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.

DNAzymes and cardiovascular disease.

Gene silencing techniques are gaining increasing popularity in the literature, both as a tool for unravelling gene function and to potentially deliver therapeutic benefit, especially in the context of cardiovascular disease. Gene-specific catalytic DNA molecules, or DNAzymes, have shown promise in ameliorating the effects of myocardial ischaemia reperfusion injury and in-stent restenosis in various animal models, demonstrating that these agents may be useful in a clinical setting. A review of the recent advances in the use of DNAzymes in treating cardiovascular disease is therefore essential given the increasing clinical burden of cardiovascular disease worldwide. We have thus sought to firstly provide background into the construct and mechanism of action of DNAzymes, with a discussion of recent improvements in design. Secondly, we have examined the effects of DNAzyme-mediated gene inhibition in in vitro studies of both endothelial and smooth muscle migration and proliferation, as well as in vivo models of acute myocardial infraction and neointima formation. Lastly we compare DNAzymes with other gene silencing tools and discuss issues involved in successfully delivering these drugs in a clinical setting.

DNAzymes targeting the transcription factor Egr-1 reduce myocardial infarct size following ischemia-reperfusion in rats.

BACKGROUND AND AIM: The transcription factor and immediate-early gene Egr-1 is widely viewed as a key upstream activator in a variety of settings within cardiovascular pathobiology. The role that Egr-1 plays in myocardial ischemia-reperfusion (IR) injury is unknown. We hypothesized that Egr-1 upregulation is of pathophysiologic importance in myocardial IR injury. METHODS AND RESOURCES: First, abrogation of Egr-1 mRNA upregulation using Egr-1 targeting DNAzymes in a rat cardiomyocyte in vitro model was demonstrated. Egr-1 mRNA and protein upregulation following myocardial IR in rats were then selectively suppressed by locally delivered DNAzyme. Furthermore, myocardial neutrophil infiltration, intercellular adhesion molecule 1 mRNA and protein expression, and myocardial infarct size were all attenuated in DNAzyme-treated animals. CONCLUSIONS: These data support the hypothesis that Egr-1 is a key contributor to myocardial IR injury, and that Egr-1 targeting strategies have therapeutic potential in this context.

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.

Catalytic oligodeoxynucleotides define a key regulatory role for early growth response factor-1 in the porcine model of coronary in-stent restenosis.

Early growth response factor-1 (Egr-1) controls the expression of a growing number of genes involved in the pathogenesis of atherosclerosis and postangioplasty restenosis. Egr-1 is activated by diverse proatherogenic stimuli. As such, this transcription factor represents a key molecular target in efforts to control vascular lesion formation in humans. In this study, we have generated DNAzymes targeting specific sequences in human EGR-1 mRNA. These molecules cleave in vitro transcribed EGR-1 mRNA efficiently at preselected sites, inhibit EGR-1 protein expression in human aortic smooth muscle cells, block serum-inducible cell proliferation, and abrogate cellular regrowth after mechanical injury in vitro. These DNAzymes also selectively inhibit EGR-1 expression and proliferation of porcine arterial smooth muscle cells and reduce intimal thickening after stenting pig coronary arteries in vivo. These findings demonstrate that endoluminally delivered DNAzymes targeting EGR-1 may serve as inhibitors of in-stent restenosis.

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.