Gene Expression Changes In Cells Research Articles

The FITNESS study: longitudinal geriatric assessment, treatment toxicity, and biospecimen collection to assess functional disability among older adults with lung cancer.

Older adults with chronic disease prioritize functional independence. We aimed to describe the feasibility of capturing functional disability and treatment toxicity among older adults with lung cancer using a longitudinal comprehensive geriatric assessment (CGA) and molecular biomarkers of aging. This prospective study included adults ≥60years with any newly diagnosed non-small-cell lung cancer. Participants were recruited from central Ohio (2018-2020). Study assessments included the Cancer and Aging Research Group CGA (CARG-CGA), short physical performance battery (SPPB), and the blessed orientation-memory concentration (BOMC) test at baseline, 3, 6, and 12months. Activities of daily living (ADLs) and instrumental ADLs (IADLs), quality of life (QoL, PROMIS 10), and treatment toxicity were captured monthly. Stool and blood were collected to characterize the gut microbiome and age-related blood biomarkers. This study enrolled 50 participants with an average age of 71.7years. Ninety-two percent of participants were Caucasian, 58% were male, and all were non-Hispanic. Most had advanced stage (stage III/IV: 90%; stage I/II: 10%), with adenocarcinoma the predominant histologic subtype (68% vs. 24% squamous). First-line treatments included chemotherapy (44%), immune checkpoint inhibitors (ICIs, 22%), chemotherapy and ICIs (30%), or tyrosine kinase inhibitors (4%). The median baseline CARG toxicity score was 8 (range 2-12). Among patients with treatment-related toxicity (n = 49), 39 (79.6%) cases were mild (grade 1-2), and 10 (20.4%) were moderate to severe (≥ grade 3). Treatment toxicity was greater among those with a CARG score ≥8 (28.0% vs. 13.6%). Higher IADL independence, QoL, and SPPB scores at baseline were positively associated with Candidatus Gastranaerophilales bacterium, Lactobacillus rogosae, and Enterobacteria phage P4. Romboutsia ilealis, Streptococcus, and Lachnoclostridium sp An138 and T cell lag3 and cd8a were associated with worse IADLs, QoL, and SPPB scores at baseline. A longitudinal CGA and biomarker collection is feasible among older adults undergoing lung cancer treatment. Gut microbe and T cell gene expression changes correlated with subjective and objective functional status assessments. Future research will test causality in these associations to improve outcomes through novel supportive care interventions to prevent functional disability.

Single-Cell RNA Sequencing Identifies Response of Renal Lymphatic Endothelial Cells to Acute Kidney Injury.

The renal lymphatic vasculature and the lymphatic endothelial cells that make up this network play important immunomodulatory roles during inflammation. How lymphatics respond to AKI may affect AKI outcomes. The authors used single-cell RNA sequencing to characterize mouse renal lymphatic endothelial cells in quiescent and cisplatin-injured kidneys. Lymphatic endothelial cell gene expression changes were confirmed in ischemia-reperfusion injury and in cultured lymphatic endothelial cells, validating renal lymphatic endothelial cells single-cell RNA sequencing data. This study is the first to describe renal lymphatic endothelial cell heterogeneity and uncovers molecular pathways demonstrating lymphatic endothelial cells regulate the local immune response to AKI. These findings provide insights into previously unidentified molecular pathways for lymphatic endothelial cells and roles that may serve as potential therapeutic targets in limiting the progression of AKI. The inflammatory response to AKI likely dictates future kidney health. Lymphatic vessels are responsible for maintaining tissue homeostasis through transport and immunomodulatory roles. Owing to the relative sparsity of lymphatic endothelial cells in the kidney, past sequencing efforts have not characterized these cells and their response to AKI. Here, we characterized murine renal lymphatic endothelial cell subpopulations by single-cell RNA sequencing and investigated their changes in cisplatin AKI 72 hours postinjury. Data were processed using the Seurat package. We validated our findings by quantitative PCR in lymphatic endothelial cells isolated from both cisplatin-injured and ischemia-reperfusion injury, by immunofluorescence, and confirmation in in vitro human lymphatic endothelial cells. We have identified renal lymphatic endothelial cells and their lymphatic vascular roles that have yet to be characterized in previous studies. We report unique gene changes mapped across control and cisplatin-injured conditions. After AKI, renal lymphatic endothelial cells alter genes involved in endothelial cell apoptosis and vasculogenic processes as well as immunoregulatory signaling and metabolism. Differences between injury models were also identified with renal lymphatic endothelial cells further demonstrating changed gene expression between cisplatin and ischemia-reperfusion injury models, indicating the renal lymphatic endothelial cell response is both specific to where they lie in the lymphatic vasculature and the kidney injury type. In this study, we uncover lymphatic vessel structural features of captured populations and injury-induced genetic changes. We further determine that lymphatic endothelial cell gene expression is altered between injury models. How lymphatic endothelial cells respond to AKI may therefore be key in regulating future kidney disease progression.

Transcriptomic comparison of primary human lung cells with lung tissue samples and the human A549 lung cell line highlights cell type specific responses during infections with influenza A virus

Influenza A virus (IAV) causes pandemics and annual epidemics of severe respiratory infections. A better understanding of the molecular regulation in tissue and cells upon IAV infection is needed to thoroughly understand pathogenesis. We analyzed IAV replication and gene expression induced by IAV strain H3N2 Panama in isolated primary human alveolar epithelial type II cells (AECIIs), the permanent A549 adenocarcinoma cell line, alveolar macrophages (AMs) and explanted human lung tissue by bulk RNA sequencing. Primary AECII exhibit in comparison to AM a broad set of strongly induced genes related to RIG-I and interferon (IFN) signaling. The response of AECII was partly mirrored in A549 cells. In human lung tissue, we observed induction of genes unlike in isolated cells. Viral RNA was used to correlate host cell gene expression changes with viral burden. While relative induction of key genes was similar, gene abundance was highest in AECII cells and AM, while weaker in the human lung (due to less IAV replication) and A549 cells (pointing to their limited suitability as a model). Correlation of host gene induction with viral burden allows a better understanding of the cell-type specific induction of pathways and a possible role of cellular crosstalk requiring intact tissue.

Correlates of vaccine protection against Mycobacterium avium sub-species paratuberculosis infection revealed in a transcriptomic study of responses in Gudair® vaccinated sheep.

A critical hindrance in the development of effective vaccine strategies to combat infectious disease is lack of knowledge about correlates of protection and of the host responses necessary for successful adaptive immunity. Often vaccine formulations are developed by stepwise experimentation, with incomplete investigation of the fundamental mechanisms of protection. Gudair® is a commercially available vaccine registered for use in sheep and goats for controlling spread of Mycobacterium avium sub-species paratuberculosis (MAP) infections and reduces mortality by up to 90%. Here, using an experimental infection model in sheep, we have utilized a transcriptomics approach to identify white blood cell gene expression changes in vaccinated, MAP-exposed Merino sheep with a protective response in comparison to those vaccinated animals that failed to develop immunity to MAP infection. This methodology facilitated an overview of gene-associated functional pathway adaptations using an in-silico analysis approach. We identified a group of genes that were activated in the vaccine-protected animals and confirmed stability of expression in samples obtained from naturally exposed commercially maintained sheep. We propose these genes as correlates of vaccine induced protection.

Inhibition of FOXP3 by stapled alpha-helical peptides dampens regulatory T cell function

Despite continuing advances in the development of novel cellular-, antibody-, and chemotherapeutic-based strategies to enhance immune reactivity, the presence of regulatory T cells (Treg cells) remains a complicating factor for their clinical efficacy. To overcome dosing limitations and off-target effects from antibody-based Treg cell deletional strategies or small molecule drugging, we investigated the ability of hydrocarbon stapled alpha-helical (SAH) peptides to target FOXP3, the master transcription factor regulator of Treg cell development, maintenance, and suppressive function. Using the crystal structure of the FOXP3 homodimer as a guide, we developed SAHs in the likeness of a portion of the native FOXP3 antiparallel coiled-coil homodimerization domain (SAH-FOXP3) to block this key FOXP3 protein-protein interaction (PPI) through molecular mimicry. We describe the design, synthesis, and biochemical evaluation of single- and double-stapled SAHs covering the entire coiled-coil expanse. We show that lead SAH-FOXP3s bind FOXP3, are cell permeable and nontoxic to T cells, induce dose-dependent transcript and protein level alterations of FOXP3 target genes, impede Treg cell function, and lead to Treg cell gene expression changes invivo consistent with FOXP3 dysfunction. These results demonstrate a proof of concept for rationally designed FOXP3-directed peptide therapeutics that could be used as approaches to amplify endogenous immune responsiveness.

Implications of estrogen and its receptors in colorectal carcinoma.

Estrogens have been implicated in the pathogenesis of various cancer types, including colorectal carcinoma (CRC). Estrogen receptors such as ERα and ERβ activate intracellular signaling cascades followed by binding to estrogen, resulting in important changes in cellular behaviors. The nuclear estrogen receptors, i.e. ERβ and ERα are responsible for the genomic actions of estrogens, whereas the other receptor, such as G protein-coupled estrogen receptor (GPER) regulates rapid non-genomic actions, which lead to secondary gene expression changes in cells. ERβ, the predominant estrogen receptor expressed in both normal and non-malignant colonic epithelium, has protective roles in colon carcinogenesis. ERβ may exert the anti-tumor effect through selective activation of pro-apoptotic signaling, increasing DNA repair, inhibiting expression of oncogenes, regulating cell cycle progression, and also by changing the micro-RNA pool and DNA-methylation. Thus, a better understanding of the underlying mechanisms of estrogen and its receptors in CRC pathogenesis could provide a new horizon for effective therapeutic development. Furthermore, using synthetic or natural compounds as ER agonists may induce estrogen-mediated anti-cancer activities against colon cancer. In this study, we report the most recent pre-clinical and experimental evidences related to ERs in CRC development. Also, we reviewed the actions of naturally occurring and synthetic compounds, which have a protective role against CRC development by acting as ER agonist.

Gene Expression Profile Analyses of the Skin Response of Balb/c-Nu Mice Model Injected by Staphylococcus aureus.

BackgroundPathogenesis and persistence of many skin diseases are related to Staphylococcus aureus (S. aureus) colonization. S. aureus infection can cause varying degrees of changes in cell gene expression, resulting in complex changes in cell phenotype and finally changes in cell life activities.Materials and MethodsThe transcriptomes of healthy and Staphylococcus aureus (S. aureus)-infected murine skin tissues were analyzed. We identified 638 differentially expressed genes (DEGs) in the infected tissues compared to the control samples, of which 324 were upregulated and 314 were downregulated, following the criteria of P < 0.01 and |log2FC| > 3. The DEGs were functionally annotated by Gene Ontology (GO), KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway and the protein–protein interaction (PPI) network analyses.ResultsThe upregulated DEGs were mainly enriched in GO terms, such as response to stimulus, immune system process and signal transduction, as well as in the complement and coagulation cascade pathway. Thus, S. aureus infection likely activates these pathways to limit the influx of neutrophils and prevent skin damage. Four clusters were identified in the PPI network, and the major hubs were mainly related to cell cycle and proliferation, and mostly downregulated. The expression levels of Nox4, Mmrn1, Mcm5, Msx1 and Fgf5 mRNAs were validated by qRT-PCR and found to be consistent with the RNA-Seq data, confirming a strong correlation between the two approaches.ConclusionThe identified genes and pathways are potential drug targets for treating skin inflammation caused by S. aureus and should be investigated further.

ZNF146/OZF and ZNF507 target LINE-1 sequences.

Repetitive sequences including transposable elements and transposon-derived fragments account for nearly half of the human genome. While transposition-competent transposable elements must be repressed to maintain genomic stability, mutated and fragmented transposable elements comprising the bulk of repetitive sequences can also contribute to regulation of host gene expression and broader genome organization. Here, we analyzed published ChIP-seq data sets to identify proteins broadly enriched on transposable elements in the human genome. We show 2 of the proteins identified, C2H2 zinc finger-containing proteins ZNF146 (also known as OZF) and ZNF507, are targeted to distinct sites within LINE-1 ORF2 at thousands of locations in the genome. ZNF146 binding sites are found at old and young LINE-1 elements. In contrast, ZNF507 preferentially binds at young LINE-1 sequences correlated to sequence changes in LINE-1 elements at ZNF507’s binding site. To gain further insight into ZNF146 and ZNF507 function, we disrupt their expression in HEK293 cells using CRISPR/Cas9 and perform RNA sequencing, finding modest gene expression changes in cells where ZNF507 has been disrupted. We further identify a physical interaction between ZNF507 and PRMT5, suggesting ZNF507 may target arginine methylation activity to LINE-1 sequences.

Inhibitory Effect of Astaxanthin on Gene Expression Changes in Helicobacter pylori-Infected Human Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) infection promotes gastric carcinogenesis by increasing oxidative stress, inflammation, and dysregulation of cell survival and proliferation of gastric epithelial cells. Astaxanthin (ASTX), a bioactive carotenoid, exhibits antioxidant and anticancer effects by modulating aberrant signaling pathways that lead to dysregulation of cell death and proliferation. To elucidate the molecular mechanism of H. pylori-induced gastric carcinogenesis and to examine the inhibitory effect of ASTX on H. pylori-induced gastric epithelial cell gene expression changes, we performed comparative RNA-sequencing (RNA-Seq) analysis for H. pylori-infected gastric epithelial cells treated with or without ASTX. RNA-Seq results reveal that differentially expressed genes (DEGs) in H. pylori-infected cells were mainly associated with the Wnt/β-catenin signaling pathway, which is related to cell proliferation. ASTX significantly reversed H. pylori-induced transcriptional alterations of the key mediators involved in β-catenin signaling, notably, porcupine (gene symbol, PORCN), spermine oxidase (SMOX), bone morphogenetic protein (BMP) and activin membrane-bound inhibitor (BAMBI), SMAD family member 4 (SMAD4), transforming growth factor-β1 (TGFB1), Fos-like 1 (FOSLI), and c-myc (MYC). We suggest that ASTX may be a potential therapeutic agent that can suppress H. pylori-induced proliferation-associated gene expression changes, in part, by counter-regulating the Wnt/β-catenin signaling pathway.

A Signaling View into the Inflammatory Tumor Microenvironment

The development of tumors requires an initiator event, usually exposure to DNA damaging agents that cause genetic alterations such as gene mutations or chromosomal abnormalities, leading to deregulated cell proliferation. Although the mere stochastic accumulation of further mutations may cause tumor progression, it is now clear that an inflammatory microenvironment has a major tumor-promoting influence on initiated cells, in particular when a chronic inflammatory reaction already existed before the initiated tumor cell was formed. Moreover, inflammatory cells become mobilized in response to signals emanating from tumor cells. In both cases, the microenvironment provides signals that initiated tumor cells perceive by membrane receptors and transduce via downstream kinase cascades to modulate multiple cellular processes and respond with changes in cell gene expression, metabolism, and morphology. Cytokines, chemokines, and growth factors are examples of major signals secreted by immune cells, fibroblast, and endothelial cells and mediate an intricate cell-cell crosstalk in an inflammatory microenvironment, which contributes to increased cancer cell survival, phenotypic plasticity and adaptation to surrounding tissue conditions. Eventually, consequent changes in extracellular matrix stiffness and architecture, coupled with additional genetic alterations, further fortify the malignant progression of tumor cells, priming them for invasion and metastasis. Here, we provide an overview of the current knowledge on the composition of the inflammatory tumor microenvironment, with an emphasis on the major signals and signal-transducing events mediating different aspects of stromal cell-tumor cell communication that ultimately lead to malignant progression.

Post-symptomatic Delivery of Brain-Derived Neurotrophic Factor (BDNF) Ameliorates Spinocerebellar Ataxia Type 1 (SCA1) Pathogenesis

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an abnormal expansion of CAG repeats in the Ataxin1 (ATXN1) gene. SCA1 is characterized by motor deficits, cerebellar neurodegeneration, and gliosis and gene expression changes. Expression of brain-derived neurotrophic factor (BDNF), growth factor important for the survival and function of cerebellar neurons, is decreased in ATXN1[82Q] mice, the Purkinje neuron specific transgenic mouse model of SCA1. As this decrease in BDNF expression may contribute to cerebellar neurodegeneration, we tested whether delivery of extrinsic human BDNF via osmotic ALZET pumps has a beneficial effect on disease severity in this mouse model of SCA1. Additionally, to test the effects of BDNF on established and progressing cerebellar pathogenesis and motor deficits, we delivered BDNF post-symptomatically. We have found that post-symptomatic delivery of extrinsic BDNF ameliorated motor deficits and cerebellar pathology (i.e., dendritic atrophy of Purkinje cells, and astrogliosis) indicating therapeutic potential of BDNF even after the onset of symptoms in SCA1. However, BDNF did not alter Purkinje cell gene expression changes indicating that certain aspects of disease pathogenesis cannot be ameliorated/slowed down with BDNF and that combinational therapies may be needed.

Transcriptome Analysis as a Tool for Investigation of Pathogenesis of Chromosomal Diseases

The aspects of transcriptional profiles of cells with chromosomal imbalance are discussed. There are certain difficulties in the assessment of phenotypic manifestations of chromosomal and genomic mutations. The data on the use of whole transcriptome analysis as an important new tool for investigation of the pathogenesis of chromosomal diseases related to aneuploidies, as well as microdeletion and microduplication syndromes, are described. The general patterns of gene expression changes in cells with chromosomal imbalance are highlighted: global dysregulation of gene expression; the similarity of transcriptional changes in cells with different aneuploidies or reciprocal copy number variations; common biological pathways and processes affected by mutations; the accumulation of transcriptional changes during ontogenesis.

Abstract 3826: CCS1477, a potent and selective p300/CBP bromodomain inhibitor, is targeted &amp; differentiated from BET inhibitors in prostate cancer cell lines in vitro

Abstract Background: CCS1477 is a potent and selective p300/CBP bromodomain inhibitor, currently in a Ph1 trial for patients with metastatic castration resistant prostate cancer (mCRPC). CCS1477 works by inhibiting the expression and function of the androgen receptor (AR), as well as inhibiting c-Myc. Bromodomain and extraterminal domain (BET) protein inhibitors are also being developed in mCRPC. We have established a BET inhibitor (BETi) resistant 22Rv1 prostate cancer cell line and used this, alongside parental 22Rv1 cells, to characterise the differential effects of p300/CBP vs BET bromodomain inhibition. Methods: 22Rv1 cells which express both the wild-type and splice variant forms of AR were cultured in the presence of increasing concentrations (30-500nM) of JQ1 for several months. A parallel set of 22Rv1 cells were cultured in the presence of vehicle (0.1% DMSO). The anti-proliferative effects of JQ1, iBet762, OTX-015 and CCS1477 (10 nm-10 µM dose range) was determined in resistant and parental 22Rv1 cells in a 5d CellTitre Glo assay. The effects of combining CCS1477 with JQ1 was measured in parental 22Rv1 cells. Protein biomarker (AR, AR-splice variant, c-Myc) responses were measured by Western blot and qPCR was used to determine changes in the expression of selected genes (AR, AR-V7, c-Myc, KLK3, TMPRSS2). Gene expression microarrays (Clariom D) were used to assess global gene expression changes in cells treated for 24h with 500nM CCS1477 or JQ1. Results: JQ1 resistant 22Rv1 cells were significantly less sensitive to JQ1 compared with parental cells. (IC50; Res, 7.3 µM vs parental, 0.06 µM). There was also cross-resistance to other chemically distinct BET inhibitors, iBET762 and OTX-015. JQ1 potently inhibited c-Myc protein and gene expression in parental cells, a response that was abrogated in the JQ1 resistant line. The inhibitory effects of JQ1 on AR gene and protein expression were reduced in the resistant line. In contrast potent anti-proliferative effects of CCS1477 were retained in JQ1 resistant cells, as was the inhibitory effect on c-Myc and AR. Combination of CCS1477 &amp; JQ1 resulted in a highly synergistic inhibitory effect on proliferation in normal 22Rv1 cells. Global gene expression analysis revealed significantly fewer altered genes after CCS1477 (27 up, 119 down) compared to JQ1 (196 up, 655 down). Conclusions: These studies provide three lines of evidence for a differentiated mode of action of CCS1477 vs BETi. First, CCS1477 continues to inhibit proliferation and relevant response biomarkers in a cell line that is resistant to BETi. Second, there is a synergistic, rather than additive effect of combining CCS1477 with JQ1. Third, there are significantly fewer genes and a distinct pattern of gene change after CCS1477 vs. JQ1. Collectively, these data point to a differentiated and more selective profile after p300/CBP inhibition with CCS1477. Citation Format: Nigel Brooks, Amy Prosser, Barbara Young, Luke Gaughan, Paul Elvin, Neil Pegg. CCS1477, a potent and selective p300/CBP bromodomain inhibitor, is targeted &amp; differentiated from BET inhibitors in prostate cancer cell lines in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3826.

Transcriptomic analysis reveals novel downstream regulatory motifs and highly transcribed virulence factor genes of Entamoeba histolytica

BackgroundPromoter motifs in Entamoeba histolytica were earlier analysed using microarray data with lower dynamic range of gene expression. Additionally, previous transcriptomic studies did not provide information on the nature of highly transcribed genes, and downstream promoter motifs important for gene expression. To address these issues we generated RNA-Seq data and identified the high and low expressing genes, especially with respect to virulence potential. We analysed sequences both upstream and downstream of start site for important motifs.ResultsWe used RNA-Seq data to classify genes according to expression levels, which ranged six orders of magnitude. Data were validated by reporter gene expression. Virulence-related genes (except AIG1) were amongst the highly expressed, while some kinases and BspA family genes were poorly expressed. We looked for conserved motifs in sequences upstream and downstream of the initiation codon. Following enrichment by AME we found seven motifs significantly enriched in high expression- and three in low expression-classes. Two of these motifs (M4 and M6) were located downstream of AUG, were exclusively enriched in high expression class, and were mostly found in ribosomal protein, and translation-related genes. Motif deletion resulted in drastic down regulation of reporter gene expression, showing functional relevance. Distribution of core promoter motifs (TATA, GAAC, and Inr) in all genes revealed that genes with downstream motifs were not preferentially associated with TATA-less promoters. We looked at gene expression changes in cells subjected to growth stress by serum starvation, and experimentally validated the data. Genes showing maximum up regulation belonged to the low or medium expression class, and included genes in signalling pathways, lipid metabolism, DNA repair, Myb transcription factors, BspA, and heat shock. Genes showing maximum down regulation belonged to the high or medium expression class. They included genes for signalling factors, actin, Ariel family, and ribosome biogenesis factors.ConclusionOur analysis has added important new information about the E. histolytica transcriptome. We report for the first time two downstream motifs required for gene expression, which could be used for over expression of E. histolytica genes. Most of the virulence-related genes in this parasite are highly expressed in culture.

Myt1 and Myt1l transcription factors limit proliferation in GBM cells by repressing YAP1 expression

Myelin transcription factor 1 (Myt1) and Myt1l (Myt1-like) are zinc finger transcription factors that regulate neuronal differentiation. Reduced Myt1l expression has been implicated in glioblastoma (GBM), and the related St18 was originally identified as a potential tumor suppressor for breast cancer. We previously analyzed changes in gene expression in a human GBM cell line with re-expression of either Myt1 or Myt1l. This revealed largely overlapping gene expression changes, suggesting similar function in these cells. Here we show that re-expression of Myt1 or Myt1l reduces proliferation in two different GBM cell lines, activates gene expression programs associated with neuronal differentiation, and limits expression of proliferative and epithelial to mesenchymal transition gene-sets. Consistent with this, expression of both MYT1 and MYT1L is lower in more aggressive glioma sub-types. Examination of the gene expression changes in cells expressing Myt1 or Myt1l suggests that both repress expression of the YAP1 transcriptional coactivator, which functions primarily in the Hippo signaling pathway. Expression of YAP1 and its target genes is reduced in Myt-expressing cells, and there is an inverse correlation between YAP1 and MYT1/MYT1L expression in human brain cancer datasets. Proliferation of GBM cell lines is reduced by lowering YAP1 expression and increased with YAP1 over-expression, which overcomes the anti-proliferative effect of Myt1/Myt1l expression. Finally we show that reducing YAP1 expression in a GBM cell line slows the growth of orthotopic tumor xenografts. Together, our data suggest that Myt1 and Myt1l directly repress expression of YAP1, a protein which promotes proliferation and GBM growth.

Leydig cell genes change their expression and association with polysomes in a stage-specific manner in the adult mouse testis.

Spermatogenesis in mammals occurs in a very highly organized manner within the seminiferous epithelium regulated by different cell types in the testis. Testosterone produced by Leydig cells regulates blood-testis barrier formation, meiosis, spermiogenesis, and spermiation. However, it is unknown whether Leydig cell function changes with the different stages of the seminiferous epithelium. This study utilized the WIN 18,446 and retinoic acid (RA) treatment regime combined with the RiboTag mouse methodology to synchronize male germ cell development and allow for the in vivo mapping of the Leydig cell translatome across the different stages of one cycle of the seminiferous epithelium. Using microarrays analysis, we identified 11 Leydig cell-enriched genes that were expressed in stage-specific manner such as the glucocorticoid synthesis and transport genes, Cyp21a1 and Serpina6. In addition, there were nine Leydig cell transcripts that change their association with polysomes in correlation with the different stages of the spermatogenic cycle including Egr1. Interestingly, the signal intensity of EGR1 and CYP21 varied among Leydig cells in the adult asynchronous testis. However, testosterone levels across the different stages of germ cell development did not cycle. These data show, for the first time, that Leydig cell gene expression changes in a stage-specific manner during the cycle of the seminiferous epithelium and indicate that a heterogeneous Leydig cell population exists in the adult mouse testis.

Exercise training improves obesity-related lymphatic dysfunction.

Key points Obesity results in perilymphatic inflammation and lymphatic dysfunction.Lymphatic dysfunction in obesity is characterized by decreased lymphatic vessel density, decreased collecting lymphatic vessel pumping frequency, decreased lymphatic trafficking of immune cells, increased lymphatic vessel leakiness and changes in the gene expression patterns of lymphatic endothelial cells.Aerobic exercise, independent of weight loss, decreases perilymphatic inflammatory cell accumulation, improves lymphatic function and reverses pathological changes in gene expression in lymphatic endothelial cells. Although previous studies have shown that obesity markedly decreases lymphatic function, the cellular mechanisms that regulate this response remain unknown. In addition, it is unclear whether the pathological effects of obesity on the lymphatic system are reversible with behavioural modifications. The purpose of this study, therefore, was to analyse lymphatic vascular changes in obese mice and to determine whether these pathological effects are reversible with aerobic exercise. We randomized obese mice to either aerobic exercise (treadmill running for 30 min per day, 5 days a week, for 6 weeks) or a sedentary group that was not exercised and analysed lymphatic function using a variety of outcomes. We found that sedentary obese mice had markedly decreased collecting lymphatic vessel pumping capacity, decreased lymphatic vessel density, decreased lymphatic migration of immune cells, increased lymphatic vessel leakiness and decreased expression of lymphatic specific markers compared with lean mice (all P < 0.01). Aerobic exercise did not cause weight loss but markedly improved lymphatic function compared with sedentary obese mice. Exercise had a significant anti‐inflammatory effect, resulting in decreased perilymphatic accumulation of inflammatory cells and inducible nitric oxide synthase expression. In addition, exercise normalized isolated lymphatic endothelial cell gene expression of lymphatic specific genes, including VEGFR‐3 and Prox1. Taken together, our findings suggest that obesity impairs lymphatic function via multiple mechanisms and that these pathological changes can be reversed, in part, with aerobic exercise, independent of weight loss. In addition, our study shows that obesity‐induced lymphatic endothelial cell gene expression changes are reversible with behavioural modifications.

Fibroblast growth factor receptor splice variants are stable markers of oncogenic transforming growth factor β1 signaling in metastatic breast cancers

IntroductionEpithelial–mesenchymal transition (EMT) and mesenchymal–epithelial transition (MET) facilitate breast cancer (BC) metastasis; however, stable molecular changes that result as a consequence of these processes remain poorly defined. Therefore, with the hope of targeting unique aspects of metastatic tumor outgrowth, we sought to identify molecular markers that could identify tumor cells that had completed the EMT:MET cycle.MethodsAn in vivo reporter system for epithelial cadherin (E-cad) expression was used to quantify its regulation in metastatic BC cells during primary and metastatic tumor growth. Exogenous addition of transforming growth factor β1 (TGF-β1) was used to induce EMT in an in situ model of BC. Microarray analysis was employed to examine gene expression changes in cells chronically treated with and withdrawn from TGF-β1, thus completing one full EMT:MET cycle. Changes in fibroblast growth factor receptor type 1 (FGFR1) isoform expression were validated using PCR analyses of patient-derived tumor tissues versus matched normal tissues. FGFR1 gene expression was manipulated using short hairpin RNA depletion and cDNA rescue. Preclinical pharmacological inhibition of FGFR kinase was employed using the orally available compound BGJ-398.ResultsMetastatic BC cells undergo spontaneous downregulation of E-cad during primary tumor growth, and its expression subsequently returns following initiation of metastatic outgrowth. Exogenous exposure to TGF-β1 was sufficient to drive the metastasis of an otherwise in situ model of BC and was similarly associated with a depletion and return of E-cad expression during metastatic progression. BC cells treated and withdrawn from TGF-β stably upregulate a truncated FGFR1-β splice variant that lacks the outermost extracellular immunoglobulin domain. Identification of this FGFR1 splice variant was verified in metastatic human BC cell lines and patient-derived tumor samples. Expression of FGFR1-β was also dominant in a model of metastatic outgrowth where depletion of FGFR1 and pharmacologic inhibition of FGFR kinase activity both inhibited pulmonary tumor outgrowth. Highlighting the dichotomous nature of FGFR splice variants and recombinant expression of full-length FGFR1-α also blocked pulmonary tumor outgrowth.ConclusionThe results of our study strongly suggest that FGFR1-β is required for the pulmonary outgrowth of metastatic BC. Moreover, FGFR1 isoform expression can be used as a predictive biomarker for therapeutic application of its kinase inhibitors.

Genotoxic changes to rodent cells exposed in vitro to tungsten, nickel, cobalt and iron.

Tungsten-based materials have been proposed as replacements for depleted uranium in armor-penetrating munitions and for lead in small-arms ammunition. A recent report demonstrated that a military-grade composition of tungsten, nickel, and cobalt induced a highly-aggressive, metastatic rhabdomyosarcoma when implanted into the leg muscle of laboratory rats to simulate a shrapnel wound. The early genetic changes occurring in response to embedded metal fragments are not known. In this study, we utilized two cultured rodent myoblast cell lines, exposed to soluble tungsten alloys and the individual metals comprising the alloys, to study the genotoxic effects. By profiling cell transcriptomes using microarray, we found slight, yet distinct and unique, gene expression changes in rat myoblast cells after 24 h metal exposure, and several genes were identified that correlate with impending adverse consequences of ongoing exposure to weapons-grade tungsten alloy. These changes were not as apparent in the mouse myoblast cell line. This indicates a potential species difference in the cellular response to tungsten alloy, a hypothesis supported by current findings with in vivo model systems. Studies examining genotoxic-associated gene expression changes in cells from longer exposure times are warranted.

Leukemia cells induce changes in human bone marrow stromal cells

BackgroundBone marrow stromal cells (BMSCs) are multipotent cells that support angiogenesis, wound healing, and immunomodulation. In the hematopoietic niche, they nurture hematopoietic cells, leukemia, tumors and metastasis. BMSCs secrete of a wide range of cytokines, growth factors and matrix proteins which contribute to the pro-tumorigenic marrow microenvironment. The inflammatory cytokines IFN-γ and TNF-α change the BMSC secretome and we hypothesized that factors produced by tumors or leukemia would also affect the BMSC secretome and investigated the interaction of leukemia cells with BMSCs.MethodsBMSCs from healthy subjects were co-cultured with three myeloid leukemia cell lines (TF-1, TF-1α and K562) using a trans-well system. Following co-culture, the BMSCs and leukemia cells were analyzed by global gene expression analysis and culture supernatants were analyzed for protein expression. As a control, CD34+ cells were also cocultured with BMSCs.ResultsCo-culture induced leukemia cell gene expression changes in stem cell pluripotency, TGF-β signaling and carcinoma signaling pathways. BMSCs co-cultured with leukemia cells up-regulated a number of proinflammatory genes including IL-17 signaling-related genes and IL-8 and CCL2 levels were increased in co-culture supernatants. In contrast, purine metabolism, mTOR signaling and EIF2 signaling pathways genes were up-regulated in BMSCs co-cultured with CD34+ cells.ConclusionsBMSCs react to the presence of leukemia cells undergoing changes in the cytokine and chemokine secretion profiles. Thus, BMSCs and leukemia cells both contribute to the creation of a competitive niche more favorable for leukemia stem cells.