Transoral robotic resection of oropharyngeal synovial sarcoma in a pediatric patient.

Localized synovial cell sarcomas are treated with surgical resection followed by chemo-radiation. Surgical resection of synovial sarcoma of the oropharynx and hypopharynx involves lip-splitting mandibulotomy resulting in treatment related morbidity. We report the successful use of Trans Oral Robotic Surgery for resection of localized synovial sarcoma of the lateral pharyngeal wall in a 15 year old patient. We were able to achieve negative surgical margins and avoid open surgery with its associated morbidity. At 2 years follow-up, patient is disease free, with no deficits in speech or swallowing functions and no cosmetic deformity.

Immune cell infiltration patterns and survival in head and neck squamous cell carcinoma.

PURPOSE: This study examines the tumour-host immune interactions in head and neck squamous cell carcinoma (HNSCC) and their relationship to human papillomavirus (HPV) infectivity and patient survival. METHODS: The adaptive and innate immune profile of surgical tumour specimens obtained from HNSCC patients was determined using qRT-PCR and immunohistochemistry. Intratumoural and invading margin leukocyte populations (CD3, CD8, CD16, CD20, CD68, FoxP3 and HLA-DR) were quantified and compared with patient disease-specific survival. Additionally, the expression of 41 immune activation- and suppression-related genes was evaluated in the tumour microenvironment. Tumour cells were also assessed for expression of HLA-A, HLA-G and HLA-DR. HPV infectivity of tumour biopsies was determined using HPV consensus primers (MY09/MY11 and GP5+/GP6+) and confirmed with p16 immunohistochemistry. RESULTS: HPV+ patient samples showed a significantly increased infiltration by intratumoural CD20+ B cells, as well as by invasive margin FoxP3+Treg, compared with HPV- patient samples. There was also a trend towards increased intratumoural CD8+ T cells and HLA-G expression on tumour cells in HPV+ samples. qRT-PCR data demonstrated a general pattern of increased immune activation and suppression mechanisms in HPV+ samples. Additionally, a combined score of intratumoural and invasive margin FoxP3 infiltration was significantly associated with disease-specific survival (P < 0.05). CONCLUSIONS: These data demonstrate significant differences in the immune cell profile of HPV+ and HPV- HNSCC. This study identifies several possible targets for immunotherapy and possible prognostic markers (FoxP3 and HLA-G) that may be specific to HNSCC.

Taxol induces apoptosis in cortical neurons by a mechanism independent of Bcl-2 phosphorylation.

Bcl-2, an antiapoptotic protein, protects cells against many but not all forms of apoptosis. For example, Bcl-2 does not protect non-neuronal cells against taxol, a microtubule-stabilizing agent. The underlying mechanism for the ineffectiveness of Bcl-2 against taxol has been the subject of intense interest. Data from non-neuronal cells indicate that taxol-induced apoptosis requires activation of N-terminal c-Jun protein kinase (JNK) that phosphorylates and inactivates Bcl-2. This suggests the interesting possibility that the apoptotic activity of JNK may be caused by phosphorylation of Bcl-2 and inhibition of the antiapoptotic activity of Bcl-2. Here we report that taxol induces apoptosis in cortical neurons but by a mechanism significantly different from that in non-neuronal cells. In contrast to human embryonic kidney 293 cells, expression of wild-type Bcl-2 in cortical neurons protected against taxol-induced apoptosis, and taxol did not induce Bcl-2 phosphorylation. Furthermore, cortical neurons express high basal JNK activity, and taxol did not stimulate total JNK activity. However, taxol activated a subpool of JNK in the nucleus and stimulated c-Jun phosphorylation. JNK inhibition or expression of a dominant-negative c-Jun abrogated taxol-induced apoptosis in cortical neurons, suggesting a role for JNK and JNK-mediated transcription in taxol-stimulated apoptosis. Furthermore, taxol-induced apoptosis in cortical neurons required inhibition of phosphatidylinositol 3-kinase signaling. These data suggest that taxol induces apoptosis in neurons by a mechanism quite distinct from that of non-neuronal cell lines and emphasize the importance of elucidating apoptotic mechanisms specific for neurons in the CNS.

Macrophage activation by polycyclic aromatic hydrocarbons: evidence for the involvement of stress-activated protein kinases, activator protein-1, and antioxidant response elements.

Polycyclic aromatic hydrocarbons (PAH) contained in fossil fuel combustion particles enhance the allergic response to common environmental Ags. A key question is: what are molecular pathways in the immune system by which PAH and conversion products drive allergic inflammation? Circumstantial evidence suggests that macrophages are involved in PAH-induced responses. We demonstrate that a representative PAH, beta-napthoflavone (BNF), and a representative quinone metabolite, tert-butylhydroxyquinone (tBHQ), induce Jun kinase and p38 mitogen-activated protein kinase activities in parallel with the generation of activator protein-1 (AP-1) mobility shift complexes in THP-1 and RAW264.7 macrophage cell lines. Activation of mitogen-activated protein kinases was dependent on generation of oxidative stress, and could be inhibited by N-acetylcysteine. Another genetic response pathway linked to PAH is the antioxidant response element (ARE), which regulates expression of detoxifying enzymes. BNF and tBHQ activated a human ARE (hARE) reporter gene in RAW264.7 cells. Interestingly, bacterial lipopolysaccharide also induced hARE/chloramphenicol acetyltransferase activity. While the hARE core, GTGACTCAGC, contains a consensus AP-1 sequence (underlined), AP-1 was not required for hARE activation. This suggests that PAH and their conversion products operate via ARE-specific transcription factors in the immune system. BNF and tBHQ did, however, induce AP-1 binding to the hARE, while constitutively active Jun kinase interfered in hARE/chloramphenicol acetyltransferase activation. This suggests that AP-1 proteins negatively regulate the hARE. These data establish important activation pathways for PAH in the immune system and provide us with targets to modulate the effect of environmental pollutants on allergic inflammation.

The c-Jun N-terminal kinase cascade plays a role in stress-induced apoptosis in Jurkat cells by up-regulating Fas ligand expression.

T lymphocytes undergo apoptosis in response to cellular stress, including UV exposure and gamma irradiation. However, the mechanism by which stress stimuli induce apoptosis is not well understood. While stress stimuli induce the activation of the c-Jun N-terminal kinase (JNK) pathway, it is not clear whether the JNK cascade is activated as a result of cell death or whether the cascade participates in inducing apoptosis. Using a Jurkat T cell line transfected with dominant active (DA)-mitogen-activated protein kinase kinase kinase (MEKK1) in a tetracycline-regulated expression system, we found that expression of DA-MEKK1 results in the apoptosis of Jurkat cells in parallel with prolonged JNK activation. Moreover, DA-MEKK1 induced Fas ligand (FasL) cell surface and mRNA expression, as well as FasL promoter activation. Interference with Fas/FasL interaction prevented DA-MEKK1-mediated apoptosis. In comparing the effect of different stress stimuli to DA-MEKK1, we found that UV, gamma irradiation, and anisomycin prolonged JNK activation in parallel with FasL expression and onset of cell death. In addition, these stimuli also enhance cell surface expression of FasL. Interference with Fas/FasL interactions inhibited anisomycin but not UV- or gamma irradiation-induced apoptosis. Our data show that while the JNK pathway contributes to stress-induced apoptosis in T lymphocytes by regulating FasL expression, not all stress stimuli use the same cell death pathway.

Inflammatory cytokines induce the expression of basic fibroblast growth factor (bFGF) isoforms required for the growth of Kaposi's sarcoma and endothelial cells through the activation of AP-1 response elements in the bFGF promoter.

BACKGROUND: The growth of Kaposi's sarcoma (KS) spindle cells is dependent on a number of inflammatory cytokines as well as the autocrine growth factor, basic fibroblast growth factor (bFGF). Moreover, inflammatory cytokines, found at increased levels in KS lesions, promote bFGF production in KS and endothelial cells. OBJECTIVES: To determine the induction of bFGF isoforms, role of bFGF in cell growth and activation of the bFGF promoter by inflammatory cytokines. DESIGN AND METHOD: 3H-Thymidine uptake, bFGF immunoblotting and transfection of dominant-negative MAP kinase components were used to study the effect of cytokines on the bFGF promoter, bFGF isoform expression and proliferation of KS cells. RESULTS: Treatment with oncostatin M (OSM), interleukin (IL)-1 and tumor necrosis factor (TNF)-alpha induced the expression of 18, 22 and 24 kDa bFGF isoforms in KS and human umbilical vein endothelial cells (HUVEC). Antisense bFGF oligonucleotides interfered in the induction of KS cell proliferation by individual cytokines. OSM, IL-1 and TNF-alpha induced the transcriptional activation of a bFGF promoter reporter gene in parallel with the activation of an AP-1 reporter. Dominant-negative ERK and dominant-negative JNK mutants interfered in cytokine-induced activation of these reporters in accordance with the role of the MAP kinase cascades in individual cytokine signaling pathways. CONCLUSIONS: OSM, IL-1 and TNF-alpha induce KS cell growth by inducing the expression of various bFGF isoforms. Moreover, bFGF production by KS and HUVEC is dependent on the activation of the ERK and JNK cascades, which result in the transcriptional activation of the bFGF promoter.

Involvement of Stat3 in interleukin-6-induced IgM production in a human B-cell line.

Interleukin-6 (IL-6) is an important B-cell growth and differentiation factor. IL-6 treatment of the human lymphoblastoid cell line, SKW6.4, leads to increased IgM production. We have previously shown that IL-6 induces activation of JAK1 and JAK2 in human B cell lines. A chimeric IL-6 receptor, comprised of the intracellular tail of the IL-6 receptor subunit gp130 fused to the extracellular domain of the epidermal growth factor (EGF) receptor, was stably transfected into SKW6.4 cells. EGF treatment induced IgM production in cells transfected with an intact gp130 cytoplasmic tail, but not in untransfected cells or cells transfected with a cytoplasmic tail lacking all four signal transducers and activators of transcription (Stat) binding sites. Moreover, EGF treatment induced Stat3 phosphorylation in cells transfected with the intact chimeric EGF-gp130 receptor along with induction of DNA-mobility shift of a classical interferon-gamma-activated site. To define further the relation between Stat3 activation and enhanced IgM production, we determined the effect of chimeric gp130 on the transcriptional activation of a genetic element linked to immunoglobulin production, namely the immunoglobulin heavy chain enhancer (IgH-enhancer). Parental as well as transfected SKW6.4 cells were transiently transfected with an IgH-enhancer-luciferase construct. The transcriptional activity of the IgH-luciferase construct was induced upon ligation of the full-length chimeric receptor but not by truncated gp130 receptors. Moreover, the gp130-induced activity of this reporter gene was abrogated by Stat3EE, a mutant Stat3 incapable of binding DNA. These results indicate that IL-6-induced B-cell differentiation, as measured by IgM production, may be controlled by Stat3 proteins.

Regulation of interleukin-2 transcription by inducible stable expression of dominant negative and dominant active mitogen-activated protein kinase kinase kinase in jurkat T cells. Evidence for the importance of Ras in a pathway that is controlled by dual receptor stimulation.

Engagement of the T cell receptor induces the activation of several mitogen-activated protein kinase modules, including the extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK) cascades. Whereas extracellular signal-regulated kinase is activated by T cell receptor/CD3 ligation alone, activation of JNK requires co-stimulation by the CD28 receptor. Activation of MEKK-1, which acts as a mitogen-activated protein kinase kinase kinase in the JNK pathway, was also induced by CD3 plus CD28 (CD3/CD28) ligation in Jurkat cells. To study the significance of the JNK cascade in T lymphocytes, we established stable Jurkat cell lines that inducibly express dominant active (DA) or dominant negative (DN) MEKK-1. Whereas expression of DA-MEKK-1 resulted in the constitutive activation of JNK along with the transcriptional activation of the minimal interleukin-2 (IL-2) promoter, DN-MEKK-1 inhibited JNK responsiveness during CD3/CD28 co-stimulation. In addition to inhibiting CD3/CD28-induced IL-2 mRNA expression, DN-MEKK-1 abrogated the transcriptional activation of the IL-2 promoter and the distal nuclear factor of activated T cells (NFAT)-activating protein 1 (AP-1) response element in that promoter. A c-Jun mutant lacking activation sites for JNK also interfered with the activation of the distal NFAT/AP-1 complex, suggesting that the JNK pathway functions by controlling AP-1 response elements in the IL-2 promoter. Using inducible stable expression of DA- and DN-Ras in Jurkat cells, we found that Ras regulates JNK activation in these cells. Our results suggest that the dual ligation of CD3 and CD28 in T cells triggers a cascade of events that involve Ras, the JNK cascade, and one or more AP-1 response elements in the IL-2 promoter.