Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profile upon IL-22 stimulation, forced expression of Hes1, or IL-22 stimulation under forced expression of Hes1, in human colon carcinoma-derived LS174T cells

ABSTRACT: A human colon carcinoma-derived cell line LS174T was modified to overexpress Hes1, a bHLH-type transcription factor, upon doxycycline addition (designated as LS174T-tetON-Hes1 cells), using the T-rex system (Invitrogen). We have previously shown that these cells can overexpress Hes1 under the control of CMV promoter (Zheng et al, Inflamm Bowel Dis, 17;2251-2260, 2011), and the amont of the overexpressed Hes1 protein reaches to the maximal level in early as 3 hours from doxycycline addition (100ng/ml), which persists for up to 24 hours. In the present experiment, LS174T-tetON-Hes1 cells were treated with recombinant human IL-22 (20ng/ml) alone, doxycycline alone (100ng/ml), co-treated by both IL-22 and doxycycline, or left untreated (Control) for 24 hours, and subjected for analysis. Experiment was done using a modified sub-line of LS174T cell (LS174T-tetON-Hes1 cells), in which overexpression of Hes1 can be induced by a Doxycycline dependent manner. Four-condition experiment, Control vs. IL-22 stimulation, Control vs Doxycycline addition (Hes1 overexpression) and Control vs IL22 stimulation+Doxycycline addition (Hes1 overexpression). Biological replicates: One for each condition, independently grown and harvested. One replicate per array.

ORGANISM(S): Homo sapiens

SUBMITTER: Okamoto Ryuichi

PROVIDER: E-GEOD-43012 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:High levels of Hes1 expression are frequently found in BCR-ABL-positive chronic myelogenous leukemia in blast crisis (CML-BC). In mouse bone marrow transplantation (BMT) models, co-expression of BCR-ABL and Hes1 induces CML-BC–like disease; however the underlying mechanism remained elusive. Here, based on gene expression analysis, we show that MMP-9 is upregulated by Hes1 in common myeloid progenitors (CMPs). Analysis of promoter activity demonstrated that Hes1 upregulated MMP-9 by activating NF-kB. Analysis of 20 samples from CML-BC patients showed that MMP-9 was highly expressed in three, with two exhibiting high levels of Hes1 expression. Interestingly, MMP-9 deficiency impaired the cobblestone area-forming ability of CMPs expressing BCR-ABL and Hes1 that were in conjunction with a stromal cell layer. In addition, these CMPs secreted MMP-9, promoting the release of soluble Kit-ligand (sKitL) from stromal cells, thereby enhancing proliferation of the leukemic cells. In accordance, mice transplanted with CMPs expressing BCR-ABL and Hes1 exhibited high levels of sKitL as well as MMP-9 in the serum. Importantly, MMP-9 deficiency impaired the development of CML-BC–like disease induced by BCR-ABL and Hes1 in mouse BMT models. The present results suggest that Hes1 promotes the development of CML-BC, partly through MMP-9 upregulation in leukemic cells. Common myeloid progenitors (CMPs; Lineage negative, c-Kit positive, Sca-1 negative, Fc-gamma-receptor low, CD34 positive fraction) were sorted with a FACSAria cell sorter (Becton Dickinson). Retroviruses were generated by transfecting Plat-E packaging cells with retrovirus vector pMYs-Hes1-IRES-GFP or empty vector (pMYs-IRES-GFP) using FuGENE 6 (Roche Diagnostics). Infection of retrovirus harboring Hes1 (pMYs-Hes1-IRES-GFP) or empty vector (pMYs-IRES-GFP) into progenitors was performed using RetroNectin (Takara Bio). Hes1-transfected CMPs and Mock-transduced CMPs were isolated 36 hours after infection with a FACSAria cell sorter. One sample of Hes1-transfected CMPs and one sample of mock-transduced CMPs were analyzed with GeneChip Mouse Genome 430 2.0 Array.

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Gene expression profile upon IL-22 stimulation, forced expression of Hes1, or IL-22 stimulation under forced expression of Hes1, in human colon carcinoma-derived LS174T cells

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