Epstein-Barr virus (EBV) isEpstein-Barr virus (EBV) is a human herpesvirus that is estimated to infect up to 90% of the world's population (28,29). EBV infection is associated with several human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, Burkitt's lymphoma, and, in immunosuppressed patients, B-cell and T-cell lymphomas (28,29). Thus, EBV is a serious pathogen and poses a significant threat to human health.Like other herpesviruses, primary infection with EBV is followed by a persistent infection of the human host. Oropharyngeal epithelium is thought to be the primary site of EBV infection and replication and of viral spread (28-30, 51), while B cells are the major site of persistent latency (28-31). Eleven of EBV's approximately 100 viral genes are expressed during latency. These include ones encoding the EBV-encoded nuclear antigens (EBNAs 1 to 6), the latent membrane proteins (LMPs 1 and 2), two EBV-encoded small nuclear RNAs, and the BamHIA transcripts (28, 29). Expression of a subset of the latter genes is sufficient to immortalize B cells and to maintain steady-state levels of the viral genome (31). Treatment of certain latently infected B-cell populations with reagents such as phorbol esters (5, 17, 64), Ca 2ϩ ionophores (15), sodium butyrate (26, 38), and serum factors (2) or cross-linking of surface anti-immunoglobulin (12, 21, 50, 53) leads to cellular differentiation and the concomitant induction of the rest of EBV's genes, followed by viral genome replication to higher copy number and production of infectious virus particles (28-30).Two immediate-early genes, BZLF1 and BRLF1, are the first to be expressed during induction of EBV out of latency (11,23,32,52). The protein products of both of these genes are strong transcriptional transactivators (9, 16). The product of the BZLF1 gene, referred to as Zta, ZEBRA, or EB1, plays a crucial role in the disruption of latency and initiation of the viral infectious cycle (10,11,41,46,52). Thus, regulation of Zta expression is critical to the state of EBV in cells.The transcriptional regulation of the BZLF1 promoter (also referred to as Zp) has been studied extensively. Zp exhibits very low basal activity and is readily activated by inducers of the viral infectious cycle. The cis-acting elements necessary both for basal activity and for response to exogenous inducers lie within the nucleotide (nt) Ϫ221 to ϩ12 region of the promoter relative to the transcriptional initiation site (12,17,18). These elements have been divided into three classes ( Fig. 1; also, see reference 36 and references therein). Four AT-rich elements, termed ZIA to ZID, are dispersed throughout the promoter. They can bind the transcription factors Sp1 and Sp3 (34) and myocyte enhancer factor 2D (37, 44). A second type of element, ZII, shares significant homology with the consensus CRE/AP-1 binding site (1,13,25,54). Finally, a region called ZIII contains multiple binding sites for the Zta protein itself (18). It has been proposed that activation of the BZLF1 gene oc...
Summary Until there are valid identifiers that visualize stem cells in vivo, we rely upon flow cytometry to enrich for subpopulations with stem cell function. However, data reporting styles for flow cytometric analyses are typically inconsistent, creating challenges in comparing results across publications. In our view, clear reporting guidelines could improve reproducibility of stem cell analyses in solid tissues.
Hydroxyurea (HU) is a competitive inhibitor of ribonucleotide reductase that is used for the treatment of myeloproliferative disorders. HU inhibits DNA replication and induces apoptosis in a cell type-dependent manner, yet the relevant pathways that mediate apoptosis in response to this agent are not well characterized. In this study, we employed the human myeloid leukemia 1 (ML-1) cell line as a model to investigate the mechanisms of HU-induced apoptosis. Exposure of ML-1 cells to HU caused rapid cell death that was accompanied by hallmark features of apoptosis, including membrane blebbing, phosphatidylserine translocation, and caspase activation. HU-induced apoptosis required new protein synthesis, was induced by HU exposures as short as 15 min, and correlated with the accumulation of p53 and induction of the p53 target gene PUMA. p53 induction in ML-1 cells was ATR dependent and downregulation of p53 through RNAi delayed HU-induced apoptosis. HU did not induce p53 or induce apoptosis in Molt-3 leukemia cells, even though exposure to HU induced a comparable level of DNA damage and robustly activated the ATR pathway. The microtubule inhibitor nocodazole suppressed HU-induced p53 accumulation in ML-1 cells suggesting that a microtubule-dependent event contributes to p53 induction and apoptosis in this cell line. Our findings outline an HU-induced cell death pathway and suggest that activation of ATR is necessary, but not sufficient, for stabilization of p53 in response to DNA replication stress.
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