After DNA damage, many cells appear to enter a sustained arrest in the G2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, gamma radiated cells progressed into mitosis and exhibited a G2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G2 checkpoint in human cells.

The energy that sustains cancer cells is derived preferentially from glycolysis. This metabolic change, the Warburg effect, was one of the first alterations in cancer cells recognized as conferring a survival advantage. Here, we show that p53, one of the most frequently mutated genes in cancers, modulates the balance between the utilization of respiratory and glycolytic pathways. We identify Synthesis of Cytochrome c Oxidase 2 (SCO2) as the downstream mediator of this effect in mice and human cancer cell lines. SCO2 is critical for regulating the cytochrome c oxidase (COX) complex, the major site of oxygen utilization in the eukaryotic cell. Disruption of the SCO2 gene in human cancer cells with wild-type p53 recapitulated the metabolic switch toward glycolysis that is exhibited by p53-deficient cells. That SCO2 couples p53 to mitochondrial respiration provides a possible explanation for the Warburg effect and offers new clues as to how p53 might affect aging and metabolism.

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We elucidate the cellular and molecular kinetics of the stepwise differentiation of human embryonic stem cells (hESCs) to primitive and definitive erythromyelopoiesis from human embryoid bodies (hEBs) in serum-free clonogenic assays. Hematopoiesis initiates from CD45 hEB cells with emergence of semiadherent mesodermalhematoendothelial (MHE) colonies that can generate endothelium and form organized, yolk sac-like structures that secondarily generate multipotent primitive hematopoietic stem progenitor cells (HSPCs), erythroblasts, and CD13 ؉ CD45 ؉ macrophages. A first wave of hematopoiesis follows MHE colony emergence and is predominated by primitive erythropoiesis characterized by a brilliant red hemoglobinization, CD71/CD325a (glycophorin A) expression, and exclusively embryonic/ fetal hemoglobin expression. A second wave of definitive-type erythroid burstforming units (BFU-e's), erythroid colonyforming units (CFU-e's), granulocytemacrophage colony-forming cells (GMCFCs), and multilineage CFCs follows next from hEB progenitors. These stages of hematopoiesis proceed spontaneously from hEB-derived cells without requirement for supplemental growth factors during hEB differentiation. Gene expression analysis of differentiating hEBs revealed that initiation of hematopoiesis correlated with increased levels of SCL/TAL1, GATA1, GATA2, CD34, CD31, and the homeobox gene-regulating factor CDX4 These data indicate that hematopoietic differentiation of hESCs models the earliest events of embryonic and definitive hematopoiesis in a manner resembling human yolk sac development, thus providing a valuable tool for dissecting the earliest events in human HSPC genesis. IntroductionClassic and contemporary anatomic studies of human embryos have revealed that human hematopoiesis begins in the second to third embryonic weeks with formation of mesoderm-derived blood islands in the extraembryonic mesoderm of the developing secondary yolk sac. 1,2 Blood islands develop foci of nucleated erythroblasts ("megaloblasts"), 3 intimately associated with and surrounded by endothelium. Yolk sac (primitive) blood cells consist of nucleated primitive erythrocytes expressing exclusively embryonic hemoglobins (eg, ⑀ 2 2 globin chains) and primitive macrophages that arise without detectable monocytic precursors. Following the onset of circulation at about 21 days of development, yolk sac cells are found in embryonic blood. The fetal liver subsequently replaces the yolk sac as the main hematopoietic organ 4,5 with appearance of definitive enucleate, macrocytic erythrocytes expressing fetal hemoglobins (eg, ␣ 2 ␥ 2 globin chains). Definitive blood cells and hematopoietic stem progenitor cells (HSPCs) can be detected in the fetal liver and embryo beginning at 5 to 6 weeks but have also been assayed as early as 4 to 5 weeks from human yolk sac, suggesting a gradual yolk sac/fetal liver HSPC transition. 1,6 The human adult long-term repopulating HSPC that ultimately seeds the fetal bone marrow and thymus is the legacy of fetal liver hematopoiesis.Both ...

Nuclear factor erythroid-2 related factor-2 (Nrf2) is a redox-sensitive transcription factor that regulates the expression of electrophile and xenobiotic detoxification enzymes and efflux proteins, which confer cytoprotection against oxidative stress and apoptosis in normal cells. Loss of function mutations in the Nrf2 inhibitor, Kelch-like ECH-associated protein (Keap1), results in constitutive activation of Nrf2 function in non-small-cell lung cancer (NSCLC). In this study, we demonstrate that constitutive activation of Nrf2 in lung cancer cells promotes tumorigenicity and contributes to chemoresistance by upregulation of glutathione, thioredoxin and the drug efflux pathways involved in detoxification of electrophiles and broad spectrum of drugs. RNAi-mediated reduction of Nrf2 expression in lung cancer cells induces generation of reactive oxygen species, suppresses tumor growth and results in increased sensitivity to chemotherapeutic drug induced cell death in vitro and in vivo. Inhibiting Nrf2 expression using naked siRNA duplexes in combination with carboplatin significantly inhibits tumor growth in a subcutaneous model of lung cancer. Thus, targeting Nrf2 activity in lung cancers, particularly those with Keap1 mutations, could be a promising strategy to inhibit tumor growth and circumvent chemoresistance.

Abnormalities of chromosome number are the most common genetic aberrations in cancer. The mechanisms regulating the fidelity of mitotic chromosome transmission in mammalian cells are therefore of great interest. Here we show that human cells without an hSecurin gene lose chromosomes at a high frequency. This loss was linked to abnormal anaphases during which cells underwent repetitive unsuccessful attempts to segregate their chromosomes. The abnormal mitoses were associated with biochemical defects in the activation of separin, the sister-separating protease, rendering it unable to cleave the cohesin subunit Scc1 efficiently. These results illuminate the function of mammalian securin and show that it is essential for the maintenance of euploidy.

Through targeted homologous recombination, we developed a panel of matched colorectal cancer cell lines that differ only with respect to their endogenous TP53 status. We then used these lines to define the genes whose expression was altered after DNA damage induced by ionizing radiation. Transcriptome analyses revealed a consistent upregulation of polo-like kinase 1 (PLK1) as well as other genes controlling the G 2/M transition in the cells whose TP53 genes were inactivated compared with those with WT TP53 genes. This led to the hypothesis that the viability of stressed cells without WT TP53 depended on PLK1. This hypothesis was validated by demonstrating that stressed cancer cells without WT TP53 alleles were highly sensitive to PLK1 inhibitors, both in vivo and in vitro.

Loss of p53 gene function, which occurs in most colon cancer cells, has been shown to abolish the apoptotic response to 5-fluorouracil (5-FU). To identify genes downstream of p53 that might mediate these effects, we assessed global patterns of gene expression following 5-FU treatment of isogenic cells differing only in their p53 status. The gene encoding mitochondrial ferredoxin reductase (protein, FR; gene, FDXR) was one of the few genes significantly induced by p53 after 5-FU treatment. The FR protein was localized to mitochondria and suppressed the growth of colon cancer cells when over-expressed. Targeted disruption of the FDXR gene in human colon cancer cells showed that it was essential for viability, and partial disruption of the gene resulted in decreased sensitivity to 5-FU-induced apoptosis. These data, coupled with the effects of pharmacologic inhibitors of reactive oxygen species, indicate that FR contributes to p53-mediated apoptosis through the generation of oxidative stress in mitochondria.5-fluorouracil (5-FU) has been in clinical use for several decades and is still the most effective adjuvant therapy for patients with colon cancer. Metabolically, it acts by blocking the enzyme thymidylate synthase and by inhibiting both RNA and DNA synthesis. Like most chemotherapeutic agents, 5-FU induces marked apoptosis in sensitive cells. However, the molecular mechanisms underlying this apoptosis are only beginning to be elucidated.Several studies have suggested a correlation between inactivation of p53 and resistance to 5-FU, both in patients and in experimental systems 1,2 . For example, a profound resistance to 5-FU-induced apoptosis was observed in a human colon cancer cell line with a targeted disruption of the p53 gene (Trp53) 3 . Such observations support a genetic basis for 5-FU chemosensitivity and indicate that isogenic cells differing only in p53 status represent a useful system for studying chemosensitivity in vitro.© 2001 Nature Publishing Group Correspondence should be addressed to B.V.; vogelbe@welch.jhu.edu. Supplementary information is available on the Nature Medicine website (http://medicine.nature.com/supplementary_info/). NIH Public Access NIH-PA Author ManuscriptThe mechanisms through which p53 induces apoptosis are the focus of much current research [4][5][6][7] . Several studies have implicated mitochondria-derived reactive oxygen species (ROS) in the process [8][9][10][11] . Though pro-apoptotic members of the Bcl-2 family of proteins have been shown to be induced by p53 and affect mitochondrial permeability, the molecular mechanisms responsible for the formation of mitochondrial ROS remain unclear 12,13 .In an effort to gain further insight into this process, we examined global patterns of gene expression using the serial analysis of gene expression (SAGE) approach 14 . We were particularly interested in finding p53-induced genes that encode mitochondrial proteins with the potential to directly stimulate ROS production. Here we identify mammalian ferredoxin reductase (FR) ...