Basis Of Differential Expression Research Articles

Abstract We002: A natural loss-of-function deletion of the cytohesin 1 (Cyth1) gene in BALB/cByJ mice does not impact cardiomyocyte polyploidy

The adult mammalian heart has limited regenerative capacity due in part to the low level of diploid cardiomyocytes (CMs). Multiple polymorphic genes determine the final level of diploid CMs, however, the relevant genes need to be identified. There is a large variation in CM ploidy between the highly related sister strains BALB/cJ and BALB/cByJ. We undertook an RNA-Seq comparison of neonatal ventricular heart tissue from BALB/cJ and BALB/cByJ to identify gene expression differences that might underlie this difference in CM ploidy. Cytohesin-1 (Cyth1) and Dynein heavy chain 17 (Dnah17) were particularly different in expression: in BALB/cJ (as in most other mouse strains), Cyth1 is expressed in the heart and Dnah17 isn’t expressed, whereas the opposite profile (Cyth1 off, Dnah17 on) was observed in BALB/cByJ heart. These two genes are immediately adjacent to each other in the mouse genome. We found that the basis of differential expression is a large deletion in the locus of BALB/cByJ mice spanning from the Cyth1 first intron to shortly downstream of the last Cyth1 exon. This creates a complete null Cyth1 allele and at the same time results in ectopic transcription of the downstream gene Dnah17, although this transcript is unlikely to encode a protein. Cyth1 encodes a guanine nucleotide exchange factor that was a plausible candidate for being involved in CM ploidy. Using the natural Cyth1 mutant locus plus an engineered null allele, we found no difference in CM ploidy in the absence of Cyth1 function on a cJ-cByJ F1 background or on an inbred C57BL/6J background. This study successfully identified a natural loss-of-function variant in the Cyth1 gene between BALB/cJ and BALB/cByJ but showed that this does not impact CM ploidy, at least by itself. It may underlie other phenotypic differences between these two strains.

Understanding the regulatory relationship of abscisic acid and bZIP transcription factors towards amylose biosynthesis in wheat.

Starch is biosynthesized during seed development and this process is regulated by many bZIP proteins in bread wheat. Abscisic acid (ABA), an important phyto-hormone involved in various physiological processes mediated by bZIPs in plants including seed development. The 'Group A' TabZIP transcription factors play important roles in the ABA signaling pathway in Arabidopsis, rice and other cereal crops but their role in regulation of amylose biosynthesis in wheat is limited. In this study 83 'Group A' TabZIPs were characterized by gene expression analysis in wheat amylose mutants. A set of 17 TabZIPs was selected on the basis of differential expression (> 2 fold) in low and high amylose mutants from RNA-seq data and validated by qRT PCR. Based on qRT PCR and correlation analysis out of the 17 TabZIPs six differentially expressed candidate TabZIPs were identified, involving in high amylose biosynthesis. The TabZIP175.2, identified as upregulated in all high amylose lines and TabZIP90.2, TabZIP129.1, TabZIP132.2, TabZIP143 and TabZIP159.2 were found downregulated in all low amylose lines, after exogenous supply of ABA. Proximal promoter analysis of starch pathway genes revealed the presence of ABA-responsive elements (ABREs) that are putative binding sites for bZIPs. Collectively, these findings indicated the involvement of putative six candidate TabZIPs as transcriptional regulators of amylose related genes via an ABA-dependent pathway in wheat. This study could help the investigators to make an informed decision to edit wheat genome for high/low amylose content using gene-editing technologies.

Abstract 282: MicroRNAs located at chromosome 8p21-22 region as novel factors underlying racial disparities in prostate cancer aggressiveness

Abstract The mechanistic basis of racial disparities in prostate cancer (PCa) aggressiveness between African American (AA) and Caucasian (CA) men are not yet fully understood. Further, molecular biomarkers to predict aggressive disease at its onset have been studied primarily in CAs, the relevance of these biomarkers to AAs are largely unknown. We propose a novel, paradigm-shifting hypothesis that expression of microRNA (miRNA) genes-miR-3622a and miR-383-located within frequently deleted chr8p21-22 region constitute important molecular factors contributing to racial disparities in PCa aggressiveness in AA vs CA men. Chromosome 8p deletions are a frequent alteration of PCa genome, with a common region of loss of heterozygosity at the chr8p21-22 locus. We previously showed that this region is associated with a set of tumor suppressive miRNAs (miR-3622a/b, -383, -4288) that are downregulated in PCa with important causal roles in tumor progression, recurrence and metastasis. Chr8p deletions increase significantly with tumor grade and are associated with poor prognosis. These deletions are well studied in CA men. However, there have been conflicting reports on frequency of chr8p deletions in AA men. In view of these conflicting studies and in light of our paradigm-shifting studies establishing the link between chr8p deletions and loss of miRNA genes in this region, we examined the expression of these miRNAs in AA vs CA clinical PCa tissues. We found that miR-3622a and miR-383 expression is significantly lower in AA men as compared to age, tumor stage and grade matched CAs. To examine if these miRNAs constitute causal factors contributing to PCa racial disparities, we overexpressed miR-3622a/miR-383/control miR in AA and CA PCa cell lines followed by functional assays. We found that these miRNAs exert more prominent effects on cellular proliferation and apoptosis in AA cell lines. To understand molecular mechanisms contributing to disparate functional effects of these miRNAs in AA vs CA cell lines, we examined potential miRNA target genes and found that miR-3622a regulates PCa stem cell marker, CD44 while miR-383 represses LEF1 and ZEB1 in a race-specific manner. Further, we determined the mechanistic basis of differential expression of miR-3622a in AAs vs CAs by examining copy number alterations (CNAs) and methylation at this locus using PCa clinical samples. Our analyses showed that while this locus is genomically altered at a lower frequency in AAs, significantly increased frequency of miRNA promoter hypermethylation was found in AAs as compared to CAs. In conclusion, our data suggests that miRNAs located in frequently deleted chr8p21-22 region constitute important causal factors contributing to racial disparities in PCa aggressiveness and clinical outcomes and are potential biomarkers to predict PCa aggressiveness and tumor recurrence in AA men. Citation Format: Divya Bhagirath, Nikhil Patel, Santu Ghosh, Roni Bollag, Sharanjot Saini. MicroRNAs located at chromosome 8p21-22 region as novel factors underlying racial disparities in prostate cancer aggressiveness [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 282.

Azoospermic infertility is associated with altered expression of DNA repair genes

Compelling evidence suggest that germs cells are predominantly sensitive to DNA damaging agents in comparison to other cells. High fidelity DNA repair in testicular cells thus becomes indispensable to preserve the genomic integrity for passing on to the progeny. Compromised DNA repair machinery in the testicular cells may result in impaired spermatogenesis and infertility. It remains unclear if the alterations in the expression of DNA repair genes correlate with azoospermia and male infertility. In the present study, 54 non-obstructive azoospermic infertile patients with hypospermatogenesis (HS, n = 26), maturation arrest (MA, n = 15), Sertoli cell only syndrome (SCOS, n = 13) and 14 controls with obstructive azoospermia, but normal spermatogenesis were recruited. Expression profiling of 84 DNA repair genes in testicular biopsy samples was performed using PCR array. Out of 84 genes, 27, 64 and 28 genes showed >5 fold down-regulation in the HS, MA and SCOS groups, respectively. On the basis of differential expression and their functional significance in spermatogenesis, ten genes (MSH2, BRIP1, CCNH, LIG4, MGMT, NTHL1, PMS1, DMC1, POLB and XPA) were selected for validation of transcript levels in a higher number of cases using RT-PCR, which corroborated the findings of array. Four genes (MSH2, LIG4, PMS1 and DMC1) were analyzed for protein levels using immunohistochemistry, which further validated the loss of DNA repair gene expression. Caspase-3 immunostaining showed that the loss of DNA repair correlated with increased testicular apoptosis in patients. Maturation arrest showed the highest apoptotic index with maximum number of downregulated genes. We conclude that the loss of DNA repair genes expression in testis correlates with increased apoptosis, azoospermia and infertility.

Abstract 2998: Identification of AURKA- and PTPRJ-interacting human colorectal cancer susceptibility alleles.

Abstract Colorectal cancer (CRC) causes nearly 50,000 deaths in the United States each year and therefore represents the third leading cause of cancer-related deaths. The identification of susceptibility variants that interact to modulate risk for sporadic CRC would have immense value as a tool to identify individuals with increased genetic predisposition to disease. Polymorphisms in Aurora Kinase A (AURKA) and Protein Tyrosine Phosphatase Receptor Type J (PTPRJ) have been shown to increase CRC risk in human case-control studies. Crosses between mice resistant and susceptible to chemically induced skin cancer have led to the identification of two loci, Skts1 and Skts5, each of which statistically interacts with the Aurka locus to promote skin cancer in a murine model. Using a similar approach, the mouse Ptprj locus has been shown to interact synergistically with the loci Scc5 and Scc13 to increase colon cancer susceptibility. Array comparative genomic hybridization studies in human colorectal tumor DNAs have shown high frequencies of genomic copy number gains or losses at the human orthologs of Skts1, Skts5, Scc5, and Scc13. Our goal is to identify sporadic CRC risk variants and to characterize genetic interactions at the AURKA- and PTPRJ-interacting loci. We hypothesize that the human orthologs of these mouse CRC susceptibility loci will interact genetically with AURKA and PTPRJ to influence CRC risk. By generating normal colon transcriptome data from CRC-susceptible and CRC–resistant mouse strains using RNA-Seq, we prioritized candidate genes on the basis of differential expression, differential splicing or potentially functional polymorphisms. Tagging single nucleotide polymorphisms (tagSNPs) for the human orthologs of these genes were tested for allele-specific imbalance by Sequenom MassARRAY quantitative genotyping of 194 paired human colorectal tumor/non-tumor DNA samples. TagSNPs exhibiting point-wise p-values < 0.1 were further tested in a replication sample set of 296 paired colorectal tumor/non-tumor DNA samples. Among ∼900 tagSNPs tested, 75 polymorphisms reproducibly showed evidence of allele-specific imbalance (point-wise p-values < 0.05). Our data indicate that allele-specific imbalance occurs in candidate genes (e.g. KLF13, HDAC9, FBN2, SNX10 and PRDM5) whose murine orthologs map to cancer susceptibility loci. Our next step is to explore expression patterns of these candidate genes in human colorectal tumor and non-tumor tissues and to investigate the effects of gene silencing or overexpression on critical cancer phenotypes such as proliferation, apoptosis, and migration in mouse and human colon cancer cell lines. Additionally, an ongoing human case-control association study of 6000 colorectal cancer cases and 6000 controls is expected to yield results shortly, which will be presented at the 2013 AACR meeting. Citation Format: Madelyn M. Gerber, Nathan P. Schulz, Mehmet Deveci, Heather L. Hampel, Umit V. Catalyurek, Albert de la Chapelle, Amanda Ewart Toland. Identification of AURKA- and PTPRJ-interacting human colorectal cancer susceptibility alleles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2998. doi:10.1158/1538-7445.AM2013-2998

Mammosphere Culture of Established Cell Lines Does Not Enrich for a More Tumorigenic Breast Cancer Stem Cell Population.

Abstract Background:The cancer stem cell model suggests that tumors arise from and are maintained by a relatively small proportion of cells, termed cancer stem cells. According to this model, only these cells have the capacity for self-renewal and the ability to repopulate the tumor bulk by giving rise to the phenotypically heterogeneous populations of cells that comprise the primary tumor. Evidence to support the cancer stem cell model has been provided by isolation of subpopulations of cells on the basis of differential expression of cell surface markers, followed by functional transplantation into animal models. These studies reveal that only a small subset of CD44+/CD24-/low cancer cells retain the ability to form new tumors after transplantation in immunodeficient mice, consistent with the cancer stem cell model. These results indicate that it may be necessary to target and eliminate cancer stem cells in order to eradicate tumors.Material and Methods:Previous studies have suggested that only breast stem cells have the capacity to proliferate in suspension as spherical colonies termed mammospheres, and that mammosphere derived breast cancer cells are CD44+/CD24-/low and able to form tumors that reproduce the heterogeneity of the original primary tumor when injected into the mammary fat pad of immunodeficient mice. This indicates that mammospheres are comprised of breast cancer stem cells. To identify and characterize breast cancer stem cells more completely, the mammosphere culture system was used to isolate and propagate breast cancer stem cells from three basal breast cancer cell lines (BT549, MDA231, MDA436) and three luminal breast cancer cell lines (MCF7, SKBR3, T47D). The tumorigenicity of the cultured mammospheres and parental cell lines was examined using dilution studies in immunodeficient mice and gene expression profiles were studied by microarray analysis.Results and Discussion:No significant differences in tumorigenicity were observed between breast cancer cells cultured as mammospheres versus standard culture conditions. Furthermore, no consistent differences in gene expression profiles were observed between mammosphere and parental cells across the panel of cancer cell lines. These results suggest that mammosphere culture is not capable of significantly enriching for a more tumorigenic breast cancer stem cell population from established breast cancer cell lines. This may indicate that cancer subpopulations that exist in primary tumors are lost during the long term culture of breast cancer cell lines and that these systems may not be a suitable model for breast cancer stem cell studies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 502.

Bayesian ranking and selection methods using hierarchical mixture models in microarray studies

The main purpose of microarray studies is screening to identify differentially expressed genes as candidates for further investigation. Because of limited resources in this stage, prioritizing or ranking genes is a relevant statistical task in microarray studies. In this article, we develop 3 empirical Bayes methods for gene ranking on the basis of differential expression, using hierarchical mixture models. These methods are based on (i) minimizing mean squared errors of estimation for parameters, (ii) minimizing mean squared errors of estimation for ranks of parameters, and (iii) maximizing sensitivity in selecting prespecified numbers of differential genes, with the largest effect. Our methods incorporate the mixture structures of differential and nondifferential components in empirical Bayes models to allow information borrowing across differential genes, with separation from nuisance, nondifferential genes. The accuracy of our ranking methods is compared with that of conventional methods through simulation studies. An application to a clinical study for breast cancer is provided.

Early gene expression in the organ of Corti exposed to gentamicin

Studies have demonstrated different pathogenetic key factors in gentamicin-induced hair cell death. The production of reactive oxygen species (ROS), as well as apoptosis-related genes, play a critical role. However, a coordinated large-scale investigation of gene expression in the organ of Corti (OC) exposed to gentamicin has not yet been conducted. Here we used DNA microarray technology to compare the expression profile of OC exposed to gentamicin to the expression profile of untreated OC. The OCs of Sprague Dawley rats were dissected and the basal turns were cultured. Two-thirds of the explants were then exposed to l00 μM gentamicin, for 4 and 8 h, while one-third of the explants remained in culture medium alone. Gene expression was analyzed using DNA microarray technology and the dChip software package. Based on the results, the 4-h time-point was chosen for further analysis. In these assays, out of 8800 genes, 12 genes were identified on the basis of differential expression in the OC exposed to gentamicin vs. control OC. The identity of these genes suggests that the response of the OC to the gentamicin challenge involves down-regulation of specific gene families in order to alleviate ROS and N-methyl- d-aspartate (NMDA) receptor-mediated cellular stress.

Collagenase-3 expression is associated with advanced local invasion in human squamous cell carcinomas of the larynx.

Collagenase-3 (MMP-13) is a matrix metalloproteinase recently identified on the basis of differential expression in normal breast tissues and in breast carcinoma. To date, collagenase-3 expression has been reported only in breast carcinomas and in articular cartilage of arthritic patients; the presence and possible implication of this enzyme in the progression of other malignant tumours are unknown. In this study collagenase-3 mRNA expression has been analysed by northern blot in a series of 35 matched squamous cell carcinomas of the larynx and the corresponding adjacent non-neoplastic tissues. In addition, mRNA expression of membrane type 1-matrix metalloproteinase (MT1-MMP) and gelatinase A, two matrix metalloproteinases which have the ability to activate collagenase-3 in vitro, was also examined in the same cases. No collagenase-3 expression was detected in any of the 35 normal mucosae, but collagenase-3 mRNA was observed in 20 of the 35 carcinomas (57 per cent). Western blot analysis revealed the presence of collagenase-3 protein in those carcinomas with high levels of mRNA expression, whereas no protein was detected in the carcinomas with negative mRNA expression, or in any of the normal tissues. The protein was localized predominantly in tumour epithelial cells. Collagenase-3 expression correlated significantly with better histological differentiation of the tumours (p = 0.026), as well as with advanced local invasion (p = 0.026). Collagenase-3 upregulation was also significantly associated with MT1-MMP and gelatinase A overexpression. These findings suggest that collagenase-3 expression may contribute to the progression of a significant subset of squamous cell carcinomas of the larynx and that its coordinate overexpression with MT1-MMP and gelatinase A may have a cooperative effect in the progression of the tumours.

Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells

The aim of the present study was to determine whether stem cell factor (SCF) and erythropoietin (EPO) act differently on defined subsets of progenitor cells, and if potential differences correlate with the receptor density on each subset. To investigate this possibility directly, we optimized conditions for the identification and purification of homogeneous progenitor cell subpopulations from human bone marrow. Populations containing 40% and 44% colony forming cells (CFCs) with 99% and 95% purity for the granulomonocytic and erythroid lineage, respectively, were sorted on the basis of differential expression of CD34, CD64, and CD71. In addition, a population containing 67% CFCs, of which 29-43% were CFU-MIX, was sorted from CD34hi CD38loCD50+ cells. Purified progenitor cell subsets were compared directly for responsiveness to SCF and EPO using a short-term proliferation assay. Expression of the receptors for SCF and EPO were then examined on each subset using a flow cytometer modified for high-sensitivity fluorescence measurements. The results show that EPO induces extensive proliferation of erythroid progenitor cells, but has no effect on the proliferation or survival of primitive or granulomonocytic progenitors, even when used in combination with other cytokines. The majority of erythroid progenitor cells furthermore stained positively with anti-EPO receptor (EPO-R) monoclonal antibodies, whereas other progenitor cells were negative. SCF alone induced extensive proliferation of erythroid progenitor cells, and had a stronger synergistic effect on primitive than on granulo-monocytic progenitors. In spite of these differences in SCF activity, there were no significant differences in SCF-R expression between the progenitor subsets. These results suggest that the selective action of EPO on erythropoiesis is determined by lineage-restricted receptor expression, whereas there are additional cell-type specific factors that influence progenitor cell responses to SCF.

Cell surface antigens of human renal cancer defined by mouse monoclonal antibodies: identification of tissue-specific kidney glycoproteins.

Seventeen monoclonal antibodies derived from fusions with spleen cells of mice immunized with established culture lines of renal cancers identified nine cell-surface antigenic systems. Six of the systems (gp160, S25, gp120r, gp120nr, gp115, and V1) represent antigens not previously described. The other three systems are related to HLA-A, -B, and -C heavy chain and A and B blood group antigens. The most restricted of the newly described antigens are gp160, S25, and gp120r. These determinants are found only on cells of renal origin, both normal and malignant, and represent differentiation antigens of human kidney. In addition to the difference in the molecular weight of two of these antigens, gp160, S25, and gp120r can be distinguished on the basis of differential expression on a panel of cultured renal cancers and normal kidney epithelium and fetal kidney cells. Glycoproteins bearing gp120r share a determinant with renal gp120nr (as indicated by sequential precipitations with monoclonal antibodies that detect gp120r and gp120nr), but gp120nr is found on a broader range of cell types, including fibroblasts and cell lines derived from lung, bladder, and colon cancers. The two other new systems, gp115 and V1, have characteristics of broadly occurring differentiation antigens but can be distinguished from each other and from gp120nr by differences in molecular weight, heat stability (V1 is a heat-stable determinant), and differential expression on cell types of diverse origin.