Comparative Gene Profiling Research Articles

Spheres Derived from Lung Adenocarcinoma Pleural Effusions: Molecular Characterization and Tumor Engraftment

Malignant pleural effusions (MPEs) could represent an excellent source to culture a wide variety of cancer cells from different donors. In this study, we set up culture conditions for cancer cells deriving from MPEs of several patients affected by the most frequent form of lung cancer, namely the subset of non small cell lung cancers (NSCLC) classified as Lung Adenocarcinomas (AdenoCa) which account for approximately 40% of lung cancer cases. AdenoCa malignant pleural effusions gave rise to in vitro cultures both in adherent and/or in spheroid conditions in almost all cases analyzed. We characterized in greater detail two samples which showed the most efficient propagation in vitro. In these samples we also compared gene profiles of spheroid vs adherent cultures and identified a set of differentially expressed genes. Finally we achieved efficient tumor engraftment in recipient NOD/SCID mice, also upon inoculation of small number of cells, thus suggesting indirectly the presence of tumor initiating cells.

Gene Profiling of Mta1 Identifies Novel Gene Targets and Functions

BackgroundMetastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1.MethodsGene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes.Significance/ConclusionThis study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define “bona fide” Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions.

Abstract 5326: CCL2 is critical for immunoresistance in association with cancer EMT

Abstract Purpose: Epitherial-to-mesenchymal transition (EMT) is a key step toward cancer metastasis, which is still a critical issue in cancer therapy. We previously reported that Snail-induced EMT accelerates cancer metastasis through multiple immunosuppressions using murine and human melanoma cells. However, the molecular mechanism is not completely understood. In this study, we confirmed the Snail-induced immunosuppressions using various human cancer cell lines, and further elucidated the mechanisms by identifying the specific mediators. Results: We first established human pancreatic cancer cell line Panc-1 cells showing typical EMT features such as reduced adhesion and enhanced invasion after transduction with snail gene, which is a transcription factor governing EMT. When human PBMCs were cocultured with the Snail transfectants, CD11c+ cell differentiation was significantly inhibited, and immunosuppressive CD4+FOXP3+ Treg cells were increased as similarly observed using Snail+ melanoma, suggesting the general features of the Snail-induced immunosuppressive mechanisms. We next conducted GeneChip analysis to identify other possible mechanisms by comparing gene profiles between the Snail transfectant and the parental cells. CCL2 was significantly upregulated in various Snail+ tumor cells after transduction with snail or treatment with TGFβ, a potent EMT inducer. CCL2 not only enhanced tumor invasion but also caused immunosuppressions through induction of Lipocalin 2 (LCN2), which was reported to induce apoptosis in various cells. CCL2 also increased LCN2 receptor Megalin and immunosuppressive molecules IDO and PD-L1 in DCs; in turn, LCN2 increased CCR2 in DCs. Utilizing the cooperative mechanism induced by CCL2 and LCN2, Snail+ cancer cells severely hampered APC function through induction of apoptosis and PD-L1/IDO expressions followed by Treg generation and then CTL attenuation. CCL2 blockade utilizing the specific siRNA or antibody significantly inhibited both tumor invasion and those immunosuppressions in vitro. Intratumoral injection with these anti-CCL2 reagents significantly inhibited tumor growth and metastasis by enhancing anti-tumor response systemically in vivo. Conclusion: These results demonstrate that CCL2 plays a vital role in the Snail-induced immunosuppressions. CCL2 blockade in tumor microenvironment may be an attractive strategy to overcome metastasis and immunoresistance of Snail+ cancers by restoring immunocompetence in cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5326.

Gene expression profiling of human oocytes at different maturational stages and after in vitro maturation

The purpose of this study was to catalog genes expressed in human oocytes at germinal vesicle (GV) or metaphase II (MII) stage and to compare gene profiles between oocytes matured in vivo (in vivo-MII) and in vitro (IVM-MII). University-based research utilizing unfertilized oocytes analyzed for > 29,000 genes with RNA amplification and microarray. GV, in vivo-MII, and IVM-MII oocytes expressed 12,219, 9735, and 8510 genes, respectively. There was extensive overlap among the 3 groups, but also some significant differences. In particular, in vivo-MII and IVM-MII oocytes shared very similar patterns of gene expression. However, some immature patterns of expression, reminiscent of GVs, persisted in IVM-MIIs. In vitro maturation is an attractive strategy for IVF treatment; however, current IVM methods produce oocytes that perform poorly in the context of IVF. Data from the current study suggest that although IVM-MII oocytes closely resemble in vivo-MII oocytes for cellular pathways related to nuclear maturity, several pathways associated with cytoplasmic functions continue to be expressed in an immature manner. Additionally, IVM-MII oocytes have differences in the expression of genes related to cellular storage and homeostasis. Differentially expressed genes/pathways provide clues for the optimization of IVM techniques.

Comparative Gene Profiling of Acute Myeloid Leukemia (AML) and Malignant Melanoma (MEL) Cell Lines Exposed to the MEK Inhibitor PD0325901 Reveals Common Effectors of the MEK/ERK Kinase Module.

The MEK/ERK kinase module is frequently deregulated in human malignancies, including AML and MEL, and represents a promising therapeutic target. We have recently demonstrated that PD0325901, a selective MEK inhibitor, potently inhibits the growth of both AML and MEL cell lines (IC50 5–20 nM), through a combination of inhibition of cell cycle progression and induction of apoptosis. In order to identify functionally relevant targets of the activity of the MEK/ERK kinase module, we compared the gene expression profiles of the PD0325901-sensitive cell lines OCI-AML3 (AML, harboring a NPM gene mutation) and M14 (MEL, harboring a BRAF mutation), after exposure to vehicle control or 10 nM PD0325901 for 6 and 24h, using the Affymetrix U133A 2.0 and U133 Plus 2.0 gene chips, respectively. All analyses were conducted using the dChip software. In both models, the unsupervised approach showed a tight clustering that was both treatment- and time-dependent; unsupervised and supervised analyses highlighted that the greatest PD0325901-induced variations in gene expression were observed at 24h and 6h in the OCI-AML3 and M14 models, respectively. Comparison of the results obtained in the OCI-AML3 and M14 cell lines showed that 5 genes were consistently modulated after 6h, including CDC25A, cyclin G2, and DUSP6. Cyclin G2 and DUSP6 were also modulated after 24h, together with cyclin D1, TIMP3, and SEMA6A. On the contrary, MAFB, selected in both models, displayed a discordant behavior, being down-regulated upon treatment in OCI-AML3 and up-regulated in M14: this is consistent with the role of MAFB as an important regulator of hematopoiesis, while it does not appear to play a role in melanogenesis. Given the ability of PD0325901 to cause apoptosis, partly through induction of mitochondrial dysfunction in both AML and MEL models, we next investigated whether MEK inhibition specifically modulated the expression of genes related to mitochondrial homeostasis: also in this specific context, the effects were more rapidly induced in M14 than in OCI-AML3, with the greatest changes observed within 6h in the M14 cell line. Two genes were down-regulated after 24h in both models: PPIF, supposedly involved in apoptosis induction, and GRPEL1, on which little is currently known. In addition to mitochondrion-specific changes in gene expression, expression of the mitochondrial protein CPT1A significantly decreased (2-fold), as assessed by WB, after 6 h of exposure of OCI-AML3 cells to 10 nM PD0325901, under which conditions ß-oxidation was also suppressed by the drug. Overall, these results indicate that MEK inhibition by PD0325901 results in the modulation of the expression of a small number of genes that are common to ontogenetically distant tumor models, thereby representing attractive candidates for a shared, functionally relevant, MAPK activation signature; moreover, MEK inhibition results in profound intracellular metabolic changes that might importantly contribute to the anti-neoplastic effect of PD0325901 in AML and, possibly, in other tumor models. MM and SC equally contributed to the work presented.

Critical Care: Applying Genomics to Inflammation Outcomes

What do gunshot wounds, burns, heart attacks, arthritis, asthma, and cancer all share in common? Apart from inflicting misery, these conditions—and others too—involve inflammation, an immune response to injury and infection that normally protects, but sometimes endangers or kills patients. Caused by immune cells accumulating at a site of injury, inflammation typically guards against infection and speeds recovery; it is a critical process and, per se, does not cause disease. But unchecked inflammation that spreads or fails to subside poses chronic and acute health risks for millions of people. Asthma patients, for instance, can’t breathe because inflammatory compounds cause airway linings to swell and mucus to spread in the lungs. Inflammation also exacerbates cancer, scientists believe, by facilitating the proliferation of abnormal cells. An acute condition called sepsis—caused when infection or inflammation spills into the bloodstream—produces organ failure and shock in critically ill patients. Up to 215,000 Americans die from sepsis every year, according to the National Institute of General Medical Sciences. Worldwide, sepsis is estimated to kill 1,400 people each day, according to a consensus document published in the June 1992 issue of Chest. In light of its implications, inflammation has become one of the hottest areas in biomedical research. J. Perren Cobb, a professor of surgery and genetics at Washington University in St. Louis, says a wide array of medical specialties stand to benefit from these investigations. “Inflammation is a major unifying syndrome, the investigation of which provides opportunities for multidisciplinary convergence,” he explains. “Studies of inflammation cut across all the domains at the NIH; it’s a fundamental process in human biology that ties everything together.” Growing evidence suggests that genetic factors drive key aspects of an individual’s inflammatory outcome. Scientists studying inflammation are trying to identify the genes that drive inflammation as well as biomarkers from throughout the course of inflammation. Stephen Chanock, who heads the Section on Genomic Variation in the Pediatric Oncology Branch at the National Cancer Institute, emphasizes that the current critical care orientation of this research has broad multidisciplinary implications that extend to environmental health. “Injuries represent the ultimate gene–environment interactions,” he explains. “Usually environmental health focuses on chronic exposures, but in this case we’re studying environmental insults that are more dangerous and intense. So, the ‘environment’ in environmental health isn’t just about pollution, it’s also experiential. We’re developing practical methods for looking at inflammation that will ultimately be applied to larger public health issues.”

167 ISOLATION AND COMPARATIVE PROFILING OF HUMAN ADIPOSE-DERIVED ADULT STEM CELLS

The stromal compartment of mesenchymal tissues is thought to harbor stem cells that display extensive proliferative capacity and multilineage potential. However, despite their potential impact in the field of regenerative medicine, little is known about the biology of stromal stem cells prior to culture. After removing adipocytes and erythrocytes from collagenase digested human adipose tissue, we identified two cell populations using flow cytometry which shared expression of stem cell markers SH2 and CD34, but lacked the phenotypic characteristics of leukocytes (CD45−). However, they were found to be discernible based on CD31 expression, a marker for endothelial cells. Using CD31 conjugated magnetic beads, we separated these cells (CD45-CD31− and CD45-CD31+) from three patients and compared global gene expression profiles using an Affymetrix platform. The prominant feature of CD45-CD31+ cells was the up-regulation of genes associated with endothelial cells. By contrast, CD45-CD31− cells were found to overexpress transcripts involved in cell cycle quiescence and cell signaling elements including those of the WNT pathway thought to be important for maintaining the stem cell state. Upon culture in DMEM/F12 with 20% FCS, only CD45-CD31− cells were capable of adhering to plastic and forming colonies. These cells with fibroblastic morphology met the key criterion of stem cells, the ability to proliferate while retaining the capacity to differentiate into mature tissues. Under appropriate inductive conditions, they were found to exclusively form bone, cartilage, adipose and neuronal-like tissues in vitro. Clonal cell lines generated from individually cultured CD45-CD31− cells displayed multilineage and proliferative capacity, validating our conclusion that they are true stem cells and not simply committed progenitors. We then undertook extensive comparative profiling of CD45-CD31− cells with their cultured counterparts to examine changes that stromal stem cells undergo during culture. Except for the disappearance of CD34, flow cytometry analysis using 52 antibodies revealed little change in cell surface phenotype as a result of culture. However, comparative global gene profiling revealed extensive down-regulation of many genes during culture. These included cell cycle arresting genes, as expected, and genes encoding elements involved in cell signaling including those belonging to the tumor necrosis factor, interleukin, transforming growth factor and chemokine families. The consequences of these changes remain unknown, but ultimately may affect the potential use of adipose tissue stem cells in regenerative medicine.

Analysis of Gene Expression in Patients with Acute Graft Versus Host Disease (GVHD) Following Hematopoietic Stem Cell Transplantation (HSCT).

To better understand the cellular events that precede onset of clinically significant acute GVHD, a complication of allogeneic HSCT, we compared global gene expression profiles in patients 3 (days 18–22) and 4 (days 28–32) weeks after transplant. Patients in this study underwent myeloablative-conditioning regimen prior to receiving a T cell replete PBSCT from a related (n=9) or unrelated donor (5 HLA matched and 4 mismatched). Blood was obtained prospectively at scheduled times (prior to administration of glucocorticosteroids). RNA was isolated from nucleated blood cells and biotin-labeled cRNA hybridized on Affymetrix HG-U133A chips. MAS 5.0 software was used to extract gene expression values.We initially compared gene expression profiles between 15 patients 3 weeks post-HSCT and 10 normal controls. A total of 1176 genes were differentially expressed with statistical criterion of NFD (number of false discovery) equal to 10. Gene profiles for these 1176 genes were compared between 8 patients who subsequently developed GVHD within 1–5 days and 7 patients who remained GVHD free for 90 days. A limited number of genes were differentially expressed with NFD=1: 3 genes in GVHD patients showed increased expression and 6 showed decreased expression.A second set of experiments was performed to compare changes occurring within individual patients over an interval of 7 days (between weeks 3 and 4) prior to diagnosis of clinically significant GVHD (onset between days 27–32). We used a pair-wise comparison with selection criterion NFD=1. Increased expression prior to GVHD was observed in 55 genes and decreased expression in 88 genes. Approximately 50 of these genes were associated with inflammation and cellular stress response. Using the same statistical criterion we compared gene profiles between weeks 3 and 4 for 3 patients who remained GVHD-free for at least 90 days. Fewer changes were observed with increased expression occurring in 6 genes and decreased expression in 14 genes. These differentially expressed genes did not overlap with the candidate genes associated with the development of GVHD. Genes showing expression changes in GVHD included:IncreasedDecreasedInflamamtory ResponseIFN-α10, IL8, IL17Transcription FactorsNFATC1GATA3Cell Surface/Signal TransductionCD6, CD7, CD8, TCR-interacting molecule, MAP4K1, TNFRSF25,Effectors MoleculesGRMMAICD/ApoptosisTOSO, BAXCellular Stress ResponseDDAH1DLAT, PKC1, COX5BThese results suggest that extensive complex gene expression changes occur among nucleated blood cells during the early post-transplant period presumably due to extensive alterations in cellular activation occurring during reconstitution. The preliminary results of the longitudinal analysis of changes occurring within individual patients indicate that early post-transplant studies are feasible and that they may be informative for yielding insight into the molecular events associated with development of clinically significant GVHD. These data also indicate a paradoxical decrease in certain T cell associated genes in GVHD. However alloimmune induced T cell activation may lead to AICD and previous studies have demonstrated increased apoptosis among peripheral blood T cells in GVHD patients. Further studies including gene expression profiling of isolated T cells will be necessary to determine if this approach can be useful in identifying a molecular “signature” for GVHD that may be useful for diagnosis and monitoring.

Cooperative signaling between cytokine receptors and the glucocorticoid receptor in the expansion of erythroid progenitors: molecular analysis by expression profiling

AbstractErythroid progenitors undergo renewal (proliferation without apparent differentiation) in response to erythropoietin (Epo), stem cell factor (SCF), and glucocorticoids (dexamethasone) (Dex). SCF and Dex cooperate with Epo to promote proliferation and inhibit differentiation of erythroid progenitors, while Epo alone is required to protect erythroid cells from apoptosis during terminal red cell maturation. To examine the mechanism of the synergistic interactions of Epo, SCF, and Dex, we analyzed gene expression patterns using DNA chip–based large-scale comparative gene profiling using microarrays enriched in hematopoietic transcripts or containing randomly selected genes. Differentially regulated genes were validated by real-time reverse transcription–polymerase chain reaction (RT-PCR). The results reveal cooperative regulation of gene expression by glucocorticoids and Epo/SCF on a number of genes, such as CIS, BTG1, VDUP1, CXCR4, GILZ, and RIKEN29300106B05. While Epo and SCF never showed opposite effects on gene expression, Dex either enhanced or attenuated the effect of Epo and/or SCF. Several glucocorticoid receptor (GR)–target genes were regulated by Dex only in the presence of Epo and/or SCF, suggesting that the GR functions in the context of a larger transactivation complex to regulate these genes. The data also suggest that modulation of cytokine-induced signals by the GR is an important mechanism in erythroid progenitor renewal.

Transcriptome profiling to identify genes involved in peroxisome assembly and function.

Yeast cells were induced to proliferate peroxisomes, and microarray transcriptional profiling was used to identify PEX genes encoding peroxins involved in peroxisome assembly and genes involved in peroxisome function. Clustering algorithms identified 224 genes with expression profiles similar to those of genes encoding peroxisomal proteins and genes involved in peroxisome biogenesis. Several previously uncharacterized genes were identified, two of which, YPL112c and YOR084w, encode proteins of the peroxisomal membrane and matrix, respectively. Ypl112p, renamed Pex25p, is a novel peroxin required for the regulation of peroxisome size and maintenance. These studies demonstrate the utility of comparative gene profiling as an alternative to functional assays to identify genes with roles in peroxisome biogenesis.