LDOC1 silenced by cigarette exposure and involved in oral neoplastic transformation

Previously, we identified global epigenetic aberrations in smoking-associated oral squamous cell carcinoma (OSCC). We hypothesized that cigarette exposure triggers OSCC through alteration of the methylome of oral cells. Here we report that cigarette smoke condensate (CSC) significantly changes the genomic 5-methyldeoxycytidine content and nuclear accumulation of DNA methyltransferase 1 (DNMT1) and DNMT3A in human untransformed oral cells. By using integrated analysis of cDNA and methylation arrays of the smoking-associated dysplastic oral cell line and OSCC tumors, respectively, we identified four epigenetic targets—UCHL1, GPX3, LXN, and LDOC1—which may be silenced by cigarette. Results of quantitative methylation-specific PCR showed that among these four genes, LDOC1 promoter was the most sensitive to CSC. LDOC1 promoter hypermethylation and gene silencing followed 3 weeks of CSC treatment. LDOC1 knockdown led to a proliferative response and acquired clonogenicity of untransformed oral cells. Immunohistochemistry showed that LDOC1 was downregulated in 53.3% (8/15) and 57.1% (20/35) of premalignant oral tissues and early stage OSCCs, respectively, whereas 76.5% (13/17) of normal oral tissues showed high LDOC1 expression. Furthermore, the microarray data showed that LDOC1 expression had decreased in the lung tissues of current smokers compared with that in those of never smokers and had significantly decreased in the lung tumors of smokers compared with that in normal lung tissues. Our data suggest that CSC-induced promoter methylation may contribute to LDOC1 downregulation, thereby conferring oncogenic features to oral cells. These findings also imply a tumor suppressor role of LDOC1 in smoking-related malignancies such as OSCC and lung cancer.     

Endogenous adenosine differentially modulates 5-hydroxytryptamine release from a human enterochromaffin cell model

BACKGROUND & AIMS: The aim was to determine whether adenosine receptors modulate cAMP, intracellular free calcium ([Ca(2+)](i)), and 5-hydroxytryptamine (5-HT) release in human carcinoid BON cells. METHODS: Adenosine receptor (R) mRNA, proteins, and function were identified by Western blots, immunofluorescent labeling, Fluo-4/AM [Ca(2+)](i) imaging, and pharmacologic/physiologic techniques. RESULTS: A1, A2, and A3Rs were present in BON cells and carcinoid tumors. Baseline 5-HT levels increased with adenosine deaminase, activation of A2Rs, and inhibition of A3Rs, whereas A3R activation decreased 5-HT. A2R antagonists or blockade of adenosine reuptake that elevates extracellular adenosine reduced mechanically evoked 5-HT release. In single BON cells, touch elevated [Ca(2+)](i) responses were augmented by adenosine deaminase, A1, and A3R antagonists. CONCLUSIONS: Tonic or mechanically evoked release of endogenous adenosine is a critical determinant of differential activation of adenosine receptors and may have important implications for gut mechanosensory reflexes.     

Projections of the zona incerta in the cat, with stimulation controls.

In eight cats single electrolytic lesions were placed in the zona incerta, and resultant fiber degeneration studies were made. In seven additional cats, stimulating electrodes were chronically implanted bilaterally into the zona incerta, H₂ (lenticular fasciculus), or H (prerubral) fields of Forel. The animals were placed in a two-compartment shuttle box, and a routinely established procedure of subthalamic stimulation was instituted. When the sensory (nociceptive) or motor manifestations and reactions were established, small lesions were made through both poles of the electrodes. The brains were studied by silver techniques for degenerating axons and terminals. Findings in the latter group of animals with physiologic substrates, compared to those in the first group, indicated that the zona incerta contains at least two major physiologic-anatomic components with differential fiber projections. The first component is a medial zona incerta proper or caudalis, paleospinothalamic, nociceptive-conducting system which causes typical escape responses. Its unequivocal projections are to the nucleus of the H₁ field, posterior and dorsal hypothalamus, part of the intralaminar system, ventromedial and ventralis anterior nuclei, nucleus reuniens, reticular nucleus, pulvinar, posterior nucleus, central gray, red nucleus, and the central tegmental tract. The second constitutent concerns pyramidal-extrapyramidal motor type responses that arise with avoidance reactions from other portions of the zona incerta. In these cases there is heavy projection to the caudate, entopenduncular, globus pallidus, and putamen nuclei. In contrast, degeneration from the nociceptive part of the zona incerta or H₂ and H fields to these nuclei is minimal.     

Isoproterenol inhibition of isolated human neutrophil function

The human PMN can contribute to the inflammatory response. Several neutrophil responses can be inhibited by agonists that increase the cellular levels of cyclic AMP. In the following article, we compared the effects of ISO on lysosomal beta-glucuronidase release, superoxide generation, and CL in isolated human PMNs. ISO inhibited the neutrophil CL response to opsonized zymosan in a dose-dependent fashion with maximal effects at 10(-4)M. ISO inhibition of CL was not enhanced by the addition of theophylline, nor was CL inhibited by the exogenous addition cyclic AMP except at a very high concentration of 10(-3)M. ISO also suppressed beta-glucuronidase release and superoxide generation in neutrophils during an incubation with opsonized zymosan particles. For ISO to inhibit beta-glucuronidase release and superoxide generation, theophylline (5 X 10(-4)M) was necessary. ISO effectively inhibits three neutrophil functions that are capable of causing tissue inflammation. Although ISO suppressed all three neutrophil responses, the inhibitory mechanisms appear to be variable.     

Genotoxicity testing of cigarette-smoke condensate in the SCE and HGPRT assays with V79 Chinese hamster cells

The genotoxicity of cigarette-smoke condensate (CSC) was investigated using V79 Chinese hamster fibroblasts in the sister-chromatid exchange (SCE) and HGPRT (hypoxanthine-guanine-phosphoribosyl-transferase) tests. CSC was negative in the SCE test both with and without metabolic activation (co-cultivation with chick-embryo primary hepatocytes). In the HGPRT test no direct mutagenicity of CSC was observed but after metabolic activation there was a considerable increase in the number of mutants. Comparison of different metabolizing systems showed that non-induced chick hepatocytes, liver homogenate from non-induced chick embryos and liver homogenate from rats pretreated with Aroclor 1254 generated similar numbers of mutants in cells treated with CSC. In addition the capacity of CSC to inhibit metabolic co-operation between V79 cells was studied. A dose-related increase in the inhibition of metabolic co-operation was observed.     

Stromal modulation of bladder cancer-initiating cells in a subcutaneous tumor model

The development of new cancer therapeutics would benefit from incorporating efficient tumor models that mimic human disease. We have developed a subcutaneous bladder tumor regeneration system that recapitulates primary human bladder tumor architecture by recombining benign human fetal bladder stromal cells with SW780 bladder carcinoma cells. As a first step, SW780 cells were seeded in ultra low attachment cultures in order to select for sphere-forming cells, the putative cancer stem cell (CSC) phenotype. Spheroids were combined with primary human fetal stromal cells or vehicle control and injected subcutaneously with Matrigel into NSG mice. SW780 bladder tumors that formed in the presence of stroma showed accelerated growth, muscle invasion, epithelial to mesenchymal transition (EMT), decreased differentiation, and greater activation of growth pathways compared to tumors formed in the absence of fetal stroma. Tumors grown with stroma also demonstrated a greater similarity to typical malignant bladder architecture, including the formation of papillary structures. In an effort to determine if cancer cells from primary tumors could form similar structures in vivo using this recombinatorial approach, putative CSCs, sorted based on the CD44⁺CD49f⁺ antigenic profile, were collected and recombined with fetal bladder stromal cells and Matrigel prior to subcutaneous implantation. Retrieved grafts contained tumors that exhibited the same structure as the original primary human tumor. Primary bladder tumor regeneration using human fetal bladder stroma may help elucidate the influences of stroma on tumor growth and development, as well as provide an efficient and accessible system for therapeutic testing.