Repression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but not its receptors during oral cancer progression.

BACKGROUND: TRAIL plays an important role in host immunosurveillance against tumor progression, as it induces apoptosis of tumor cells but not normal cells, and thus has great therapeutic potential for cancer treatment. TRAIL binds to two cell-death-inducing (DR4 and DR5) and two decoy (DcR1, and DcR2) receptors. Here, we compare the expression levels of TRAIL and its receptors in normal oral mucosa (NOM), oral premalignancies (OPM), and primary and metastatic oral squamous cell carcinomas (OSCC) in order to characterize the changes in their expression patterns during OSCC initiation and progression. METHODS: DNA microarray, immunoblotting and immunohistochemical analyses were used to examine the expression levels of TRAIL and its receptors in oral epithelial cell lines and in archival tissues of NOM, OPM, primary and metastatic OSCC. Apoptotic rates of tumor cells and tumor-infiltrating lymphocytes (TIL) in OSCC specimens were determined by cleaved caspase 3 immunohistochemistry. RESULTS: Normal oral epithelia constitutively expressed TRAIL, but expression was progressively lost in OPM and OSCC. Reduction in DcR2 expression levels was noted frequently in OPM and OSCC compared to respective patient-matched uninvolved oral mucosa. OSCC frequently expressed DR4, DR5 and DcR1 but less frequently DcR2. Expression levels of DR4, DR5 and DcR1 receptors were not significantly altered in OPM, primary OSCC and metastatic OSCC compared to patient-matched normal oral mucosa. Expression of proapoptotic TRAIL-receptors DR4 and DR5 in OSCC seemed to depend, at least in part, on whether or not these receptors were expressed in their parental oral epithelia. High DR5 expression in primary OSCC correlated significantly with larger tumor size. There was no significant association between TRAIL-R expression and OSSC histology grade, nodal status or apoptosis rates of tumor cells and TIL. CONCLUSION: Loss of TRAIL expression is an early event during oral carcinogenesis and may be involved in dysregulation of apoptosis and contribute to the molecular carcinogenesis of OSCC. Differential expressions of TRAIL receptors in OSCC do not appear to play a crucial role in their apoptotic rate or metastatic progression.

Analyses of pp60$\sp{{\rm c}-src}$ and epidermal growth factor receptor protein tyrosine kinases in a human colon adenocarcinoma cell line following induction of goblet cell-like characteristics by tumor necrosis factor-alpha

Regulation of colonic epithelial cell proliferation and differentiation remains poorly understood due to the inability to design a model system which recapitulates these processes. Currently, properties of "differentiation" are studied in colon adenocarcinoma cell lines which can be induced to express some, but not all of the phenotypes of normal cells. In this thesis, the DiFi human colon adenocarcinoma cell line is utilized as an in vitro model system in which to study mucin production. In response to treatment with tumor necrosis factor-alpha, DiFi cells acquire some properties of mucin-producing goblet cells including altered morphology, increased reactivity to wheat germ agglutinin, and increased mucin production as determined by RNA expression as well as reactivity with the MUC-1 antibodies, HMFG-1 and SM-3. Thus, TNF-treated DiFi cells represent one of the few in vitro systems in which mucin expression can be induced.^ DiFi cells express an activated pp60$\sp{{\rm c}-src},$ as do most colon adenocarcinomas and derived cell lines, as well as an amplified epidermal growth factor (EGF) receptor. To assess potential changes in these enzymes during induction of differentiation characteristics, potential changes in the levels and activities of these enzymes were examined. For pp60$\sp{{\rm c}-src},$ no changes were observed in protein levels, specific activity of the kinase, cellular localization, or phosphorylation pattern as determined by Staphylococcus aureus V8 protease partial proteolytic mapping after induction of goblet cell-like phenotypic changes. These results suggest that pp60$\sp{{\rm c}-src}$ is regulated differentially in goblet cells than in absorptive cells, as down-modulation of pp60$\sp{{\rm c}-src}$ kinase occurs in the latter. Therefore, effects on pp60$\sp{{\rm c}-src}$ may be critical in colon regulation, and may be important in generating the various colonic epithelial cell types.^ In contrast to pp60$\sp{{\rm c}-src},$ EGF receptor tyrosine kinase activity decreased ($<$5-fold) after TNF treatment and at the time in which morphologic changes were observed. Similar decreases in tyrosine phosphorylation of EGF receptor were observed as assessed by immunoblotting with an anti-phosphotyrosine antibody. In addition, ($\sp{125}$I) -EGF cell surface binding was reduced approximately 3-fold following TNF treatment with a concomitant reduction in receptor affinity ($<$2-fold). These results suggest that modulation of EGF receptor may be important in goblet cell differentiation. In contrast, other published studies have demonstrated that increases in EGF receptor mRNA and in ($\sp{125}$I) -EGF binding accompany differentiation toward the absorptive cell phenotype. Therefore, differential regulation of both EGF receptor and pp60$\sp{{\rm c}-src}$ occur along the goblet cell and absorptive cell differentiation pathways. Thus, my results suggest that TNF-treated DiFi cells represent a unique system in which to study distinct patterns of regulation of pp60$\sp{{\rm c}-src}$ and EGF receptor in colonic cells, and to determine if increased MUC-1 expression is an early event in goblet cell differentiation. ^

The processing of human pro-tumor necrosis factor

Human pro-TNF-$\alpha$ is a 26 kd type II transmembrane protein, and it is the precursor of 17 kd mature TNF. Pro-TNF release mature from its extracellular domain by proteolytic cleavage between resideu Ava ($-$1) and Val (+1). Both forms of TNF are biologically active and the native form of mature TNF is a bell-shaped trimer. The structure of pro-TNF was studied both in intact cell system and in an in vitro translation system by chemical crosslinking. We found that human pro-TNF protein exist as a trimer in intact cells (LPS-induced THP-1 cells and TNF cDNA transfected COS-3 cells) and this trimeric structure is assembled intracellularly, possibly in the ER. By analysis several deletion mutants, we observed a correlation between expression of pro-TNF cytotoxicity in a juxtacrine fashion and detection of the trimer, suggesting the trimeric structure is very important for its biologic activity. With a series of deletion mutants in the linking domain, we found that the small deletion did not block the cleavage and large deletion did regardless of the presence or absence of the native cleavage site, suggesting that the length of the residues between the plasma membrane and the base of the trimer determines the rate of the cleavage, possibly by blocking the accessibility of the cleavage enzyme to its action site. Our data also suggest that the native cleavage site is not sufficient for the release of mature TNF and alternative cleavage site(s) exists. ^

Ras signaling in tumor necrosis factor-induced apoptosis

Tumor necrosis factor (TNF)-induced apoptosis is important in immunologic cytotoxicity, autoimmunity, sepsis, normal embryonic development, and wound healing. TNF exerts cytotoxicity on many types of tumor cells but not on normal cells. The molecular events leading to cell death triggered by TNF are still poorly understood. We found that enforced expression of an activated H-ras oncogene converted the non-tumorigenic TNF-resistant C3H 10T1/2 fibroblasts into tumorigenic cells (10TEJ) that also became very sensitive to TNF-induced apoptosis. This finding suggested that the oncogenic form of H-Ras, in which the p21 is locked in the GTP-bound form, could play a role in TNF-induced apoptosis of these cells. To investigate whether Ras activation is an obligatory step in TNF-induced apoptosis, we introduced two different molecular antagonists of Ras, namely the Rap1A tumor suppressor gene or the dominant-negative rasN17 gene, into H-ras transformed 10TEJ cells. Expression of either Rap1A or RasN17 in 10TEJ cells resulted in abrogation of TNF-induced apoptosis. Similar results were obtained by expression of either Ras antagonist in L929 cells, a fibroblast cell line that is sensitive to TNF-induced apoptosis but does not have a ras mutation. The effects of Rap-1A and RasN17 appear to be specific to TNF, since cytotoxicity induced by doxorubicin and thapsigargin are unaffected. Additionally, constitutive apoptosis sensitivity in isolated nuclei, as measured by activation of Ca$\sp{2+}$-dependent endogenous endonuclease, is not affected by Rap-1A or RasN17. Moreover, TNF treatment of L929 cells increased Ras-bound GTP, indicating that Ras activation is triggered by TNF. Thus, Ras activation is required for TNF-induced apoptosis in mouse cells. ^

Interferon-gamma release assays for latent tuberculosis infection in child and young adult candidates or recipients of tumor necrosis factor inhibitor therapy

Background. Inhibition of tumor necrosis factor (TNF) is associated with progression of latent tuberculosis infection (LTBI) to active disease. LTBI screening prior to starting TNF inhibitor therapy is recommended. Blood tests, collectively known as interferon-gamma release assays (IGRAs), offer a means other than the tuberculin skin test (TST) of screening for LTBI. However, in the setting of immune compromise, anergy may limit the clinical utility of IGRAs. ^ Methods. A cross-sectional study was conducted in children and young adults ≤ 21 years of age who were cared for at Texas Children's Hospital in Houston, TX, during 2011 and who were candidates for, or were receiving, tumor necrosis factor (TNF)-inhibitor therapy. All subjects answered a risk factor questionnaire and were tested for LTBI by two commercially available IGRAs (QuantiFERON-Gold In-Tube assay and the T-SPOT.TB assay), along with the TST. T-cell phenotypes were evaluated through flow cytometry, both at baseline and after antigen stimulation. ^ Results. Twenty-eight subjects were enrolled. All were TST negative and none were IGRA positive. Results were negative for the 27 subjects who were tested with QuantiFERON-Gold In-Tube. However, 26% of subjects demonstrated anergy in the T-SPOT.T. Patients with T-SPOT. TB anergy had lower quantitative IFN-γ responses to mitogen in the QFT assay—the mean IFN-γ level to mitogen in patients without T-SPOT.TB anergy was 9.84 IU/ml compared to 6.91 IU/ml in patients with T-SPOT.TB anergy (P = 0.046). Age and use of TNF inhibitors, corticosteroids, or methotrexate use were not significantly associated with T-SPOT.TB anergy. Antigen stimulation revealed depressed expression of intracellular IFN-γ in subjects with T-SPOT. TB anergy. ^ Conclusions. The frequency of anergy in this population is higher than would be expected from studies in adults. There appears to be inappropriate IFN-γ responses to antigen in subjects with T-SPOT. TB anergy. This immune defect was detected by the T-SPOT. TB assay but not by the QuantiFERON-Gold In-Tube assay. Further data are needed to clarify the utility of IGRAs in this population.^

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

A distinct element involved in the lipopolysaccharide activation of the tumor necrosis factor -alpha promoter in a promonocytic cell line, THP-1

TNF-α is a pleiotropic cytokine involved in normal homeostasis and plays a key role in defending the host from infection and malignancy. However when deregulated, TNF-α can lead to various disease states. Therefore, understanding the mechanisms by which TNF-α is regulated may aid in its control. In spite of the knowledge gained regarding the transcriptional regulation of TNF-α further characterization of specific TNF-α promoter elements remains to be elucidated. In particular, the T&barbelow;NF-α A&barbelow;P-1/C&barbelow;RE-like (TAC) element of the TNF-α promoter has been shown to be important in the regulation of TNF-α in lymphocytes. Activating transcription factor-2 (ATF-2) and c-Jun were shown to bind to and transactivate the TAC element However, the role of TAC and transcription factors ATF-2 and c-Jun in the regulation of TNF-α in monocytes is not as well characterized. Lipopolysaccharide (LPS), a potent activator of TNF-α in monocytes, provides a good model to study the involvement of TAC in TNF-α regulation. On the other hand, all-tram retinoic acid (ATRA), a physiological monocyte-differentiation agent, is unable to induce TNF-α protein release. ^ To delineate the functional role of TAC, we transfected the wildtype or the TAC deleted TNF-α promoter-CAT construct into THP-1 promonocytic cells before stimulating them with LPS. CAT activity was induced 17-fold with the wildtype TNF-α promoter, whereas the CAT activity was uninducible when the TAC deletion mutant was used. This daft suggests that TAC is vital for LPS to activate the TNF-α promoter. Electrophoretic mobility shift assays using the TAC element as a probe showed a unique pattern for LPS-activated cells: the disappearance of the upper band of a doublet seen in untreated and ATRA treated cells. Supershift analysis identified c-Jun and ATF-2 as components of the LPS-stimulated binding complex. Transient transfection studies using dominant negative mutants of JNK, c-Jun, or ATF-2 suggest that these proteins we important for LPS to activate the TNF-α promoter. Furthermore, an increase in phosphorylated or activated c-Jun was bound to the TAC element in LPS-stimulated cells. Increased c-Jun activation was correlated with increased activity of Jun N-terminal kinase (JNK), a known upstream stimulator of c-Jun and ATF-2, in LPS-stimulated monocytes. On the other hand, ATRA did not induce TNF-α protein release nor changes in the phosphorylation of c-Jun or JNK activity, suggesting that pathways leading to ATRA differentiation of monocytic cells are independent of TNF-α activation. Together, the induction of TNF-α gene expression seems to require JNK activation, and activated c-Jun binding to the TAC element of the TNF-α promoter in THP-1 promonocytic cells. ^