Cell-related Gene Expression Research Articles

The E3 Ubiquitin Ligase GRAIL Regulates T Cell Tolerance and Regulatory T Cell Function by Mediating T Cell Receptor-CD3 Degradation

T cell activation is tightly regulated to avoid autoimmunity. Gene related to anergy in lymphocytes (GRAIL, encoded by Rnf128) is an E3 ubiquitin ligase associated with T cell tolerance. Here, we generated and analyzed GRAIL-deficient mice and found they were resistant to immune tolerance induction and exhibited greater susceptibility to autoimmune diseases than wild-type mice. GRAIL-deficient naive T cells, after activation, exhibited increased proliferation and cytokine expression than controls and did not depend on costimulation for effector generation. Moreover, GRAIL-deficient regulatory T (Treg) cells displayed reduced suppressive function, associated with increased Th17 cell-related gene expression. GRAIL-deficient naive and Treg cells were less efficient in downregulating T cell receptor (TCR)-CD3 expression after activation and exhibited increased NFATc1 transcription factor expression; GRAIL expression promoted CD3 ubiquitinylation. Our results indicate that GRAIL, by mediating TCR-CD3 degradation, regulates naive T cell tolerance induction and Treg cell function.

Abstract 3331: New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in colon and prostate CSCs

Abstract In vast majority of human cancers, malignant stem cells (CSCs) are responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that colon and prostate CSCs are not only functionally and morphologically different from their bulk counterparts, but themselves may represent heterogeneous phenotypic populations. In contrast to the major (bulk) colon and prostate cancer cells expressing low levels of CD133 and CD44, cells with a high combined expression of these markers, as well as colon cancer cells lacking CD133, possessed several characteristic stem cell features, including high tumor initiating potential, profound self-renewal capacity, and an ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating multicellular spheroids induced by candidate phenotypic cell populations; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214 in 3D culture. Experimental: Selected CSC phenotypes were isolated from highly invasive colon cancer HCT116 cells and prostate cancer PC3MM2 cells. A stem cell-specific PCR array (SABiosciences) revealed that 3D spheroids induced by either colon CD133high/CD44high or CD133neg cells, as well as by CD133high/CD44high prostate cancer cells expressed profound up-regulation of stem cell-related genes compared to their differentiated counterparts (68%, 56% and 61%, correspondently). The FACS analysis has shown that both colon and prostate spheroids contained minority cell populations with high levels of expression of Oct4, Sox2, c-Myc and Nanog, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 0.01-0.1 μg/ml for 48 hr not only induced growth inhibition and apoptotic cell death in these two types of cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities. Summay: We report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon and prostate cancer spheroids induced by and enriched with drug resistant tumorigenic cell populations and efficiently inhibited expression of the majority of stem cell-related genes. Conclusion: Our data indicates that the long-term efficacy of the SB-T-1214 against several drug resistant tumors in vivo may be explained by down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells. 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 3331.

New-generation taxoid SB-T-1214 inhibits stem cell-related gene expression in 3D cancer spheroids induced by purified colon tumor-initiating cells

BackgroundGrowing evidence suggests that the majority of tumors are organized hierarchically, comprising a population of tumor-initiating, or cancer stem cells (CSCs) responsible for tumor development, maintenance and resistance to drugs. Previously we have shown that the CD133high/CD44high fraction of colon cancer cells is different from their bulk counterparts at the functional, morphological and genomic levels. In contrast to the majority of colon cancer cells expressing moderate levels of CD133, CD44 and CD166, cells with a high combined expression of CD133 and CD44 possessed several characteristic stem cell features, including profound self-renewal capacity in vivo and in vitro, and the ability to give rise to different cell phenotypes. The present study was undertaken for two aims: a) to determine stem cell-related genomic characteristics of floating 3D multicellular spheroids induced by CD133high/CD44high colon cancer cells; and b) to evaluate CSC-specific alterations induced by new-generation taxoid SB-T-1214.ResultsSelected CSC phenotype was isolated from three independent invasive colon cancer cell lines, HCT116, HT29 and DLD-1. A stem cell-specific PCR array assay (SABiosciences) revealed that colonospheres induced by purified CD133high/CD44high expressing cells display profound up-regulation of stem cell-related genes in comparison with their bulk counterparts. The FACS analysis has shown that the 3D colonospheres contained some minority cell populations with high levels of expression of Oct4, Sox2, Nanog and c-Myc, which are essential for stem cell pluripotency and self-renewal. Single administration of the SB-T-1214 at concentration 100 nM-1 μM for 48 hr not only induced growth inhibition and apoptotic cell death in these three types of colon cancer spheroids in 3D culture, but also mediated massive inhibition of the stem cell-related genes and significant down-regulation of the pluripotency gene expression. PCR array and FACS data were confirmed with western blotting. Importantly, viable cells that survived this treatment regimen were no longer able to induce secondary floating spheroids and exhibited significant morphological abnormalities.ConclusionsWe report here that a new-generation taxoid SB-T-1214 possesses significant activity against colon cancer spheroids induced by and enriched with drug resistant tumorigenic CD133high/CD44high cells and efficiently inhibited expression of the majority of stem cell-related genes. Our data indicates that the previously observed long-term efficacy of SB-T-1214 against drug resistant colon tumors in vivo may be explained by the down-regulation of multiple stem cell-related genes in the tumorigenic cell population, in addition to its known efficacy as a mitotic poison against proliferating cancer cells.

MMP-13 Plays a Role in Keratinocyte Migration, Angiogenesis, and Contraction in Mouse Skin Wound Healing

Matrix metalloproteinases (MMPs) have been implicated in wound healing. To analyze the roles of MMP-9 and MMP-13 in wound healing, we generated full-thickness cutaneous wounds in MMP-9 knockout (KO), MMP-13 KO, MMP-9/13 double KO, and wild-type mice. Macroscopic wound closure was delayed in all of the KO mice, as compared with wild-type mice. The rate of re-epithelialization was significantly delayed in MMP-9 KO and MMP-13 KO mice and remarkably delayed in MMP-9/13 double KO mice, as compared with wild-type mice. Both MMP-9 and MMP-13 were expressed by the leading edges of epidermal cells in wild-type mice, and the migration of keratinocytes was suppressed by treatment with an MMP inhibitor or transfection of small interfering RNAs for MMP-9 or MMP-13, as compared with controls. The vascular density in wound granulation was significantly lower in both MMP-13 KO and MMP-9/13 double KO mice than in wild-type mice. Degradation of connective tissue growth factor in wound tissue was transiently prevented in MMP-13 KO mice. Morphometric analyses demonstrated a reduction in both wound contraction and myofibroblast formation in both MMP-13 KO and MMP-9/13 double KO mice. Proliferation and transforming growth factor-beta1-induced myofibroblast differentiation of dermal fibroblasts from MMP-13 KO mice were decreased, as compared with wild-type dermal fibroblasts. These data suggest that MMP-13 plays a role in keratinocyte migration, angiogenesis, and contraction in wound healing, while MMP-9 functions in keratinocyte migration.

Corticortophin releasing factor 2 receptor agonist treatment significantly slows disease progression in mdx mice

BackgroundDuchenne muscular dystrophy results from mutation of the dystrophin gene, causing skeletal and cardiac muscle loss of function. The mdx mouse model of Duchenne muscular dystrophy is widely utilized to evaluate the potential of therapeutic regimens to modulate the loss of skeletal muscle function associated with dystrophin mutation. Importantly, progressive loss of diaphragm function is the most consistent striated muscle effect observed in the mdx mouse model, which is the same as in patients suffering from Duchenne muscular dystrophy.MethodsUsing the mdx mouse model, we have evaluated the effect that corticotrophin releasing factor 2 receptor (CRF2R) agonist treatment has on diaphragm function, morphology and gene expression.ResultsWe have observed that treatment with the potent CRF2R-selective agonist PG-873637 prevents the progressive loss of diaphragm specific force observed during aging of mdx mice. In addition, the combination of PG-873637 with glucocorticoids not only prevents the loss of diaphragm specific force over time, but also results in recovery of specific force. Pathological analysis of CRF2R agonist-treated diaphragm muscle demonstrates that treatment reduces fibrosis, immune cell infiltration, and muscle architectural disruption. Gene expression analysis of CRF2R-treated diaphragm muscle showed multiple gene expression changes including globally decreased immune cell-related gene expression, decreased extracellular matrix gene expression, increased metabolism-related gene expression, and, surprisingly, modulation of circadian rhythm gene expression.ConclusionTogether, these data demonstrate that CRF2R activation can prevent the progressive degeneration of diaphragm muscle associated with dystrophin gene mutation.

Stem cell-related cardiac gene expression early after murine myocardial infarction

Clinical experimental stem cell therapy after myocardial infarction appears feasible, but its use has preceded the understanding of the working mechanism. The ischemic recipient cardiac environment is determinative for the attraction and subsequent fate of stem cells. Here, we studied expression levels of genes that are anticipated to be essential for adequate stem cell-based cardiac repair at various time-points during the 1 month period following myocardial infarction (MI). Gene expression in the hearts of mice that underwent MI by permanent or transient (30 min) ligation of the coronary artery was monitored using quantitative RT-PCR analysis of mRNA isolated from whole heart sections as well as from specific, laser micro-dissected, regions of sections. Protein expression was performed by immunohistochemical stainings and Western blot analysis. Many inflammatory genes were highly expressed for at least 1 week after MI. The expression of pro-angiogenic genes such as bFGF, VEGF-A and VEGF-R2 changed only marginally post-MI. Markers used to test stem cell gene expression remained unchanged post-MI with the exception of G-CSF and GM-CSF, which are genes that are also known to enhance the inflammatory response. Analysis of micro-dissected regions revealed that SDF-1, SCF (both stem cell attractants) and VEGF-R2 (involved in angiogenesis) gene expression was slightly decreased especially in the infarcted region. Genes that are generally considered to participate in stem cell-related processes and angiogenesis were not upregulated after MI, whereas the inflammatory gene expression dominated. Modulation of this imbalance might be of value for stem cell-mediated therapy.

PPARγ2 Regulates a Molecular Signature of Marrow Mesenchymal Stem Cells

Bone formation and hematopoiesis are anatomically juxtaposed and share common regulatory mechanisms. Bone marrow mesenchymal stromal/stem cells (MSC) contain a compartment that provides progeny with bone forming osteoblasts and fat laden adipocytes as well as fibroblasts, chondrocytes, and muscle cells. In addition, marrow MSC provide an environment for support of hematopoiesis, including the development of bone resorbing osteoclasts. The PPAR2 nuclear receptor is an adipocyte-specific transcription factor that controls marrow MSC lineage allocation toward adipocytes and osteoblasts. Increased expression of PPAR2 with aging correlates with changes in the MSC status in respect to both their intrinsic differentiation potential and production of signaling molecules that contribute to the formation of a specific marrow micro-environment. Here, we investigated the effect of PPAR2 on MSC molecular signature in respect to the expression of gene markers associated exclusively with stem cell phenotype, as well as genes involved in the formation of a stem cell supporting marrow environment. We found that PPAR2 is a powerful modulator of stem cell-related gene expression. In general, PPAR2 affects the expression of genes specific for the maintenance of stem cell phenotype, including LIF, LIF receptor, Kit ligand, SDF-1, Rex-1/Zfp42, and Oct-4. Moreover, the antidiabetic PPAR agonist TZD rosiglitazone specifically affects the expression of “stemness” genes, including ABCG2, Egfr, and CD44. Our data indicate that aging and anti-diabetic TZD therapy may affect mesenchymal stem cell phenotype through modulation of PPAR2 activity. These observations may have important therapeutic consequences and indicate a need for more detailed studies of PPAR2 role in stem cell biology.