Feasibility of a Hybrid Web-Based and In-Person Self-management Intervention Aimed at Preventing Acute to Chronic Pain Transition After Major Lower Extremity Trauma (iPACT-E-Trauma): A Pilot Randomized Controlled Trial.

Objective 1) To assess the feasibility of research methods to test a self-management intervention aimed at preventing acute to chronic pain transition in patients with major lower extremity trauma (iPACT-E-Trauma) and 2) to evaluate its potential effects at three and six months postinjury. Design A pilot randomized controlled trial (RCT) with two parallel groups. Setting A supraregional level 1 trauma center. Methods Fifty-six adult patients were randomized. Participants received the intervention or an educational pamphlet. Several parameters were evaluated to determine the feasibility of the research methods. The potential efficacy of iPACT-E-Trauma was evaluated with measures of pain intensity and pain interference with activities. Results More than 80% of eligible patients agreed to participate, and an attrition rate of ≤18% was found. Less than 40% of screened patients were eligible, and obtaining baseline data took 48 hours postadmission on average. Mean scores of mild pain intensity and pain interference with daily activities (<4/10) on average were obtained in both groups at three and six months postinjury. Between 20% and 30% of participants reported moderate to high mean scores (≥4/10) on these outcomes at the two follow-up time measures. The experimental group perceived greater considerable improvement in pain (60% in the experimental group vs 46% in the control group) at three months postinjury. Low mean scores of pain catastrophizing (Pain Catastrophizing Scale score < 30) and anxiety and depression (Hospital Anxiety and Depression Scale scores ≤ 10) were obtained through the end of the study. Conclusions Some challenges that need to be addressed in a future RCT include the small proportion of screened patients who were eligible and the selection of appropriate tools to measure the development of chronic pain. Studies will need to be conducted with patients presenting more serious injuries and psychological vulnerability or using a stepped screening approach.

Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB.

TRAIL (tumour-necrosis factor-related apoptosis ligand or Apo2L) triggers apoptosis through engagement of the death receptors TRAIL-R1 (also known as DR4) and TRAIL-R2 (DR5). Here we show that the c-Rel subunit of the transcription factor NF-kappaB induces expression of TRAIL-R1 and TRAIL-R2; conversely, a transdominant mutant of the inhibitory protein IkappaBalpha or a transactivation-deficient mutant of c-Rel reduces expression of either death receptor. Whereas NF-kappaB promotes death receptor expression, cytokine-mediated activation of the RelA subunit of NF-kappaB also increases expression of the apoptosis inhibitor, Bcl-xL, and protects cells from TRAIL. Inhibition of NF-kappaB by blocking activation of the IkappaB kinase complex reduces Bcl-x L expression and sensitizes tumour cells to TRAIL-induced apoptosis. The ability to induce death receptors or Bcl-xL may explain the dual roles of NF-kappaB as a mediator or inhibitor of cell death during immune and stress responses.

An optimal range of transcription potency is necessary for efficient cell transformation by c-Rel to ensure optimal nuclear localization and gene-specific activation.

c-Rel is overexpressed in several B-cell lymphomas and c-rel gene overexpression can transform primary chicken lymphoid cells and induce tumors in animals. Although c-Rel is generally a stronger transcriptional activator than its viral derivative v-Rel, its oncogenic activity is significantly weaker. Among the mutations acquired during c-Rel's evolution into v-Rel are deletion of c-Rel's transactivation domain 2 (cTAD2) and mutations in cTAD1. Given the critical role of the Rel TADs in cell transformation, we investigated how mutations in c-Rel's cTAD1 and cTAD2 contribute to its oncogenicity and that of v-Rel. Mutations in cTAD2 noticeably increased c-Rel's transforming activity by promoting its nuclear localization and gene-specific transactivation, despite an overall decrease in kappaB site-dependent transactivation potency. Conversely, substitution of vTAD by cTAD1 increased v-Rel's transactivation and transforming efficiencies, whereas its substitution by the stronger cTAD2 compromised activation of mip-1beta but not irf-4 and was detrimental to cell transformation. These results suggest that the Rel TADs differentially contribute to gene-specific activation and that an optimal range of transcription potency is necessary for efficient transformation. These findings may have important implications for understanding how Rel TAD mutations can lead to a more oncogenic phenotype.

Current insights into the regulation of programmed cell death by NF-kappaB.

The nuclear factor-kappaB (NF-kappaB) transcription factors have emerged as major regulators of programmed cell death (PCD) whether via apoptosis or necrosis. In this context, NF-kappaB's activity has important ramifications for normal tissue development, homoeostasis and the physiological functions of various cell systems including the immune, hepatic, epidermal and nervous systems. However, improper regulation of PCD by NF-kappaB can have severe pathologic consequences, ranging from neurodegeneration to cancer, where its activity often precludes effective therapy. Although NF-kappaB generally protects cells by inducing the expression genes encoding antiapoptotic and antioxidizing proteins, its role in apoptosis and necrosis can vary markedly in different cell contexts, and NF-kappaB can sensitize cells to death-inducing stimuli in some instances. This article describes our current knowledge of the role of NF-kappaB in apoptosis and necrosis, and focuses on the many advances since we last reviewed this rapidly evolving topic in Oncogene 3 years ago. There has been substantial progress in understanding NF-kappaB's mode of action in apoptosis and necrosis and the mechanisms that regulate its anti- vs proapoptotic activities. These recent developments shed new light on the role of NF-kappaB in many disease conditions including tumor development, tumor progression and anticancer treatment.

Distinct domains of IkappaBalpha regulate c-Rel in the cytoplasm and in the nucleus.

IkappaBalpha is a critical regulator of Rel/NF-KB-mediated gene activation. It controls the induction of NF-KB factors by retaining them in the cytoplasm and also functions in the nucleus to terminate the induction process. In this study, we show that IkappaBalpha regulates the transcriptional activity of c-Rel in the nuclear compartment. We also demonstrate that discrete functional domains of IkappaBalpha are responsible for the cytoplasmic and nuclear regulation of c-Rel. We show that the determinants for the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin regions of IkappaBalpha and that the N-terminal domain of IkappaBalpha is required to mask the c-Rel nuclear localization signal. Importantly, IkappaBalpha sequences necessary to regulate c-Rel in the nucleus map to its central ankyrin domain and to a few negatively charged amino acids that immediately follow in the C-terminal IkappaBalpha PEST domain. The mapping of the IkappaBalpha determinants that control the cytoplasmic and nuclear activities of c-Rel to specific regions of the molecule suggests that IkappaBalpha inhibitors could be designed to antagonize Rel/NF-kappaB activity in different subcellular compartments or at defined stages of activation.

The Rel/NF-kappaB family directly activates expression of the apoptosis inhibitor Bcl-x(L).

The transcription factors of the Rel/NF-kappaB family are key regulators of immune and inflammatory responses and contribute to lymphocyte proliferation, survival, and oncogenesis. The absolute correlation between the antiapoptotic and oncogenic activities of the Rel/NF-kappaB oncoprotein v-Rel emphasizes the importance of characterizing the death antagonists under NF-kappaB control. Our recent finding that the prosurvival Bcl-2 homolog Bfl-1 (also called A1) is a direct transcriptional target of NF-kappaB raised the issue of whether NF-kappaB is a specific or global regulator of death antagonists in the Bcl-2 family. Here, we demonstrate that NF-kappaB differentially regulates the expression of particular Bcl-2-related death inhibitors and that it directly activates the expression of Bcl-x(L). While Bcl-x(L) was significantly upregulated by c-Rel and RelA, Bcl-2 was not. Importantly, stimuli that activate endogenous NF-kappaB factors also upregulated bcl-x gene expression and this effect was antagonized by an inhibitor of NF-kappaB activity. The expression of bcl-x suppressed apoptosis in the presence or absence of NF-kappaB activity. Functional analysis of the bcl-x promoter demonstrated that it is directly controlled by c-Rel. These results establish that NF-kappaB directly regulates the expression of distinct prosurvival factors in the Bcl-2 family, such as Bcl-x(L) and Bfl-1/A1. These findings raise the possibility that some of these factors may contribute to oncogenesis associated with aberrant Rel/NF-kappaB activity.

The RxxRxRxxC motif conserved in all Rel/kappa B proteins is essential for the DNA-binding activity and redox regulation of the v-Rel oncoprotein.

The v- and c-Rel oncoproteins bind to oligonucleotides containing kappa B motifs, form heterodimers with other members of the Rel family, and modulate expression of genes linked to kappa B motifs. Here, we report that the RxxRxRxxC motif conserved in all Rel/kappa B family proteins is absolutely required for v-Rel protein-DNA contact and its resulting transforming activity. We also demonstrate that serine substitution of the cysteine residue conserved within this motif enables v-Rel to escape redox control, thereby promoting overall DNA binding. These mutant proteins retained the ability to competitively inhibit kappa B-mediated transcriptional activation of the human immunodeficiency virus long terminal repeat but failed to efficiently transform chicken lymphoid cells both in vitro and in vivo. Our data indicate that reduction of the conserved cysteine residue in the RxxRxRxxC motif may be required for optimal DNA-protein interactions. These results provide direct biochemical evidence that the DNA-binding activity of v-Rel is subject to redox control and that the conserved cysteine residue in the RxxRxRxxC motif is critical for this regulation. These studies suggest that the DNA-binding, transcriptional, and biological activities of Rel family proteins may also be subject to redox control in vivo.

Role of the three polyoma virus early proteins in tumorigenesis.

A modified polyoma virus genome which can encode the middle T protein but not the large or small T proteins transforms rat cells in culture with an efficiency about 20% that of the wild-type genome. Although middle T-transformed cells grow as tumors when transplanted into nude mice or syngeneic rats, the middle T gene alone is totally inactive when used in a more stringent and rigorous assay for tumorigenicity such as the injection of DNA into newborn rats. Thus, functions other than those expressed by middle T antigen are required for the elaboration of all the properties associated with tumorigenesis. To assess whether a complementary function could be exerted by the large or the small T antigen, we constructed plasmids containing two modified early regions which independently encoded middle T and one of the two other proteins. Both recombinants were tumorigenic in newborn rats. Cell lines derived by transfer of these plasmids under no special selective conditions did not acquire the property of growth in low-serum medium but exhibited the same tumorigenic properties as wild-type polyoma DNA-transformed cells. Furthermore, a recombinant which encoded the middle and small T antigens, but not the large T antigen, was tumorigenic in newborn rats. Although the small T antigen provides a complementary function for tumorigenicity, it cannot complement the middle T antigen for an efficient induction of transformation of cultured cells. This suggests that the complementary function exerted by the small T antigen is different from that of the N-terminal fragment of the large T protein.

c-Rel arrests the proliferation of HeLa cells and affects critical regulators of the G1/S-phase transition.

A tetracycline-regulated system was used to characterize the effects of c-Rel on cell proliferation. The expression of c-Rel in HeLa cells led to growth arrest at the G1/S-phase transition, which correlated with its nuclear localization and the induction of endogenous IkappaB alpha expression. These changes were accompanied by a decrease in E2F DNA binding and the accumulation of the hypophosphorylated form of Rb. In vitro kinase assays showed a reduction in Cdk2 kinase activity that correlated with elevated levels of p21WAF1 Cdk inhibitor and p53 tumor suppressor protein. While the steady-state levels of WAF1 transcripts were increased, pulse-chase analysis revealed a sharp increase in p53 protein stability. Importantly, the deletion of the C-terminal transactivation domains of c-Rel abolished these effects. Together, these studies demonstrate that c-Rel can affect cell cycle control and suggest the involvement of the p21WAF1 and p53 cell cycle regulators.

Mapping of a serine-rich domain essential for the transcriptional, antiapoptotic, and transforming activities of the v-Rel oncoprotein.

The v-Rel oncoprotein belongs to the Rel/NF-kappaB family of transcription factors and induces aggressive lymphomas in chickens and transgenic mice. Current models for cell transformation by v-Rel invoke the combined activation of gene expression and the dominant inhibition of transcription mediated by its cellular homologs. Here, we mapped a serine-rich transactivation domain in the C terminus of v-Rel that is necessary for its biological activity. Specific serine-to-alanine substitutions within this region impaired the transcriptional activity of v-Rel, whereas a double mutant abolished its function. In contrast, substitutions with phosphomimetic aspartate residues led to a complete recovery of the transcriptional potential. The transforming activity of v-Rel mutants correlated with their ability to inhibit programmed cell death. The transforming and antiapoptotic activities of v-Rel were abolished by defined Ser-to-Ala mutations and restored by most Ser-to-Asp substitutions. However, one Ser-to-Asp mutant showed wild-type transactivation ability but failed to block apoptosis and to transform cells. These results show that the transactivation function of v-Rel is necessary but not sufficient for cell transformation, adding an important dimension to the transformation model. It is possible that defined protein-protein interactions are also required to block apoptosis and transform cells. Since v-Rel is an acutely oncogenic member of the Rel/NF-kappaB family, our data raise the possibility that phosphorylation of its serine-rich transactivation domain may regulate its unique biological activity.

Polyoma middle T antigen requires cooperation from another gene to express the malignant phenotype in vivo.

The oncogenic potential of polyomavirus in newborn hamsters can be expressed by a recombinant encoding only the middle T protein. However, polyoma middle T requires the cooperation from small T to induce tumors in newborn rats. Similar complementary functions such as cocarcinogens or tumor promotors can be exerted by the simian virus 40 T antigens as well as by one or several products of the early region 1A of adenovirus 2.

Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB.

Rel family proteins regulate the expression of genes linked to kappa B-binding motifs. Little is known, however, of the mechanism by which they enhance transcription. We have investigated the ability of the v-Rel and c-Rel oncoproteins to interact with components of the basal transcription machinery. Here we report that both the acidic transcription activation domain mapping to the unique C terminus of chicken c-Rel and the F9 cell-specific activation region common to both v-Rel and c-Rel interact with the TATA-binding protein (TBP) and transcription factor IIB (TFIIB) in vitro and in vivo. We also demonstrate that TPB interaction with Rel activation regions leads to synergistic activation of transcription of a kappa B-linked reporter gene. Combined with the observation that the mouse c-Rel and human RelA proteins also interact with TBP and TFIIB in vitro, these results suggest that association with basal transcription factors is important for the transcriptional activities of Rel family proteins.

Constitutive proteasome-mediated turnover of Bfl-1/A1 and its processing in response to TNF receptor activation in FL5.12 pro-B cells convert it into a prodeath factor.

Bfl-1/A1 is generally recognized as a Bcl-2-related inhibitor of apoptosis. We show that Bfl-1 undergoes constitutive ubiquitin/proteasome-mediated turnover. Moreover, while Bfl-1 suppresses apoptosis induced by staurosporine or cytokine withdrawal, it is proapoptotic in response to tumor necrosis factor (TNF) receptor activation in FL5.12 pro-B cells. Its anti- versus proapoptotic effect is regulated by two proteolytic events: (1) its constitutive proteasome-mediated turnover and (2) its TNF/cycloheximide (CHX)-induced cleavage by mu-calpain, or a calpain-like activity, coincident with acquisition of a proapoptotic phenotype. In vitro studies suggest that calpain-mediated cleavage of Bfl-1 occurs between its Bcl-2 homology (BH)4 and BH3 domains. This would be consistent with the generation of a proapoptotic Bax-like BH1-3 molecule. Overall, our studies uncovered two new regulatory mechanisms that play a decisive role in determining Bfl-1's prosurvival versus prodeath activities. These findings might provide important clues to counteract chemoresistance in tumor cells that highly express Bfl-1.

The effect of tailored Web-based interventions on pain in adults: a systematic review protocol.

BACKGROUND: Information technologies can facilitate the implementation of health interventions, especially in the case of widespread conditions such as pain. Tailored Web-based interventions have been recognized for health behavior change among diverse populations. However, none of the systematic reviews looking at Web-based interventions for pain management has specifically addressed the contribution of tailoring. METHODS: The aims of this systematic review are to assess the effect of tailored Web-based pain management interventions on pain intensity and physical and psychological functions. Randomized controlled trials including adults suffering from any type of pain and involving Web-based interventions for pain management, using at least one of the three tailoring strategies (personalization, feedback, or adaptation), will be considered. The following types of comparisons will be carried out: tailored Web-based intervention with (1) usual care (passive control group), (2) face-to-face intervention, and (3) standardized Web-based intervention. The primary outcome will be pain intensity measured using a self-report measure such as the numeric rating scale (e.g., 0-10) or visual analog scale (e.g., 0-100). Secondary outcomes will include pain interference with activities and psychological well-being. A systematic review of English and French articles using MEDLINE, Embase, CINAHL, PsycINFO, Web of Science, and Cochrane Library will be conducted from January 2000 to December 2015. Eligibility assessment will be performed independently in an unblinded standardized manner by two reviewers. Extracted data will include the following: sample size, demographics, dropout rate, number and type of study groups, type of pain, inclusion and exclusion criteria, study setting, type of Web-based intervention, tailoring strategy, comparator, type of pain intensity measure, pain-related disability and psychological well-being outcomes, and times of measurement. Disagreements between reviewers at the full-text level will be resolved by consulting a third reviewer, a senior researcher. DISCUSSION: This systematic review is the first one looking at the specific ingredients and effects of tailored and Web-based interventions for pain management. Results of this systematic review could contribute to a better understanding of the mechanisms by which Web-based interventions could be helpful for people facing pain problems. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42015027669.

A mutant Rel-homology domain promotes transcription by p50/NFkappaB1.

Previous studies showed that the binding of p50/NFkappaB1 to particular kappaB DNA sites altered its conformation in a way that correlated with transcriptional activation. Here, we investigated the effects of protein-protein interactions on the transcriptional activity of p50. We show that the association of p50 with a mutant Rel-homology domain (RHD) defective for DNA binding led to synergistic activation of kappaB site-dependent transcription, whereas neither protein alone had any effect. Partial proteolytic analysis showed that the conformation of p50 in these complexes differed from that in wild-type c-Rel-RHD/p50 complexes, and correlated with activated transcription. These results suggest that the Rel-homology domain can act as an allosteric effector to promote transcription by p50/NFkappaB1 and that the configuration of p50 is important for its activity. This also suggests that Rel proteins can promote transcription by other Rel-family members without binding to their DNA recognition site. These studies emphasize the important role of protein-protein interactions in Rel and NFkappaB-mediated transcription.

Aberrant rel/nfkb genes and activity in human cancer.

Rel/NF-kappaB transcription factors are key regulators of immune, inflammatory and acute phase responses and are also implicated in the control of cell proliferation and apoptosis. Remarkable progress has been made in understanding the signal transduction pathways that lead to the activation of Rel/NF-kappaB factors and the consequent induction of gene expression. Evidence linking deregulated Rel/NF-kappaB activity to oncogenesis in mammalian systems has emerged in recent years, consistent with the acute oncogenicity of the viral oncoprotein v-Rel in animal models. Chromosomal amplification, overexpression and rearrangement of genes coding for Rel/NF-kappaB factors have been noted in many human hematopoietic and solid tumors. Persistent nuclear NF-kappaB activity was also described in several human cancer cell types, as a result of constitutive activation of upstream signaling kinases or mutations inactivating inhibitory IkappaB subunits. Studies point to a correlation between the activation of cellular gene expression by Rel/NF-kappaB factors and their participation in the malignant process. Experiments implicating NF-kappaB in the control of the apoptotic response also support a role in oncogenesis and in the resistance of tumor cells to chemotherapy. This review focuses on the status of the rel, nfkb and ikb genes and their activity in human tumors and their association with the onset or progression of malignancies.

The v-rel oncogene encodes a cell-specific transcriptional activator of certain promoters.

Transformation by the v-rel oncogene of avian reticuloendotheliosis virus strain T (Rev-T) is primarily cell-specific. While v-rel efficiently transforms chicken spleen and bone marrow stem cells in vitro and induces rapid lethal lymphomas in young birds, it does not rapidly transform chicken embryo fibroblasts. The nuclear localization of the v-rel gene product in non-transformed fibroblasts along with its ability to function as a transforming protein in the nucleus of chicken spleen cells suggests that p59v-rel might belong to the family of nuclear oncoproteins and thus may express an immortalizing function in fibroblasts. To gain insight into the specificity of cell transformation by the v-rel oncogene, we determined whether v-rel could immortalize primary rat fibroblasts. Our experiments have shown that, unlike other nuclear oncoproteins, p59v-rel did not immortalize primary rat embryo fibroblasts. However p59v-rel was able to cooperate in a synergistic way with the polyomavirus middle T protein in inducing efficient transformation of established rat fibroblasts by increasing the steady-state level of middle T RNA, indicating that p59v-rel might function as a transactivator. Cotransfection of cells from different species with the v-rel gene along with constructs expressing the chloramphenicol acetyl transferase gene under the control of different promoters revealed that p59v-rel is a cell-specific transcriptional transactivator of certain promoters. Moreover, the extent of cell-specific transactivation by v-rel correlated with its toxic effect in these same cells.

N-terminal determinants of I kappa B alpha necessary for the cytoplasmic regulation of c-Rel.

I kappa B alpha is a dual regulator of Rel/NF-kappa B transcription factors. I kappa B alpha retains inactive NF-kappa B dimers in the cytoplasm, and inhibits their DNA-binding and transcriptional activities in the nucleus. Our previous studies identified discrete functional domains in I kappa B alpha responsible for the cytoplasmic and nuclear regulation of c-Rel. Determinants necessary for regulating c-Rel in the nucleus mapped to the central ankyrin domain of I kappa B alpha and a few negatively-charged amino acids that follow in the C-terminal PEST region. In contrast, sequences involved in the cytoplasmic regulation of c-Rel reside in the N-terminal and central ankyrin domains of I kappa B alpha. Here, we present a refined mapping of the N-terminal determinants of I kappa B alpha necessary for the cytoplasmic regulation of c-Rel homodimers. We demonstrate that amino acids 48 - 58 in p40/I kappa B alpha are essential to block the nuclear localization of c-Rel dimers. These data define a region of I kappa B alpha that may be required for optimal masking of the c-Rel NLS, or for the nuclear export of c-Rel/I kappa B alpha complexes. These findings highlight a novel function for the N-terminus of I kappa B alpha in the control of the subcellular localization of Rel/NF-kappa B dimers. Given the implication of deregulated NF-kappa B activity in hematopoietic and solid tumors, our findings predict that certain alterations in this domain of I kappa B alpha may have severe biological repercussions.

Two novel functions associated with the Rel oncoproteins: DNA replication and cell-specific transcriptional activation.

The v-Rel oncoprotein and its cellular homolog c-Rel belong to the Rel/kappa B family of transcription factors. Members of this family share extensive sequence similarity in their N-terminal halves, a region referred to as the Rel Homology Region (RHR), bind to NF-kappa B DNA motifs and form heterodimers with one another. Whereas c-Rel activates transcription of kappa B-linked genes, v-Rel behaves as a dominant-interfering mutant of c-Rel- and kappa B-mediated transcription activation. Here we describe two novel activities of the Rel oncoproteins. One induces kappa B-site dependent stimulation of polyomavirus (Py) DNA replication and maps to the N-terminus of the RHR, a region where no transcription activation function was detected. This activity is common to v-Rel, c-Rel, p52 (p49/lyt10), RelA (p65) and the p50 subunit of NF-kappa B. The second promotes transcriptional activation in undifferentiated F9 cells and maps 3' to the RHR, a region essential for the transforming activity of v-Rel.

The viral and cellular Rel oncoproteins induce the differentiation of P19 embryonal carcinoma cells.

Rel proteins have been implicated in the differentiation and transformation of lymphoid cells. Despite their significant sequence homology to the Drosophila Dorsal morphogen, a functional role for Rel proteins in early development has yet to be established. Here we show that expression of the v- or c-rel genes in P19 embryonal carcinoma (EC) cells leads to drastic morphological changes accompanied by the expression of several differentiation-specific antigens. In contrast, v-Rel mutants defective for DNA-binding as well as those lacking the F9 cell-specific transcription activation region of v-Rel were equally unable to promote P19 cell differentiation. Our results demonstrate that transcriptional activation by Rel proteins correlates with Rel-induced differentiation of EC cells, and raise the intriguing possibility that Rel proteins may play an important role in early developmental processes.