Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia

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Research paper

Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia Ritam Chatterjee, Sujata Law

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Stem Cell Research and Application Unit, Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, 108, C.R Avenue, Kolkata, 700073, West Bengal, India, India

A R T I C L E I N F O

A B S T R A C T

Keywords: Aplastic anemia Hematopoiesis Hematopoietic stem/progenitor cells Niche ROS Epigenetics

Aplastic anemia or bone marrow failure often develops as an effect of chemotherapeutic drug application for the treatment of various pathophysiological conditions including cancer. The long-term bone marrow injury affects the basic hematopoietic population including hematopoietic stem/progenitor cells (HSPCs). The present study aimed in unearthing the underlying mechanisms of chemotherapeutics mediated bone marrow aplasia with special focus on altered redox status and associated effects on hematopoietic microenvironment and epigenetic status of hematopoietic cells. The study involves the development of busulfan and cyclophosphamide mediated mouse model for aplastic anemia, characterization of the disease with blood and marrow analysis, cytochemical examinations of bone marrow, flowcytometric analysis of hematopoietic population and microenvironmental components, determination of ROS generation, apoptosis profiling, expressional studies of Notch-1 signaling cascade molecules, investigation of epigenetic modifications including global CpG methylation of DNA, phosphorylation of histone-3 with their effects on bone marrow kinetics and expressional analysis of the anti-oxidative molecules viz; SOD-2 and Sdf-1. Severe hematopoietic catastrophic condition was observed during aplastic anemia which involved peripheral blood pancytopenia, marrow hypocellularity and decreased hematopoietic stem/progenitor population. Generation of ROS was found to play a central role in the cellular devastation in aplastic marrow which on one hand can be correlated with the destruction of hematopoiesis supportive niche components and alteration of vital Notch-1 signaling and on other hand was found to be associated with the epigenetic chromatin modifications viz; global DNA CpG hypo-methylation, histone-3 phosphorylation promoting cellular apoptosis. Decline of anti-oxidant components viz; Sdf-1 and SOD-2 hinted towards the irreversible nature of the oxidative damage during marrow aplasia. Collectively, the findings hinted towards the mechanistic correlation among ROS generation, microenvironmental impairment and epigenetic alterations that led to hematopoietic catastrophe under aplastic stress. The findings may potentiate successful therapeutic strategy development for the dreadful condition concerned.

1. Introduction Long term and short term hematopoietic stem cells (HSCs) are at the top of the hematopoietic hierarchy which has the ability of self-renewal, proliferation and differentiation into different lineages of blood cells passing through various transient progenitor stages (Mikkola and Orkin, 2006; Weissman et al., 2001). Hematopoietic disruption due to bone marrow injury is a common late effect of chemotherapeutic tumor management which involves the development of myelosuppressive condition leading to aplastic anemia (Mauch et al., 1995; Testa et al., 1984; Wang et al., 2006; Shao et al., 2013; Young, 1988; Scatena et al., 2010; Bowcock et al., 1989; Shepherd et al., 1994). Under homeostatic condition the osteoblastic niche, adjacent to

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endosteal bone surface provides a special environment to the HSCs that facilitates self-renewal (Shao et al., 2013; Guerrouahen et al., 2011; Zhang et al., 2003; Calvi et al., 2003a). The intricate-interactions promote self renewal of hematopoietic cells in a part by keeping them quiescent as due to low metabolic activities HSCs produces less reactive oxygen species (ROS) which would otherwise cause oxidative damage to hematopoietic mass (Takubo et al., 2010). Moreover, endosteal osteoblastic niche is mostly hypoxic as it is relatively remote from blood flow (Winkler et al., 2010). Increase of ROS can cause oxidative damage to HSC population impairing self-renewal ability and inducing HSC senescence which promotes premature exhaustion of HSC leading to long term bone marrow suppression (Shao et al., 2013; Wang et al., 2010; Ito et al., 2006, 2004). Apart from affecting basic hematopoietic

Corresponding author at: Department of Biochemistry and Medical Biotechnology, Calcutta School of Tropical Medicine, Kolkata-700073, India. E-mail address: [email protected] (S. Law).

https://doi.org/10.1016/j.ejcb.2017.11.003 Received 5 September 2017; Received in revised form 4 November 2017; Accepted 20 November 2017 0171-9335/ © 2017 Elsevier GmbH. All rights reserved.

Please cite this article as: Chatterjee, R., European Journal of Cell Biology (2017), https://doi.org/10.1016/j.ejcb.2017.11.003

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following the methodologies of Pereira and Law, 2017. In brief, 200 μl of blood was collected in heparinized vial by tail vein puncture from both the control and experimental groups of mice. Total RBC and WBC counts were determined by standard laboratory techniques using hemocytometer chamber (Rohem, India). Reticulocyte count was determined by brilliant Cresyl blue staining. Estimation of hemoglobin content was done by colorimetric method using Drabkin’s reagent (Stanbio Reagent, India). Differential leukocyte counts were determined by the analysis of blood films after Leishman staining.

population, redox imbalance in bone marrow is also reported to have negative impact on hematopoiesis supportive microenvironment as well (Mangialardi et al., 2014; Khatri et al., 2016). In addition to the stemstromal disruption in bone marrow, ROS has the generalized property of altering epigenetic regulation over cell. On one hand, ROS is associated with global CpG hypomethylation of DNA and on the other hand can facilitate aberrant histone-3 (H3) phosphorylation that leads to apoptotic elimination of cell (Wu and Ni, 2015; Tikoo et al., 2001). It is a well established fact that cellular exposure to certain stresses viz; ionizing radiation or chemotherapeutic agents results in the increase in ROS production and this initial oxidative stress not only produces immediate damage to cells, but more importantly perturbs cellular metabolism that disturbs the normal oxidation/reduction (redox) reactions, leading to a persistent and prolonged elevation in ROS production (Shao et al., 2013; Balaban et al., 2005; Richardson et al., 2015). Our present study aims in establishing the correlation between the alteration of bone marrow redox status and the shift of stem-stromal equilibrium as well as epigenetic modification in hematopoietic stem/progenitor cell (HSPC) compartment during chemotherapeutic drug induced bone marrow aplasia. Mouse model can be a powerful tool to screen the underlying complexities of drug mediated aplastic anemia. Alkylating drugs viz; busulfan (BU), cyclophosphamide (CP) are used to develop the hematopoietic catastrophic condition in experimental animals for decades (Morley and Blake, 1974; Fried et al., 1977; Ottolander et al., 1982; Boyd et al., 1986; Gibson et al., 2003a; Gibson et al., 2003b; Chen, 2005; Chatterjee et al., 2008; Chen et al., 2014; Chatterjee et al., 2016a). BU and CP mediated aplastic anemic condition in mice recapitulates many features of chemotherapy mediated long term marrow failure in human (Chatterjee et al., 2016a; Chatterjee et al., 2009; Chatterjee et al., 2010). In the present study, the novel mouse model was used for the extrapolation of the mentioned mechanistic interventions.

2.4. Bone marrow histology Histological preparation of bone marrow was done as described previously (Chatterjee et al., 2016c). In brief, long bones were collected immediately after the sacrifice of mice, fixed with 10% buffered formalin, decalcified with 10% formic acid, dehydrated with ascending grades of alcohol, embedded in paraffin, cut into 5 μm thick sections and finally subjected to routine H & E staining. Stained tissue sections from both the groups of mice were analysed by light microscopic observations. Tissues where < 30% of intertrabecular space remained occupied by hematopoietic cells were considered to be hypo-cellular (Kong et al., 2013). Tissue sections were also subjected to Mallory’s trichrome staining following the protocol of Chatterjee et al., 2016b; Humason, 1979. 2.5. Marrow isolation and single cell preparation Marrow cells flushed out from the long bones of 6 mice in RPMI1640 (Sigma, USA) using 26 gauge needled syringe were pooled and made into single cells by repeat pipetting. Cells were thoroughly washed in ice-cold media several times to remove the debris. Finally, the cell suspension was passed through 100 μm cell strainer. 2.6. Oil red O staining

2. Materials and methods Assessment of bone marrow adipocyte content in control and aplastic mice was done by Oil Red O staining according to the protocol used by Chattopadhyay et al., 2016a. In brief, air dried bone marrow smears from both the groups were subjected to 10% formaldehyde fixation for 10 min and thereafter stained with Oil Red O solution (Oil Red O: water; 3:2) for 15 min followed by the removal of excess stain by washing and counter staining with Harris Hematoxiline for microscopic analysis. Adipocytes were identified by red coloration.

2.1. Animals Healthy, inbred Swiss albino mice weighing 20–25 gm, aged 8 weeks of both the sexes were maintained in the animal house of the Calcutta School of Tropical Medicine in proper conditions (12 h light–dark cycle, 22 ± 2 °C temperatures, and humidity) and provided with standard food and water ad libitum. Animal maintenance and all the experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) of Calcutta School of Tropical Medicine.

2.7. Measurement of bone marrow ROS ROS level in the bone marrow was measured by Dihydroethidium (DHE; Sigma Aldrich, USA) staining (Rothe and Valet, 1990). In brief, bone marrow cells at a density of 106cells/ml were treated with 20 μl of 30 mM DHE and incubated for 30 min in dark. The intensity of the resultant red fluorescence was determined by FACS analysis (BD-FACS Calibur, Cell Quest Pro software; BD Bioscience, USA).

2.2. Induction of aplastic anemia Use of chemotherapeutic drugs like busulfan and cyclophosphamide is in practice to develop mouse model of aplastic anemia for decades (Morley and Blake, 1974; Boyd et al., 1986; Gibson et al., 2003a; Gibson et al., 2003b; Chen, 2005; Chatterjee et al., 2008; Chen et al., 2014; Chatterjee et al., 2016a; Chatterjee et al., 2009; Chatterjee et al., 2010; Chatterjee et al., 2016b). In our experimental study, mice were divided into control and aplastic anemia groups each containing 30 animals. Aplastic anemia was induced in mice by i.p. injections of two doses each of busulfan (20 mg kg−1 b.w.) and cyclophosphamide (80 mg kg−1 b.w.) at an interval of 28 days. The animals were kept for three months post second dose for the development of long term marrow injury condition as per the protocol of Chatterjee et al., 2016a. Control group of mice received comparable volume of aqueous saline solution. (Fig.1A–C)

2.8. Nuclear chromatin condensation study To access the chromatin condensation status, 1 × 106 marrow cells from both the group of mice were transferred on glass slides, mounted with DAPI shield (Sigma Aldrich, USA) and examined under laser scanning confocal microscope (FV1200, Olympus). Observations of 20 random fields were taken into consideration in both the cases. 2.9. Global DNA CpG methylation analysis in HSPC population Global DNA CpG methylation was evaluated by the classical method of staining cells with monoclonal antibody against 5-methylcytosine (Cell signaling Technology, USA) (Milutinovic et al., 2003). Briefly,

2.3. Peripheral blood hemogram Total count and differential count of blood cells were done 2

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Fig.1. Hematopoietic catastrophe in aplastic marrow. Experimental procedure (A) of disease development involves challenging of mice with two doses of each BU (20 mg kg−1 b.w) and CP (20 mg kg−1 b.w) at an interval of 28 days. Aplastic condition developed after 3 months of second dosing (B). Control group of mice received comparable doses of saline and used for experimental purpose after similar time interval (C). Flowcytometric studies revealed significant decrease of hematopoietic stem cells (CD 150+ cells) as well as entire hematopoietic stem/progenitor population (c-Kit+ cells) in aplastic marrow as compared to that of control (D, E). Histological analysis of entire microscopic fields of control marrow showed the presence of adequate hematopoietic space (F) which was found to be decreased considerably in aplastic marrow by the presence of fat cells (G). Hematopoietic disruption under chemotherapeutic stress was manifested by the decrease of all the hematological parameters viz; WBC count (H), RBC count (I), Platelet count (J), hemoglobin content (K) reticulocyte count (L) in aplastic blood as compared to that of control. Moreover severe neutropenic condition (M) appeared in aplastic mice. Panel N consists of schematic representation of the drug induced hematopoietic disruption that led to the peripheral blood pancytopenia in aplastic anemic condition. * [Magnification; F, G = 400X]. (P values; **P < 0.01, ***P < 0.001, ****P < 0.0001).

bone marrow cells from control and aplastic anemic mice were fixed in 1.5% PFA, permeabilized with 90% chilled methanol, thoroughly washed with PBS and finally suspended in FACS fluid. 2 μl of anti- 5-methylcytosine antibody was added per 2 × 106 cells followed by incubation at 37 °C for 30 min. After that, 2 μl of alexa fluor-488 tagged

secondary antibody (Invitrogen, USA) was added to the cells followed by similar incubation at 37 °C for 30 min in dark. Mean fluorescence intensity (MFI) of 5-methylcytosine staining was determined flowcytometrically (10,000 events) by applying virtual gating on the low SSC/ low FSC region which was earlier reported to be rich in Sca-1 positive 3

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HSPCs (Chatterjee et al., 2008; Chatterjee et al., 2016a; Chatterjee et al., 2016c; Chattopadhyay et al., 2016a; Schuurhuis et al., 2013; Gajkowska et al., 2006; Papadaki et al., 2005).

significant. Each experiment was performed three times.

2.10. Histone-3 (H3) phosphorylation study with fluorescence imaging

3.1. Hematopoietic catastrophe in aplastic anemic condition

H3 phosphorylation at Ser10 was determined by immune-fluorescence (IF) analysis by staining the bone marrow cells with anti-phospho H3 (Ser10) antibody and alexa fluor-488 tagged secondary antibody. Immunostaining was done following the protocol of Chatterjee et al., 2017 (primary antibody 1: 200 dilution and secondary antibody 1: 600 dilution) (Chatterjee et al., 2017). DAPI shield (Sigma-Aldrich, USA) was used to stain the nuclei. Slides were studied under fluorescent microscope (Axio Scope.A1, Zeiss) and the images obtained were analysed using NIH Image J software.

Severe hematopoietic disruption due to aplastic stress was evident from blood and marrow studies. Flowcytometric analysis revealed significant decrease in the population of CD 150+ (1.72% ± 0.89 versus 8.91% ± 0.35; P < 0.001) and c-Kit+ (3.22% ± 0.64 versus 13.74% ± 1.13; P < 0.001) cells in bone marrow (Figs 1D,E, 5A). CD 150 is the marker for both long term and short term HSC in murine bone marrow and c-Kit denotes the entire HSPC population (Kiel et al., 2005; Kiel et al., 2008; Yilmaz et al., 2006; Chen et al., 2008; Kim et al., 2006; Oguro et al., 2013; Wilson et al., 2008; van Galen et al., 2014; Nadri et al., 2002; Chatterjee et al., 2014; Ikuta and Weissman, 1992; Lkuto et al., 1991). The severe decrease in the basic hematopoietic population was found to be associated with overall decrease in bone marrow cellularity as depicted by the histological analysis of bone marrow which showed considerable shrinkage of hematopoietic space in aplastic marrow as compared to that of control (Fig. 1F,G). Manifestation of the hematopoietic catastrophe in aplastic mice was evident from peripheral blood analysis where all the parameters viz; WBC count (Fig.1H), RBC count (Fig. 1I), platelet count (Fig. 1J), hemoglobin content (Fig.1J), reticulocyte count (Fig. 1L), were found to be significantly decreased. Significant reduction of neutrohil population (Fig. 1M) was found in aplastic blood which depicted severe myelosupression due to chemotherapeutic insults. The findings hinted towards the severe hematopoietic catastrophe produced under aplastic stress (Fig. 1N).

3. Results

2.11. Analysis of cellular apoptosis profile in HSPC compartment Detection of apoptosis was done flowcytometrically by measuring the MFI of cleaved-caspase-3 (ultimate apoptosis executer) expression in the low SSC/low FSC region of scatterogram after staining the bone marrow cells with anti-cleaved-caspase-3 antibody (Cell Signaling Technology, USA) and respective fluorochrome tagged secondary antibody as described in our previous publication (Chatterjee et al., 2016a). 2.12. Flowcytometric protein expression study Flowcytometry was performed for the protein expression analysis according to our previously published protocol (Chatterjee et al., 2016a; Chatterjee et al., 2016c; Chatterjee et al., 2017; Daw et al., 2016). Brief accounts for the same are as follows:

3.2. Redox imbalance under aplastic stress

2.12.1. Cell surface proteins For the analysis of cell surface proteins, bone marrow cells from both the groups were fixed in 1.5% PFA, washed with PBS and suspended in FACS fluid. Analysis of CD 150, c-Kit, CD 45, Jagged-1 was performed by incubating the cells with respective primary antibodies (anti-CD 150 PE, anti-c-Kit FITC from BD-Bioscience, USA; anti-CD 45 FITC from eBioscience, USA; anti-Jagged-1 from Cell Signaling Technology, USA) at 37 °C in dark for 30 min. Cells for the analysis of Jagged-1 were further incubated for another 30 min at similar condition after the addition of flurochrome tagged secondary antibody. Cells were finally subjected to FACS analysis (10,000 events) on BD FACS Calibur (Becton-Dickinson, USA) using Cell Quest Pro software (BDBioscience, USA).

DHE staining revealed significant increase in the level of ROS in aplastic bone marrow as compared to that of control (Mean fluorescent intensity or MFI; 411.96 ± 24.46 versus 127.63 ± 13.55; P < 0.0001) (Fig. 2A and B). The phenomenon was in agreement with the previous findings depicting the generation of oxidative disbalance due to the effect of chemotherapeutic drugs (Chen et al., 2007; Gilliam and St Clair, 2011; Angsutararux et al., 2015; Conklin, 2004). 3.3. ROS associated microenvironmental disruption in aplastic marrow Production of ROS is capable to cause oxidative damage to HSPCs (Shao et al., 2013; Wang et al., 2010; Ito et al., 2006; Ito et al., 2004). Osteoblastic niche provides protective microenvironment to the HSPCs that promote self renewal in hypoxic condition (Shao et al., 2013; Guerrouahen et al., 2011; Zhang et al., 2003; Calvi et al., 2003a; Takubo et al., 2010; Winkler et al., 2010). Generation of ROS is known to have adverse effect on osteoblasts (Almeida et al., 2007; Arai et al., 2007; Jung, 2014; Jin et al., 2017). Active osteoblasts can be identified by the presence of type-I collagen which takes blue coloration during Mallory‘s trichrome staining (Rodan et al., 1988; Kini and Nandeesh, 2012; Clarke, 2008; Huang et al., 2007; Domitrović and Jakovac, 2010). A significant decrease of osteoblast lining in endosteal bone surface was evident from Mallory‘s trichrome stained histological section of aplastic marrow (Fig. 2C) in contrast to that of control where prominent blue lining was observed in the endosteal region (Fig. 2D). Moreover, flowcytometric analysis also revealed the significant decrease of Runx-2 (11.4% ± 1.63 versus 19.76% ± 1.78; P < 0.01) and Alp-1 (17.11% ± 3.04 versus 29.44% ± 1.92; P < 0.01) positive population in aplastic marrow (Figs 2E,F 5A). Runx-2 has been identified as the markers for maturing osteoblasts while Alp-1 is expressed by the both preosteoblasts and matured osteoblasts (Chatterjee et al., 2017; Huang et al., 2007; Komori, 2009; Khlusov et al., 2011; Lin et al., 2010). Apart from affecting osteoblastic niche components, ROS

2.12.2. Intra-cellular proteins For the analysis of intra-cellular proteins viz; Runx-2, Alkaline phosphatise-1 (Alp-1), Stro-1, Sdf-1, Notch-1, Presenellin-1 (PS-1), cMyc, Protein phosphatise-1 (PP-1), SOD-2, bone marrow cells were fixed in 1.5% PFA, permeabilized with 90% chilled methanol, thoroughly washed in PBS and finally suspended in FACS fluid. Cells were then incubated with respective primary antibodies (anti-Runx-2, antiSdf-1, anti-Notch-1, anti-PS-1, anti-SOD-2, anti-PP-1 from Cell Signaling Technology, USA; anti-Alp-1 from Genetex, USA; anti-Stro-1, anti-c-Myc from Biolegend, USA) at 37 °C and thereafter incubated with flurochrome tagged secondary antibodies at similar condition in dark. Cells were finally subjected to FACS analysis by early mentioned process. 2.13. Statistics All the quantitative values were expressed as mean ± SD (standard deviation). Statistical analysis was done by unpaired Student‘s T-test where probabilities of P < 0.05 and P < 0.0001 were considered as 4

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Fig. 2. ROS associated alterations of hematopoietic microenvironmental components during aplastic anemia. Flowcytometric analysis revealed a significant increase of ROS level in aplastic marrow (A). Graphical representation of the same is depicted in panel B. Mallory’s trichrome staining of histological sections of control bone marrow (C) showed the presence of prominent osteoblastic lining (marked with blue arrows) in the endosteal region that took blue coloration as well as adequate hematopoietic region (marked with black arrows). In contrary, the aplastic marrow (D) showed considerable decrease of endosteal osteoblastic lining and hematopoietic mass together with concomitant increase of adipocytes (marked with yellow arrows). Furthermore, flowcytometry revealed the significant decrease of Runx-2+ (E), Alp-1+ (F), CD 45− (G), Stro-1+ (H) populations in aplastic marrow as compared to that of control. In comparison with control, Sdf-1 was also found to be downregulated in aplastic marrow (I). Oil Red O staining depicted an increase of adipocytes as identified by red coloration (marked with black arrows) in aplastic marrow (K) as compared to that of control (J). Panel L represents the correlative association of ROS elevation, hematopoiesitic microenvironmental disruption and increased adipogenesis in aplastic marrow due to chemotherapeutic stress. [Magnification; C, D = 400X; J, K = 100X].

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is also known to affect CD 45−ve marrow cells which correspond to stromal population- a heterogeneous mixture of fibroblasts, osteoblasts, and endothelial cells (Khatri et al., 2016). Significant decrease in the percentage of CD 45−ve population (25.3% ± 1.57 versus 39.26% ± 1.74; P < 0.001) (Figs. 2G, 5A) in aplastic marrow hinted towards the ROS associated stromal devastation under chemotherapeutic insult. Stro-1 positive marrow population which is reported to be enriched with CFU-F (Simmons and Torok-Storb, 1991; Simmons et al., 1994; Dennis et al., 2002; Ramakrishnan et al., 2013) was also found to be negatively affected (9.58% ± 1.5 versus 16.4% ± 1.97; P < 0.01) (Figs 2H, 5A) in aplastic condition which reinforced the scenario of ROS associated microenvironmental calamity. CXCL-12 or Sdf-1 which is secreted by various niche components is known to protect HSPCs from oxidative stress (Dar et al., 2005; Zhang et al., 2016). Severe decline of marrow Sdf-1 level (MFI; 114.95 ± 4.09 versus 210.55 ± 6.09; P < 0.0001) (Figs 2I 5B) under aplastic stress might have added to the susceptibility of hematopoietic population to the oxidative damage. In addition to impairment of the hematopoiesis supportive niche, ROS is also reported to increase adipogenesis in bone marrow (Younce and Kolattukudy, 2012; Gummersbach et al., 2009; Liu et al., 2012a; Liu et al., 2012b). Adipocytes are the negative components of bone marrow microenvironment that hinders proper hematopoiesis (Naveiras et al., 2009). Along with the elevation of ROS level, concomitant rise of adipocyte population was observed in aplastic marrow (Fig. 2K) in comparison to that of control (Fig. 2J) as identified by the red coloration after Oil Red O staining. All the findings reflected an overall negative impact of ROS over hematopoietic micoenvironment during aplastic anemia (Fig. 2L).

(Fig. 3F). Moreover, Notch-1 signaling is reported to have protective role against oxidative stress through the up-regulation of the ROS scavenger- SOD-2 (Cai et al., 2016). Expressional downregulation of SOD-2 (MFI; 245.32 ± 3.13 versus 335.55 ± 5.62; P < 0.0001) (Figs 3E, 5B) was found in aplastic HSPCs that signified the irreversibility of the redox imbalance which can be correlated with the deregulation of Notch-1 signaling under aplastic stress (Fig. 3F). 3.5. ROS related epigenetic modifications and apoptosis in hematopoietic population during aplastic anemia Oxidative disbalance can alter epigenetic chromatin modifications leading to cellular catastrophe. One of the potent ROS driven epigenetic changes involves the alteration of global DNA CpG methylation pattern. ROS is reported to promote global CpG hypo-methylation in DNA which is associated with increased cellular apoptosis (Wu and Ni, 2015; Fan et al., 2001; Notley et al., 2017). Along with the increased ROS in aplastic bone marrow, flowcytometric analysis revealed an expressional decline of 5-methylecytosine (MFI; 47.5 ± 1.63 versus 52.22 ± 1.11; P < 0.05) (Figs 4A, 5B ) that corresponds to the decrease of global DNA CpG methylation which is in agreement with the previous findings regarding DNA hypo-methylation related to the cellular exposure to alkylating agents (Nyce, 1989). Expression of DNMT-1, enzyme that mediates CpG methylation of DNA (Bestor, 2000; Hermann et al., 2004) was also found to be significantly down-regulated in aplastic HSPCs (MFI; 40.41 ± 2.95 versus 55.26 ± 2.89; P < 0.01) (Figs 4B; 5B). Moreover, increased percentage of cells (37.4% ± 3.85 versus 3.48% ± 2.05; P < 0.001) showing the expression of the apoptotic marker- cleaved-Caspase-3 (Gown and Willingham, 2002) in the aplastic HSPC compartment (Figs 4F, 5A) hinted towards the correlation between ROS associated global DNA CpG hypo-methylation and increased cellular apoptosis under chemotherapeutic stress. ROS can also trigger cellular apoptosis by inducing premature chromatin condensation through the phosphorylation of Ser-10 residue of Histone-3 (H-3) (Tikoo et al., 2001). Phosphorylation of H-3 at Ser-10 which can occur during mitosis under the influence of mitogens as well as during apoptosis due to the effects of diverse death stimuli including ROS generation (Tikoo et al., 2001; Kang et al., 2007; Crosio et al., 2002; Chattopadhyay et al., 2016b; Prigent and Dimitrov, 2003; Park and Kim, 2012; Wang and Lippard, 2004). A major impact of phospho-H-3 (Ser-10) on cell involves chromatin condensation, a common step for both the initiation of apoptosis and cell division (Tikoo et al., 2001; Park and Kim, 2012; Rossetto et al., 2012). Fluorescence microscopy revealed the elevation of phospho-H-3 (Ser-10) expression pattern in aplastic hematopoietic cells (Fig. 4C). Our previous findings depicted severe mitotic catastrophe in aplastic HSPCs along with downregulation of Aurora kinase, a key molecule required for H-3 phosphorylation at Ser-10 prior to mitosis (Chatterjee et al., 2016a). Moreover, in the present study confocal microscopy of DAPI stained cells following the previously described protocol by Muñoz et al. (2011); Manna et al. (2012) together (Muñoz et al., 2011; Manna et al., 2012) with the 3D surface plotting of the obtained images by NIH Image J software, depicted increase of chromatin condensation in aplastic marrow cells (reflected by the increased DAPI staining intensity) as compared to that of control (Fig. 4D). Association of chromatin condensation with elevated cleaved-caspase-3 expression pattern and increased ROS under aplastic stress hinted towards the correlation of oxidative stress related hematopoietic cellular death with the increased phosphorylation of H-3 at Ser-10. On the other hand, PP-1 which is known to dephosphorylate phospho-H-3 at Ser-10 (Prigent and Dimitrov, 2003; Adhvaryu and Selker, 2008), was found to be significantly downregulated in aplastic HSPCs (MFI; 23.47 ± 3.57 versus 54.32 ± 2.66; P < 0.001) (4E, 5B) which might have resulted in the sustained H-3 phosphorylation promoting chromatin condensation. The findings identified the ROS associated epigenetic alterations as one of the driving forces towards hematopoietic catastrophe during chemotherapeutics mediated bone

3.4. Oxidative stress associated alteration of notch-1 signaling in HSPC rich marrow compartment during aplastic anemia Hematopoietic microenvironment provides HSPCs with special milieu that supports their self-renewal and this is likely achieved in part by extensive interactions between hematopoietic cells and the niche via Jagged/Notch axis (Calvi et al., 2003b; Varnum-Finney et al., 2000; Ohishi et al., 2003; Blank et al., 2008; Weber and Calvi, 2010). Activation of the pathway involves interaction of Notch ligand viz, Jagged1 expressed on the surface of a signaling cell with the Notch receptor viz; Notch-1 expressed on the surface of a receiving cell (Crabtree et al., 2016). The interaction promotes the liberation of the Notch intracellular domain (NICD) via multi-step proteolytic event where the final executor cleavage for NICD liberation is mediated by the γ-secretase complex (Shih and Wang, 2007). The crucial proteolytic function of γ-secretase complex is exerted by the master molecule Presenelin-1 (PS-1) (De Strooper et al., 1999; De Strooper et al., 2012). Cleaved NICD translocates to the nucleus for the transcriptional regulations of the target genes (Hansson et al., 2004; Struhl and Adachi, 1998; Mumm and Kopan, 2000). In hematopoietic system, Notch ligands are identified on stroma while HSPCs are enriched with active notch-1 intracellular domain (NICD) (Weber and Calvi, 2010; VarnumFinney et al., 1998). During steady state hematopoiesis Notch-1 driven expression of c-Myc is crucial for the self-renewal maintenance of HSPCs (Satoh et al., 2004). c-Myc activity is also required for the survivability of hematopoietic cells (Laurenti et al., 2008). In aplastic marrow, significant decline of Jagged+ cells (21.58% ± 1.65 versus 35.45% ± 3; P < 0.01) (Figs. 3A 5A ) was found which corresponds to the ROS associated decrease of the hematopoietic microenvironmental components. On the other hand, the hematopoietic compartment (low SSC/low FSC) showed a concomitant decline in the expressions of cleaved-Notch-1 i.e; NICD (MFI; 29.24 ± 1.53 versus 36.28 ± 2.16; P < 0.01) (Figs 3B, 5B), PS-1 (MFI; 13.53 ± 0.9 versus 17.38 ± 1.59; P < 0.05) (Figs 3C, 5B) and c-Myc (MFI; 17.27 ± 2.07 versus 26.59 ± 2.01; P < 0.01) (Figs 3D, 5B) which reflected the disruption of Notch-1 signaling under aplastic stress due to the reduction of Jagged-1 expressing microenvironmental components 6

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Fig. 3. Alteration of Jagged-1/Notch-1 signaling axis under aplastic stress. Flowcytometric studies showed decreased Jagged-1+ cells (A) in aplastic bone marrow as well as downregulations of cleaved-Notch-1 (B), PS-1 (C) and c-Myc (D) in the HSPC compartment during aplastic anemia. SOD-2 expression was also found to be decreased in aplastic marrow cells as compared to that of control (E). Panel F depicted the schematic representation of the association of ROS and decreased stromal population (Jagged-1+) resulting in the deregulations of the Notch-1 signaling components in aplastic HSPCs which caused the disruption of c-Myc mediated hematopoietic self-renewal and survivality as well as decrease of SOD-2 mediated antioxidant activity.

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Fig. 4. Epigenetic modifications under oxidative stress in aplastic marrow. Flowcytometry revealed the concomitant decrease of 5-methylcytosine level (A) and DNMT-1 expression (B) in aplastic HSPC rich marrow compartment as compared to that of control that hinted towards the ROS associated decrease of global DNA CpG methylation pattern under chemotherapeutic stress. Fluorescence imaging (C) showed increased expression pattern of phospho-H-3 (Ser 10) in aplastic marrow as compared to that of control. Confocal microscopic (D) analysis revealed increased chromatin condensation in aplastic marrow cells in comparison with that of control. In addition, flowcytometric analysis also showed a decline of PP-1 expression (E) as well as increase of cleaved-caspase-3+ cells (F) in aplastic HSPC compartment. Panel G represents the association of aplastic stress related oxidative imbalance and epigenetic alterations in hematopoietic population that exerted negative effects on hematopoietic cells during bone marrow aplasia. [Magnification; C = 400X; D = 1000X].

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Fig. 5. Graphical representations of the studied molecules. Panel A represents the comparative scenario regarding the percentages of CD 150+, c-Kit+, Runx2+, Alp-1+, CD 45−, Stro-1+, Jagged-1+ and cleaved-caspase-3+ cells between control and aplastic marrow cells. Panel B depicted the comparative MFI values regarding the expressions of Sdf-1, NICD, Ps-1, c-Myc, SOD-2, 5-Methylcytosine, DNMT-1 and PP-1 in the hematopoietic population from control and aplastic mice. (P values; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)

reduction of microenvironmental components could have made HSPC population more vulnerable to the oxidative damage. Nevertheless, decrease in Sdf-1 level in aplastic marrow hinted towards the loss of HSPC protective system against oxidative insults during aplastic condition making the hematopoietic population more susceptible to stress related damage. Apart from disrupting the hematopoiesis supportive niche components during aplastic anemia, ROS was found to be associated with increased marrow adipocytes, a negative component of hematopoietic niche. All the findings suggested an overall negative impact of ROS generation on the microenvironmental support to steady state hematopoiesis during bone marrow aplasia. After unveiling the scenario of ROS associated microenvironmental disruption in aplastic marrow, the study aimed in establishing the mechanistic correlation between the micrenvironmental impairment and disrupted hematopoiesis under aplastic stress. Since, Notch-1 signaling driven c-Myc expression plays a crucial role in sustaining selfrenewing hematopoietic population (Satoh et al., 2004), our study focused on the ROS associated niche disruption related alteration of the vital signaling axis in aplastic marrow. Jagged-1, the ligand for Notch-1 is expressed by the stromal cells and ROS associated damage to the stromal mass was clearly manifested by the decreased Jagged-1+ cells in aplastic marrow. The effect of the decreased Jagged-1 expression pattern in aplastic marrow was exerted in the HSPC population which showed expressional down-regulations of cleaved-Notch-1, the activated Notch-1 form as well as Presenellin-1, the Notch activating

marrow aplasia (Fig. 4G). 4. Discussion One of the common cancer treatment related late effects involves long-term bone marrow injury leading to marrow failure or aplastic anemic condition resulting from chemotherapy induced damage to hematopoietic stem/progenitor cells (HSPCs) (Testa et al., 1984; Wang et al., 2006; Shao et al., 2013; Young, 1988; Scatena et al., 2010; Bowcock et al., 1989). The goal of the study is to find the underlying mechanisms of chemotherapeutics induced marrow aplasia using mouse model which may facilitate the research to develop new therapeutic strategies against the dreadful condition. The cytopenic condition in marrow as well as in peripheral blood under aplastic stress was the manifestation of the severe damage to the basic hematopoietic population which involves the reduction of hematopoietic stem/progenitor population (c-kit+ cells) as a whole as well as the HSC population (CD 150+ cells) in particular. The study identified increased ROS generation as a central player in driving the catastrophic features during bone marrow aplasia. The osteoblastic niche, which maintains HSPC pool in hypoxic environment, was found to be severely affected by the generation of oxidative marrow disbalanced condition under aplastic stress. Moreover, the entire stromal mass (CD 45−cells) including CFU-F enriched Stro+ cells was found to be greatly reduced due to oxidative assault in aplastic condition. This 9

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Fig. 6. Schematic representation of the study outcome. Aplastic anemic stress generated in bone marrow due to chemotherapeutic insults resulted in the elevation of ROS level which was associated with the decrease of osteoblastic niche as well as stromal components and increase of adipocytes which had negative effects on hematopoietic population. ROS associated microenvironmental disruption in aplastic marrow can be correlated with the deregulation of Notch-1 signaling which also affected the hematopoietic physiology. Moreover, epigenetic alterations under oxidative stress resulted in the increased hematopoietic catastrophe. Downregulations of Sdf-1 and SOD-2 in aplastic marrow hinted towards the loss of the cellular protective mechanisms against ROS.

proteolytic component of γ-secretase complex. Deregulation of Notch-1 signaling in aplastic marrow was manifested in two ways − firstly, by expressional decline of c-Myc resulting in the impaired HSPC‘s self-renewal activity as well survivality and secondly, by decrease of SOD-2 level promoting the reduced antioxidant activity against ROS. This alteration of Notch-1 signaling can be regarded as a vital factor behind the reduction of HSPC population under the oxidative stress during aplastic anemia. In addition to the disruption of hematopoiesis supportive microenvironment and signaling regulation over hematopoietic population, ROS was also found to be associated with the altered epigenetic status of hematopoietic cells during bone marrow aplasia. Due to the well established phenomenon of ROS associated global DNA CpG hypomethylation (Wu and Ni, 2015), decline in the expression of 5-methylcytosine in aplastic HSPCs corresponding to the decrease of global DNA CpG methylation along with the downregulation of methylating enzyme DNMT-1 can be correlated with the generation of oxidative disbalance under chemotherapeutic stress. DNA hypo-methylation is associated with cellular apoptosis and increased expression of cleavedCaspase-3 in aplastic HSPCs which hinted towards the correlative association of the elevated ROS, decreased CpG methylation status of DNA and increased apoptosis rate in the hematopoietic marrow compartment during aplastic anemia. Moreover, apoptosis and associated chromatin condensation in aplastic marrow can be well correlated with increased phosphorylation of H-3 at Ser-10 domain which is a well established epigenetic phenomenon under the influence of various death stimuli, ROS being one of them (Tikoo et al., 2001; Prigent and Dimitrov, 2003; Park and Kim, 2012; Wang and Lippard, 2004). In summary, it can be demonstrated that the chemotherapeutics mediated bone marrow failure or aplastic anemia involved the generation of ROS which was associated with the exertion of negative impact on the hematopoietic niche, deregulation of microenvironment related Notch-1 signaling and alteration of epigenetic status of the HSPCs leading to an irreversible hematopoietic devastation (Fig. 6). We

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