Ester M. Hammond

Regulation of p53 by Hypoxia: Dissociation of Transcriptional Repression and Apoptosis from p53-Dependent Transactivation

ABSTRACT Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

ATR/ATM Targets Are Phosphorylated by ATR in Response to Hypoxia and ATM in Response to Reoxygenation

The ATR kinase phosphorylates both p53 and Chk1 in response to extreme hypoxia (oxygen concentrations of less than 0.02%). In contrast to ATR, loss of ATM does not affect the phosphorylation of these or other targets in response to hypoxia. However, hypoxia within tumors is often transient and is inevitably followed by reoxygenation. We hypothesized that ATR activity is induced under hypoxic conditions because of growth arrest and ATM activity increases in response to the oxidative stress of reoxygenation. Using the comet assay to detect DNA damage, we find that reoxygenation induced significant amounts of DNA damage. Two ATR/ATM targets, p53 serine 15 and histone H2AX, were both phosphorylated in response to hypoxia in an ATR-dependent manner. These phosphorylations were then maintained in response to reoxygenation-induced DNA damage in an ATM-dependent manner. The reoxygenation-induced p53 serine 15 phosphorylation was inhibited by the addition ofN-acetyl-l-cysteine (NAC), indicating that free radical-induced DNA damage was mediated by reactive oxygen species. Taken together these data implicate both ATR and ATM as critical roles in the response of hypoxia and reperfusion in solid tumors.

DNA Damage during Reoxygenation Elicits a Chk2-Dependent Checkpoint Response

ABSTRACT Due to the abnormal vasculature of solid tumors, tumor cell oxygenation can change rapidly with the opening and closing of blood vessels, leading to the activation of both hypoxic response pathways and oxidative stress pathways upon reoxygenation. Here, we report that ataxia telangiectasia mutated-dependent phosphorylation and activation of Chk2 occur in the absence of DNA damage during hypoxia and are maintained during reoxygenation in response to DNA damage. Our studies involving oxidative damage show that Chk2 is required for G2 arrest. Following exposure to both hypoxia and reoxygenation, Chk2−/− cells exhibit an attenuated G2 arrest, increased apoptosis, reduced clonogenic survival, and deficient phosphorylation of downstream targets. These studies indicate that the combination of hypoxia and reoxygenation results in a G2 checkpoint response that is dependent on the tumor suppressor Chk2 and that this checkpoint response is essential for tumor cell adaptation to changes that result from the cycling nature of hypoxia and reoxygenation found in solid tumors.

Temporal and spatial expression of two isoforms of the Dutt1/Robo1 gene in mouse development

The mammalian homologue of the Drosophila axonal guidance receptor roundabout is expressed in a wide range of tissues. Here we show that alternative splicing of the Dutt1/Robo1 gene results in two mRNA transcripts with different signal peptides, which are differentially expressed throughout mouse embryogenesis. Since mice with a targeted deletion in the Dutt1/Robo1 gene have abnormal lung pathology, immunohistochemistry was used to identify the cellular expression pattern of Dutt1/Robo1 during lung development. Dutt1/Robo1 expression was widespread and diffuse in the lung at embryonic day 17.5 but became increasingly localised to the bronchial epithelium in newborn and adult mice.

Bringing H2AX into the angiogenesis family.

The cells ability to sense and respond to DNA damage is critical to maintain homeostasis and prevent the development of cancer. Paradoxically, Economopoulou et al. recently reported that a DNA damage response protein, H2AX, promotes tumor growth and angiogenesis.

The induction and suppression of apoptosis by viruses

Publisher Summary This chapter discusses how viruses induce and/or suppress apoptosis, focusing on the viruses that can transform cells and cause cancers in mammals or those that have provided invaluable insights into the mechanism of apoptosis. Apoptosis is an opposing process to transformation in vivo, as cells that fail to apoptose correctly can then grow inappropriately, giving rise to tumors. A consideration of the ways in which viral oncogenes regulate apoptosis, both positively and negatively, will provide insights into some of the mechanisms by which virally-induced and naturally occurring tumors might arise. After viral infection, there is pressure on the host cell to apoptose rapidly as a way of combating the virus. Most viruses express one or more proteins to inhibit apoptosis, such that optimal production of progeny can occur at an appropriate time. At a late stage during infection, a process akin to, but perhaps distinct from apoptosis occurs, such that cell lysis can take place releasing the viral progeny. The study of virus-induced apoptosis has provided considerable insight into how the mechanism of apoptosis is regulated, both positively and negatively. These studies have increased the knowledge of many aspects of normal cellular functioning.