Bromodeoxyuridine Induces Senescence in Neural Stem and Progenitor Cells
ABSTRACT Bromodeoxyuridine (BrdU) is a halogenated pyrimidine that incorporates into newly synthesized DNA during the S phase. BrdU is used ubiquitously in cell birthdating studies and as a means of measuring the proliferative index of various cell populations. In the absence of secondary stressors, BrdU is thought to incorporate relatively benignly into replicating DNA chains. However, we report here that a single, low-dose pulse of BrdU exerts a profound and sustained antiproliferative effect in cultured murine stem and progenitor cells. This is accompanied by altered terminal differentiation, cell morphology, and protein expression consistent with the induction of senescence. There is no evidence of a significant increase in spontaneous cell death; however, cells are rendered resistant to chemically induced apoptosis. Finally, we show that a brief in vivo BrdU regimen reduces the proliferative potential of subsequently isolated subependymal zone neurosphere-forming cells. We conclude, therefore, that BrdU treatment induces a senescence pathway that causes a progressive decline in the replication of rapidly dividing stem/progenitor cells, suggesting a novel and uncharacterized effect of BrdU. This finding is significant in that BrdU-incorporating neural stem/progenitor cells and their progeny should not be expected to behave normally with respect to proliferative potential and downstream functional parameters. This effect highlights the need for caution when results based on long-term BrdU tracking over multiple rounds of replication are interpreted. Conversely, the reliable induction of senescence in stem/progenitor cells in vitro and in vivo may yield a novel platform for molecular studies designed to address multiple aspects of aging and neurogenesis.Disclosure of potential conflicts of interest is found at the end of this article.
Article: Neural stem cells exposed to BrdU lose their global DNA methylation and undergo astrocytic differentiation.[show abstract] [hide abstract]
ABSTRACT: Bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) is a halogenated nucleotide of low toxicity commonly used to monitor DNA replication. It is considered a valuable tool for in vitro and in vivo studies, including the detection of the small population of neural stem cells (NSC) in the mammalian brain. Here, we show that NSC grown in self-renewing conditions in vitro, when exposed to BrdU, lose the expression of stem cell markers like Nestin, Sox2 and Pax6 and undergo glial differentiation, strongly up-regulating the astrocytic marker GFAP. The onset of GFAP expression in BrdU exposed NSC was paralleled by a reduced expression of key DNA methyltransferases (DNMT) and a rapid loss of global DNA CpG methylation, as we determined by our specially developed analytic assay. Remarkably, a known DNA demethylating compound, 5-aza-2'-deoxycytidine (Decitabine), had similar effect on demethylation and differentiation of NSC. Since our key findings apply also to NSC derived from murine forebrain, our observations strongly suggest more caution in BrdU uses in stem cells research. We also propose that BrdU and its related substances may also open new opportunities for differentiation therapy in oncology.Nucleic Acids Research 02/2012; 40(12):5332-42. · 8.03 Impact Factor
Article: Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury.[show abstract] [hide abstract]
ABSTRACT: Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury. C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation. BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation. Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.Respiratory research 01/2011; 12:78. · 3.36 Impact Factor
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ABSTRACT: The last decade witnessed an explosion of interest in cancer stem cells (CSCs). The realization of epithelial ovarian cancer (EOC) as a CSC-related disease has the potential to change approaches in the treatment of this devastating disease dramatically. The etiology and early events in the progression of these carcinomas are among the least understood of all major human malignancies. Compared to the CSCs of other cancer types, the identification and study of EOC stem cells (EOCSCs) is rather difficult due to several major obstacles: the heterogeneity of tumors comprising EOCs, unknown cells of origin, and lack of knowledge considering the normal ovarian stem cells. This poses a major challenge for urgent development in this research field. This review summarizes and evaluates the current evidence for the existence of candidate normal ovarian epithelial stem cells as well as EOCSCs, emphasizing the requirement for a more definitive laboratory approach for the isolation, identification, and enrichment of EOCSCs. The present review also revisits the ongoing debate regarding other cells and tissues of origin of EOCs, and discusses early events in the pathogenesis of this disease. Finally, this review discusses the signaling pathways that are important regulators of candidate EOCSC maintenance and function, their potential role in the distinct pathogenesis of different EOC subtypes, as well as potential mechanisms and clinical relevance of EOCSC involvement in drug resistance.TheScientificWorldJOURNAL 01/2011; 11:1243-69. · 1.66 Impact Factor