A versatile mouse model of epitope-tagged histone H3.3 to study epigenome dynamics.

The variant histone H3.3 is incorporated into the genome in a transcription-dependent manner. This histone is thus thought to play a role in epigenetic regulation. However, our understanding of how H3.3 controls gene expression and epigenome landscape has remained incomplete. This is partly because precise localization of H3.3 in the genome has been difficult to decipher particularly for cells in vivo To circumvent this difficulty, we generated knockin mice, by homologous recombination, to replace both of the two H3.3 loci (H3f3a and H3f3b) with the hemagglutinin-tagged H3.3 cDNA cassette, which also contained a GFP gene. We show here that the hemagglutinin-tagged H3.3 and GFP are expressed in the majority of cells in all adult tissues tested. ChIP-seq data, combined with RNA-seq, revealed a striking correlation between the level of transcripts and that of H3.3 accumulation in expressed genes. Finally, we demonstrate that H3.3 deposition is markedly enhanced upon stimulation by interferon on interferon-stimulated genes, highlighting transcription-coupled H3.3 dynamics. Together, these H3.3 knockin mice serve as a useful experimental model to study epigenome regulation in development and in various adult cells in vivo.

Comparative proteomic analysis of human peripheral blood mononuclear cells indicates adaptive response to low-dose radiation in individuals from high background radiation areas of Kerala.

There remain significant uncertainties in estimation of risks with low doses of radiation. The small coastal belt in the southwestern state of Kerala, India, extending from Neendakara in the south to Purakkad in the north is one of the most extensively studied high-level natural background radiation areas (HLNRAs) of the world to address these concerns. The natural radioactivity here is due to occurrence of monazite sand bearing placer deposits along the coastline. In this study, proteomic approach was employed to study the response of human peripheral blood mononuclear cells (PBMCs) from individuals residing in HLNRA (N = 10; mean radiation dose: 15.60 ± 3.04 mGy/y) vis-à-vis responses in individuals from adjoining normal-level natural background radiation areas (NLNRA; N = 10; mean radiation dose: ≤1.50 mGy/y) using two-dimensional gel electrophoresis coupled with mass spectrometry. A total of 15 proteins were found to be statistically altered in individuals from HLNRA when compared to individuals from NLNRA (P ≤ 0.05). Most of the changes in expression were small. The mean coefficient of variation for the differentially altered proteins was found to be ~34%. Pathway enrichment analysis with Database for Annotation, Visualization and Integrated Discovery distinguished 44 biological processes significantly (P ≤ 0.05) modulated in HLNRA samples. More importantly, when challenged with an ex vivo dose of 2 Gy, HLNRA PBMCs responded with an up-regulation of many protective pro-survival proteins such as protein disulfide-isomerase A1 (PDIA1), peroxiredoxin 6 (PRDX6) and glucose-regulated protein 78 kDa (GRP78). PDIA1 and PRDX6 are known to play an important role in redox homeostasis. GRP78 is considered the master regulator of unfolded protein response that aims to restore endoplasmic reticulum homeostasis and thus, regulate cell survival. Principal component analysis identified clear clusters based on radiation dose. The expression changes of key proteins were validated by western blotting using additional samples from HLNRA and NLNRA. This indicates that the human cells respond to low dose of ionising radiation through dynamic changes in the proteome to maintain adaptive homeostasis. These findings emphasise that the dose-response relationship at low doses of radiation may not be linear and, thus, provide mechanistic challenge to the linear-no-threshold hypothesis.

Gene expression of immediate early genes of AP-1 transcription factor in human peripheral blood mononuclear cells in response to ionizing radiation.

Ionizing radiation (IR) is considered ubiquitous in nature. The immediate early genes are considered the earliest nuclear targets of IR and are induced in the absence of de novo protein synthesis. Many of these genes encode transcription factors that constitute the first step in signal transduction to couple cytoplasmic effects with long-term cellular response. In this paper, coordinated transcript response of fos and jun family members which constitute activator protein 1 transcription factor was studied in response to IR in human peripheral blood lymphocytes at the G0 stage. Gene expression was monitored 5 min, 1 h and 4 h post-irradiation with Co60 γ-rays (dose rate of 0.417 Gy/min) and compared with sham-irradiated controls. When gene expression was analyzed at the early time point of 5 min post-irradiation with 0.3 Gy, the studied samples showed two distinct trends. Six out of ten individuals (called 'Group I responders') showed transient, but significant up-regulation for fosB, fosL1, fosL2 and c-jun with an average fold change (FC) ≥1.5 as compared to sham-irradiated controls. The Students's t test p value for all four genes was ≤0.001, indicating strong up-regulation. The remaining four individuals (called Group II responders) showed down-regulation for these same four genes. The average FC with 0.3 Gy in Group II individuals was 0.53 ± 0.22 (p = 0.006) for fosB, 0.60 ± 0.14 (p = 0.001) for fosL1, 0.52 ± 0.16 (p = 0.001) for fosL2 and 0.59 ± 0.28 (p = 0.03) for c-jun. The two groups could be clearly distinguished at this dose/time point using principal component analysis. Both Group I and Group II responders did not show any change in expression for three genes (c-fos, junB and junD) as compared to sham-irradiated controls. Though a similar trend was seen 5 min post-irradiation with a relatively high dose of 1 Gy, the average FC was lower and change in gene expression was not statistically significant (at p < 0.05), except for the down-regulation at fosL2 for Group II individuals (mean FC = 0.70 ± 0.15, p = 0.008). Both groups of individuals did not show any differential change in expression (FC ~ 1.0) for most loci at the late time points of 1 and 4 h, neither with 0.3 Gy nor with 1 Gy.

Biological and cellular responses of humans to high-level natural radiation: A clarion call for a fresh perspective on the linear no-threshold paradigm.

There remains considerable uncertainty in obtaining risk estimates of adverse health outcomes of chronic low-dose radiation. In the absence of reliable direct data, extrapolation through the linear no-threshold (LNT) hypothesis forms the cardinal tenet of all risk assessments for low doses (≤ 100 mGy) and for the radiation protection principle of As Low As Reasonably Achievable (ALARA). However, as recent evidences demonstrate, LNT assumptions do not appropriately reflect the biology of the cell at the low-dose end of the dose-response curve. In this regard, human populations living in high-level natural radiation areas (HLNRA) of the world can provide valuable insights into the biological and cellular effects of chronic radiation to facilitate improved precision of the dose-response relationship at low doses. Here, data obtained over decades of epidemiological and radiobiological studies on HLNRA populations is summarized. These studies do not show any evidence of unfavourable health effects or adverse cellular effects that can be correlated with high-level natural radiation. Contrary to the assumptions of LNT, no excess cancer risks or untoward pregnancy outcomes have been found to be associated with cumulative radiation dose or in-utero exposures. Molecular biology-driven studies demonstrate that chronic low-dose activates several cellular defence mechanisms that help cells to sense, recover, survive, and adapt to radiation stress. These mechanisms include stress-response signaling, DNA repair, immune alterations and most importantly, the radiation-induced adaptive response. The HLNRA data is consistent with the new evolving paradigms of low-dose radiobiology and can help develop the theoretical framework of an alternate dose-response model. A rational integration of radiobiology with epidemiology data is imperative to reduce uncertainties in predicting the potential health risks of chronic low doses of radiation.

Effect of GNE Mutations on Cytoskeletal Network Proteins: Potential Gateway to Understand Pathomechanism of GNEM.

GNE myopathy is an inherited neuromuscular disorder caused by mutations in GNE (UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase) gene catalyzing the sialic acid biosynthesis pathway. The characteristic features include muscle weakness in upper and lower extremities, skeletal muscle wasting, and rimmed vacuole formation. More than 200 GNE mutations in either epimerase or kinase domain have been reported worldwide. In Indian subcontinent, several GNE mutations have been recently identified with unknown functional correlation. Alternate role of GNE in various cellular processes such as cell adhesion, migration, apoptosis, protein aggregation, and cytoskeletal organization have been proposed in recent studies. We aim to understand and compare the effect of various GNE mutations from Indian origin on regulation of the cytoskeletal network. In particular, F-actin dynamics was determined quantitatively by determining F/G-actin ratios in immunoblots for specific proteins. The extent of F-actin polymerization was visualized by immunostaining with Phalloidin using confocal microscopy. The proteins regulating F-actin dynamics such as RhoA, cofilin, Arp2, and alpha-actinin were studied in various GNE mutants. The altered level of cytoskeletal organization network proteins affected cell migration of GNE mutant proteins as measured by wound healing assay. The functional comparison of GNE mutations will help in better understanding of the genotypic severity of the disease in the Indian population. Our study offers a potential for identification of therapeutic molecules regulating actin dynamics in GNE specific mutations.

Activation of DNA damage response signaling in mammalian cells by ionizing radiation.

Cellular responses to DNA damage are fundamental to preserve genomic integrity during various endogenous and exogenous stresses. Following radiation therapy and chemotherapy, this DNA damage response (DDR) also determines development of carcinogenesis and therapeutic outcome. In humans, DNA damage activates a robust network of signal transduction cascades, driven primarily through phosphorylation events. These responses primarily involve two key non-redundant signal transducing proteins of phosphatidylinositol 3-kinase-like (PIKK) family - ATR and ATM, and their downstream kinases (hChk1 and hChk2). They further phosphorylate effectors proteins such as p53, Cdc25A and Cdc25C which function either to activate the DNA damage checkpoints and cell death mechanisms, or DNA repair pathways. Identification of molecular pathways that determine signaling after DNA damage and trigger DNA repair in response to differing types of DNA lesions allows for a far better understanding of the consequences of radiation and chemotherapy on normal and tumor cells. Here we highlight the network of DNA damage response pathways that are activated after treatment with different types of radiation. Further, we discuss regulation of cell cycle checkpoint and DNA repair processes in the context of DDR in response to radiation.

Chronic exposure of humans to high level natural background radiation leads to robust expression of protective stress response proteins.

Understanding exposures to low doses of ionizing radiation are relevant since most environmental, diagnostic radiology and occupational exposures lie in this region. However, the molecular mechanisms that drive cellular responses at these doses, and the subsequent health outcomes, remain unclear. A local monazite-rich high level natural radiation area (HLNRA) in the state of Kerala on the south-west coast of Indian subcontinent show radiation doses extending from ≤ 1 to ≥ 45 mGy/y and thus, serve as a model resource to understand low dose mechanisms directly on healthy humans. We performed quantitative discovery proteomics based on multiplexed isobaric tags (iTRAQ) coupled with LC-MS/MS on human peripheral blood mononuclear cells from HLNRA individuals. Several proteins involved in diverse biological processes such as DNA repair, RNA processing, chromatin modifications and cytoskeletal organization showed distinct expression in HLNRA individuals, suggestive of both recovery and adaptation to low dose radiation. In protein-protein interaction (PPI) networks, YWHAZ (14-3-3ζ) emerged as the top-most hub protein that may direct phosphorylation driven pro-survival cellular processes against radiation stress. PPI networks also identified an integral role for the cytoskeletal protein ACTB, signaling protein PRKACA; and the molecular chaperone HSPA8. The data will allow better integration of radiation biology and epidemiology for risk assessment [Data are available via ProteomeXchange with identifier PXD022380].

Integrated transcriptomic and proteomic analysis of microplasts derived from macrophage-conditioned medium-treated MCF-7 breast cancer cells.

Microplasts are large extracellular vesicles originating from migratory, invasive, and metastatic cancer cells. Here, to gain insight into the role of microplasts in cancer progression, we performed a proteomic and transcriptomic characterization of microplasts isolated from MCF-7 breast cancer cells treated with macrophage-conditioned medium. These cells were found to be viable, highly migratory, and metabolically active, indicating that microplasts derived from these cells are not apoptotic bodies. Transcriptomic/proteomic analyses identified 10273 mRNAs and 821 proteins in microplasts. Interestingly, 377 microplast mRNAs coded for corresponding microplast proteins. Microplast mRNAs and proteins were mainly associated with binding and catalytic activities. Microplasts showed enrichment of mRNAs involved in transcription regulation and proteins involved in processes such as cell-cell adhesion and translation. Pathway analysis showed enrichment of ribosomes and carbon metabolism. These results suggest a close resemblance between microplasts and parent cells, with mRNA and protein cargo relevant in intercellular signaling.

Early antioxidant responses via the concerted activation of NF-κB and Nrf2 characterize the gamma-radiation-induced adaptive response in quiescent human peripheral blood mononuclear cells.

The radiation-induced adaptive response (RI-AR) is a non-targeted effect which is outside the scope of the classical Linear-No-Threshold (LNT) dose-response paradigm. However, the mechanisms of the RI-AR are not well understood. We have studied the RI-AR in quiescent human peripheral blood mononuclear cells (PBMCs). PBMCs in G0 phase were 'primed' with a low dose (100 mGy gamma radiation) and then, after an 'adaptive window' of 4 h, 'challenged' with a high dose (2 Gy). A small (5.7%) increase in viability and a decrease in DNA strand breaks were seen in primed cells, compared to non-primed cells. This was consistent with lower levels of reactive oxygen species, higher mitochondrial membrane potential, and increased activity of antioxidant enzymes such as catalase, superoxide dismutase, thioredoxin reductase, and glutathione peroxidase, in the primed cells. Reduced oxidative stress in primed PBMCs correlated with greater nuclear translocation of the redox-sensitive transcription factors Nuclear factor kappa B (NF-κB) and Nuclear factor E2-related factor 2 (Nrf2). Distinct differences in responses were seen in PBMCs irradiated with low dose (100 mGy) and high dose (2 Gy). These findings provide insight into the mechanisms of radioadaptation in human cells.

Simultaneous extraction of nuclear and mitochondrial DNA from human blood.

A method of simultaneous isolation of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) from human blood has been proposed by improvising Lahiri's method of isolation of nuclear DNA. The approach presented here provides selectively enriched fractions and eliminates the need for two different methods or separate reagent sets for the extraction of nDNA and mtDNA. It employs an initial nuclear/ cytoplasm partitioning, followed by the similar procedural steps for the two fractions separately. It gives good quality and quantity of the nDNA as well as the mtDNA, suitable for processes like PCR amplification and sequencing and may prove to be useful for people studying population genetics and evolution using molecular markers maximizing the available resources, especially in cases where a large database needs to be generated from limited amount of blood sample. From 3 ml of blood, the yields of mtDNA salvaged from the supernatant were sufficient to set approximately 4x10(5) reactions (starting with 250 fg DNA per reactions) of mtDNA loci which otherwise would have been discarded as per original Lahiri's procedure. The quality of mtDNA from the mitochondrial fraction was suitable for all major downstream processes as confirmed by locus specific PCR amplifications and sequencing. Through this procedure, the wastage of nDNA can be avoided when mtDNA loci is studied.

Dynamic changes in the proteome of human peripheral blood mononuclear cells with low dose ionizing radiation.

Humans are continually exposed to ionizing radiation from natural as well as anthropogenic sources. Though biological effects of high dose radiation exposures have been well accepted, studies on low-to-moderate dose exposures (in the range of 50-500 mGy) have been strongly debated even as researchers continue to search for elusive 'radiation signatures' in humans. Proteins are considered as dynamic functional players that drive cellular responses. However, there is little proteomic information available in context of human exposure to ionizing radiation. In this study, we determined differential expressed proteins in G0 peripheral blood mononuclear cells (PBMCs) from healthy individuals 1h and 4h after 'ex vivo' exposure with two radiation doses (300 mGy and 1 Gy). Twenty-three proteins were found to be significantly altered in irradiated cells when compared to sham irradiated cells with fold change ± 1.5-fold (p ≤ 0.05), with only three proteins showing ≥ 2.5-fold change, either with dose or with time. Mass spectrometry analyses identified redox sensor protein, chloride intracellular channel protein 1 (CLIC-1), the antioxidant protein, peroxiredoxin-6 and the pro-survival molecular chaperone 78 KDa glucose regulated protein (GRP78) among the 23 modulated proteins. The mean coefficient of variation (CV) for the twenty-three radiation responsive protein spots was found to be 33.7% for 300 mGy and 48.3% for 1 Gy. We thus, conclude that the radiation proteomic response of G0 human PBMCs, which are in the resting stage of the cell cycle, involves moderate upregulation of protective mechanisms, with low inter-individual variability. This study will help further our understanding of cellular effects of low dose acute radiation in humans and contribute toward differential biomarker discovery.

Molecular Understanding of Growth Inhibitory Effect from Irradiated to Bystander Tumor Cells in Mouse Fibrosarcoma Tumor Model.

Even though bystander effects pertaining to radiation risk assessment has been extensively studied, the molecular players of radiation induced bystander effect (RIBE) in the context of cancer radiotherapy are poorly known. In this regard, the present study is aimed to investigate the effect of irradiated tumor cells on the bystander counterparts in mouse fibrosarcoma (WEHI 164 cells) tumor model. Mice co-implanted with WEHI 164 cells γ-irradiated with a lethal dose of 15 Gy and unirradiated (bystander) WEHI 164 cells showed inhibited tumor growth, which was measured in terms of tumor volume and Luc+WEHI 164 cells based bioluminescence in vivo imaging. Histopathological analysis and other assays revealed decreased mitotic index, increased apoptosis and senescence in these tumor tissues. In addition, poor angiogenesis was observed in these tumor tissues, which was further confirmed by fluorescence imaging of tumor vascularisation and CD31 expression by immuno-histochemistry. Interestingly, the growth inhibitory bystander effect was exerted more prominently by soluble factors obtained from the irradiated tumor cells than the cellular fraction. Cytokine profiling of the supernatants obtained from the irradiated tumor cells showed increased levels of VEGF, Rantes, PDGF, GMCSF and IL-2 and decreased levels of IL-6 and SCF. Comparative proteomic analysis of the supernatants from the irradiated tumor cells showed differential expression of total 24 protein spots (21 up- and 3 down-regulated) when compared with the supernatant from the unirradiated control cells. The proteins which showed substantially higher level in the supernatant from the irradiated cells included diphosphate kinase B, heat shock cognate, annexin A1, angiopoietin-2, actin (cytoplasmic 1/2) and stress induced phosphoprotein 1. However, the levels of proteins like annexin A2, protein S100 A4 and cofilin was found to be lower in this supernatant. In conclusion, our results provided deeper insight about the damaging RIBE in an in vivo tumor model, which may have significant implication in improvement of cancer radiotherapy.

Role of ATM in bystander signaling between human monocytes and lung adenocarcinoma cells.

The response of a cell or tissue to ionizing radiation is mediated by direct damage to cellular components and indirect damage mediated by radiolysis of water. Radiation affects both irradiated cells and the surrounding cells and tissues. The radiation-induced bystander effect is defined by the presence of biological effects in cells that were not themselves in the field of irradiation. To establish the contribution of the bystander effect in the survival of the neighboring cells, lung carcinoma A549 cells were exposed to gamma-irradiation, 2Gy. The medium from the irradiated cells was transferred to non-irradiated A549 cells. Irradiated A549 cells as well as non-irradiated A549 cells cultured in the presence of medium from irradiated cells showed decrease in survival and increase in γ-H2AX and p-ATM foci, indicating a bystander effect. Bystander signaling was also observed between different cell types. Phorbol-12-myristate-13-acetate (PMA)-stimulated and gamma-irradiated U937 (human monocyte) cells induced a bystander response in non-irradiated A549 (lung carcinoma) cells as shown by decreased survival and increased γ-H2AX and p-ATM foci. Non-stimulated and/or irradiated U937 cells did not induce such effects in non-irradiated A549 cells. Since ATM protein was activated in irradiated cells as well as bystander cells, it was of interest to understand its role in bystander effect. Suppression of ATM with siRNA in A549 cells completely inhibited bystander effect in bystander A549 cells. On the other hand suppression of ATM with siRNA in PMA stimulated U937 cells caused only a partial inhibition of bystander effect in bystander A549 cells. These results indicate that apart from ATM, some additional factor may be involved in bystander effect between different cell types.

Effect of proton and gamma irradiation on human lung carcinoma cells: Gene expression, cell cycle, cell death, epithelial-mesenchymal transition and cancer-stem cell trait as biological end points.

Proton beam therapy is a cutting edge modality over conventional gamma radiotherapy because of its physical dose deposition advantage. However, not much is known about its biological effects vis-a-vis gamma irradiation. Here we investigated the effect of proton- and gamma- irradiation on cell cycle, death, epithelial-mesenchymal transition (EMT) and "stemness" in human non-small cell lung carcinoma cells (A549). Proton beam (3MeV) was two times more cytotoxic than gamma radiation and induced higher and longer cell cycle arrest. At equivalent doses, numbers of genes responsive to proton irradiation were ten times higher than those responsive to gamma irradiation. At equitoxic doses, the proton-irradiated cells had reduced cell adhesion and migration ability as compared to the gamma-irradiated cells. It was also more effective in reducing population of Cancer Stem Cell (CSC) like cells as revealed by aldehyde dehydrogenase activity and surface phenotyping by CD44(+), a CSC marker. These results can have significant implications for proton therapy in the context of suppression of molecular and cellular processes that are fundamental to tumor expansion.

Characterization of TP53 microsatellite locus among selected ethnic populations of India.

POPULATION: A total of 253 individuals belonging to five ethnic populations of India were analyzed for pentanucleotide microsatellite TP53. These included Konkanasthas and Marathas (from Maharashtra, western India) representing Indo-Aryan lineage and Ezhavas, Nairs and Muslims (from Kerala, southwest India) representing Indo-Dravidian lineage. To the best of our knowledge, allele frequency data at TP53 microsatellite locus exists only for German Caucasians (1,2), Northern Portuguese (3) and West African from S. Tomé e Príncipe (4); the present study is the first report on Asian populations.

STR polymorphism among two tribal populations of India.

Two tribal populations of India, Bison Horn Maria and Muria from Bastar district of Madhya Pradesh in Central India were studied for DNA polymorphisms at tetranucleotide short tandem repeat (STR) loci (F13A01 and HUMvWA). A total of 63 random adult individuals for F13A01 locus and 53 samples for HUMvWA were analyzed in the present study.