Human Papilloma Virus Positive Oropharyngeal Squamous Cell Carcinoma and the Immune System: Pathogenesis, Immunotherapy and Future Perspectives.

Oropharyngeal squamous cell carcinoma (OPSCC), a subset of head and neck squamous cell carcinoma (HNSCC), involves the palatine tonsils, soft palate, base of tongue, and uvula, with the ability to spread to adjacent subsites. Personalized treatment strategies for Human Papillomavirus-associated squamous cell carcinoma of the oropharynx (HPV+OPSCC) are yet to be established. In this article, we summarise our current understanding of the pathogenesis of HPV+OPSCC, the intrinsic role of the immune system, current ICI clinical trials, and the potential role of small molecule immunotherapy in HPV+OPSCC.

Egr-1 functions as a master switch regulator of remote ischemic preconditioning-induced cardioprotection.

Despite improved treatment options myocardial infarction (MI) is still a leading cause of mortality and morbidity worldwide. Remote ischemic preconditioning (RIPC) is a mechanistic process that reduces myocardial infarction size and protects against ischemia reperfusion (I/R) injury. The zinc finger transcription factor early growth response-1 (Egr-1) is integral to the biological response to I/R, as its upregulation mediates the increased expression of inflammatory and prothrombotic processes. We aimed to determine the association and/or role of Egr-1 expression with the molecular mechanisms controlling the cardioprotective effects of RIPC. This study used H9C2 cells in vitro and a rat model of cardiac ischemia reperfusion (I/R) injury. We silenced Egr-1 with DNAzyme (ED5) in vitro and in vivo, before three cycles of RIPC consisting of alternating 5 min hypoxia and normoxia in cells or hind-limb ligation and release in the rat, followed by hypoxic challenge in vitro and I/R injury in vivo. Post-procedure, ED5 administration led to a significant increase in infarct size compared to controls (65.90 ± 2.38% vs. 41.00 ± 2.83%, p < 0.0001) following administration prior to RIPC in vivo, concurrent with decreased plasma IL-6 levels (118.30 ± 4.30 pg/ml vs. 130.50 ± 1.29 pg/ml, p < 0.05), downregulation of the cardioprotective JAK-STAT pathway, and elevated myocardial endothelial dysfunction. In vitro, ED5 administration abrogated IL-6 mRNA expression in H9C2 cells subjected to RIPC (0.95 ± 0.20 vs. 6.08 ± 1.40-fold relative to the control group, p < 0.05), resulting in increase in apoptosis (4.76 ± 0.70% vs. 2.23 ± 0.34%, p < 0.05) and loss of mitochondrial membrane potential (0.57 ± 0.11% vs. 1.0 ± 0.14%-fold relative to control, p < 0.05) in recipient cells receiving preconditioned media from the DNAzyme treated donor cells. This study suggests that Egr-1 functions as a master regulator of remote preconditioning inducing a protective effect against myocardial I/R injury through IL-6-dependent JAK-STAT signaling.

EGFR and the complexity of receptor crosstalk in the cardiovascular system.

Signaling pathways play a critical role in the maintenance of cellular structure and function. These pathways can act together with synergistic or antagonistic outcome. Cooperative and integrative cellular communication networks, otherwise known as crosstalk can amplify signaling cascades. Here, we focus on receptor crosstalk in the context of cardiovascular pathologies, mainly involving the epidermal growth factor receptor (EGFR), a critical mediator of multiple receptor pathways in normal physiological and pathophysiological processes. Considerable experimental evidence suggests that the uncontrolled expression of EGFR contributes to tumorigenesis through inhibition of apoptosis, angiogenesis, anchorage-independent growth and tumor-associated inflammation. Abnormal activation of the intrinsic tyrosine kinase of EGFR through mutation or overexpression is observed in various human cancer types. On the other hand, the role of EGFR in vascular biology is not well understood. In cardiovascular pathologies, such as atherosclerosis and restenosis, vascular smooth muscle cells (SMCs) migrate and proliferate, contributing to neointima formation, whilst apoptosis may cause plaque instability. EGFR can be transactivated by numerous pathologic stimuli that regulate SMC behaviour. This review describes our current understanding of the role of EGFR in SMC biology and pathology.

In vitro and in vivo proliferation, differentiation and migration of cardiac endothelial progenitor cells (SCA1+/CD31+ side-population cells).

BACKGROUND: Side-population (SP) cells are a select population identified by a capacity to efflux Hoechst dye and are enriched for stem/progenitor cell activity. Previous studies suggested that cardiac SP (CSP) cells could be divided into SCA1(+)/CD31(-) and SCA1(+)/CD31(+) CSP cells. SCA1(+)/CD31(-) CSP cells have been shown to be cardiac stem/progenitor cells. However, SCA1(+)/CD31(+) CSP cells have not been fully characterized. OBJECTIVE: The aim of the present study was to characterize SCA1(+)/CD31(+) CSP cells in the adult mouse heart, and investigate their abilities to proliferate, differentiate, vascularize and migrate in vitro and in vivo. RESULTS: Using fluorescence-activated cell sorting (FACS), RT-PCR, and assays of cell proliferation, differentiation and migration, and a murine model of myocardial infarction (MI), we showed that SCA1(+)/CD31(+) CSP cells express stem cell and endothelial-specific genes, and reside in the blood vessels. These cells were able to proliferate, differentiate, migrate and vascularize in vitro and in vivo. After MI, SDF-1α and CXCR4 were up-regulated in the damaged myocardium and on SCA1(+)/CD31(+) CSP cells, respectively. Our results further showed that SDF-1α induced migration of these cells in vitro. Importantly, we found that SCA1(+)/CD31(+) CSP cells could migrate into the ischemic region from the non-ischemic area within the myocardium and form a vascular tube-like structure after MI. CONCLUSIONS: Based on the gene expression profile, localization of SCA1(+)/CD31(+) CSP cells, and their ability to proliferate, differentiate, migrate and vascularize in vitro and in vivo, we postulate that SCA1(+)/CD31(+) CSP cells may represent endothelial progenitor cells in the mouse heart.

A monopartite sequence is essential for p45 NF-E2 nuclear translocation, transcriptional activity and platelet production.

BACKGROUND: p45 NF-E2 is a bZIP transcription factor crucial for thrombopoiesis, as indicated by the fact that loss of p45 NF-E2 function results in dramatic embryonic lethal thrombopoietic defects and its overexpression boosts platelet release. OBJECTIVES: In the present study, we set out to identify the sequences responsible for p45 NF-E2 nuclear import, evaluate its transport mechanism and ascertain its functional significance. METHODS: A series of p45 NF-E2 deletion constructs fused to green fluorescent protein (GFP) was created and their cellular localization examined in mammalian cells, with the factor responsible for nuclear import identified using an in vitro transport assay. A p45 NF-E2 derivative mutated in the nuclear targeting sequence (NLS) was generated and its biological activity compared with wild type (wt) in luciferase assays, and proplatelet and platelet production measured in murine megakaryocytes transduced with a retroviral vector. RESULTS: Here we show that residues 271-273 are essential for nuclear import of p45 NF-E2 in COS-7 and in primary bone marrow cells. The p45 NF-E2 NLS facilitates nuclear import specifically via importin (IMP) 7. Although within the DNA-binding domain of p45 NF-E2, the NLS is not essential for DNA-binding, but is crucial for transcriptional activation and biological activity; where, in contrast to wt, a mutant derivative with a mutated NLS failed to promote proplatelet and platelet production in murine megakaryocytes. CONCLUSIONS: The NLS is critical for p45 NF-E2 function, with the present study being the first to demonstrate the importance of NLS-dependent nuclear import of p45 NF-E2 for platelet development.

DNAzymes and their therapeutic possibilities.

Our increasing understanding of the regulatory mechanisms involved in the pathogenesis of disease is opening up opportunities for therapeutic intervention. To tackle the unmet disease burden, the last decade has seen the emergence of gene-targeting small-molecule nucleic acid-based strategies, such as antisense oligodeoxynucleotides, ribozymes, small interfering RNA and DNAzymes. DNAzymes represent promising candidates for drug therapy in a wide range of diseases, such as cancer and cardiovascular disorders. This brief review will discuss recent developments in DNAzymes and their therapeutic potential.

Squamous cell carcinoma growth in mice and in culture is regulated by c-Jun and its control of matrix metalloproteinase-2 and -9 expression.

Squamous cell carcinoma (SCC) is an invasive malignancy of epidermal keratinocytes. Surgical excision is currently the main treatment; however, this can cause scarring and disfigurement. There is accordingly, an acute need for alternative strategies to treat SCC. The transcription factor c-Jun is expressed in human SCC and another common form of invasive skin cancer, basal cell carcinoma together with the mitogenic marker-proliferating cell nuclear antigen. Here, we have employed DNAzymes (catalytic DNA molecules) targeting c-Jun (Dz13) to inhibit c-Jun expression in SCC cells. Dz13 inhibits SCC proliferation and suppresses solid SCC tumor growth and tumor angiogenesis in severe combined immunodeficient mice. We further demonstrate that Dz13 inhibits c-Jun, together with matrix metalloproteinase (MMP)-2 and MMP-9 expression in the tumors, consistent with DNAzyme inhibition of MMP-2 and MMP-9 gelatinolytic activity by zymography. Dz13 also suppressed the expression of vascular endothelial growth factor and fibroblast growth factor-2 in the tumors. These findings demonstrate that c-Jun regulates SCC growth and suggest that DNAzymes targeting this transcription factor may potentially be useful as inhibitors of cutaneous carcinoma.

Vessel wall apoptosis and atherosclerotic plaque instability.

Atherosclerotic cardiovascular disease remains the leading cause of death in the industrialized world. Most cardiovascular deaths result from acute coronary syndromes, including unstable angina pectoris and acute myocardial infarction. Coronary syndromes often arise from acute coronary thrombosis, itself commonly a result of disruption or rupture of the fibrous cap of a lipid-laden atherosclerotic plaque. Despite this huge clinical burden of atherosclerotic plaque instability, our understanding of the molecular mechanisms mediating atherosclerotic plaque disruption and rupture, at a cellular level, remains limited. Placed in a clinical context, this review discusses our current understanding of the molecular basis for atherosclerotic plaque instability, with particular emphasis on the process of apoptosis-or programmed cell death-seen increasingly as playing a key role in a number of cell types within the vessel wall.

Signaling and transcriptional control of Fas ligand gene expression.

Fas ligand (FasL), a member of the tumor necrosis factor family, initiates apoptosis by binding to its surface receptor Fas. As a consequence, there is sequential activation of caspases and the release of cytochrome c from the mitochondria, with additional caspase activation followed by cellular degradation and death. Recent studies have shed important insight into the molecular mechanisms controlling FasL gene expression at the level of transcription. Nuclear factors such as nuclear factor in activated T cells, nuclear factor-kappa B, specificity protein-1, early growth response factor, interferon regulatory factor, c-Myc and the forkhead transcriptional regulator, alone or cooperatively, activate FasL expression. These factors are often coexpressed with FasL in pathophysiologic settings including human atherosclerotic lesions. Here, we review these important advances in our understanding of the signaling and transcriptional mechanisms controlling FasL gene expression.

Beyond angioplasty: novel developments in interventional cardiology.

Two specific areas in interventional cardiology have, until recently, remained problematic. First is the emerging issue of the so-called 'no option' patient, considered untreatable by conventional percutaneous coronary intervention (PCI) or surgery. Second is the long-standing dilemma of restenosis following PCI. Strategies addressing these two critical areas have been the subject of intense research efforts recently. Several important breakthroughs are being made in the important areas of novel revascularization techniques, antirestenotic agents and stent-based delivery methods. It is conceivable that these novel developments will soon mean that a broader range of patients can be treated, and that the issue of restenosis will now be seriously challenged.

Inducible expression of the megakaryocyte-specific gene glycoprotein IX is mediated through an Ets binding site and involves upstream activation of extracellular signal-regulated kinase.

Glycoprotein IX is a megakaryocyte-specific gene crucial for adequate and functional expression of the Glycoprotein Ib-IX complex. This study used phorbol 12-myristate 13-acetate (PMA) and thrombopoietin (TPO)-induced differentiation of Dami and UT-7 cells, respectively, to investigate the regulation of inducible Glycoprotein IX expression during megakaryocyte differentiation. PMA and TPO were able to modulate GPIX expression at mRNA and protein levels. Transient transfection studies using nested 5'-deletions and mutations of the GPIX promoter demonstrated the absolute requirement of an inverted Ets site 5'-ACTTCCT-3' for inducible reporter gene expression. The upstream signaling events associated with PMA and TPO-inducible expression of GPIX were also investigated. The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase inhibitor PD98059 inhibited both PMA and TPO-inducible reporter activity in a dose-dependent manner, whereas inhibition of p38/MAPK had no significant effect. The protein kinase C inhibitor GF109203X failed to inhibit TPO-activation of the GPIX promoter in UT-7 cells. This study demonstrates that inducible expression in response to either PMA or TPO is mediated through the Ets site in the proximal promoter of GPIX and is dependent upon the upstream activation of MAPK/extracellular signal-regulated kinase.

Induction of the transcriptional repressor Yin Yang-1 by vascular cell injury. Autocrine/paracrine role of endogenous fibroblast growth factor-2.

Yin Yang-1 (YY1) is a multifunctional transcription factor that can repress the expression of many growth factor, hormone, and cytokine genes implicated in atherogenesis. YY1 expression is activated in rat vascular smooth muscle cells shortly after injury. YY1 DNA binding activity paralleled elevated protein levels in the nucleus. Smooth muscle cell injury triggered the rapid extracellular release of immunoreactive fibroblast growth factor-2 (FGF-2). YY1 induction after injury was blocked by neutralizing antibodies directed against FGF-2. This growth factor increased YY1 mRNA and protein expression and stimulated YY1 binding and transcriptional activity. Overexpression of YY1 inhibited smooth muscle cell replication. Immunohistochemical analysis demonstrated YY1 staining in medial smooth muscle cells, coincident with FGF-2 expression. Proliferating cell nuclear antigen staining, in contrast, was confined mainly to the atherosclerotic intima. This is the first demonstration that YY1 is induced by either injury or FGF-2, is differentially expressed in normal and diseased human arteries, and that its overexpression inhibits vascular smooth muscle but not endothelial cell replication.

Sp1 phosphorylation regulates apoptosis via extracellular FasL-Fas engagement.

Apoptosis of smooth muscle cells (SMC) in atherosclerotic vessels can destabilize the atheromatus plaque and result in rupture, thrombosis, and sudden death. In efforts to understand the molecular processes regulating apoptosis in this cell type, we have defined a novel mechanism involving the ubiquitously expressed transcription factor Sp1. Subtypes of SMC expressing abundant levels of Sp1 produce the death agonist, Fas ligand (FasL) and undergo greater spontaneous apoptosis. Sp1 activates the FasL promoter via a distinct nucleotide recognition element whose integrity is crucial for inducible expression. Inducible FasL promoter activation is also inhibited by a dominant-negative form of Sp1. Increased SMC apoptosis is preceded by Sp1 phosphorylation, increased FasL transcription, and the autocrine/paracrine engagement of FasL with its cell-surface receptor, Fas. Inducible FasL transcription and apoptosis are blocked by dominant-negative protein kinase C-zeta, whose wild-type counterpart phosphorylates Sp1. Thus, Sp1 phosphorylation is a proapoptotic transcriptional event in vascular SMC and, given the wide distribution of this housekeeping transcription factor, may be a common regulatory theme in apoptotic signal transduction.

Regulation of apolipoprotein E production in macrophages (review).

Apolipoprotein E (apo E), a 34 kDa component of lipoproteins produced by the liver and in circulating macrophages, plays a critical role in the reverse transport of cholesterol to the liver via the circulation. Cholesterol-rich macrophages (macrophage foam cells) are a major cell type in human atherosclerotic lesions. Apo E deficiency in mice leads to the formation of atherosclerotic lesions. Conversely, macrophage-specific expression of apo E in these deficient mice can reduce the extent of atherosclerosis. These observations, together with the anti-inflammatory and anti-proliferative properties of Apo E, demonstrate an atheroprotective role for the apolipoprotein. Agents that regulate macrophage metabolism are also able to modulate apo E expression. Sterol loading, for example, enhances apo E synthesis and secretion. Additionally, exposure of macrophage foam cells to cholesterol acceptors such as apo A-1, the protein component of high density lipoprotein, further enhance apo E secretion. Cytokines can have a negative regulatory effect on apo E production in macrophages. Apo E expression is controlled at the transcriptional, post-transcriptional and post-translational level. Here, we review the cellular and molecular mechanisms modulating apo E synthesis and secretion in macrophages.

The role of platelet alpha-granular proteins in the regulation of thrombopoietin messenger RNA expression in human bone marrow stromal cells.

Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.

Induction of platelet-derived growth factor B-chain expression by transforming growth factor-beta involves transactivation by Smads.

Transforming growth factor-beta (TGF-beta) regulates a diverse array of biological processes, such as proliferation, differentiation, extracellular matrix production, and apoptosis. In cultured vascular endothelial cells, TGF-beta induces the expression of platelet-derived growth factor (PDGF) B-chain, a mitogen and chemoattractant, at the level of transcription. The molecular mechanism(s) underlying this process are not presently understood. In this study, we performed serial 5' deletion and transient transfection analysis to define a region in the PDGF-B promoter mediating inducible responsiveness to TGF-beta. This region contains an atypical nucleotide recognition element for the Smad family of transcriptional regulators. Electrophoretic mobility shift analysis revealed that nuclear proteins bound to this site in a transient and specific manner. Supershift studies demonstrated the physical association of Smad4 with the promoter. Overexpression of Smad4 activated the PDGF-B promoter and superinduced PDGF-B promoter-dependent expression in cells exposed to TGF-beta. Moreover, simultaneous cotransfection of Smad3 and Smad4 activated the PDGF-B promoter. This effect was attenuated when Smad4 was substituted with its dominant negative counterpart. Mutation of the (-81)CAGA(-78) motif in the PDGF-B promoter abrogated Smad-inducible promoter-dependent expression. Overexpression of Smad2 and Smad3 transactivated the PDGF-B promoter in a synergistic manner. These findings demonstrate the existence of a novel, functional binding element in the proximal region of the PDGF-B promoter mediating responsiveness to TGF-beta.

Vascular smooth muscle cells express the transcriptional corepressor NAB2 in response to injury.

The early growth response 1 (Egr-1 or NGFI-A) gene product is a zinc finger protein transcription factor which has been implicated in the regulation of genes differentially expressed during the development of vascular disease. Egr-1 activity is regulated by alterations in the amount of protein, as well as protein-protein interactions with positive and negative transcriptional cofactors. NGFI-A-binding protein 2 (NAB2) is an example of a negative transcriptional cofactor capable of binding directly to Egr-1 and repressing Egr-1-mediated transcription. In this study, we show that NAB2 is rapidly and transiently expressed in vascular smooth muscle cells (VSMC) in response to the model agonist phorbol 12-myristate 13-acetate (PMA). This induction occurs at the protein as well as mRNA level, and the time course of induction trails closely behind that of Egr-1. NAB2 expression in VSMC is capable of inhibiting Egr-1 dependent gene expression in response to either PMA or fibroblastic growth factor-2 (FGF-2). In an in vivo model of mechanical arterial injury NAB2 levels also increase transiently in VSMC at a time when Egr-1 is elevated. It is possible that NAB2 is part of a negative-feedback mechanism which serves to down-regulate Egr-1-mediated gene transcription in injured VSMC.

Early growth response factor 1: a pleiotropic mediator of inducible gene expression.

The zinc-finger transcription factor, early growth response factor-1 (Egr-1) is an immediate-early gene product inducibly expressed in response to diverse stimuli. This review examines the emerging role of Egr-1 in vascular pathobiology.

Zinc finger transcription factors mediate high constitutive platelet-derived growth factor-B expression in smooth muscle cells derived from aortae of newborn rats.

Platelet-derived growth factor (PDGF) B-chain gene is differentially expressed in smooth muscle cells (SMCs) derived from the rat aortic wall. SMCs cultured from two week-old rats (pups) express high levels of PDGF-B mRNA, whereas cells isolated from three month-old rats (adults) express low levels of PDGF-B. Nuclear run-off experiments indicate that increased PDGF-B gene expression in pups is mediated, at least in part, at the transcriptional level. We used electrophoretic mobility shift assays and Western blot analysis to demonstrate that levels of Sp1 and Sp3, two zinc finger transcription factors which mediate basal expression of the PDGF-B gene, are elevated in pup nuclei compared with adult nuclei. The immediate-early transcription factor, Egr-1, which footprints the PDGF-B promoter, is also constitutively expressed in these cells. Transient transfection and binding studies show that these factors interact with a region in the proximal PDGF-B promoter key for basal expression in pup cells. Mutation of this proximal element in transfected pup cells attenuates reporter gene expression to levels observed in adult cells. Conversely, overexpression of Sp1 in adult cells augments PDGF-B promoter-dependent expression. Elevated PDGF-B expression in cultured newborn rat SMCs may therefore require high constitutive expression of a number of zinc finger transcription factors and their specific interactions with the proximal PDGF-B promoter.

Regulation of PDGF-B in endothelial cells exposed to cyclic strain.

The present study was designed to examine the regulation by cyclic strain of endothelial cell (EC) platelet-derived growth factor-B chain (PDGF-B) expression. We demonstrate in this study that bovine aortic ECs subjected to 10% (but not 6%) average strain resulted in a 2.6-fold increase in PDGF-B steady state mRNA and immunoreactive protein. Nuclear runoff transcription assays confirmed the induction of PDGF-B transcripts. To address the regulation of PDGF-B gene expression by cyclic strain, we transfected bovine aortic ECs with a construct containing 450 bp of human PDGF-B promoter sequence coupled to chloramphenicol acetyltransferase (CAT), and found that subjecting these cells to 10% average strain resulted in a twofold increase in CAT activity by 4 hours. Analysis of nested 5' deletions of the promoter transfected into ECs demonstrated a 55% drop-off in activity between position -313 and -153, with no induction of activity with the - 101-bp minimal promoter. Since a shear stress response element (SSRE) is located at position -125, we tested the hypothesis that the SSRE site was necessary and/or sufficient for induction of PDGF-B activity with strain. Electromobility shift assays revealed that nuclear proteins from ECs exposed to strain for short intervals (30 minutes) bound to the PDGF-B SSRE. However, transfection of ECs with hybrid promoter constructs containing the SV40 sequence promoter downstream of the SSRE or the -153 PDGF-B promoter sequence bearing a mutation in the SSRE demonstrated that the SSRE was not necessary for inducible reporter gene expression in ECs exposed to cyclic strain.