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Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion.

ABSTRACT: Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory effects of prostaglandin E2 (PGE2), and evaluate progeny for p53 mutations. Resting IgM(+)IgD(+)CD27(-) B cells from human tonsils were labeled with CFSE and stimulated in vitro with complement-coated antigen surrogate, IL-4, and BAFF ± exogenous PGE2 (50 nM) or an analog specific for the EP2 PGE2 receptor. We use flow cytometry to measure p53 and AID protein within variably divided blasts, qRT-PCR of p53 mRNA from cultures with or without actinomycin D to monitor mRNA transcription/stability, and single-cell p53 RT-PCR/sequencing to assess progeny for p53 mutations. We report that EP2 signaling triggers increased p53 gene transcriptional activity in AID(+) cycling blasts (P<0.01). Progeny exhibit p53 mutations at a frequency (8.5 × 10(-4)) greater than the baseline error rate (<0.8 × 10(-4)). We conclude that, devoid of the repressive influences of Bcl-6, dividing B lymphoblasts in inflamed tissues should display heightened p53 transcription and increased risk of p53 mutagenesis.

SUBMITTER: Haque S

PROVIDER: S-EPMC3898655 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion.

Haque Shabirul S Yan Xiao Jie XJ Rosen Lisa L McCormick Steven S Chiorazzi Nicholas N Mongini Patricia K A PK

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20131021 2

Within T-cell-dependent germinal centers, p53 gene transcription is repressed by Bcl-6 and is thus less vulnerable to mutation. Malignant lymphomas within inflamed extranodal sites exhibit a relatively high incidence of p53 mutations. The latter might originate from normal B-cell clones manifesting activation-induced cytosine deaminase (AID) and up-regulated p53 following T-cell-independent (TI) stimulation. We here examine p53 gene transcription in such TI clones, with a focus on modulatory eff ...[more]

PMID: 24145719

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Project description:Background & aimsInflammation may contribute to the formation, maintenance, and expansion of cancer stem cells (CSCs), which have the capacity for self-renewal, differentiation, and resistance to cytotoxic agents. We investigated the effects of the inflammatory mediator prostaglandin E2 (PGE2) on colorectal CSC development and metastasis in mice and the correlation between levels of PGE2 and CSC markers in human colorectal cancer (CRC) specimens.MethodsColorectal carcinoma specimens and matched normal tissues were collected from patients at the Mayo Clinic (Scottsdale, AZ) and analyzed by mass spectrometry and quantitative polymerase chain reaction. Human primary CRC cells and mouse tumor cells were isolated using microbeads or flow cytometry and analyzed for sphere-formation and by flow cytometry assays. LS-174T cells were sorted by flow cytometry (for CD133(+)CD44(+) and CD133(-)CD44(-) cells) and also used in these assays. NOD-scidIL-2Rγ(-/-) (NSG) mice were given cecal or subcutaneous injections of LS-174T or human primary CRC cells. Apc(Min/+) mice and NSG mice with orthotopic cecal tumors were given vehicle (controls), PGE2, celecoxib, and/or Ono-AE3-208. PGE2 downstream signaling pathways were knocked down with small hairpin RNAs, expressed from lentiviral vectors in LS-174T cells, or blocked with inhibitors in human primary CRC cells.ResultsLevels of PGE2 correlated with colonic CSC markers (CD133, CD44, LRG5, and SOX2 messenger RNAs) in human colorectal carcinoma samples. Administration of PGE2 to Apc(Min/+) mice increased tumor stem cells and tumor burden, compared with controls. NSG mice given PGE2 had increased numbers of cecal CSCs and liver metastases compared with controls after intracecal injection of LS-174T or human primary CRC cells. Alternatively, celecoxib, an inhibitor of prostaglandin-endoperoxide synthase 2, reduced polyp numbers in Apc(Min/+) mice, liver metastasis in NSG mice with orthotopic tumors, and numbers of CSCs in Apc(Min/+) and NSG mice. Inhibitors or knockdown of PGE2 receptor 4 (EP4), phosphoinositide 3-kinase (PI3K) p85α, extracellular signal-regulated kinase 1 (ERK1), or nuclear factor (NF)-κB reduced PGE2-induced sphere formation and expansion of LS-174T and/or human primary CRC cells. Knockdown of ERK1 or PI3K p85α also attenuated PGE2-induced activation of NF-κB in LS-174T cells. An EP4 antagonist reduced the ability of PGE2 to induce CSC expansion in orthotopic tumors and to accelerate the formation of liver metastases. Knockdown experiments showed that NF-κB was required for PGE2 induction of CSCs and metastasis in mice.ConclusionsPGE2 induces CSC expansion by activating NF-κB, via EP4-PI3K and EP4-mitogen-activated protein kinase signaling, and promotes the formation of liver metastases in mice. The PGE2 signaling pathway therefore might be targeted therapeutically to slow CSC expansion and colorectal cancer progression.

Dataset Information

Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion.

Publications

Effects of prostaglandin E2 on p53 mRNA transcription and p53 mutagenesis during T-cell-independent human B-cell clonal expansion.

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