Human glioblastoma cells: Tet-on(mutant p53 R175H expression) vs Tet-off(p53-null)
ABSTRACT: TP53 R175H mutation is one of the most common mutations in human cancers and cancer cells with this mutation stably express R175H protein in the nucleus. To identify the synthetic lethal gene interacted with the R175H, we conducted the high throughput screening by using tetracyclin inducible R175H expression system in SF126 cells and comprehensive shRNA library carried by lenti virus. We identified 906 candidate gene suppressions that may lead to accelerated cell growth inhibition under the presence of R175H. Inhibitor of differentiation 1 (ID1) was one of the candidate genes and suppression of ID1 by siRNA resulted in acceleration of growth inhibition of cell lines expressing endogenous R175H but not in TP53 null cell lines. The transient expression of R175H in TP53 null cell lines and suppression of ID1 and/or TP53 R175H in cell lines with endogenous R175H revealed that the cell growth inhibition by ID1 suppression was dependent on R175H expression but not other common p53 mutants (R273H). Flow cytometric (FACS) analysis exhibited that ID1 suppression resulted in G1 arrest and the arrest was accelerated by suppression of R175H. In conclusion, ID1 is a synthetic lethal gene interacted with R175H and is considered to be a novel molecular target of cancer therapy for R175H expressing cells. R175H expression(Tet-on) group was labeled by Cy5, and p53-null(Tet-off) group was labeled by Cy3. Three independent experiments were conducted (We have triplicate data).
Project description:We used microarray to determine the genes whose expression was changed at six hours after doxycycline treatment Cells were transfected with β-globin let7 wt constructs. Two-condition experiment; HeLa tet off cells overexpressed control vs HeLa tet off cells treated with doxycycline for six hours
Project description:We investigated the role of the transcriptional regulator Id2 in the context of MLL-rearranged acute myeloid leukemia (AML). Using an AML mouse model driven by tet-regulated MLL-AF9 co-expressed with oncogenic NRASG12D (Tet-off MLL-AF9), we demonstrated that MLL-AF9 regulates the E protein pathway by suppressing Id2, while activating the expression of its target E2-2. Moreover, we found that Id2 over-expression in Tet-Off MLL-AF9 AML cells in vitro partially phenocopies MLL-AF9 depletion and results inhibition of leukemia growth, loss of leukemia stem cell-associated gene expression pattern and induction of differentiation. To compare gene expression changes associated with enforced Id2 expression and MLL-AF9 withdrawal, RNA sequencing analysis was performed on Tet-off MLL-AF9 cells transduced with an Id2 over-expressing or a control vector, or upon MLL-AF9 dox-inducible knock-down. Primary AMLs driven by Tet-off inducible MLL/AF9 expression linked to dsRED reporter, in association with oncogenic NRASG12D (Tet-off MLL-AF9) were generated by reconstituting lethally irradiated congenic mice with foetal liver cells co-transduced with a Tet-Off-MLL-AF9-dRED retroviral vector and a second vector co-expressing NRASG12D together with the Tet-Off responsive transcriptional activator. RNA sequencing analysis sequencing analysis was performed on Tet-Off MLL-AF9/dsRED+ AML cells treated in vitro with doxycycline (DOX) for 4 days to inactivate MLL-AF9 expression or left untreated (UT). For the Id2 over-expression experiment, Tet-Off MLL-AF9/dsRED+ AML cells were transduced in vitro with an Id2-GFP or a control-GFP retroviral vector. Viable GFP-positive cells were FACS-sorted 2 days after transduction and used for RNA sequencing analysis.
Project description:In order to identify novel genes regulated by p53, stable line containing tet-on inducible p53 construct was generated and used for gene expression analysis. Tet-on p53 inducible stable line was induced by doxycyclin for 24 hours before total RNA was extracted for array analysis.
Project description:Alteration of the PTEN/PI3K pathway is associated with late stage and castrate resistant prostate cancer (CRPC). However, how PTEN loss involves in CRPC development is not clear. Here we show that castration-resistant growth is an intrinsic property of Pten-null prostate cancer (CaP) cells, independent of cancer development stage.PTEN loss suppresses androgen-responsive gene expressions by modulating androgen receptor (AR) transcription factor activity. Conditional deletion of AR in the epithelium promotes the proliferation of Pten-null cancer cells, at least in part, by down-regulating androgen-responsive gene FKBP5 and preventing PHLPP-mediated AKT inhibition. Our findings identify PI3K and AR pathway crosstalk as a mechanism of CRPC development, with potentially important implications for CaP etiology and therapy Mouse embryonic fibroblasts (MEFs) carrying a tet-inducible Pten transgene were generated by retro viral infection and antibiotic selection. Cells were treated with 2 ug/ml doxycycline for 24 or 48 hours in tet-free FBS (5%)/MEF media (n=2). Reference samples were either cells before treatment (n=2). After each time point cells were washed twice with PBS and RNA trizol extracted. WT samples (n =2) were also included as a control.
Project description:To identify genes involved in raditaion resistence in glioblastoma, Decode shRNA Viral screening was performed on U87MG and LN229 glioblastoma cell lines. Cells were irraidated with 2 Gy and then compared to unirradiated cell genomic DNA on Decode custom Agilent barcode arrays.
Project description:Reporter genes integrated into the genome are a powerful tool to reveal effects of regulatory elements and local chromatin context on gene expression. However, so far such reporter assays have been of low throughput. Here we describe a multiplexing approach for the parallel monitoring of transcriptional activity of thousands of randomly integrated reporters. More than 27,000 distinct reporter integrations in mouse embryonic stem cells, obtained with two different promoters, show ~1,000-fold variation in expression levels. Data analysis indicates that lamina-associated domains act as attenuators of transcription, likely by reducing access of transcription factors to binding sites. Furthermore, chromatin compaction is predictive of reporter activity. We also found evidence for cross-talk between neighboring genes, and estimate that enhancers can influence gene expression on average over ~20 kb. The multiplexed reporter assay is highly flexible in design and can be modified to query a wide range of aspects of gene regulation. TRIP assay of tet-Off promotor; 4 experiments, each with 2 technical replicates. Each experiment was induced at 4 different concentrations of doxycycline.
Project description:The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is the prototype downstream effector of the tumor suppressor protein p53. Yet, evidence from human cancer and mice models, imply that p21WAF1/Cip1, under certain conditions, can exercise oncogenic activity. The mechanism behind this behavior is still obscure. Within this context we unexpectedly noticed, predominantly in p53 mutant human cancers, that a subset of highly atypical cancerous cells expressing strongly p21WAF1/Cip1 demonstrated also signs of proliferation. This finding suggests either tolerance to high p21WAF1/Cip1 levels or that p21WAF1/Cip1 per se guided a selective process that led to more aggressive off-springs. To address the latter scenario we employed p21WAF1/Cip1-inducible p53-null cellular models and monitored them over a prolonged time period, using high-throughput screening means. After an initial phase characterized by stalled growth, mainly due to senescence, a subpopulation of p21WAF1/Cip1 cells emerged, demonstrating increased genomic instability, aggressiveness and chemo-resistance. At the mechanistic level unremitted p21WAF1/Cip1 production “saturates” the CRL4CDT2 and SCFSkp2 ubiquitin ligase complexes reducing the turn-over of the replication licensing machinery. Deregulation of replication licensing triggered replication stress fuelling genomic instability. Conceptually, the above notion should be considered when anti-tumor strategies are designed, since p21WAF1/Cip1 responds also to p53-independent signals, including various chemotherapeutic compounds. We used microarrays to compare the non-induced and “escaped” Saos2-p21WAF1/Cip1 Tet-ON cells gene expression profile Multiple arrays for non-induced and “escaped” Saos2-p21WAF1/Cip1 Tet-ON cells
Project description:Myocilin, a causative gene for open-angle glaucoma, encodes a secreted glycoprotein of unknown function. To elucidate its function(s), we produced a stably transfected HEK293 cell line expressing myocilin and compared the expression profiles between the myocilin-expressing cell line and a vector control cell line using Affymetrix GeneChip U133 plus 2.0 array. A significant portion of differentially-expressed genes in the myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have an important role regulating cell growth/survival.. We developed vector control and myocilin-expressing Tet On HEK293 cell lines. The gene expression profiles in two stable cell lines were compared.
Project description:We generated tetracycline-inducible myc-GABPα-expressing Gabpα conditional knockout embryonic stem cells (Tet-Gabbpα cKO ES cells), and then compared the transcriptional profiles between control (-Tet) and Gabpα-null (+Tet) cells by an oligo DNA microarray analysis. Overall design: Tet-Gabpα cKO ES cells were cultured with or without Tet for 1 day. Total RNAs were isolated and subjected to microarray analysis.