scholarly journals Insights into Intra-Tumoral Heterogeneity: Transcriptional Profiling of Chemoresistant MPM Cell Subpopulations Reveals Involvement of NFkB and DNA Repair Pathways and Contributes a Prognostic Signature

2021 ◽  
Vol 22 (21) ◽  
pp. 12071
Author(s):  
Mario Cioce ◽  
Andrea Sacconi ◽  
Harvey I. Pass ◽  
Claudia Canino ◽  
Sabrina Strano ◽  
...  
Keyword(s):  
Gene Expression ◽  
Prognostic Significance ◽  
Cell Number ◽  
Cell Subpopulation ◽  
Mrna Levels ◽  
Tumor Resistance ◽  
Cell Subpopulations ◽  
Chemoresistant Cell

Chemoresistance is a hallmark of malignant pleural mesothelioma (MPM) management and the expression of ALDH1A3 is responsible for the survival and activity of MPM chemoresistant cell subpopulations (ALDHbright cells). We enriched mesothelioma ALDHbright cells to near homogeneity by FACS sorting and an Aldefluor assay and performed unbiased Affymetrix gene expression profiling. Viability and ELISA assays were used to rule out significant apoptosis in the sorted cell subpopulations and to assess target engagement by butein. Statistical analysis of the results, pathway enrichment and promoter enrichment were employed for the generation of the data. Q-RTPCR was used to validate a subset of the identified, modulated mRNAs In this work, we started from the observation that the mRNA levels of the ALDH1A3 isoform could prognostically stratify MPM patients. Thus, we purified MPM ALDHbright cells from NCI-H2595 cells and interrogated their gene expression (GES) profile. We analyzed the GES of the purified cells at both a steady state and upon treatment with butein (a multifunctional tetrahydroxy-chalcone), which abates the ALDHbright cell number, thereby exerting chemo-sensitizing effects in vitro and in vivo. We identified 924 genes modulated in a statistically significant manner as a function of ALDH status and of the response to the inhibitor. Pathway and promoter enrichment identified the molecular determinant of high ALDH status and how butein treatment altered the molecular portrait of those chemoresistant cell subpopulations. Further, we unraveled an eighteen-gene signature with high prognostic significance for MPM patients, and showed that most of the identified prognostic contributors escaped the analysis of unfractionated samples. This work proves that digging into the unexplored field of intra-tumor heterogeneity (ITH) by working at the cell subpopulation level may provide findings of prognostic relevance, in addition to mechanistic insights into tumor resistance to therapy.

scholarly journals Simultaneous gene quantitation of multiple genes in individual bovine nuclear transfer blastocysts

Reproduction ◽  
2007 ◽  
Vol 133 (1) ◽  
pp. 231-242 ◽  
Author(s):  
Craig Smith ◽  
Debbie Berg ◽  
Sue Beaumont ◽  
Neil T Standley ◽  
David N Wells ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Transfer ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Ectopic Expression ◽  
Cell Number ◽  
Transcript Levels ◽  
Multiple Genes

During somatic cell nuclear transfer (NT), the transcriptional status of the donor cell has to be reprogrammed to reflect that of an embryo. We analysed the accuracy of this process by comparing transcript levels of four developmentally important genes (Oct4,Otx2,Ifitm3,GATA6), a gene involved in epigenetic regulation (Dnmt3a) and three housekeeping genes (β-actin, β-tubulinandGAPDH) in 21 NT blastocysts with that in genetically half-identicalin vitroproduced (IVP,n=19) andin vivo(n=15) bovine embryos. We have optimised an RNA-isolation and SYBR-green-based real-time RT-PCR procedure allowing the reproducible absolute quantification of multiple genes from a single blastocyst. Our data indicated that transcript levels did not differ significantly between stage and grade-matched zona-free NT and IVP embryos except for Ifitm3/Fragilis, which was expressed at twofold higher levels in NT blastocysts.Ifitm3expression is confined to the inner cell mass at day 7 blastocysts and to the epiblast in day 14 embryos. No ectopic expression in the trophectoderm was seen in NT embryos. Gene expression in NTand IVP embryos increased between two- and threefold for all eight genes from early to late blastocyst stages. This increase exceeded the increase in cell number over this time period indicating an increase in transcript number per cell. Embryo quality (morphological grading) was correlated to cell number for NT and IVP embryos with grade 3 blastocysts containing 30% fewer cells. However, only NT embryos displayed a significant reduction in gene expression (50%) with loss of quality. Variability in gene expression levels was not significantly different in NT, IVP orin vivoembryos but differed among genes, suggesting that the stringency of regulation is intrinsic to a gene and not affected by culture or nuclear transfer.Oct4levels exhibited the lowest variability. Analysing the total variability of all eight genes for individual embryos revealed thatin vivoembryos resembled each other much more than did NT and IVP blastocysts. Furthermore,in vivoembryos, consisting of 1.5-fold more cells, generally contained two- to fourfold more transcripts for the eight genes than did their cultured counterparts. Thus, culture conditions (in vivoversusin vitro) have greater effects on gene expression than does nuclear transfer when minimising genetic heterogeneity.

282 GENE EXPRESSION PATTERNS AND QUALITY OF BOVINE EMBRYOS PRODUCED UNDER DIFFERENT OXYGEN CONCENTRATIONS

2007 ◽  
Vol 19 (1) ◽  
pp. 256
Author(s):  
W. J. Son ◽  
M. K. B. ◽  
Y. J. Jeong ◽  
S. Balasubramanian ◽  
S. Y. Choe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryo Development ◽  
Expression Patterns ◽  
Cell Number ◽  
Blastocyst Stage ◽  
Total Cell ◽  
Blastocyst Development ◽  
Glut 1

Various factors are known to influence the survival and development of in vitro-produced embryos, including co-culture with somatic cells, antioxidants, and O2 tension. Studies in several species report that embryo development and quality were enhanced at low O2 concentrations. This study compared the effects of 2 O2 concentrations on IVP embryo development, embryo quality, and gene expression to those of in vivo counterparts. Cumulus–oocyte complexes were matured in vitro in TCM-199 with hormones and 10% FCS, and inseminated in TALP medium. Presumptive zygotes were cultured in SOF medium under either 5% or 20% O2 in air. In triplicate, sets of 5 embryos at the 2-cell, 4-cell, 8-cell, 16-cell, morula, and Day 7 blastocyst stages were used for analyzing the expression patterns of apoptotic (Bax and Bcl2), metabolism (Glut-1 and Glut-5), stress (Sox, Hsp70, and G6PDH), compaction (Cx43), oxidation (PRDX5, NADH, and MnSOD), and implantation (VEGF and IFN-tau) genes using real-time quantitative PCR. The expression of each gene was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Statistical analysis was performed with Bonferroni and Duncan tests by ANOVA (P < 0.05). Cleavage rates did not differ among groups. Blastocyst and hatched blastocyst development in 5% O2 was significantly (P < 0.05) higher than in 20% O2. Total cell number of in vivo blastocysts was significantly (P < 0.05) higher than that of IVP blastocysts. ICM ratio and apoptosis of in vivo blastocysts were significantly (P < 0.05) lower than for IVP blastocysts. The relative abundances (RAs) of Glut-1, Glut-5, MnSOD, NADH, PRDX5, Cx43, Bcl2, and IFN-τ were significantly (P < 0.05) higher in in vivo embryos, whereas the RAs of Sox, G6PDH, Hsp70, Bax, and VEGF were significantly (P < 0.05) lower than for IVP counterparts. In conclusion, culture at 5% O2 concentration resulted in higher rates of development to the blastocyst stage, higher total cell numbers, and decreased apoptosis. Furthermore, differences in expression of genes including Glut-1, Glut-5, Sox, G6PDH, Hsp70, Bax, Bcl2, Cx43, PRDX5, NADH, MnSOD, VEGF, and IFN-τ may prove useful in determining optimal culture conditions. This work was supported by ARPC (204119-03-SB010), Republic of Korea.

6 GENE EXPRESSION ANALYSIS OF IN VIVO- AND IN VITRO-MATURED PORCINE METAPHASE II OOCYTES

2014 ◽  
Vol 26 (1) ◽  
pp. 117 ◽  
Author(s):  
L. Cox ◽  
G. Saunders ◽  
J. Stevens ◽  
S. C. Isom
Keyword(s):  
Gene Expression ◽  
Rna Degradation ◽  
Functional Gene ◽  
Gene Set Enrichment Analysis ◽  
Developmental Competence ◽  
Mrna Levels ◽  
Multiple Testing Correction ◽  
Metaphase Ii Oocytes

In vitro-matured (IVM) oocytes lack the same developmental competence as oocytes that are matured in vivo (IVV), yet no compelling explanation for this discrepancy has been provided at the molecular level. The aim of this study was to quantify and compare mRNA levels in IVM and IVV oocytes for genes from a wide variety of functional gene categories, including RNA degradation, pluripotency, epigenome modification, oocyte-specific, and apoptosis. Quantitative real-time PCR (qPCR) was used to evaluate the relative gene expression levels of 70 genes in each of 33 individual IVM oocytes from 4 different collection days and 29 individual IVV oocytes from 4 different donor animals. The qPCR data were analysed using ANOVA and significance was assigned at P < 0.05. After a multiple testing correction was applied, relative transcript abundances for 32 of the 70 genes tested were found to be significantly different (q < 0.05) between the IVM and IVV oocytes. Of these significantly different genes, 23 were higher in the IVM oocytes and only 9 were higher in the IVV oocytes. The 32 significantly differentially expressed genes were then evaluated in relation to their corresponding functional gene categories. Of particular interest, transcripts for 7/14 RNA degradation-related genes (CNOT3, DCP1A, DDX6, LSM1, PABPN1, PABPN1L, PARN) and 3/9 oocyte specific genes (BMP15, YBX2, H1FOO) were significantly more abundant in the IVM oocytes. In contrast, transcripts for 4/8 epigenetic related transcripts (ASH2l, DNMT1, EHMT2, EZH2), 2/2 apoptosis related genes (BCL2, XIAP), and 1/4 pluripotency factors (LIN28) were significantly more abundant in the IVV oocytes. Gene set enrichment analysis confirmed that, within the context of this experimental design, RNA degradation and chromatin remodelling pathways are significantly perturbed in IVM oocytes. We conclude that in vitro maturation has profound effects on transcript populations of metaphase-II oocytes, with most transcripts being higher in IVM oocytes. We expect that this data will lead to a better understanding of how we can improve the quality of oocytes that are matured in vitro as well as provide information to help to identify markers that could be indicative of oocyte quality.

scholarly journals DNA Hypermethylation Downregulates Telomerase Reverse Transcriptase (TERT) during H. pylori-Induced Chronic Inflammation

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Françoise I. Bussière ◽  
Valérie Michel ◽  
Julien Fernandes ◽  
Lionel Costa ◽  
Vania Camilo ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reverse Transcriptase ◽  
Chronic Inflammation ◽  
Telomerase Activity ◽  
Telomerase Reverse Transcriptase ◽  
Mrna Levels ◽  
Dna Hypermethylation ◽  
H Pylori

Helicobacter pylori infection causes chronic gastritis and is the major risk factor of gastric cancer. H. pylori induces a chronic inflammation-producing reactive oxygen species (ROS) which is a source of chromosome instabilities and contributes to the development of malignancy. H. pylori also promotes DNA hypermethylation, known to dysregulate essential genes that maintain genetic stability. The maintenance of telomere length by telomerase is essential for chromosome integrity. Telomerase reverse transcriptase (TERT) is the catalytic component of telomerase activity and an important target during host-pathogen interaction. We aimed to investigate the consequences of H. pylori on the regulation of TERT gene expression and telomerase activity. In vitro, hTERT mRNA levels and telomerase activity were analysed in H. pylori-infected human gastric epithelial cells. In addition, C57BL/6 and INS-GAS mice were used to investigate the influence of H. pylori-induced inflammation on TERT levels. Our data demonstrated that, in vitro, H. pylori inhibits TERT gene expression and decreases the telomerase activity. The exposure of cells to lycopene, an antioxidant compound, restores TERT levels in infected cells, indicating that ROS are implicated in this downregulation. In vivo, fewer TERT-positive cells are observed in gastric tissues of infected mice compared to uninfected, more predominantly in the vicinity of large aggregates of lymphocytes, suggesting an inflammation-mediated regulation. Furthermore, H. pylori appears to downregulate TERT gene expression through DNA hypermethylation as shown by the restoration of TERT transcript levels in cells treated with 5′-azacytidine, an inhibitor of DNA methylation. This was confirmed in infected mice, by PCR-methylation assay of the TERT gene promoter. Our data unraveled a novel way for H. pylori to promote genome instabilities through the inhibition of TERT levels and telomerase activity. This mechanism could play an important role in the early steps of gastric carcinogenesis.

scholarly journals Beta2-adrenoceptor desensitization in non-pregnant estrogen-primed rat myometrium involves modulation of oxytocin receptor gene expression

1998 ◽  
Vol 20 (2) ◽  
pp. 261-270 ◽  
Author(s):  
T Engstrom ◽  
P Bratholm ◽  
H Vilhardt ◽  
NJ Christensen
Keyword(s):  
Gene Expression ◽  
Contractile Response ◽  
Receptor Gene ◽  
Mrna Levels ◽  
Oxytocin Receptor Gene ◽  
Pregnant Rat ◽  
Homologous Desensitization ◽  
Stimulation Of

The nona-peptide oxytocin (OT) induces contraction of the myometrium by interaction with specific plasma membrane associated OT receptors (OTR), whereas stimulation of beta2-adrenoceptors (beta2AR) causes relaxation. Homologous desensitization of the myometrium to both hormones has been described. However, a possible interaction between the two systems has not been investigated. In the present study, long-term in vivo treatment of non-pregnant estrogen-primed rats with isoproterenol decreased maximal relaxation of isolated uterine strips challenged with isoproterenol. Increased EC50 values of similarly treated animals suggest that the coupling between receptor occupancy and contractile response was impaired. Since beta2AR mRNA levels were left unchanged, we conclude that the homologous desensitization to beta2 stimulation is not due to changes in beta2AR gene expression. OT infusion did not alter beta2AR mRNA levels or isoproterenol-induced relaxation of isolated uterine strips. Treatment with OT had no effect on the amount of myometrial OTR mRNA. We have previously found that OT down-regulates OTR in the non-pregnant rat myometrium, but this therefore does not appear to take place at the level of mRNA production. Isoproterenol treatment resulted in a three-fold increase in OTR mRNA. This was accompanied by a 91% rise in OTR binding and an augmented contractile response of isolated uterine strips to OT, suggesting that the increased production of mRNA reflects formation of active receptors. Neither OTR affinity nor EC50 of in vitro strips was affected by isoproterenol treatment. We conclude that stimulation of beta2AR causes heterologous up-regulation of OTR in the non-pregnant estrogen-primed rat myometrium.

scholarly journals Interleukin 10 up-regulates elastin gene expression in vivo and in vitro at the transcriptional level

1994 ◽  
Vol 302 (2) ◽  
pp. 331-333 ◽  
Author(s):  
S Reitamo ◽  
A Remitz ◽  
K Tamai ◽  
I Ledo ◽  
J Uitto
Keyword(s):  
Gene Expression ◽  
Interleukin 10 ◽  
Transcriptional Level ◽  
Mrna Levels ◽  
Tgf Beta ◽  
Cultured Fibroblasts ◽  
Beta 2 ◽  
Elastin Gene

In immune cells, such as T cells and monocytes, interleukin 10 (IL-10) has regulatory functions on a number of cytokines, including IL-1, IL-2, IL-8 and tumour necrosis factor-alpha expression. However, the effects of IL-10 have not previously been studied in detail in connective-tissue cells. In the present study, we show that recombinant human IL-10 at physiological concentrations has direct effects on the expression of the human elastin gene both in vivo and in vitro. Transgenic mice expressing a human elastin promoter/chloramphenicol acetyltransferase (CAT) reporter gene construct were injected subcutaneously with IL-10 (1-100 ng) and the site of injection was biopsied after 24 h. CAT assay revealed an increase of up to 3.5-fold in the promoter activity with 10 ng of IL-10. Transforming growth factor-beta 2 (TGF-beta 2) is known to up-regulate elastin gene expression in cultured fibroblasts. When IL-10 was added to such cultures, the effects of TGF-beta 2 on elastin mRNA levels were synergistically potentiated. These results suggest that IL-10 has an up-regulatory effect on elastin gene expression.

Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1737-1737
Author(s):  
Liyan Pang ◽  
Xun Wang ◽  
Yuhuan Wang ◽  
Gerd Blobel ◽  
Mortimer Poncz
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Critical Role ◽  
Regulatory Region ◽  
Maturation Stage ◽  
Specific Gene ◽  
Mrna Levels ◽  
Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

Progesterone and EGF inhibit mouse mammary gland prolactin receptor and beta-casein gene expression

1994 ◽  
Vol 267 (5) ◽  
pp. C1467-C1472 ◽  
Author(s):  
S. Nishikawa ◽  
R. C. Moore ◽  
N. Nonomura ◽  
T. Oka
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Prolactin Receptor ◽  
Mammary Epithelium ◽  
Mouse Mammary Gland ◽  
Mrna Levels ◽  
Casein Gene ◽  
Beta Casein

Regulation of mouse mammary gland long-form prolactin receptor (PRL-RL) mRNA levels by progesterone and epidermal growth factor (EGF) and the relationship between PRL-RL and beta-casein gene expression were examined in vivo and in vitro. PRL-RL and beta-casein mRNA levels increased approximately 6- and 15-fold from the pregnant to the lactating period, respectively, when normalized to the level of beta-actin mRNA. Ovariectomy of pregnant mice rapidly reduced the serum concentration of progesterone and increased the level of PRL-RL and beta-casein mRNAs approximately three- and fourfold compared with sham-operated animals 24 h after the operation. Injection of progesterone, but not estrogen, inhibited the increase in both mRNA levels. PRL-RL and beta-casein mRNA levels in cultured mammary epithelium increased in response to insulin, hydrocortisone, and prolactin, whereas progesterone or EGF caused inhibition. The combination of EGF and progesterone produced a greater inhibition than either hormone alone. These results indicate that both progesterone and EGF serve as negative regulators of lactogenesis.

scholarly journals Copper-dependent activation of hypoxia-inducible factor (HIF)-1: implications for ceruloplasmin regulation

Blood ◽  
2005 ◽  
Vol 105 (12) ◽  
pp. 4613-4619 ◽  
Author(s):  
Falk Martin ◽  
Tobias Linden ◽  
Dörthe M. Katschinski ◽  
Felix Oehme ◽  
Ingo Flamme ◽  
...  
Keyword(s):  
Gene Expression ◽  
Glucose Transporter ◽  
Iron Concentration ◽  
Hypoxia Inducible Factor ◽  
Sensory System ◽  
Mrna Levels ◽  
Copper Binding ◽  
Glucose Transporter 1

Abstract Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)α subunits. Upon hydroxylation under normoxic conditions, HIFα is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1α under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)

The Pro-Differentiation Effect of Doxycycline on Human SZ95 Sebocytes

Dermatology ◽  
2020 ◽  
pp. 1-5
Author(s):  
Christos C. Zouboulis ◽  
Síona Ní Raghallaigh ◽  
Gerd Schmitz ◽  
Frank C. Powell
Keyword(s):  
Lipid Content ◽  
Acne Vulgaris ◽  
Cell Number ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Sebaceous Glands ◽  
Peroxisome Proliferator Activated Receptor ◽  
New Findings

Background: Despite their widespread clinical use in both acne vulgaris and rosacea, the effects of tetracyclines on sebocytes have not been investigated until now. Sebaceous glands are central to the pathogenesis of acne and may be important in the development of rosacea. Objective: The aim of this study was to assess the effects of doxycycline on the immortalized SZ95 sebaceous gland cell line as a model for understanding possible effectiveness on the sebaceous glands in vivo. Methods: The effects of doxycycline on SZ95 sebocyte numbers, viability, and lipid content as well as its effects on the mRNA levels of peroxisome proliferator-activated receptors α and γ, in comparison to the peroxisome proliferator-activated receptor γ agonist troglitazone, were investigated. Results: Doxycycline reduced the cell number and increased the lipid content of SZ95 sebocytes in vitro after 2 days of treatment. These doxycycline effects may be explained by an upregulation of peroxisome proliferator-activated receptor γ mRNA levels at 12 and 24 h, whereas troglitazone already upregulated peroxisome proliferator-activated receptor γ levels after 6 h. Both compounds did not influence peroxisome proliferator-activated receptor α mRNA levels. Conclusion: These new findings illustrate a previously unknown effect of doxycycline on sebocytes, which may be relevant to their modulation of disorders of the pilosebaceous unit, such as acne vulgaris and rosacea.

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