Biomedical subjects

Neil D Perkins

Publications and source records attributed to Neil D Perkins.

At least 19 recordsLinked to original sources

PubMed2006

Regulation of p53 tumour suppressor target gene expression by the p52 NF-kappaB subunit.

The p52/p100 nuclear factor kappa B (NF-kappaB) subunit (NF-kappaB2) is aberrantly expressed in many tumour types and has been implicated as a regulator of cell proliferation. Here, we demonstrate that endogenous p52 is a direct regulator of Cyclin D1 expression. However, stimulation of Cyclin D1 expression alone cannot account for all the cell cycle effects of p52/p100 and we also find that p52 represses expression of the Cyclin-dependent kinase inhibitor p21(WAF/CIP1). Significantly, this latter effect is dependent upon basal levels of the tumour suppressor p53. By contrast, p52 cooperates with p53 to regulate other known p53 target genes such as PUMA, DR5, Gadd45alpha and Chk1. p52 associates directly with these p53-regulated promoters where it regulates coactivator and corepressor binding. Moreover, recruitment of p52 is p53 dependent and does not require p52-DNA-binding activity. These results reveal a complex role for p52 as regulator of cell proliferation and p53 transcriptional activity. Furthermore, they imply that in some cell types, p52 can regulate p53 function and influence p53-regulated decision-making following DNA damage and oncogene activation.

Apoptosis Regulatory Proteins↗

PubMed2006

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

BACKGROUND: While many common chemotherapeutic drugs and other inducers of DNA-damage result in both NF-kappaB nuclear translocation and DNA-binding, we have previously observed that, depending on the precise stimulus, there is great diversity of the function of NF-kappaB. 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-kappaB that repressed rather than induced NF-kappaB 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-kappaB DNA-binding and transcriptional repression in U-2 OS cells. RESULTS: The 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-kappaB 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-kappaB. CONCLUSION: Induction of NF-kappaB 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-kappaB, they are not required for the particular ability of NF-kappaB to repress rather than activate transcription. Together with our previous data, these results demonstrate that the nature of the NF-kappaB 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-kappaB function could have both diagnostic and prognostic value.

Antibiotics, Antineoplastic↗

PubMed2006

Cisplatin mimics ARF tumor suppressor regulation of RelA (p65) nuclear factor-kappaB 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.

Antineoplastic Agents↗

PubMed2006

Regulation of NF-kappaB function.

In the 20 years since its discovery, research into the NF-kappaB (nuclear factor-kappaB) family of transcription factors has revealed an amazing diversity of functions. NF-kappaB proteins are regulators of the immune, inflammatory, stress, proliferative and apoptotic responses of a cell to a very large number of different stimuli. NF-kappaB complexes can be found in all cell types, indicating that the number of different contexts in which NF-kappaB can become induced is enormous. Moreover, many reports suggest apparently opposing or contradictory functions for NF-kappaB. It is clear that it is not simply enough to understand the pathways leading to nuclear localization and DNA binding of NF-kappaB subunits. It is also important that we comprehend the regulation of NF-kappaB subunit functionality if we are to understand the NF-kappaB pathway as a whole. These issues include the mechanisms controlling the specificity and timing of genes regulated by NF-kappaB under particular circumstances. They also include the reasons why NF-kappaB subunits can sometimes repress rather than activate transcription and how the NF-kappaB response is integrated with other important transcription factor pathways in the cell, such as the induction of the p53 tumour suppressor following DNA damage or oncogene activation. Understanding the mechanisms that regulate NF-kappaB function has important implications for our understanding of the role that NF-kappaB subunits play in human inflammatory diseases and cancer, and could also impact on the use of future NF-kappaB-based clinical therapies.

Animals↗

PubMed2005

p21WAF1/CIP1 regulates the p300 sumoylation motif CRD1 through a C-terminal domain independently of cyclin/CDK binding.

Although best known for its ability to inhibit Cyclin/Cdk complexes and the replication protein PCNA, p21(WAF1/CIP1) is a multifunctional protein that interacts with many cellular binding partners, including a number of transcriptional regulators. Previously, we characterized p21 derepression of the p300 sumoylation-dependent transcriptional repression domain, CRD1. Such repression domains are at least partially dependent upon recruitment of histone deacetylase (HDAC) complexes but the mechanism through which p21 selectively disrupts CRD1 activity remains unknown. Here, we demonstrate that distinct motifs in the C-terminus of p21 are required for regulation of p300 CRD1 function and that this effect does not correlate with Cyclin or PCNA binding. Through the creation of N-terminal glutathione-s-transferase fusion proteins, which also overcome the problems of instability that result from many p21 mutations, we investigated p21 binding to HDACs. Although p21 binds both Class I and Class II HDACs in vitro, only weak association with HDAC1 and 2 is seen in cells. Mutation of the p21 PCNA binding domain significantly increases this interaction suggesting that binding is mutually exclusive and only naturally occurs under certain conditions. Binding of HDACs also failed to correlate with CRD1 inducibility, suggesting that p21 targets other transcriptional repression complexes to mediate this effect.

Amino Acid Motifs↗

PubMed2005

ARF the integrator: linking NF-kappaB, p53 and checkpoint kinases.

The ARF tumor suppressor initiates the cellular response to aberrant oncogene activation through binding to and inhibiting the activity of Hdm2/Mdm2, the inhibitor of p53. However, many pathways also active in the cell will oppose p53 function if left unchecked. An example of this, is the RelA (p65) NF-kappaB subunit. Frequently activated by oncogenes, RelA is a potent inducer of anti-apoptotic gene expression, which has the potential to inhibit the pro-apoptotic functions of p53. We have recently discovered that by inducing the activity of the checkpoint kinases ATR and Chk1, ARF neutralises this opposing pathway. ARF-induced Chk1 phosphorylates RelA on threonine 505, a residue in its transactivation domain, thus inhibiting NF-kappaB's ability to stimulate anti-apoptotic gene expression. Furthermore, ARF-induced ATR is required for efficient induction and activation of p53. We propose that this pathway will target other proteins with pro-proliferative or anti-apoptotic functions. Therefore, through this mechanism, ARF can integrate the cellular response to an oncogene, thus maximising the effectiveness of the p53 tumor suppressor pathway.

ADP-Ribosylation Factors↗

PubMed2005

Cyclin D1 represses p300 transactivation through a cyclin-dependent kinase-independent mechanism.

Cyclin D1 encodes a regulatory subunit, which with its cyclin-dependent kinase (Cdk)-binding partner forms a holoenzyme that phosphorylates and inactivates the retinoblastoma protein. In addition to its Cdk binding-dependent functions, cyclin D1 regulates cellular differentiation in part by modifying several transcription factors and nuclear receptors. The molecular mechanism through which cyclin D1 regulates the function of transcription factors involved in cellular differentiation remains to be clarified. The histone acetyltransferase protein p300 is a co-integrator required for regulation of multiple transcription factors. Here we show that cyclin D1 physically interacts with p300 and represses p300 transactivation. We demonstrated further that the interaction of the two proteins occurs at the peroxisome proliferator-activated receptor gamma-responsive element of the lipoprotein lipase promoter in the context of the local chromatin structure. We have mapped the domains in p300 and cyclin D1 involved in this interaction. The bromo domain and cysteine- and histidine-rich domains of p300 were required for repression by cyclin D1. Cyclin D1 repression of p300 was independent of the Cdk- and retinoblastoma protein-binding domains of cyclin D1. Cyclin D1 inhibits histone acetyltransferase activity of p300 in vitro. Microarray analysis identified a signature of genes repressed by cyclin D1 and induced by p300 that promotes cellular differentiation and induces cell cycle arrest. Together, our results suggest that cyclin D1 plays an important role in cellular proliferation and differentiation through regulation of p300.

Cyclin D1↗

PubMed2005

Regulation of NF-kappaB 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-kappaB 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 alpha-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-kappaB 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.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2005

Differential regulation of c-Jun-dependent transcription by SUMO-specific proteases.

c-Jun is a transcription factor that plays an important role in regulating cell growth, apoptosis, differentiation, and transformation. The transcriptional activity of c-Jun can be regulated by both phosphorylation and sumoylation. It has also been shown that c-Jun transcription can be regulated by SuPr-1, an alternatively spliced form of SUMO-specific protease 2 (SENP2). However, the ability of SuPr-1 to enhance c-Jun transcription is dependent on promyelocytic leukemia but is independent of the desumoylation activity of SuPr-1. Here, we show that SUMO-specific protease 1 (SENP1) also markedly enhances the transcription activity of c-Jun. The action of SENP1 on c-Jun transcription is independent of the sumoylation and phosphorylation status of c-Jun but is critically dependent on the desumoylation activity of SENP1. We further show that p300 is essential for SENP1 to enhance c-Jun-dependent transcription because SENP1 can desumoylate the CRD1 domain of p300, thereby releasing the cis-repression of CRD1 on p300. Thus, two SUMO-specific proteases regulate c-Jun-dependent transcription through entirely different mechanisms.

Animals↗

PubMed2005

The DEAD box protein p68: a novel transcriptional coactivator of the p53 tumour suppressor.

The DEAD box RNA helicase, p68, has been implicated in various cellular processes and has been shown to possess transcriptional coactivator function. Here, we show that p68 potently synergises with the p53 tumour suppressor protein to stimulate transcription from p53-dependent promoters and that endogenous p68 and p53 co-immunoprecipitate from nuclear extracts. Strikingly, RNAi suppression of p68 inhibits p53 target gene expression in response to DNA damage, as well as p53-dependent apoptosis, but does not influence p53 stabilisation or expression of non-p53-responsive genes. We also show, by chromatin immunoprecipitation, that p68 is recruited to the p21 promoter in a p53-dependent manner, consistent with a role in promoting transcriptional initiation. Interestingly, p68 knock-down does not significantly affect NF-kappaB activation, suggesting that the stimulation of p53 transcriptional activity is not due to a general transcription effect. This study represents the first report of the involvement of an RNA helicase in the p53 response, and highlights a novel mechanism by which p68 may act as a tumour cosuppressor in governing p53 transcriptional activity.

Apoptosis↗

PubMed2005

SIRT1 deacetylation and repression of p300 involves lysine residues 1020/1024 within the cell cycle regulatory domain 1.

The SIR2 family of nicotinamide adenosine dinucleotide (NAD)-dependent deacetylases modulates diverse biological functions in different species, including longevity, apoptosis, cell cycle exit, and cellular differentiation. SIRT1, the closest mammalian ortholog of the yeast SIR2 (silent information regulator 2) gene, represses several transcription factors, including p53, NFkappaB and forkhead proteins. The p300 protein serves as a rate-limiting transcriptional cointegrator of diverse transcription factors either to activate or to repress transcription through modular subdomains. Herein, SIRT1 physically interacted with and repressed p300 transactivation, requiring the NAD-dependent deacetylase activity of SIRT1. SIRT1 repression involved the CRD1 transcriptional repression domain of p300. Two residues within the CRD1 domain (Lys-1020 and Lys-1024) were required for SIRT1 repression and served as substrates for SIRT1 deacetylation. These residues also serve as acceptor lysines for modification by the ubiquitin-like SUMO protein. The SUMO-specific protease SSP3 relieved SIRT1 repression of p300. SSP3 antagonism of SIRT1 required the SUMO-deconjugating function of SSP3. Thus, p300 serves as a deacetylase substrate for SIRT1 through a conserved SUMO consensus motif. Because p300 is a limiting transcriptional cofactor, deacetylation and repression of p300 by SIRT1 may serve an important integration point during metabolism and cellular differentiation.

Amino Acid Motifs↗

PubMed2004

The FHA domain protein SNIP1 is a regulator of the cell cycle and cyclin D1 expression.

Smad nuclear interacting protein 1 (SNIP1) is an evolutionarily conserved protein containing a forkhead-associated (FHA) domain that regulates gene expression through interactions with multiple transcriptional regulators. Here, we have used short interfering RNAs (siRNAs) to knockdown SNIP1 expression in human cell lines. Surprisingly, we found that reduction in SNIP1 levels resulted in significantly reduced cell proliferation and accumulation of cells in the G1 phase of the cell cycle. Consistent with this result, we observed that cyclin D1 protein and mRNA levels were reduced. Moreover, SNIP1 depletion results in inhibition of cyclin D1 promoter activity in a manner dependent upon a previously characterized binding site for the AP-1 transcription factor family. SNIP1 itself is induced upon serum stimulation immediately prior to cyclin D1 expression. These effects were independent of the tumour suppressors p53 and retinoblastoma (Rb), but were consistent with an interaction with BRG1, a component of the ATP-dependent chromatin remodelling complex, Swi/Snf. These results define both a new function for SNIP1 and identify a previously unrecognized regulator of the cell cycle and cyclin D1 expression.

Base Sequence↗

PubMed2004

The p68 and p72 DEAD box RNA helicases interact with HDAC1 and repress transcription in a promoter-specific manner.

BACKGROUND: p68 (Ddx5) and p72 (Ddx17) are highly related members of the DEAD box family and are established RNA helicases. They have been implicated in growth regulation and have been shown to be involved in both pre-mRNA and pre-rRNA processing. More recently, however, these proteins have been reported to act as transcriptional co-activators for estrogen-receptor alpha (ER alpha). Furthermore these proteins were shown to interact with co-activators p300/CBP and the RNA polymerase II holoenzyme. Taken together these reports suggest a role for p68 and p72 in transcriptional activation. RESULTS: In this report we show that p68 and p72 can, in some contexts, act as transcriptional repressors. Targeting of p68 or p72 to constitutive promoters leads to repression of transcription; this repression is promoter-specific. Moreover both p68 and p72 associate with histone deacetylase 1 (HDAC1), a well-established transcriptional repression protein. CONCLUSIONS: It is therefore clear that p68 and p72 are important transcriptional regulators, functioning as co-activators and/or co-repressors depending on the context of the promoter and the transcriptional complex in which they exist.

Adenocarcinoma↗

PubMed2004

Induction of transcription by p21Waf1/Cip1/Sdi1: role of NFkappaB and effect of non-steroidal anti-inflammatory drugs.

p21(Waf1/Cip1/Sdi1) is the primary mediator of cell cycle arrest in response to different forms of stress and in the programs of senescence and differentiation. p21 interacts with many regulatory proteins and has broad effects on cellular gene expression. p21 was previously shown to stimulate NFkappaB transcriptional activity through its effect on the p300/CBP transcription cofactor family. p21 expression in human cells increases mRNA levels of different genes, some of which have been implicated in carcinogenesis and age-related diseases. Here we report that p21 expression stimulates promoters of six p21-responsive human genes and the cytomegalovirus promoter, as well as an artificial promoter containing NFkappaB response elements. The IkappaBalpha super-repressor blocked the effect of p21 on all but one of the promoters, and the response to p21 was abrogated by the mutagenesis of an NFkappaB element. p21 inducibility of all the tested promoters and of the endogenous p21-responsive genes was strongly inhibited by adenoviral E1A protein and its deletion mutants that bind p300/CBP but not p21 or Rb. Sulindac and some other non-steroidal anti-inflammatory drugs that inhibit NFkappaB decrease the effects of p21 on the responsive promoters and endogenous genes. These findings suggest the feasibility of developing agents that will counteract p21-mediated induction of disease-associated genes.

Adenovirus E1A Proteins↗

PubMed2004

Reprogramming RelA.

The diversity of activators and targets of the NF-kappaB transcription factor family demands that there be regulatory mechanisms in place to control the specificity with which genes under their control are induced. In part this can be achieved through selective induction of different NF-kappaB subunits and through co-operative interactions with heterologous DNA-binding proteins and co-activators. Recent work from our laboratory indicates another critical mechanism regulating NF-kappaB. We find that the RelA(p65) NF-kappaB subunit does not always function as an inducer of gene expression, but under certain circumstances can be programmed to actively repress these same target genes. This repressor form of NF-kappaB appears to be induced by distinct, atypical pathways of activation and also through the action of some tumor suppressors. The identification of these pathways not only allows a reinterpretation of NF-kappaB function in normal cells and during tumorigenesis but could also have implications for both traditional and NF-kappaB based cancer therapy.

Animals↗

PubMed2004

Active repression of antiapoptotic gene expression by RelA(p65) NF-kappa B.

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.

Animals↗

PubMed2004

NF-kappaB: tumor promoter or suppressor?

A role for the NF-kappaB family of transcription factors as tumor promoters is firmly established. However, other data suggest that NF-kappaB can also inhibit tumor growth. Moreover, NF-kappaB activity is modulated by tumor suppressors, such as p53 and ARF, whereby NF-kappaB subunits repress, rather than activate, the expression of tumor-promoting genes. This suggests a dual function of NF-kappaB during tumor progression - in the early stages, NF-kappaB inhibits tumor growth but, as further mutations lead to a loss of tumor suppressor expression, the oncogenic functions of NF-kappaB become unleashed, allowing it to actively contribute to tumorigenesis. Here, I discuss this hypothesis, its implications for NF-kappaB function, and how this might influence the use of NF-kappaB-based anticancer therapies.

Animals↗

PubMed2003

Oncogenes, tumor suppressors and p52 NF-kappaB.

A role for the p52 NF-kappaB subunit in tumorigenesis has been steadily emerging since its discovery as a gene associated with chromosomal translocations in B- and T-cell lymphomas. Now Eliopoulos and co-workers have extended these studies to examine the effect of the Epstein-Barr virus (EBV)-encoded latent infection membrane protein 1 (LMP1) on p52. They find that LMP1 stimulates the processing of p100 to p52 NF-kappaB. Moreover, nuclear p52 is also associated with LMP1 expression in tumor tissue biopsies. They also demonstrate that the pathway leading to p100/p52 processing is distinct from that engaged by LMP1 to activate other NF-kappaB subunits through IkappaBalpha degradation. A clearer picture is now developing of the important role that p52 NF-kappaB plays during normal cell growth and how subverting its function can contribute to oncogenesis.

Gene Expression Regulation, Neoplastic↗