ABSTRACT
Unintended outcomes of cancer therapy include ionizing radiation (IR)-induced stem cell depletion, diminished regenerative capacity, and accelerated aging. Stem cells exhibit attenuated DNA damage response (DDR) and are hypersensitive to IR, as compared to differentiated non-stem cells. We performed genomic discovery research to compare stem cells to differentiated cells, which revealed Phosphoprotein phosphatase 2A (PP2A) as a potential contributor to susceptibility in stem cells. PP2A dephosphorylates pATM, γH2AX, pAkt etc. and is believed to play dual role in regulating DDR and apoptosis. Although studied widely in cancer cells, the role of PP2A in normal stem cell radiosensitivity is unknown. Here we demonstrate that constitutively high expression and radiation induction of PP2A in stem cells plays a role in promoting susceptibility to irradiation. Transient inhibition of PP2A markedly restores DNA repair, inhibits apoptosis, and enhances survival of stem cells, without affecting differentiated non-stem and cancer cells. PP2Ai-mediated stem cell radioprotection was demonstrated in murine embryonic, adult neural, intestinal, and hematopoietic stem cells.
Subject(s)
Enzyme Inhibitors/pharmacology , Oxazoles/pharmacology , Protein Phosphatase 2/antagonists & inhibitors , Radiation Tolerance/drug effects , Stem Cells/drug effects , Stem Cells/radiation effects , Animals , Apoptosis , Ataxia Telangiectasia Mutated Proteins/metabolism , Cell Line, Tumor , Cells, Cultured , DNA Repair , Hematopoietic Stem Cells/drug effects , Hematopoietic Stem Cells/enzymology , Hematopoietic Stem Cells/pathology , Hematopoietic Stem Cells/radiation effects , Humans , Male , Marine Toxins , Mice , Mice, Inbred C57BL , Mouse Embryonic Stem Cells/drug effects , Mouse Embryonic Stem Cells/enzymology , Mouse Embryonic Stem Cells/pathology , Mouse Embryonic Stem Cells/radiation effects , Neural Stem Cells/drug effects , Neural Stem Cells/enzymology , Neural Stem Cells/pathology , Neural Stem Cells/radiation effects , Protein Phosphatase 2/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Stem Cells/enzymology , Stem Cells/pathology , Time Factors , Tissue Culture TechniquesABSTRACT
Dynamic spatiotemporal modification of chromatin around DNA damage is vital for efficient DNA repair. Normal stem cells exhibit an attenuated DNA damage response (DDR), inefficient DNA repair, and high radiosensitivity. The impact of unique chromatin characteristics of stem cells in DDR regulation is not yet recognized. We demonstrate that murine embryonic stem cells (ES) display constitutively elevated acetylation of histone H3 lysine 9 (H3K9ac) and low H3K9 tri-methylation (H3K9me3). DNA damage-induced local deacetylation of H3K9 was abrogated in ES along with the subsequent H3K9me3. Depletion of H3K9ac in ES by suppression of monocytic leukemia zinc finger protein (MOZ) acetyltransferase improved ATM activation, DNA repair, diminished irradiation-induced apoptosis, and enhanced clonogenic survival. Simultaneous suppression of the H3K9 methyltransferase Suv39h1 abrogated the radioprotective effect of MOZ inhibition, suggesting that high H3K9ac promoted by MOZ in ES cells obstructs local upregulation of H3K9me3 and contributes to muted DDR and increased radiosensitivity.
Subject(s)
Embryonic Stem Cells/metabolism , Embryonic Stem Cells/radiation effects , Histones/metabolism , Lysine/metabolism , Radiation Tolerance , Radiation, Ionizing , Acetylation , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA Breaks/radiation effects , DNA Repair/radiation effects , Down-Regulation/radiation effects , Histone Acetyltransferases/metabolism , Methylation , Mice, Inbred C57BL , Neural Stem Cells/metabolism , Neural Stem Cells/radiation effects , Radiation Tolerance/radiation effects , Up-Regulation/genetics , Up-Regulation/radiation effectsABSTRACT
Normal tissue injury resulting from cancer radiotherapy is often associated with diminished regenerative capacity. We examined the relative radiosensitivity of normal stem cell populations compared with non-stem cells within several radiosensitive tissue niches and culture models. We found that these stem cells are highly radiosensitive, in contrast to their isogenic differentiated progeny. Of interest, they also exhibited a uniquely attenuated DNA damage response (DDR) and muted DNA repair. Whereas stem cells exhibit reduced ATM activation and ionizing radiation-induced foci, they display apoptotic pannuclear H2AX-S139 phosphorylation (γH2AX), indicating unique radioresponses. We also observed persistent phosphorylation of H2AX-Y142 along the DNA breaks in stem cells, which promotes apoptosis while inhibiting DDR signaling. In addition, down-regulation of constitutively elevated histone-3 lysine-56 acetylation (H3K56ac) in stem cells significantly decreased their radiosensitivity, restored DDR function, and increased survival, signifying its role as a key contributor to stem cell radiosensitivity. These results establish that unique epigenetic landscapes affect cellular heterogeneity in radiosensitivity and demonstrate the nonubiquitous nature of radiation responses. We thus elucidate novel epigenetic rheostats that promote ionizing radiation hypersensitivity in various normal stem cell populations, identifying potential molecular targets for pharmacological radioprotection of stem cells and hopefully improving the efficacy of future cancer treatment.
