Department of Cancer Genetics, Royal College of Surgeons in Ireland, Dublin, Ireland.
MiRNAs regulate gene expression at a post-transcriptional level and their dysregulation can play major roles in the pathogenesis of many different forms of cancer, including neuroblastoma, an often fatal paediatric cancer originating from precursor cells of the sympathetic nervous system. We have analyzed a set of neuroblastoma (n = 145) that is broadly representative of the genetic subtypes of this disease for miRNA expression (430 loci by stem-loop RT qPCR) and for DNA copy number alterations (array CGH) to assess miRNA involvement in disease pathogenesis. The tumors were stratified and then randomly split into a training set (n = 96) and a validation set (n = 49) for data analysis. Thirty-seven miRNAs were significantly over- or under-expressed in MYCN amplified tumors relative to MYCN single copy tumors, indicating a potential role for the MYCN transcription factor in either the direct or indirect dysregulation of these loci. In addition, we also determined that there was a highly significant correlation between miRNA expression levels and DNA copy number, indicating a role for large-scale genomic imbalances in the dysregulation of miRNA expression. In order to directly assess whether miRNA expression was predictive of clinical outcome, we used the Random Forest classifier to identify miRNAs that were most significantly associated with poor overall patient survival and developed a 15 miRNA signature that was predictive of overall survival with 72.7% sensitivity and 86.5% specificity in the validation set of tumors. We conclude that there is widespread dysregulation of miRNA expression in neuroblastoma tumors caused by both over-expression of the MYCN transcription factor and by large-scale chromosomal imbalances. MiRNA expression patterns are also predicative of clinical outcome, highlighting the potential for miRNA mediated diagnostics and therapeutics.
The combination of 5-Fluorouracil plus p53 pathway restoration is associated with depletion of p53-deficient or mutant p53-expressing putative colon cancer stem cells.
Laboratory of Molecular Oncology and Cell Cycle Regulation, Departments of Medicine (Hematology/Oncology), Genetics and Pharmacology, the Institute for Translational Medicine and Therapeutics, and the Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
The cancer stem cell hypothesis suggests that rare populations of tumor-initiating cells may be resistant to therapy, lead to tumor relapse and contribute to poor prognosis for cancer patients. We previously demonstrated the feasibility of p53 pathway restoration in p53-deficient tumor cell populations using small molecules including ellipticine or its derivatives. We now establish a single cell p53-regulated green fluorescent protein (EGFP)-reporter system in human DLD1 colon tumor cells expressing mutant p53 protein. We use these p53-EGFP reporter DLD1 cells to investigate the status of p53 transcriptional activity in putative colon cancer stem cell populations following exposure to p53 pathway-restoring drugs and/or classical chemotherapy. We demonstrate induction of p53-specific EGFP reporter fluorescence following overexpression of p53 family member p73 by an Adenovirus vector. We further show that p53-reporter activity is induced in DLD1 putative cancer stem cell side-populations analyzed by their Hoechst dye efflux properties following treatment with the p53 pathway restoring drug ellipticine. Combination of ellipticine with the cytotoxic agent 5-fluorouracil resulted in increased cytotoxicity as compared to either agent alone and this was associated with depletion of putative cancer stem cell populations as compared with 5-FU alone treatment. Our results support the feasibility of therapeutic targeting of mutant p53 in putative cancer stem cells as well as the potential to enhance cytotoxic chemotherapy.
PMID: 19923910 [PubMed - as supplied by publisher]
Visualization and enrichment of live putative cancer stem cell populations following p53 inactivation or Bax deletion using non-toxic fluorescent dyes.
Laboratory of Molecular Oncology and Cell Cycle Regulation, Departments of Medicine (Hematology/Oncology), Genetics and Pharmacology, the Institute for Translational Medicine and Therapeutics, and the Abramson Comprehensive Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, PA, USA; Biochemistry and Molecular Biophysics Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
Putative cancer stem cell (CSC) populations efflux dyes such as Hoechst 33342 giving rise to side populations (SP) that can be analyzed or isolated by flow cytometry. However, Hoechst 33342 is highly toxic, more so to non-SP cells, and thus presents difficulties in interpreting in vivo studies where non-SP cells appear less tumorigenic than SP cells in immunodeficient mice. We searched for non-toxic dyes to circumvent this problem as well as to image these putative CSCs. We found that the fluorescent dye calcein, a product of intracellular Calcein AM cleavage, is effluxed by a small subpopulation, calcein low population (C(lo)P). This population overlaps with SP and demonstrated long term cell viability, lack of cell stress and proliferation in several cancer cell lines when stained whereas Hoechst 33342 staining caused substantial apoptosis and ablated proliferation. We also found that the effluxed dye D-luciferin exhibits strong UV-fluorescence that can be imaged at cellular resolution and spatially overlaps with Calcein AM. In order to evaluate the hypothesis that p53 loss promotes enrichment of putative CSC populations we used Calcein AM, D-luciferin and Mitotracker Red FM as a counterstain to visualize dye-effluxing cells. Using fluorescence microscopy and flow cytometry we observed increased dye-effluxing populations in DLD-1 colon tumor cells with mutant p53 versus wild-type (WT) p53-expressing HCT116 cells. Deletion of the wild-type p53 or pro-apoptotic Bax genes induced the putative CSC populations in the HCT116 background to significant levels. Restoration of WT p53 in HCT116 p53(-/-) cells by an adenovirus vector eliminated the putative CSC populations whereas a control adenovirus vector, Ad-LacZ, maintained the putative CSC population. Our results suggest it is possible to image and quantitatively analyze putative CSC populations within the tumor microenvironment and that loss of pro-apoptotic and tumor suppressing genes such as Bax or p53 enrich such tumor-prone populations.
PMID: 19923899 [PubMed - as supplied by publisher]
Mesenchymal Stem Cells Derived from Human Gingiva Are Capable of Immunomodulatory Functions and Ameliorate Inflammation-Related Tissue Destruction in Experimental Colitis.
*Center for Craniofacial Molecular Biology, University of Southern California, School of Dentistry, Los Angeles, CA 90033.
Aside from the well-established self-renewal and multipotent differentiation properties, mesenchymal stem cells exhibit both immunomodulatory and anti-inflammatory roles in several experimental autoimmune and inflammatory diseases. In this study, we isolated a new population of stem cells from human gingiva, a tissue source easily accessible from the oral cavity, namely, gingiva-derived mesenchymal stem cells (GMSCs), which exhibited clonogenicity, self-renewal, and multipotent differentiation capacities. Most importantly, GMSCs were capable of immunomodulatory functions, specifically suppressed peripheral blood lymphocyte proliferation, induced expression of a wide panel of immunosuppressive factors including IL-10, IDO, inducible NO synthase (iNOS), and cyclooxygenase 2 (COX-2) in response to the inflammatory cytokine, IFN-gamma. Cell-based therapy using systemic infusion of GMSCs in experimental colitis significantly ameliorated both clinical and histopathological severity of the colonic inflammation, restored the injured gastrointestinal mucosal tissues, reversed diarrhea and weight loss, and suppressed the overall disease activity in mice. The therapeutic effect of GMSCs was mediated, in part, by the suppression of inflammatory infiltrates and inflammatory cytokines/mediators and the increased infiltration of regulatory T cells and the expression of anti-inflammatory cytokine IL-10 at the colonic sites. Taken together, GMSCs can function as an immunomodulatory and anti-inflammatory component of the immune system in vivo and is a promising cell source for cell-based treatment in experimental inflammatory diseases.
PMID: 19923445 [PubMed - as supplied by publisher]
Department of Obstetrics and Gynecology, The Second Affiliated Hospital of Harbin Medical University, No. 246, Xue Fu Road, Nan Gang District, 150001, Harbin, Hei Longjiang, China.
INTRODUCTION: Endostatin is the most potent inhibitor of tumor angiogenesis. However, endostatin protein has a short half-time and virus-mediated endostatin gene therapy has serious toxicity, which limits the application of endostatin in clinical therapy. Mesenchymal stem cells (MSCs) are considered to be able to accumulate at the site of cancers with high specificity and may be used as a new delivery of endostatin. MATERIALS AND METHODS: The MSCs from the human bone marrow were transfected with recombinant adenovirus encoding endostatin and EGFP (MSC-EN cells). The tropism capacity of MSCs was quantitatively assayed in vitro using the Millicell system. To investigate the impact of secreted endostatin on cancer cells, SKOV3 cells were co-cultured with MSC-EN cells in Millicell for 48 h, then apoptosis and cell cycle were analyzed on a flow cytometer. RESULTS: In contrast with 293 cells and saline, SKOV3 cells significantly stimulated migration of MSCs, the number reached 919.67 +/- 19.96 (P <>
PMID: 19921255 [PubMed - as supplied by publisher]
Institute for Drug Development, Cancer Therapy and Research Centre, University of Texas Health Science Centre at San Antonio, USA.
Chronic myeloid leukemia (CML) is the consequence of a single balanced translocation that produces the BCR-ABL fusion oncogene which is detectable in over 90% of patients at presentation. The BCR-ABL inhibitor imatinib mesylate (IM) has improved survival in all phases of CML and is the standard of care for newly diagnosed patients in chronic phase. Despite the very significant therapeutic benefits of IM, a small minority of patients with early stage disease do not benefit optimally while IM therapy in patients with advanced disease is of modest benefit in many. Diverse mechanisms may be responsible for IM failures, with point mutations within the Bcr-Abl kinase domain being amongst the most common resistance mechanisms described in patients with advanced CML. The development of novel agents designed to overcome IM resistance, while still primarily targeted on BCR-ABL, led to the creation of the high affinity aminopyrimidine inhibitor, nilotinib. Nilotinib is much more potent as a BCR-ABL inhibitor than IM and inhibits both wild type and IM-resistant BCR-ABL with significant clinical activity across the entire spectrum of BCR-ABL mutants with the exception of T315I. The selection of a second generation tyrosine kinase inhibitor to rescue patients with imatinib failure will be based on several factors including age, co-morbid medical problems and ABL kinase mutational profile. It should be noted that while the use of targeted BCR-ABL kinase inhibitors in CML represents a paradigm shift in CML management these agents are not likely to have activity against the quiescent CML stem cell pool. The purpose of this review is to summarize the pre-clinical and clinical data on nilotinib in patients with CML who have failed prior therapy with IM or dasatinib.
Stem Cells and Diabetes Section, Lab 12, National Center for Cell Science, Ganeshkhind Road, Pune 411 007, India.
Phenotypic plasticity is a phenomenon that describes the occurrence of 2 or more distinct phenotypes under diverse conditions. This article discusses the work carried out over the past few years in understanding the potential of human pancreatic islet-derived progenitors for cell replacement therapy in diabetes. The phenotypic plasticity exhibited by pancreatic progenitors during reversible epithelial-to-mesenchymal transition (EMT) and possible role of microRNAs in regulation of this process is also presented herein.
Centre for Respiratory Research, Rayne Institute, Centre for Advanced Biomedical Imaging, Department of Medicine and Institute of Child Health, and Division of Biosciences, University College London; and Davy-Faraday Research Laboratory, The Royal Institute of Great Britain, London, United Kingdom.
The ability of mesenchymal stem cells (MSC) to specifically home to tumors has suggested their potential use as a delivery vehicle for cancer therapeutics. MSC integration into tumors has been shown in animal models using histopathologic techniques after animal sacrifice. Tracking the delivery and engraftment of MSCs into human tumors will need in vivo imaging techniques. We hypothesized that labeling MSCs with iron oxide nanoparticles would enable in vivo tracking with magnetic resonance imaging (MRI). Human MSCs were labeled in vitro with superparamagnetic iron oxide nanoparticles, with no effect on differentiation potential, proliferation, survival, or migration of the cells. In initial experiments, we showed that as few as 1,000 MSCs carrying iron oxide nanoparticles can be detected by MRI one month after their coinjection with breast cancer cells that formed subcutaneous tumors. Subsequently, we show that i.v.- injected iron-labeled MSCs could be tracked in vivo to multiple lung metastases using MRI, observations that were confirmed histologically. This is the first study to use MRI to track MSCs to lung metastases in vivo. This technique has the potential to show MSC integration into human tumors, allowing early-phase clinical studies examining MSC homing in patients with metastatic tumors. [Cancer Res 2009;69(23):8862-7].
PMID: 19920196 [PubMed - as supplied by publisher]
Authors' Affiliations: Departments of Pathology and Biomedical Engineering, The University of Alabama at Birmingham, Birmingham, Alabama.
PURPOSE: Current evidence indicates that an osteoblast lesion in prostate cancer is preceded by osteolysis. Thus, prevention of osteolysis would reduce complications of bone metastasis. Bone marrow-derived mesenchymal stem cells have the ability to differentiate into osteoblast and produce osteoprotegerin, a decoy receptor for the receptor activator for nuclear factor kappaB ligand, naturally. The present study examined the potential of unmodified mesenchymal stem cells to prevent osteolytic bone lesions in a preclinical mouse model of prostate cancer. EXPERIMENTAL DESIGN: The human prostate cancer cell line PC3 was implanted in tibiae of severe combined immunodeficient mice. After establishment of the tumor, either unmodified or genetically engineered mesenchymal stem cells overexpressing osteoprotegerin was injected at the site of tumor growth. The effects of therapy were monitored by bioluminescence imaging, micro-computed tomography, immunohistochemistry, and histomorphometry. RESULTS: Data indicated significant (P <>
PMID: 19920103 [PubMed - as supplied by publisher]
Autologous mesenchymal stem cell treatment increased T regulatory cells with no effect on disease activity in two systemic lupus erythematosus patients.
Facultad de Medicina, Universidad de los Andes, Santiago, Chile.
Mesenchymal stem cells (MSCs) exert suppressive effects in several disease models including lupus prone mice. However, autologous MSC therapy has not been tested in human systemic lupus erythematosus (SLE). We evaluate the safety and efficacy of bone marrow (BM)-derived MSCs in two SLE patients; the suppressor effect of these cells in-vitro and the change in CD4+CD25+FoxP3+ T regulatory (Treg) cells in response to treatment. Two females (JQ and SA) of 19 and 25 years of age, fulfilling the 1997 American College of Rheumatology (ACR) criteria for SLE were infused with autologous BM-derived MSCs. Disease activity indexes and immunological parameters were assessed at baseline, 1, 2, 7 and 14 weeks. Peripheral blood lymphocyte (PBL) subsets and Treg cells were quantitated by flow cytometry, and MSCs tested for in-vitro suppression of activation and proliferation of normal PBLs. No adverse effects or change in disease activity indexes were noted during 14 weeks of follow-up, although circulating Treg cells increased markedly. Patient MSCs effectively suppressed in-vitro PBL function. However, JQ developed overt renal disease 4 months after infusion. MSC infusion was without adverse effects, but did not modify initial disease activity in spite of increasing CD4+CD25+FoxP3+ cell counts. One patient subsequently had a renal flare. We speculate that the suppressive effects of MSC-induced Treg cells might be dependent on a more inflammatory milieu, becoming clinically evident in patients with higher degrees of disease activity.
PMID: 19919974 [PubMed - as supplied by publisher]
