Deshpande ◽

Nicolette Schlichting ◽

Elisabeth A. Hinds ◽

Changchuin Mao ◽

...

Keyword(s):

Gene Expression ◽

Target Gene ◽

Kinase Activity ◽

Thymocyte Development ◽

Hematopoietic Stem ◽

Notch Target Gene ◽

Target Gene Expression ◽

Lymphoid Malignancies ◽

Proliferation And Differentiation

Abstract Cyclin-dependent kinase-6 (CDK6) is required for early thymocyte development and tumorigenesis. To mechanistically dissect the role of CDK6 in thymocyte development, we generated and analyzed mutant knock-in mice and found that mice expressing a kinase-dead Cdk6 allele (Cdk6K43M) had a pronounced reduction in thymocytes and hematopoietic stem cells and progenitor cells (Lin−Sca-1+c-Kit+ [LSK]). In contrast, mice expressing the INK4-insensitive, hyperactive Cdk6R31C allele displayed excess proliferation in LSK and thymocytes. However, this is countered at least in part by increased apoptosis, which may limit progenitor and thymocyte expansion in the absence of other genetic events. Our mechanistic studies demonstrate that CDK6 kinase activity contributes to Notch signaling because inactive CDK6 kinase disrupts Notch-dependent survival, proliferation, and differentiation of LSK, with concomitant alteration of Notch target gene expression, such as massive up-regulation of CD25. Further, knockout of CD25 in Cdk6K43M mice rescued most defects observed in young mice. These results illustrate an important role for CDK6 kinase activity in thymocyte development that operates partially through modulating Notch target gene expression. This role of CDK6 as a downstream mediator of Notch identifies CDK6 kinase activity as a potential therapeutic target in human lymphoid malignancies.

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Comparison of Oxidant-Antioxidant Status in Patients with Vitiligo and Healthy Population

Kathmandu University Medical Journal ◽

10.3126/kumj.v12i2.13660 ◽

2015 ◽

Vol 12 (2) ◽

pp. 132-136 ◽

Cited By ~ 7

Author(s):

S Agrawal ◽

A Kumar ◽

TK Dhali ◽

SK Majhi

Keyword(s):

Oxidative Stress ◽

Free Radicals ◽

Vitamin E ◽

Vitamin C ◽

Control Group ◽

Healthy Population ◽

Significant Difference ◽

Destruction Of Melanocytes ◽

And Control

Background Vitiligo is a well-recognized pigmentary disorder of the skin and /or mucous membrane characterized by circumscribed ivory or chalky white macules devoid of identifiable melanocytes. The pathogenesis of vitiligo is complex and still not well understood. According to autocytotoxic hypothesis, oxidative stress has been suggested to be the initial pathogenic event in melanocyte degeneration. The role of free radicals and oxidative damage in the pathophysiology of vitiligo has been documented in recent studies.Objective To evaluate the role of oxidative stress in patients with vitiligo and of healthy controls by measuring levels of the oxidant malondialdehyde (MDA) and antioxidants vitamin C and vitamin E in serum and catalase (CAT) in erythrocytes.Method A total of 80 clinically diagnosed cases of vitiligo and 80 control subjects were included in the study to assess the activity of MDA, vitamin C and vitamin E in serum and CAT in erythrocytes of patients and controls by using the spectrophotometric assay.Result There was statistically significant increase in the levels of MDA in patients with vitiligo compared to the control group (p<0.001). No significant difference was found in the levels of vitamin C (p=0.411) and vitamin E (p=0.771) between the patients with vitiligo and control group. The levels of CAT in the vitiligo patients were found to be significantly lower than those of controls (p<0.001).Conclusion Increased oxidative stress and decreased catalase have been observed in vitiligo patients and the data suggesting that the free radicals may be involved in the destruction of melanocytes or dysregulation of melanogenesis.Kathmandu University Medical Journal Vol.12(2) 2014: 132-136

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Oxidative Stress and the Epigenetics of Cell Senescence: Insights from Progeroid Syndromes

Current Pharmaceutical Design ◽

10.2174/1381612824666190114164117 ◽

2019 ◽

Vol 24 (40) ◽

pp. 4755-4770 ◽

Cited By ~ 3

Author(s):

Carlos Romá-Mateo ◽

Marta Seco-Cervera ◽

José S. Ibáñez-Cabellos ◽

Giselle Pérez ◽

Ester Berenguer-Pascual ◽

...

Keyword(s):

Oxidative Stress ◽

Epigenetic Regulation ◽

Critical Role ◽

Cell Senescence ◽

Related Factors ◽

Senescent Phenotype ◽

Progeroid Syndromes ◽

Novel Therapeutic Strategies ◽

And Control

Background: Cell senescence constitutes a critical process to respond to a variety of insults and adverse circumstances. Senescence involves the detention of DNA replication and cell proliferation, and hence, genetic programs associated with DNA damage response, chromosome stability, chromatin rearrangement, epigenetic reprogramming, and cell cycle are tightly linked to the senescent phenotype. Although senescence increases with age, the real implication of senescence regulation in the progress of aging in humans is largely discussed. In this context, reactive oxygen species (ROS) accumulation has also been postulated to play a critical role in cell homeostasis, aging processes, and control of proliferation. Methods: The previous years have produced a high increase in data that refine our understanding of the role of ROS, and their relationship with epigenetic events, in determining cellular fate. Results: The accumulating evidence regarding the epigenetic regulation of ROS-mediated processes provides promising tools to deepen in our comprehension of the process of senescence, and to develop novel therapeutic strategies. In this review, we aim to provide an overview of the relationships between oxidative stress and cell senescence. Conclusion: We provide information about the role of epigenetic regulation in senescence and aging, collecting recent data from some examples of progeroid syndromes in which cell senescence, oxidative stress and epigenetic mechanisms are severely impaired. Finally, a collection of data is presented regarding current pharmacological approaches that either target or use oxidative stress-related factors or epigenetic regulators as strategies for disease treatment.

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The Hedgehog Regulated Transcription Factor and Tumor Suppressor, Gli3, Regulates Hematopoietic Stem Cell Number, Self-Renewal and Differentiation

Blood ◽

10.1182/blood.v126.23.2381.2381 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2381-2381

Author(s):

Parvesh Chaudhry ◽

Mohan Singh ◽

Amy R McManus ◽

Aparna Jorapur ◽

Stephen James Capone ◽

...

Keyword(s):

Transcription Factor ◽

Tumor Suppressor ◽

Hedgehog Signaling ◽

Fine Tuning ◽

Myeloid Differentiation ◽

Self Renewal ◽

Hematopoietic System ◽

Hh Signaling

Abstract The Hedgehog (Hh) signaling pathway plays a critical role in embryonic development and adult tissue homeostasis and has emerged as an important therapeutic target in many cancers, including leukemia and myeloproliferative diseases. Our mechanistic understanding of Hh pathway signaling and regulation comes primarily from developmental studies in neural and limb development. Studies of Hedgehog signaling in the hematopoietic system have produced contradictory results, and no clear consensus regarding Hh signaling in normal hematopoiesis is available to inform the role of Hedgehog signaling in hematologic malignancies. In our work we have focused on understanding the downstream effectors of Hedgehog signaling, the Gli transcription factors. The three Gli proteins, Gli1, Gli2 and Gli3 have both transcriptional activator and repressor functions, which allow for regulation and fine-tuning of Hedgehog pathway output. Previous studies from our group have revealed that Gli1null HSCs had no defects in self-renewal, however myeloid differentiation and stress hematopoiesis were severely impaired (Merchant, et al., Blood 2010). In normal tissues, Hh pathway activation via Ptch/Smo causes an increase in the downstream activating transcription factor GLI1 and a decrease in the transcriptional repressor Gli3R. Our recent studies demonstrated that GLI3R has a tumor suppressor role in human acute myeloid leukemia by directly repressing AKT expression (Chaudhry et al., AACR Annual Meeting 2015). To date nothing is known about the role of Gli3 in normal hematopoiesis. In the present study, we crossed Vav-Cre transgenic mice to Gli3fl/fl mice to generate mice with a conditional loss of Gli3 (Gli3null) in the hematopoietic system. HSC self-renewal was analyzed by serial transplant. In comparison to HSCs from Gli3 wild type (Gli3WT) mice bone marrow (BM), HSCs from Gli3null BM showed decreased long-term engraftment and self-renewal. In addition, quantification of long-term HSC (LT-HSC, CD34neg Flt3neg KSL), short-term HSC (ST-HSC, CD34+ Flt3neg KSL), and multi-potent progenitor (MPP, CD34+ Flt3+ KSL) revealed that the frequency of LT-HSCs in Gli3null BM (0.004-0.007%) was lower compared to Gli3 WT BM (0.008-0.02%). In mice transplanted with Gli3null BM, myeloid expansion was observed with a block in T and B cell lineage differentiation. Analysis of the c-Kit+ Sca1neg Linneg (KL) myeloid progenitor compartment revealed a two-fold increase in the FcRγhigh CD34+ KL granulocyte-monocyte progenitors (GMPs) in Gli3null BM, suggesting an expansion of granulocytic compartment. Since Gli3R is a key negative regulator of Gli1, these are consistent with decrease in GMP and myeloid differentiation previously seen in Gli1null mice. In summary, our studies reveal a previously unknown function for Gli3 in regulating HSCs and myeloid differentiation, and help to elucidate the complex regulation of Hh signaling in the hematopoietic system. Disclosures No relevant conflicts of interest to declare.

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P6283Downregulation of Tim44 exacerbates oxidative stress-induced ROS production and cardiomyocytes death by reducing mitochondrial SOD2

European Heart Journal ◽

10.1093/eurheartj/ehz746.0881 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

S Ikeda ◽

S Matsushima ◽

K Okabe ◽

A Ishikita ◽

T Tadokoro ◽

...

Keyword(s):

Oxidative Stress ◽

Cardiac Remodeling ◽

Protein Import ◽

Mitochondrial Fraction ◽

Left Ventricular ◽

Protein Carbonylation ◽

Neonatal Rat ◽

Ros Production ◽

Protein Levels

Abstract Background Mitochondrial dysfunction has been highlighted as a critical driver of cardiac remodeling and failure. Mitochondria contains about 1500 proteins, 99% of which are encoded in the nuclear genome. Therefore, protein import into mitochondria is essential to maintain mitochondrial function. Previous reports suggest that nuclear-encoded mitochondrial precursor proteins import into mitochondria by multiple complex; translocase of outer membrane (TOM), translocase of inner membrane (TIM), and protein associated motor (PAM). However, the role of these protein import machineries of mitochondria in cardiac remodeling remains to be elucidated. Objective The purpose of this study was to elucidate the role of TOM, TIM, and PAM complex in cardiac remodeling and cardiomyocyte death. Methods and results C57BL/6J mice were subjected to myocardial infarction (MI) by permanent ligation of left anterior descending artery. Four weeks after operation, MI-mice demonstrated left ventricular (LV) dilation (LV end-diastolic dimension: 3.91 vs. 5.54 mm, n=8–11, p<0.05) and dysfunction (LV fractional shortening: 33.3 vs. 7.7%, n=8–11, p<0.05). Tim44 protein levels, a component of PAM complex, in mitochondrial fraction from non-infarcted left ventricle were significantly decreased compared with those in the heart from sham-operated mice by 39% (p<0.05), whereas other proteins related to TOM, TIM and PAM complex such as Tom20, Tom22, Tom40, Tom70, Tim22, Tim23 and mtHSP70 were not altered between MI-mice and sham-mice. In addition, blue-native polyacrylamide gel electrophoresis revealed that a protein complex associated to Tim44 was significantly decreased in non-infarcted LV by 40% (p<0.05). Superoxide dismutase 2 (SOD2), a mitochondrial matrix protein, was decreased in mitochondrial fraction from non-infarcted LV by 20% (p<0.05), accompanied by enhancing protein carbonylation, a marker of oxidative stress, by 40% (p<0.05). To assess the role of Tim44, it was downregulated by small interfering RNA in cultured neonatal rat ventricular myocytes (NRVMs). Knockdown of Tim44 significantly decreased SOD2 protein levels in mitochondrial fractionation (22%, p<0.05), with no significant changes in its mRNA levels. Furthermore, knockdown of Tim44 significantly increased protein carbonylation (20%, p<0.05) and cleaved caspase 3 (47%, p<0.05) and decreased cell viability (69%, p<0.05), assessed by cell titer assay, in H2O2-treatred NRVMs. Conclusions Downregulation of Tim44 exacerbates oxidative stress-induced ROS production and cardiomyocytes death, which is associated with a decrease in mitochondrial SOD2. Endogenous Tim44 might play a protective role in cardiac remodeling by attenuating oxidative stress and cardiomyocyte death via SOD2 import into mitochondria.

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How the Pathological Microenvironment Affects the Behavior of Mesenchymal Stem Cells in the Idiopathic Pulmonary Fibrosis

International Journal of Molecular Sciences ◽

10.3390/ijms21218140 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8140

Author(s):

Martina Bonifazi ◽

Mariangela Di Vincenzo ◽

Miriam Caffarini ◽

Federico Mei ◽

Michele Salati ◽

...

Keyword(s):

Oxidative Stress ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Chronic Disease ◽

Idiopathic Pulmonary Fibrosis ◽

Pulmonary Fibrosis ◽

Cell Types ◽

And Control ◽

And Oxidative Stress

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by fibroblasts activation, ECM accumulation, and diffused alveolar inflammation. The role of inflammation in IPF is still controversial and its involvement may follow nontraditional mechanisms. It is seen that a pathological microenvironment may affect cells, in particular mesenchymal stem cells (MSCs) that may be able to sustain the inflamed microenvironment and influence the surrounding cells. Here MSCs have been isolated from fibrotic (IPF-MSCs) and control (C-MSCs) lung tissue; first cells were characterized and compared by the expression of molecules related to ECM, inflammation, and other interdependent pathways such as hypoxia and oxidative stress. Subsequently, MSCs were co-cultured between them and with NHLF to test the effects of the cellular crosstalk. Results showed that pathological microenvironment modified the features of MSCs: IPF-MSCs, compared to C-MSCs, express higher level of molecules related to ECM, inflammation, oxidative stress, and hypoxia; notably, when co-cultured with C-MSCs and NHLF, IPF-MSCs are able to induce a pathological phenotype on the surrounding cell types. In conclusion, in IPF the pathological microenvironment affects MSCs that in turn can modulate the behavior of other cell types favoring the progression of IPF.

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