Lymphocyte Cell Lines Research Articles

Improving Proteoform Identifications in Complex Systems Through Integration of Bottom-Up and Top-Down Data.

Cellular functions are performed by a vast and diverse set of proteoforms. Proteoforms are the specific forms of proteins produced as a result of genetic variations, RNA splicing, and post-translational modifications (PTMs). Top-down mass spectrometric analysis of intact proteins enables proteoform identification, including proteoforms derived from sequence cleavage events or harboring multiple PTMs. In contrast, bottom-up proteomics identifies peptides, which necessitates protein inference and does not yield proteoform identifications. We seek here to exploit the synergies between these two data types to improve the quality and depth of the overall proteomic analysis. To this end, we automated the large-scale integration of results from multiprotease bottom-up and top-down analyses in the software program Proteoform Suite and applied it to the analysis of proteoforms from the human Jurkat T lymphocyte cell line. We implemented the recently developed proteoform-level classification scheme for top-down tandem mass spectrometry (MS/MS) identifications in Proteoform Suite, which enables users to observe the level and type of ambiguity for each proteoform identification, including which of the ambiguous proteoform identifications are supported by bottom-up-level evidence. We used Proteoform Suite to find instances where top-down identifications aid in protein inference from bottom-up analysis and conversely where bottom-up peptide identifications aid in proteoform PTM localization. We also show the use of bottom-up data to infer proteoform candidates potentially present in the sample, allowing confirmation of such proteoform candidates by intact-mass analysis of MS1 spectra. The implementation of these capabilities in the freely available software program Proteoform Suite enables users to integrate large-scale top-down and bottom-up data sets and to utilize the synergies between them to improve and extend the proteomic analysis.

A carotenoid-enriched extract from pumpkin delays cell proliferation in a human chronic lymphocytic leukemia cell line through the modulation of autophagic flux

Abstract Chronic lymphocytic leukemia (CLL) is the most frequent form of leukemia in the adult population. From an asymptomatic state, which can lay for years, CLL rapidly evolves becoming fatal. Apoptosis resistance and induction of a protective form of autophagy are possible explanations of the poor responsivity of CLL to conventional and innovative therapeutic drugs. Carotenoids, including α- and β-carotene, lycopene, and derivatives, such as retinoic acid, have been studied for their significant antiproliferative and differentiating activity in cancer. Here, we report that a carotenoid-enriched extract (CE) obtained from pumpkin by supercritical CO2 extraction showed an anti-proliferative effect on HG3 cell line derived from human B-CLL cells. CE induced a 40% delay in cell proliferation compared to untreated cells, without signs of cytotoxicity. This delay was associated with p27Kip1 over-expression, AMPK activation and modulation of autophagy flux. In HG3 cells treated with CE, we detected a 30% autophagosome intracellular increase and changes in the expression of canonical biochemical markers of autophagy (LC3-II, p62). These results suggest the presence in the CE of a pool of bioactive carotenoids acting additively or synergistically in retarding cancer cell growth.

Antibody-induced internalisation of retroviral envelope glycoproteins is a signal initiation event.

As obligate parasites, viruses highjack, modify and repurpose the cellular machinery for their own replication. Viral proteins have, therefore, evolved biological functions, such as signalling potential, that alter host cell physiology in ways that are still incompletely understood. Retroviral envelope glycoproteins interact with several host proteins, extracellularly with their cellular receptor and anti-envelope antibodies, and intracellularly with proteins of the cytoskeleton or sorting, endocytosis and recirculation pathways. Here, we examined the impact of endogenous retroviral envelope glycoprotein expression and interaction with host proteins, particularly antibodies, on the cell, independently of retroviral infection. We found that in the commonly used C57BL/6 substrains of mice, where murine leukaemia virus (MLV) envelope glycoproteins are expressed by several endogenous MLV proviruses, the highest expressed MLV envelope glycoprotein is under the control of an immune-responsive cellular promoter, thus linking MLV envelope glycoprotein expression with immune activation. We further showed that antibody ligation induces extensive internalisation from the plasma membrane into endocytic compartments of MLV envelope glycoproteins, which are not normally subject to constitutive endocytosis. Importantly, antibody binding and internalisation of MLV envelope glycoproteins initiates signalling cascades in envelope-expressing murine lymphocytic cell lines, leading to cellular activation. Similar effects were observed by MLV envelope glycoprotein ligation by its cellular receptor mCAT-1, and by overexpression in human lymphocytic cells, where it required an intact tyrosine-based YXXΦ motif in the envelope glycoprotein cytoplasmic tail. Together, these results suggest that signalling potential is a general property of retroviral envelope glycoproteins and, therefore, a target for intervention.

An RNA-Directed Gene Editing Strategy for Attenuating the Infectious Potential of Feline Immunodeficiency Virus-Infected Cells: A Proof of Concept

Modern antiretroviral therapy for immunodeficiency viruses, although remarkably effective in controlling viral transcription, and overt virus-associated morbidity, has failed to absolutely eradicate retroviruses from their infected hosts as a result of proviral integration in long-lived reservoir cells. Immunodeficiency virus-infected patients are therefore consigned to lifelong antiviral therapy as a means to control viremia, viral transmission, and infection-associated morbidity. Unfortunately, lifelong antiviral therapies can be difficult for patients to continuously maintain and may be associated with therapy-specific morbidities. Patient advocates have argued for new methods to achieve retroviral eradication. As a proof-of-concept study, a lentivirus-delivered RNA-directed gene editing strategy was utilized in a series of in vitro experiments in an attempt to attenuate the feline immunodeficiency virus (FIV) proviral load, viral transcription, and production of infectious virions. We found that a feline T lymphocyte cell line (MCH5-4) treated with an FIV-specific clustered regularly interspersed short palindromic repeats (CRISPR)-associated protein 9 (Cas9) gene editing tool resulted in a reduction of cell-free viral RNA relative to control cells. Decreased infectious potential was demonstrated in a two-step FIV infection study—naïve MCH5-4 cells infected with cell-free FIV harvested from FIV-infected and CRISPR lentivirus-treated cells had less integrated proviral DNA than control cells. This study represents the initial steps towards the development of an effective method of proviral eradication in an immunodeficiency virus-infected host.

Histone Deacetylase Inhibitors Enhance CRISPR‐Cas9 Cutting Efficiency

The CRISPR/Cas9 technology is a prominent genome‐editing tool as an adaptable nuclease capable of producing a double strand break at almost any genomic loci. However, the modification of hematopoietic stem and progenitor cells (HSPC) via the homology directed repair (HDR) pathway is still inefficient. We hypothesize that histone deacetylase inhibitors (HDACi), such as Valproic Acid (VPA) and Sodium Butyrate (NaB), could enhance HDR efficiency by increasing the accessibility of the genome‐editing machinery. To address the potential utilization of HDACi therapeutically, we began by assessing the effect of VPA and NaB on cell growth and viability. No statistically significant effect on cell growth or viability was observed at concentrations as high as 50mM in a myeloid‐erythroid leukemic cell line (K562 cells). However, K562 cells only represent one lineage of the HSPC progeny. Therefore, we also treated an immortalized T lymphocyte cell line (Jurkat cells), representative of the lymphoid lineage, with either VPA or NaB. No effect on the growth pattern or viability of Jurkat cells was evident at concentrations as high as 5mM VPA and NaB. However, Jurkat cells did appear to be more susceptible to HDACi. Therefore, we ascertained that concentrations of HDACi lower than 5mM would be tolerated in HSPCs. At concentrations as low as 0.0005mM HDACi an enhancement in CRISPR cutting efficiency was evidenced as assessed in both K562 cells and Jurkat cells. This enhancement did not appear to be locus specific, in that two sets of CRISPR guide RNAs were analyzed revealing similar trends of enhancement. If the same trend follows, treatment with HDACi can be expanded to virtually any genome editing experiment utilizing the CRISPR‐Cas9 system to enhance cutting and thus therapeutic gene editing.Support or Funding InformationCSUPERBEffect of Valproic Acid and Butyrate on the Growth Curve of Hematopoietic Cell Lines A) Schematic representation of the experimental design utilized to test the effect of HDACi on the division of two hematopoietic cell lines. B) K562 cells treated with 300mM and 100mM VPA were not observed to divide. C) K562 cells treated with 500mM NaB were not observed to divide. D) Jurkat cells appear to be more susceptible to VPA. E) Jurkat cells treated with 50mM NaB proliferated at a much slower rate.Figure 1

Connecting the microbiome to host metabolites: understanding how the microbiome controls immune activity in birds

The gastrointestinal (GI) microbiome plays an important role in the development and function not only of the GI, but also the immune system. Products such as probiotics represent promising treatments for improving animal health through the GI, but their modes of action are largely still unknown. Previous research in our laboratory has demonstrated supplementation with a lactic acid bacteria probiotic product alters host energy partitioning in immune tissues, including increased ATP production and consumption in circulating leukocytes; and was associated with a more rapid antibody response to antigen. To better understand the communication between probiotics and the immune system our laboratory has focused on characterizing how supplementation affects the microbiome and host systems. We hypothesized that the changes previously observed in immune tissue activity and energy metabolism were regulated by a probiotic‐stimulated factor in the serum. We examined the ability of serum isolated from probiotic‐fed animals to augment ATP production in vitro. Chicken lymphocyte cell lines were cultured for 4 days in media supplemented with serum from treated and control birds. Cells cultured in serum from probiotic‐fed birds had higher levels of ATP (P < 0.05) compared to controls. Transcriptomic analysis of these cells suggest an increase in genes associated with cell survival and differentiation, and signaling via TGF‐β, IFN‐γ, IL‐7, and IL‐1β. Analysis of the metabolites in serum and digesta from these animals identified several metabolite changes correlate with the change in ATP levels in lymphocytes and putative changes in pro‐ and anti‐inflammatory cytokine production.

EVALUATING THE ASSOCIATION BETWEEN MITOCHONDRIAL DYSFUNCTION AND COGNITIVE DECLINE IN ALZHEIMER'S DISEASE

Introduction Alzheimer's Disease (AD) is the 6th leading cause of death in the United States, with the current number of 5.8?million affected individuals anticipated to increase to 13.8?million by 2050 (Alzheimer's Association, 2019). As the number of older adults living with Alzheimer's rapidly increases, research continues to probe the complexities of the disease's progression. AD was originally identified by the presence of amyloid plaques and neurofibrillary tangles in brain, and most research has focused on these elements. However, targeting amyloid cascade and tau phosphorylation has not resulted in the development of drugs that have halted or reversed the cognitive decline associated with AD (Du, Wang, & Geng, 2018). AD has multiple determinants, and, in most cases, plaques and tangles may not be secondary to other underlying processes. The focus of our research is investigating the role of cellular energy dysfunction in the etiology and progression of AD. Preliminary findings from our group, Sonntag and colleagues (2017), revealed changes in energy metabolism (or bioenergetics) in the fibroblasts of 10 patients with late-onset Alzheimer's Disease, the most common form of AD, compared to 20 age-matched controls, with implications of impaired mitochondrial metabolic potential as a possible contributor to the pathophysiology of AD. The present study builds on the prior work of Sonntag et al, aiming to confirm previous findings with additional samples of fibroblasts and lymphocytes from older adults with mild to moderate Alzheimer's Disease. The cells gathered are being converted to brain cells, neurons and glia, to determine whether the abnormalities seen in peripheral lines are also seen in the brain. In collaboration with the Geriatric Psychiatry Research Program at McLean, data obtained from the collected samples will be analyzed alongside the results from brain imaging data (specifically, MRI and amyloid PET scan) and clinical measures of the participants as supporting evidence in the link between mitochondrial dysfunction and cognitive decline in AD. Methods This study aims to enroll a total of 40 subjects (ages 65 to 89): 20 control subjects and 20 subjects diagnosed with mild to moderate Alzheimer's Disease. Enrolled subjects will undergo the following scales to assess for comorbid psychiatric disorders and their severity: Montgomery-Asberg Depression Rating Scale (MADRS),Young Mania Rating Scale (YMRS), Geriatric Depression Scale (GDS) and the Mini-International Neuropsychiatric Interview (MINI). The Montreal Cognitive Assessment (MOCA) is also performed to determine study suitability. Once inclusion criteria are met for both populations, samples of skin and blood will be collected and subsequently processed to grow cell lines of fibroblasts and lymphocytes. These cells are reprogrammed to induced pluripotent stem cell lines (iPSC) and then to neuronal and glial lines. Results Though ongoing, we anticipate similar results to Sonntag et al. (2017), as genetic determinants are shared among peripheral and CNS cells. In culture, both genetic and exogenous risk factors can be studied. As a secondary result, a correlation is anticipated between the bioenergetic abnormalities observed in the cells and in the brain imaging data and clinical measures of the participants. These findings may support the association between mitochondrial dysfunction and the cognitive decline in Alzheimer's Disease among a larger sample size. Conclusions Identifying the mitochondrial dysfunction in Alzheimer's Disease not only contributes a new explanation for its complex pathophysiology, complementary to other hypotheses, but provides a new direction for the development of novel treatments to effectively target the devastating effects of cognitive decline in AD. This research was funded by: This study is funded by the Program for Neuropsychiatric Research at McLean Hospital

Ruxolitinib Regulates the Autophagy Machinery in Multiple Myeloma Cells.

Ruxolitinib is a selective JAK1/2 inhibitor approved by the FDA for myelofibrosis in 2014 and nowadays, comprehensive investigations on the potential of the agent as a targeted therapy for haematological malignancies are on the rise. In multiple myeloma which is a cancer of plasma cells, the Interleukin- 6/JAK/STAT pathway is emerging as a therapeutic target since the overactivation of the pathway is associated with poor prognosis. In this study, our purpose was to discover the potential anticancer effects of ruxolitinib in ARH-77 multiple myeloma cell line compared to NCI-BL 2171 human healthy B lymphocyte cell line. Cytotoxic effects of ruxolitinib in ARH-77 and NCI-BL 2171 cells were determined via WST-1 assay. The autophagy mechanism induced by ruxolitinib measured by detecting autophagosome formation was investigated. Apoptotic effects of ruxolitinib were analyzed with Annexin V-FITC Detection Kit and flow cytometry. We performed RT-qPCR to demonstrate the expression changes of the genes in the IL-6/JAK/STAT pathway in ARH-77 and NCI-BL 2171 cells treated with ruxolitinib. We identified the IC50 values of ruxolitinib for ARH-77 and NCI-BL 2171 as 20.03 and 33.9μM at the 72nd hour, respectively. We showed that ruxolitinib induced autophagosome accumulation by 3.45 and 1.70 folds in ARH-77 and NCI-BL 2171 cells compared to the control group, respectively. Treatment with ruxolitinib decreased the expressions of IL-6, IL-18, JAK2, TYK2, and AKT genes, which play significant roles in MM pathogenesis. All in all, ruxolitinib is a promising agent for the regulation of the IL-6/JAK/STAT pathway and interferes with the autophagy mechanism in MM.

P136 EPSTEIN-BARR VIRUS TRANSCRIPTION CO-FACTORS BIND TO MANY INFLAMMATORY BOWEL DISEASE RISK LOCI

Abstract Objective IBD is a chronic inflammatory disorder of the GI tract with complex etiology that involves both genetic variants and environmental factors. Several viruses and bacteria have been suggested as potential causes of the disease. The clinical course of IBD appears to be influenced by EBV tissue infection. With a prevalence of >90% in the adult human population, EBV infection is nearly ubiquitous. We reported that the EBV transcription co-factor EBNA2 was concentrated in the risk loci of IBD relative to the remainder of the genome (Pc=1.24E-13, (RR) = 3.33, Nature Genetics 50:699, 2018). The objective of this study was to determine whether new data and studies made available since the data used for our 2018 study further supported the possibility that EBV was related to some IBD as a potential etiologic agent. Methods Genomic approaches offer previously unavailable perspectives for understanding disease mechanisms. The previous analysis was based upon the 9 viral transcription factor and co-factor (TF) datasets available in human cells in 2015.Of the 52 now available there are 29 from EBV. The number of established IBD loci qualifying for analysis has expanded from 112 in 2015 to 324 available now. We applied simulation analysis to assess statistical significance of TF associations with IBD risk loci using RELI (Regulatory Element Locus Intersection) (Nat Genet 50:699, 2018). RELI tests the probability of the observed association by the count of intersections of the same variant from two sources. We extracted 700 SNPs in European ancestry from 93 published GWAS and 350 additional candidate gene studies for IBD. All variants with linkage disequilibrium r2>0.8 of the best variant were included, while loci with r2>0.2 with a more highly significant locus were excluded. Results Among the 52 viral TF datasets, 139 of the 324 IBD loci were occupied by EBNALP (RR=2.08793, Pc=3.7321E-23), 113 by EBNA3C (RR=2.35113, Pc=2.84678E-22), 88 loci by EBNA2 (RR=3.2, Pc=7.8E-32) and 28 loci by EBNA3ABC, which used an antibody that did not distinguish between EBNA3 subtypes. Analysis of 11,535 human TF ChIP-seq datasets produced 1578 with strong associations (p<E-6) with IBD risk loci. The locus intersections of the three EBV gene products optimally clustered at 150 IBD loci (46%) with a set of 26 human TFs (p<2E-308), thereby nominating these loci as potentially having EBV-dependent mechanisms that alter genetic risk for IBD. The human TFs were enriched for (ChIP-seq) data obtained from EBV-infected B lymphocyte cell line (OR = 10.12425329, Chi-squared = 241.2925605) Moreover, the human TFs that participate in the super-enhancers that form in the latency III EBV infected and transformed B cell are also concentrated among the statistically significant associations (p<E-6) with the IBD risk loci (OR) = 2.9448, P = 10E-7). Conclusion These preliminary findings nominate EBV for a role in the pathogenesis and etiology of IBD by mechanisms operating in transformed B cells through the latency III EBV program of viral gene expression.

Analytical Validation of Telomere Analysis Technology\xae for the High-Throughput Analysis of Multiple Telomere-Associated Variables

BackgroundA large number of studies have suggested a correlation between the status of telomeres and disease risk. High-throughput quantitative fluorescence in situ hybridization (HT Q-FISH) is a highly accurate telomere measurement technique that can be applied to the study of large cell populations. Here we describe the analytical performance testing and validation of Telomere Analysis Technology (TAT®), a laboratory-developed HT Q-FISH-based methodology that includes HT imaging and software workflows that provide a highly detailed view of telomere populations.MethodsTAT was developed for the analysis of telomeres in peripheral blood mononuclear cells (PBMCs). TAT was compared with Terminal Restriction Fragment (TRF) length analysis, and tested for accuracy, precision, limits of detection (LOD) and specificity, reportable range and reference range.ResultsUsing 6 different lymphocyte cell lines, we found a high correlation between TAT and TRF for telomere length (R2 ≥ 0.99). The standard variation (assay error) of TAT was 454 base pairs, and the limit of detection of 800 base pairs. A standard curve was constructed to cover human median reportable range values and defined its lower limit at 4700 bp and upper limits at 14,400 bp. Using TAT, up to 223 telomere associated variables (TAVs) can be obtained from a single sample. A pilot, population study, of telomere analysis using TAT revealed high accuracy and reliability of the methodology.ConclusionsAnalytical validation of TAT shows that is a robust and reliable technique for the characterization of a detailed telomere profile in large cell populations. The combination of high-throughput imaging and software workflows allows for the collection of a large number of telomere-associated variables from each sample, which can then be used in epidemiological and clinical studies.

Non-accidental structural chromosome aberrations in established monkey B-lymphocyte cell lines

Established primate lymphocyte cell lines obtained from tumour samples and from EBV-positive monkeys served us as the model system for studying the role of genetic factors and chromosomal abnormalities in malignization. The investigation of chromosome regions and genes involved in chromosomal aberrations leading to malignization in these lines was the aim of our work. Cytogenetic analysis was performed at different stages of cultivation in vitro. To determine the oncogenes and tumour suppressor genes located on aberrant chromosomes, data on mapping rhesus macaque genes, and high similarity of human and monkey karyotypes were used. We found that, in the line obtained from lymphomatous baboon tissue, the inactivation of tumour suppressor gene RB1 on chromosome 17 after chromosomal rearrangement is one of the most probable causes of in vivo malignization. Chromosomal aberrations at the region of oncogene c-Ki-ras and tumour suppressor gene TP53 change the proliferative status and differentiation in established cell lines obtained from healthy but EBV-seropositive primates. The other cause of malignization in these lines is an increase in expression of the oncogene c-myc caused by trisomy of chromosome 8 where c-myc is located. Structural aberrations in established primate cell lines affecting several chromosomal loci were identified as: (1) causing the proto-oncogene activation – the central event in the tumour clone occurrence, and (2) deactivating tumour suppressor genes. The change in the chromosome number leads to increase in oncogenic products and to damage of regulatory functions associated with cell proliferation.

NAD-linked mechanisms of gene de-repression and a novel role for CtBP in persistent adenovirus infection of lymphocytes

BackgroundAdenovirus (AdV) infection is ubiquitous in the human population and causes acute infection in the respiratory and gastrointestinal tracts. In addition to lytic infections in epithelial cells, AdV can persist in a latent form in mucosal lymphocytes, and nearly 80% of children contain viral DNA in the lymphocytes of their tonsils and adenoids. Reactivation of latent AdV is thought to be the source of deadly viremia in pediatric transplant patients. Adenovirus latency and reactivation in lymphocytes is not well studied, though immune cell activation has been reported to promote productive infection from latency. Lymphocyte activation induces global changes in cellular gene expression along with robust changes in metabolic state. The ratio of free cytosolic NAD+/NADH can impact gene expression via modulation of transcriptional repressor complexes. The NAD-dependent transcriptional co-repressor C-terminal Binding Protein (CtBP) was discovered 25 years ago due to its high affinity binding to AdV E1A proteins, however, the role of this interaction in the viral life cycle remains unclear.MethodsThe dynamics of persistently- and lytically-infected cells are evaluated. RT-qPCR is used to evaluate AdV gene expression following lymphocyte activation, treatment with nicotinamide, or disruption of CtBP-E1A binding.ResultsPMA and ionomycin stimulation shifts the NAD+/NADH ratio in lymphocytic cell lines and upregulates viral gene expression. Direct modulation of NAD+/NADH by nicotinamide treatment also upregulates early and late viral transcripts in persistently-infected cells. We found differential expression of the NAD-dependent CtBP protein homologs between lymphocytes and epithelial cells, and inhibition of CtBP complexes upregulates AdV E1A expression in T lymphocyte cell lines but not in lytically-infected epithelial cells.ConclusionsOur data provide novel insight into factors that can regulate AdV infections in activated human lymphocytes and reveal that modulation of cellular NAD+/NADH can de-repress adenovirus gene expression in persistently-infected lymphocytes. In contrast, disrupting the NAD-dependent CtBP repressor complex interaction with PxDLS-containing binding partners paradoxically alters AdV gene expression. Our findings also indicate that CtBP activities on viral gene expression may be distinct from those occurring upon metabolic alterations in cellular NAD+/NADH ratios or those occurring after lymphocyte activation.

Long non-coding RNA GAS5 regulates human B lymphocytic leukaemia tumourigenesis and metastasis by sponging miR-222.

Accumulating evidence has shown that lncRNA GAS5 is a novel tumour-promoting RNA that contributes to tumour progression by sponging miRNAs. However, the detailed role of lncRNA GAS5 in B lymphocytic leukaemia is still unclear. A qRT-PCR assay was used to examine the levels of lncRNA GAS5 and miR-222 in leukomonocytes of patients with B lymphocytic leukaemia and in healthy donors. Raji cells were transfected with GAS5 overexpression or shRNA-GAS5 plasmids for 48⁢h, and cell proliferation was assessed by the CCK-8 assay, while apoptosis and cell cycle progression were assessed using flow cytometry. The Transwell assay was applied to detect the invasion of Raji cells with GAS5 overexpression or knockdown. The dual luciferase reporter assay and regression curve were conducted to evaluate the binding interaction between lncRNA GAS5 and miR-222. The results showed that the expression of lncRNA GAS5 was decreased in B lymphocytic leukaemia patients compared with the healthy group, and the levels of lncRNA GAS5 in B lymphocytic leukaemia cell lines were significantly higher than those in the normal B cell line, whereas the levels of miR-222 were increased in B lymphocytic leukaemia patients compared with the healthy group. Moreover, cell culture experiments indicated that lncRNA GAS5 overexpression decreased B lymphocytic leukaemia cell proliferation, promoted B lymphocytic leukaemia cell apoptosis, arrested B lymphocytic leukaemia cells in the G1 phase of the cell cycle, and inhibited B lymphocytic leukaemia cell invasion. Finally, the luciferase reporter assay showed a direct target interaction between lncRNA GAS5 and miR-222. The regression analysis showed a negative correlation between the levels of lncRNA GAS5 and miR-222. Thus, our data suggested that lncRNA GAS5 could effectively sponge miR-222 to modulate human B lymphocytic leukaemia cell tumourigenesis and metastasis. This work advances our understanding of the clinical significance of lncRNA GAS5 from the perspective of lncRNA-miRNA regulation.

Elevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis.

Phosphorylated histone 2AX (γH2AX) is a long-standing marker for DNA double-strand breaks (DSBs) from ionizing radiation in the field of radiobiology. This led to the perception of γH2AX being a general marker of direct DNA damage with the treatment of other agents such as low-dose exogenous ROS that unlikely act on cellular DNA directly. Cold physical plasma confers biomedical effects majorly via release of reactive oxygen and nitrogen species (ROS). In vitro, increase of γH2AX has often been observed with plasma treatment, leading to the conclusion that DNA damage is a direct consequence of plasma exposure. However, increase in γH2AX also occurs during apoptosis, which is often observed with plasma treatment as well. Moreover, it must be questioned if plasma-derived ROS can reach into the nucleus and still be reactive enough to damage DNA directly. We investigated γH2AX induction in a lymphocyte cell line upon ROS exposure (plasma, hydrogen peroxide, or hypochlorous acid) or UV-B light. Cytotoxicity and γH2AX induction was abrogated by the use of antioxidants with all types of ROS treatment but not UV radiation. H2AX phosphorylation levels were overall independent of analyzing either all nucleated cells or segmenting γH2AX phosphorylation for each cell cycle phase. SB202190 (p38-MAPK inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) significantly inhibited γH2AX induction upon ROS but not UV treatment. Finally, and despite γH2AX induction, UV but not plasma treatment led to significantly increased micronucleus formation, which is a functional read-out of genotoxic DNA DSBs. We conclude that plasma-mediated and low-ROS γH2AX induction depends on caspase activation and hence is not the cause but consequence of apoptosis induction. Moreover, we could not identify lasting mutagenic effects with plasma treatment despite phosphorylation of H2AX.

Reduction of integrin alpha 4 activity through splice modulating antisense oligonucleotides

With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

Constructing Human Proteoform Families Using Intact-Mass and Top-Down Proteomics with a Multi-Protease Global Post-Translational Modification Discovery Database

Complex human biomolecular processes are made possible by the diversity of human proteoforms. Constructing proteoform families, groups of proteoforms derived from the same gene, is one way to represent this diversity. Comprehensive, high-confidence identification of human proteoforms remains a central challenge in mass spectrometry-based proteomics. We have previously reported a strategy for proteoform identification using intact-mass measurements, and we have since improved that strategy by mass calibration based on search results, the use of a global post-translational modification discovery database, and the integration of top-down proteomics results with intact-mass analysis. In the present study, we combine these strategies for enhanced proteoform identification in total cell lysate from the Jurkat human T lymphocyte cell line. We collected, processed, and integrated three types of proteomics data (NeuCode-labeled intact-mass, label-free top-down, and multi-protease bottom-up) to maximize the number of confident proteoform identifications. The integrated analysis revealed 5950 unique experimentally observed proteoforms, which were assembled into 848 proteoform families. Twenty percent of the observed proteoforms were confidently identified at a 3.9% false discovery rate, representing 1207 unique proteoforms derived from 484 genes.

Cytotoxic Effects of Titanium Dioxide Nanaoparticles Synthesized by Laser Technique on Peripheral Blood Lymphocytes and Hep-2 Cell Line

This study assay the toxic effects of titanium dioxide nanoparticles (TiO2NPs) on peripheral blood lymphocytes and Hep-2 cell by measuring Mitotic Index (MI), Blastogenic Index (BI), Total Chromosomal Aberration (TCA) and growth rate of cancer cells. TiO2NPs were synthesized using laser technique by ablation of titanium target immersed in distilled de-ionized water (DDW) using Q-switched pulsed Nd:YAG laser with repetition rate of 1 Hz, wavelength of 1064 nm, 300 pulses and energy of 400 mJ. Chromosomal analyses were carried out at different concentrations of synthesized TiO2 NPs (0.0, 45, 90, and 180) µg/mL. X-ray diffraction (XRD) exhibited anatase phase of prepared TiO2NPs and showed that the crystalline structures of TiO2NPs is tetragonal. Absorption spectrum of TiO2 NPs was at ultraviolet-region 215 nm due to Surface Plasmon Resonance (SPR), and the particles size distribution of the prepared TiO2NPs ranged from 40 to 110 nm. Finding showed that both MI and BI suffered from high reduction while TCA suffered from significant elevation represented by Chromatid Breaks (CB), Micronuclei (MN) and Ring Chromosome (RC). The highest proportion of (TCA) has been observed in 180 µg/mL of TiO2NPs, (significant at p ≤ 0.05). while the toxic effects of prepared TiO2NPs on Hep-2 cell line represent decline in growth rate with an increase in concentration of TiO2NPs with malformation in the cell structure and shape and placement of the nucleus. The ablation via Q-switched Nd: YAG laser has adequacy to prepare TiO2NPs with 215 nm absorption spectrum, nearly spherical particles and suitable particle size (40–110 nm). In vitro assay showed that prepared TiO2 NPs have significant cytotoxic effects on peripheral blood lymphocyte and Hep-2 cell line by reduction in both Mitotic index (MI) and blastogenic index (BI) and growth rate of cancer cells.

MTORC1/2 Inhibitor Served as a More Ideal Agent Against the Growth of Mouse Lymphocytic Leukemia Both In Vitro and In Vivo.

Chronic lymphocytic leukemia (CLL) still remains an incurable disease as the cells evade apoptosis, which is an obstacle for current therapeutic approaches. Therefore, our aim was to identify an ideal target of leukemic cell growth for developing inhibitors. Mouse lymphocytic leukemia cell line L1210, human Toledo cells and a DBA/2 mouse graft model were used to analyze the activity of dual mTORC1/2 inhibitor AZD2014s. Western blotting and flow cytometry were performed to determine the mechanism. AZD2014 inhibited L1210 and human Toledo cell proliferation. Treatment with AZD2014 reduced the phosphorylation levels of S6K1 and 4EBP1 and the protein levels of Rictor, a component of the mTORC2 pathway. AZD2014 induced cell cycle arrest at the G0-G1 phase by reducing the expression of cyclin D1 and CDK4. Oral administration of AZD2014 significantly inhibited the growth of L1210 cell grafts in DBA/2 mice. The mTORC1/2 inhibitor may be a better therapeutic agent compared to PI3K/mTORC1 inhibitors for treating patients with CLL.

Chitosan gold nanoparticles induce different ROS-dependent cell death modalities in leukemic cells.

BackgroundNanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells.Materials and methodsWe synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism.ResultsWe found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism.ConclusionCH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.

Transcriptome-wide association study of multiple myeloma identifies candidate susceptibility genes

BackgroundWhile genome-wide association studies (GWAS) of multiple myeloma (MM) have identified variants at 23 regions influencing risk, the genes underlying these associations are largely unknown. To identify candidate causal genes at these regions and search for novel risk regions, we performed a multi-tissue transcriptome-wide association study (TWAS).ResultsGWAS data on 7319 MM cases and 234,385 controls was integrated with Genotype-Tissue Expression Project (GTEx) data assayed in 48 tissues (sample sizes, N = 80–491), including lymphocyte cell lines and whole blood, to predict gene expression. We identified 108 genes at 13 independent regions associated with MM risk, all of which were in 1 Mb of known MM GWAS risk variants. Of these, 94 genes, located in eight regions, had not previously been considered as a candidate gene for that locus.ConclusionsOur findings highlight the value of leveraging expression data from multiple tissues to identify candidate genes responsible for GWAS associations which provide insight into MM tumorigenesis. Among the genes identified, a number have plausible roles in MM biology, notably APOBEC3C, APOBEC3H, APOBEC3D, APOBEC3F, APOBEC3G, or have been previously implicated in other malignancies. The genes identified in this TWAS can be explored for follow-up and validation to further understand their role in MM biology.