Cancer-related epigenome changes associated with reprogramming to induced pluripotent stem cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Leander Van Neste
PROVIDER: E-GEOD-29873 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Cancer-related epigenome changes associated with reprogramming to induced pluripotent stem cells.
Ohm Joyce E JE Mali Prashant P Van Neste Leander L Berman David M DM Liang Liang L Pandiyan Kurinji K Briggs Kimberly J KJ Zhang Wei W Argani Pedram P Simons Brian B Yu Wayne W Matsui William W Van Criekinge Wim W Rassool Feyruz V FV Zambidis Elias E Schuebel Kornel E KE Cope Leslie L Yen Jonathan J Mohammad Helai P HP Cheng Linzhao L Baylin Stephen B SB
Cancer research 20100914 19
The ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome-wide assays, we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These includ ...[more]
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Project description:The ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome-wide assays, we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These include hundreds of abnormal gene silencing events, patterns of aberrant responses to epigenetic-modifying drugs resembling those for cancer cells, and presence in iPS and partially reprogrammed cells of cancer-specific gene promoter DNA methylation alterations. Our findings suggest that by studying the process of induced reprogramming, we may gain significant insight into the origins of epigenetic gene silencing associated with human tumorigenesis, and add to means of assessing iPS for safety. Direct expression comparison of iPS lines, cultured stem cell lines and normal differentiated cells. Re-expression experiments with 5-aza-2′-deoxycytidine (AZA) and trichostatin A (TSA) to identify hypermethylated genes.
2011-06-16 | E-GEOD-29870 | biostudies-arrayexpress
Project description:Altered gene expression is a hallmark of human cancers and arises in part through abnormal epigenetic regulation of gene transcription. The best characterized epigenetic alteration involves tumor suppressor gene inactivation via transcriptional repression associated with aberrant DNA hypermethylation of promoter region CpG islands1. Despite characterization of a growing number of such genes, the majority have yet to be identified. We now describe a genome wide microarray gene expression approach for human colorectal cancer cells, which can efficiently identify hundreds of hypermethylated genes for any cancer type. We compared isogenic cells altered pharmacologically versus genetically to induce genomic demethylation, to pinpoint genes activated by DNA demethylation, but not by inhibition of class I and II histone deacetylases (HDACs). We achieve an 82% success rate in predicting genes with densely hypermethylated CpG islands and complete gene silencing. The genes are similarly hypermethylated in primary tumors and have previously undetected tumor suppressor functions. Our approach provides the first highly efficient, comprehensive, platform for defining the cancer âDNA hypermethylome" Experiment Overall Design: Cell culture and treatment. HCT116 cells and isogenic genetic knockout derivatives were maintained as previously described14. For drug treatments, log phase HCT116 cells were cultured in McCoys 5A media (Invitrogen) containing 10% BCS and 1x penicillin/streptomycin with ïµïM 5aza-deoxycytidine (DAC) (Sigma; stock solution: 1mM in PBS) for 96 hours, replacing media and DAC every 24 hours. Cell treatment with 300nM Trichostatin A (Sigma; stock solution: 1.5mM dissolved in ethanol) was performed for 18 hours. Control cells underwent mock treatment in parallel with addition of equal volume of PBS or ethanol without drugs. Experiment Overall Design: Microarray analysis. Total RNA was harvested from log phase cells using the Qiagen kit according to the manufacturers instructions, including a DNAase step. RNA was quantified using the NanoDrop ND-100 followed by quality assessment with 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for individual samples were greater than 200ng/ul, with 28s/18s ratios greater than 2.2 and RNA integrity numbers of 10 (highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturers instructions. The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification. 0.75 microgram of samples labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized, and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies. Experiment Overall Design: Data analysis. All arrays were subject to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of signal and background intensity of both red and green channels were examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. All calculations were performed using the R statistical computing platform37 and packages from Bioconductor bioinformatics software project38. The log ratio of red signal to green signal was calculated after background-subtraction and LoEss normalization as implemented in the limma package from Bioconductor39,40. Individual arrays were scaled to have the same inter-quartile range (75th percentile -25th percentile) Log fold changes were averaged over dye-swap replicate microarrays to produce a single set of expression values for each condition.
2008-06-13 | E-GEOD-4763 | biostudies-arrayexpress
Project description:Many cancers may originate in stem or early progenitor cells 1-4 which depend on epigenetic modulation of gene expression for normal function (ref). We have studied whether epigenetic states in cancers, and particularly abnormal gene silencing associated with promoter DNA hypermethylation in CpG rich regions (refs), may represent links between cancer and stem cell epigenetic patterns. We studied embryonal carcinomas (EC) which are malignancies of embryonic stem (ES) cells, but retain high potential for multi-lineage differentiation 5-8. Surprisingly, genes DNA hypermethylated and silenced in adult cancers are unmethylated in ES and EC cells. Remarkably, the promoter chromatin of the genes in EC cells is virtually identical to the same genes in adult cancers when the latter are induced to de-methylate. This chromatin, as recently reported for CpG island promoters of low expression, developmentally related, genes in ES cells (refs), is âbivalentâ and contains both active and repressive histone modifications. The latter includes trimethyl lysine 27 of histone H3 (H3K27me3), which may recruit DNA methylation (refs), and the promoter regions are enriched for polycomb group (PcG) proteins which place this H3K27me3 mark. Thus, many genes which undergo abnormal DNA methylation and permanent silencing in adult cancers may be programmed to do so by a pre-existent stem cell-like chromatin pattern inherent to the cells in which they arise. Experiment Overall Design: Data analysis. All arrays were subject to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of signal and background intensity of both red and green channels are examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. Experiment Overall Design: All calculations were performed using the R statistical computing platform, (cite R) and packages from Bioconductor bioinformatics software project (cite Bioconductor). The log ratio of red median signal to green median signal was calculated after background-subtraction and loess normalzation as implemented in the limma package from Bioconductor. ( cite Smyth) Individual arrays were scaled to have the same inter-quartile range (75th percentile -25th percentile) Log fold changes were averaged over dye-swap replicate microarrays to produce a single set of expression values for each condition.
2008-06-13 | E-GEOD-4809 | biostudies-arrayexpress
Project description:Polycomb CBX7 was stably expressed in embryonal carcinoma cells in order to identify target genes. The study was specifically geared toward unveiling gene targets that are susceptible to DNA hypermethylation in adult cancer. A number of CBX7 target tumor suppressor genes were thereby identified and used in further studies to characterize the mechanism surrounding CBX7 mediated gene suppression. Keywords: CBX7 target gene identification A cDNA encoding the ORF of polycomb CBX7 under the control of EF1alpha promoter was transfected into the Tera-2 embryonal carcinoma cell line. Empty vector was used to transfect control cells. The cells were subject to stable selection using puromycin and a pooled population of CBX7 overexpressing cells was established. Gene expression analysis was performed by comparing the gene expression profile of CBX7 cells to empty vector control cells.
2009-09-23 | E-GEOD-10541 | biostudies-arrayexpress
Project description:The identification and characterization of tumor suppressor genes has enhanced our understanding of the biology of cancer and enabled the development of new diagnostic and therapeutic modalities. Whereas in past decades, a handful of tumor suppressors have been slowly identified using techniques such as linkage analysis, large-scale sequencing of the cancer genome has enabled the rapid identification of a large number of genes that are mutated in cancer. However, determining which of these many genes play key roles in cancer development has proven challenging. Specifically, recent sequencing of human breast and colon cancers has revealed a large number of somatic gene mutations, but virtually all are heterozygous, occur at low frequency, and are tumor-type specific. Keywords: Microarray, Hypermethylome, DNA-hypermethylation, DAC, TSA, Epigenetic, Mutation, Colorectal Cancer, Breast Cancer The candidate hypermethylome genes were identified as described below. These genes were compared to the CAN genes identified by Sjöblom T et al (Science, 2006). Cell culture and treatment. For drug treatments, log phase MCF7, T-47D, MDA-MB231 and MDA-MB-468 cells were cultured in McCoys 5A media (Invitrogen) containing 10% BCS and 1x penicillin/streptomycin with 5M 5aza-deoxycytidine (DAC) (Sigma; stock solution: 1mM in PBS) for 96 hours, replacing media and DAC every 24 hours. Cell treatment with 300nM Trichostatin A (Sigma; stock solution: 1.5mM dissolved in ethanol) was performed for 18 hours. Control cells underwent mock treatment in parallel with addition of equal volume of PBS or ethanol without drugs. Microarray analysis. Total RNA was harvested from log phase cells using the Qiagen kit according to the manufacturers instructions, including a DNAase step. RNA was quantified using the NanoDrop ND-100 followed by quality assessment with 2100 Bioanalyzer (Agilent Technologies). RNA concentrations for individual samples were greater than 200ng/ul, with 28s/18s ratios greater than 2.2 and RNA integrity numbers of 10 (highest). Sample amplification and labeling procedures were carried out using the Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies) according to the manufacturers instructions. The labeled cRNA was purified using the RNeasy mini kit (Qiagen) and quantified. RNA spike-in controls (Agilent Technologies) were added to RNA samples before amplification. 0.75 microgram of samples labeled with Cy3 or Cy5 were mixed with control targets (Agilent Technologies), assembled on Oligo Microarray, hybridized, and processed according to the Agilent microarray protocol. Scanning was performed with the Agilent G2565BA microarray scanner under default settings recommended by Agilent Technologies. Data analysis. All arrays were subject to quality checks recommended by the manufacturer. Images were visually inspected for artifacts and distributions of signal and background intensity of both red and green channels were examined to identify anomalous arrays. No irregularities were observed, and all arrays were retained and used. All calculations were performed using the R statistical computing platform and packages from Bioconductor bioinformatics software project. The log ratio of red signal to green signal was calculated and Loess normalization as implemented in the limma package from Bioconductor.
2009-09-23 | E-GEOD-10613 | biostudies-arrayexpress
Project description:he ability to induce pluripotent stem cells from committed, somatic human cells provides tremendous potential for regenerative medicine. However, there is a defined neoplastic potential inherent to such reprogramming that must be understood and may provide a model for understanding key events in tumorigenesis. Using genome-wide assays, we identify cancer-related epigenetic abnormalities that arise early during reprogramming and persist in induced pluripotent stem cell (iPS) clones. These include hundreds of abnormal gene silencing events, patterns of aberrant responses to epigenetic-modifying drugs resembling those for cancer cells, and presence in iPS and partially reprogrammed cells of cancer-specific gene promoter DNA methylation alterations. Our findings suggest that by studying the process of induced reprogramming, we may gain significant insight into the origins of epigenetic gene silencing associated with human tumorigenesis, and add to means of assessing iPS for safety. Methylation was analyzed using Illumina's 27k Infinium platform for direct detection of methylation after bisulfite conversion. The overall methylation status was determined for several iPS lines and the pool cells from which they are derived. These methylation levels can be compared directly to those of cultured stem cells, differentiated cells and cancer cell lines.
2011-06-16 | E-GEOD-29872 | biostudies-arrayexpress
Project description:Reversal of gene promoter DNA hypermethylation and associated abnormal gene silencing is an attractive approach to cancer therapy. The DNA methylation inhibitor, decitabine (5-aza-2'-deoxycitidine), is proving efficacious for hematological neoplasms especially at lower, less toxic, doses. Experimentally, high doses induce rapid DNA damage and cytotoxicity, but these may not explain the prolonged time to response seen in patients. Transient exposure of leukemic and solid tumor cells to clinically-relevant nanomolar doses, without causing immediate cytotoxicity or apoptosis, produces sustained reduced tumorigenicity, and for leukemia cells, diminished long-term self-renewal. These effects appear triggered by cellular reprogramming and include sustained decreases in promoter DNA methylation with associated gene re-expression, and anti-tumor changes in multiple key cellular regulatory pathways, most of which are high priority targets for pharmacologic anti-cancer strategies. Thus, low dose decitabine regimens appear to have broad applicability for cancer management. [Gene expression profiling] Leukemia cell lines Kasumi-1 and KG1A are treated with 10nM DAC during 72 hours and gene expression was assayed at day 3, 7 and 14 after the start of the treatment. Appropriate mock treated samples were used as control in each case. In addition, Kasumi-1 cells were also treated with a higher dose of DAC (500nM), 100nM ARA-C and 300 nM TSA, again controlled against mock treated Kasumi-1 cells, to separate dose and agent dependent effects. MCF7 was studied as an example of a solid tumor cell line. Therefore MCF7 cells were treated with 100nM DAC and results were assayed at day 1, day 3 and day 10. [Methylation profiling] The effects of the demethylating agent DAC were studied in the leukemia cell line Kasumi-1 over a 28 day time course. Intermediate time points were studied at days 3, 7, 14 and 21. These results were verfied in KG1A and KG1 leukemia cell lines, at one selected time point. The effects on one primary sample were also studied. Four normal leukemia samples (PL1, 2, 4 and 5) were used as general controls. The effect of DAC was compared to ARA-C, TSA. Both mock treated and day 3 DAC treated Kasumi-1 cells were repeated. These results were verified at one selected time point for the DAC treated MCF7 breast cancer cell line.
2012-03-21 | E-GEOD-20945 | biostudies-arrayexpress
Project description:We report a novel technique to reprogram human fibroblasts into endothelial and smooth muscle cells using partial iPSC reprogramming and chemically defined media. Using appropriate media conditions for differentiation of human pluripotent cells to CD34+ vascular progenitor cells, we show that temporary expression of pluripotent transcription factors and treatment with chemically-defined media, will induce differentiation of human fibroblasts to CD34+ vascular progenitor cells. Sorted CD34+ cells can then be directed to differentiate into vascular endothelial cells expressing a variety of smooth muscle markers. We have assessed the global DNA methylation (Illumina Infinium HD 450K DNA methylationBeadChips) and transcriptional (Illumina HT12v4 Gene Expression Bead Array) profiles of transdifferentiated endothelial cells and smooth muscle, human embryonic stem cell (hESC) and human induced pluripotent stem cell (hiPSC) differentiated CD34+ angioblasts, hESCs, hiPSC, primary smooth muscle and primary human umbilical vein endothelial cells using microarrays.
2012-09-18 | E-GEOD-40909 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE36812: Epigenome analysis of cord blood samples from newborns GSE36828: Genome-wide analysis of gene expression levels in placenta and cord blood samples from newborns babies GSE36829: Epigenome analysis of placenta samples from newborns GSE36852: Epigenome analysis of newborn placenta and cord blood samples Refer to individual Series
2012-03-28 | E-GEOD-36831 | biostudies-arrayexpress
Project description:We recently showed that some human induced pluripotent stem cell (iPSC) clones were defective in neural differentiation and were marked with the activation of long term repeats (LTRs) of human endogenous retroviruses (HERVs). We herein demonstrated that these LTRs were transiently overexpressed during the generation of iPSCs and contributed to reprogramming. When the generation of iPSCs was completed, LTRs were re-suppressed to levels similar to those in human ES cells. However, differentiation-defective iPSC clones maintained high LTR expression levels, which indicated that these clones failed to complete reprogramming. lincRNA-RoR, a long intergenic non-coding RNA (lincRNA) that was previously shown to support the induction and maintenance of pluripotency, was detected among the LTR-driven transcripts. Short hairpin RNAs against the conserved sequence in LTRs or lincRNA-RoR markedly reduced the efficiency of iPSC generation. Reprogramming factors including OCT3/4, SOX2, and KLF4 bound to most LTRs. The expression of KLF4 was low in normal iPSC clones, but remained high in differentiation-defective clones. The forced expression of KLF4 in human embryonic stem cells led to the activation of LTRs and defects in neural differentiation. These results demonstrated that the transient overexpression of KLF4/LTR/lincRNA-RoR played crucial roles in reprogramming toward pluripotency in humans, whereas a failure in its re-silence resulted in differentiation defects. Four human induced pluripotent stem cell lines were subcloned into 55 separate lines. Bisulfite converted genomic DNA lysates from fibroblast, induced pluripotent stem cell, intermediate reprogrammed cell and embryonic stem cell lines were hybridized to Illumina HumanMethylation450 BeadChip.
2014-08-04 | E-GEOD-54848 | biostudies-arrayexpress