Kirsteen J Campbell

Active Repression of Antiapoptotic Gene Expression by RelA(p65) NF-κB

With the emerging role of NF-kappa B in cancer it is important that its responses to stimuli relevant to tumor progression and therapy are understood. Here, we demonstrate that NF-kappa B induced by cytotoxic stimuli, such as ultraviolet light (UV-C) and the chemotherapeutic drugs daunorubicin/doxorubicin, is functionally distinct to that seen with the inflammatory cytokine TNF and is an active repressor of antiapoptotic gene expression. Surprisingly, these effects are mediated by the RelA(p65) NF-kappa B subunit. Furthermore, UV-C and daunorubicin inhibit TNF-induced NF-kappa B transactivation, indicating that this is a dominant effect. Consistent with this, mechanistic studies reveal that UV-C and daunorubicin induce the association of RelA with histone deacetylases. RelA can therefore be both an activator and repressor of its target genes, dependent upon the manner in which it is induced. This has important implications for the role of NF-kappa B in tumorigenesis and the use of NF-kappa B inhibitors in cancer therapy.

p53- and Mdm2-Independent Repression of NF-κB Transactivation by the ARF Tumor Suppressor.

One mechanism by which a cell affords protection from the transforming effects of oncogenes is via the action of the tumor suppressor, ARF, which activates p53 by inactivating Mdm2. Many oncogenes have also been shown to activate the transcription factor NF-kappa B, which can contribute toward the malignant phenotype in many ways, including an ability to antagonize p53. Here we find that ARF inhibits NF-kappa B function and its antiapoptotic activity independent of Mdm2 and p53. ARF represses the transcriptional activation domain of the NF-kappa B family member RelA by inducing its association with the histone deacetylase, HDAC1. Further, we show that the response of NF-kappa B to the oncogene Bcr-Abl is determined by the ARF status of the cell. These results reveal an important function of ARF that can regulate the NF-kappa B response to oncogene activation.

Regulation of NF‐κB and p53 through activation of ATR and Chk1 by the ARF tumour suppressor

The ARF tumour suppressor is a central component of the cellular defence against oncogene activation. In addition to activating p53 through binding Mdm2, ARF possesses other functions, including an ability to repress the transcriptional activity of the antiapoptotic RelA(p65) NF‐κB subunit. Here we demonstrate that ARF induces the ATR‐ and Chk1‐dependent phosphorylation of the RelA transactivation domain at threonine 505, a site required for ARF‐dependent repression of RelA transcriptional activity. Consistent with this effect, ATR and Chk1 are required for ARF‐induced sensitivity to tumour necrosis factor α‐induced cell death. Significantly, ATR activity is also required for ARF‐induced p53 activity and inhibition of proliferation. ARF achieves these effects by activating ATR and Chk1. Furthermore, ATR and its scaffold protein BRCA1, but not Chk1, relocalise to specific nucleolar sites. These results reveal novel functions for ARF, ATR and Chk1 together with a new pathway regulating RelA NF‐κB function. Moreover, this pathway provides a mechanism through which ARF can remodel the cellular response to an oncogenic challenge and execute its function as a tumour suppressor.

Nud1p links astral microtubule organization and the control of exit from mitosis

The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell‐cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the γ‐tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cell‐cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1‐2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1‐2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.

Cisplatin Mimics ARF Tumor Suppressor Regulation of RelA (p65) Nuclear Factor-κB Transactivation

The RelA (p65) nuclear factor-kappaB (NF-kappaB) subunit can contribute towards tumor cell survival through inducing the expression of a variety of antiapoptotic genes. However, the NF-kappaB response can show great diversity and is not always antiapoptotic. Here, we find that cisplatin, a DNA cross-linking agent and commonly used anticancer compound, does not affect RelA nuclear translocation but modulates its transcriptional activity. Similar to other genotoxic agents, such as daunorubicin and UV light, cisplatin treatment in the U-2 OS osteosarcoma cell line represses RelA activity and inhibits expression of the NF-kappaB antiapoptotic target gene Bcl-x(L). The mechanism through which cisplatin achieves these effects is different to daunorubicin and UV light but shows great similarity to the RelA regulatory pathway induced by the ARF tumor suppressor: cisplatin regulation of RelA requires ATR/Chk1 activity, represses Bcl-x(L) but not XIAP expression, and results in phosphorylation of RelA at Thr(505). In contrast to these results, another chemotherapeutic drug etoposide activates NF-kappaB and induces expression of these target genes. Thus, within a single tumor cell line, there is great heterogeneity in the NF-kappaB response to different, commonly used chemotherapeutic drugs. These observations suggest that it might be possible to minimize the ability of RelA to inhibit cancer therapy by diagnostically predicting the type of chemotherapeutic drug most compatible with NF-kappaB functionality in a tumor cell type. Moreover, our data indicate that at least with respect to RelA, cisplatin functions as an ARF mimic. Other drugs capable of mimicking this aspect of ARF function might therefore have therapeutic potential.

The p53-inhibitor Pifithrin-α inhibits Firefly Luciferase activity in vivo and in vitro

BackgroundPifithrin-α is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-α in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-α provides a useful tool in the laboratory to investigate the function of p53 in model systems.ResultsWhile investigating the effects of pifithrin-α on the transcriptional activity of NF-κB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-α was observed both in vivo and in vitro. Pifithrin-α did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-α to active extracts inhibited this activity. Furthermore, pifithrin-α also inhibited recombinant firefly luciferase protein activity.ConclusionsAmong its other biological activities, pifithrin-α is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

The p53-inhibitor Pifithrin-α inhibits Firefly Luciferase activity in vivo

BackgroundPifithrin-α is a small molecule inhibitor of p53 transcriptional activity. It has been proposed that the use of pifithrin-α in conjunction with chemotherapeutic and radiation therapies for cancer will reduce the side effects of these treatments in normal tissue that still contains wild type p53. In addition, pifithrin-α provides a useful tool in the laboratory to investigate the function of p53 in model systems.ResultsWhile investigating the effects of pifithrin-α on the transcriptional activity of NF-κB, we observed a strong inhibition of reporter plasmids containing the firefly luciferase gene. Firefly luciferase is one of the most commonly used enzymes in reporter gene assays. In contrast, no inhibition of reporter plasmids containing Renilla luciferase or chloramphenicol acetyltransferase was observed. The inhibition of firefly luciferase activity by pifithrin-α was observed both in vivo and in vitro. Pifithrin-α did not inhibit firefly luciferase protein expression, but rather suppressed light production/emission, since addition of exogenous pifithrin-α to active extracts inhibited this activity. Furthermore, pifithrin-α also inhibited recombinant firefly luciferase protein activity.ConclusionsAmong its other biological activities, pifithrin-α is an inhibitor of firefly luciferase activity. Caution must therefore be taken when using this compound, which has been characterised as an inhibitor of p53 transcriptional activity, to investigate effects on gene expression using transiently transfected reporter plasmids. Furthermore, these results demonstrate that when using novel compounds, the choice of vectors used in the experimental procedures might be of great importance for the correct conclusions to be made.

Differential regulation of NF-κB activation and function by topoisomerase II inhibitors

BackgroundWhile many common chemotherapeutic drugs and other inducers of DNA-damage result in both NF-κB nuclear translocation and DNA-binding, we have previously observed that, depending on the precise stimulus, there is great diversity of the function of NF-κB. In particular, we found that treatment of U-2 OS osteosarcoma cells with the anthracycine daunorubicin or with ultraviolet (UV-C) light resulted in a form of NF-κB that repressed rather than induced NF-κB reporter plasmids and the expression of specific anti-apoptotic genes. Anthracyclines such as daunorubicin can induce DNA-damage though inhibiting topoisomerase II, intercalating with DNA and undergoing redox cycling to produce oxygen free radicals. In this study we have investigated other anthracyclines, doxorubicin and aclarubicin, as well as the anthracenedione mitoxantrone together with the topoisomerase II inhibitor ICRF-193, which all possess differing characteristics, to determine which of these features is specifically required to induce both NF-κB DNA-binding and transcriptional repression in U-2 OS cells.ResultsThe use of mitoxantrone, which does not undergo redox cycling, and the reducing agent epigallocatechingallate (EGCG) demonstrated that oxygen free radical production is not required for induction of NF-κB DNA-binding and transcriptional repression by these agents and UV-C. In addition, the use of aclarubicin, which does not directly inhibit topoisomerase II and ICRF-193, which inhibits topoisomerase II but does not intercalate into DNA, demonstrated that topoisomerase II inhibition is not sufficient to induce the repressor form of NF-κB.ConclusionInduction of NF-κB DNA-binding and transcriptional repression by topoisomerase II inhibitors was found to correlate with an ability to intercalate into DNA. Although data from our and other laboratories indicates that topoisomerase II inhibition and oxygen free radicals do regulate NF-κB, they are not required for the particular ability of NF-κB to repress rather than activate transcription. Together with our previous data, these results demonstrate that the nature of the NF-κB response is context dependent. In a clinical setting such effects could profoundly influence the response to chemotherapy and suggest that new methods of analyzing NF-κB function could have both diagnostic and prognostic value.

Cezanne regulates E2F1-dependent HIF2α expression

ABSTRACT Mechanisms regulating protein degradation ensure the correct and timely expression of transcription factors such as hypoxia inducible factor (HIF). Under normal O2 tension, HIFα subunits are targeted for proteasomal degradation, mainly through vHL-dependent ubiquitylation. Deubiquitylases are responsible for reversing this process. Although the mechanism and regulation of HIFα by ubiquitin-dependent proteasomal degradation has been the object of many studies, little is known about the role of deubiquitylases. Here, we show that expression of HIF2α (encoded by EPAS1) is regulated by the deubiquitylase Cezanne (also known as OTUD7B) in an E2F1-dependent manner. Knockdown of Cezanne downregulates HIF2α mRNA, protein and activity independently of hypoxia and proteasomal degradation. Mechanistically, expression of the HIF2α gene is controlled directly by E2F1, and Cezanne regulates the stability of E2F1. Exogenous E2F1 can rescue HIF2α transcript and protein expression when Cezanne is depleted. Taken together, these data reveal a novel mechanism for the regulation of the expression of HIF2α, demonstrating that the HIF2α promoter is regulated by E2F1 directly and that Cezanne regulates HIF2α expression through control of E2F1 levels. Our results thus suggest that HIF2α is controlled transcriptionally in a cell-cycle-dependent manner and in response to oncogenic signalling. Summary: Cezanne regulates HIF2α levels and activity by modulating expression of the HIF2α gene through regulation of E2F1 protein levels. Cezanne depletion impairs normal cell cycle progression and increases cell death.

Regulation of checkpoint kinase signalling and tumorigenesis by the NF-κB regulated gene, CLSPN

Inhibition of the tumour promoting activities of NF-κB by cell signalling pathways has been proposed as a natural mechanism to limit the development of cancer. However, there has been a lack of evidence for these effects in vivo. Here we report that RelAT505A mice, where a CHK1 targeted Thr505 phosphosite is mutated to alanine, display earlier onset of MYC driven lymphoma than wild type littermates. We describe a positive feedback loop in which the NF-κB subunits RelA and c-Rel, in a manner dependent upon RelA Thr505 phosphorylation, drive the expression of the ATR checkpoint kinase regulator Claspin in response to DNA replication stress in cancer cells. This in turn is required for maintenance of CHK1 activity. Loss of a single allele of the Clspn gene in mice is sufficient to drive earlier tumorigenesis and low levels of CLSPN mRNA expression are associated with worse survival in some forms of human cancer. We propose that loss of this pathway early in tumorigenesis promotes cancer development through increased genomic instability. However, in malignant cancer cells it can help promote their addiction to the checkpoint kinase signalling required for the maintenance of genomic integrity. Importantly, disruption of this pathway leads to resistance of cells to treatment with CHK1 inhibitors. Claspin expression could therefore act as a biomarker for responsiveness of patients to CHK1 inhibitors and provide a potential pathway for the development of tumour resistance.