In vitro chemosensitivity of canine mast cell tumors grades II and III to all-trans-retinoic acid (ATRA).

Mast cell tumor (MCT) is one of the most prevalent neoplasms that affect the skin and soft tissue of dogs. Because mast cell tumors present a great variety of clinical appearance and behavior, their treatment becomes a challenge. While retinoids are well recognized as promising antitumor agents, there have been only a few reports about retinoids` effect on canine cancers. The aim of this study was to investigate the chemosensitivity of MCT grades II and III to all-trans retinoic acid (ATRA). Immediately after surgical resection, MCT were prepared for primary culture. Samples of MCTs were also fixed in formalin for histopathology and grading according to the classification of Patnaik et al. (Veterinary Pathology 21(5):469-474, 1984). The best results were obtained when neoplastic mast cells were co-cultivated with fibroblasts. Cultured mast cells were, then, treated with concentrations of 10(-4) to 10(-7) M of ATRA, in order to evaluate their chemosensitivity to this retinoid. MTT assay was performed to estimate cell growth and death. The highest level of mast cell chemosensivity was obtained at the dose of 10(-4) M (p < 0,002). MCT of grades II or III were equally susceptible to the treatment with ATRA. Cell death was observed on the first 24 h until 48 h. According to these results, ATRA may be a potential chemotherapeutic agent for the treatment of canine MCT.

Ultrastructure of Motor Nerve Terminals in the Anterior Third of Wistar Rat Tongue

The nerve terminals of intrinsic muscular fibers of the tongue of adult wistar rats was studied by using silver impregnation techniques, transmission electron microscopy (TEM), and high resolution scanning electron microscopy (HRSEM) to observe the nerve fibers and their terminals. Silver impregnation was done according to Winkelman and Schmit, 1957. For TEM, small blocks were fixed in modified Karnovsky solution, postfixed in 1% buffered osmium tetroxide solution, and embedded in Spurr resin. For HRSEM, the parts were fixed in 2% osmium tetroxide solution with 1/15 M sodium phosphate buffer (pH 7.4) at 4 degrees C for 2 h, according to the technique described by Tanaka, 1989. Thick myelinated nerve bundles were histologically observed among the muscular fibers. The intrafusal nerve fiber presented a tortuous pathway with punctiform terminal axons in clusters contacting the surface of sarcolemma. Several myelinated nerve fibers involved by collagen fibers of the endoneurium were observed in HRSEM in three-dimensional aspects. The concentric lamellae of the myelin sheath and the axoplasm containing neurofilaments interspersed among the mitochondria were also noted. In TEM, myofibrils, mitochondria, rough endoplasmic reticulum, Golgi`s apparatus, and glycogen granules were observed in sarcoplasm. It is also noted that the sarcomeres constituted by myofilaments with their A, I, and H bands and the electron dense Z lines. In areas adjacent to muscular fibers, there were myelinated and unmyelinated nerve fibers involved by endoneurium and perineurium. In the region of the neuromuscular junction, the contact with the sarcolemma of the muscular cell occurs forming several terminal buttons and showing numerous evaginations of the cell membrane. In the terminal button, mitochondria and numerous synaptic vesicles were observed. Microsc. Res. Tech. 72:464-470, 2009. (C) 2009 Wiley-Liss. Inc.

Expression of Homeobox Genes in Oral Squamous Cell Carcinoma Cell Lines Treated With All-Trans Retinoic Acid

Oral squamous cell carcinoma (OSCC) may arise from potentially malignant oral lesions. All-trans retinoic acid (atRA), which plays a role in cell growth and differentiation, has been studied as a possible chemotherapeutic agent in the prevention of this progression. While the mechanism by which atRA suppresses cell growth has not been completely elucidated, it is known that homeobox genes are atRA targets. To determine if these genes are involved in the atRA-mediated OSCC growth inhibition, PCR array was performed to evaluate the expression of 84 homeobox genes in atRA-sensitive SCC-25 cells compared to atRA-resistant SCC-9 cells following 7 days with atRA treatment. Results showed that the expression of 8 homeobox genes was downregulated and expression of 4 was upregulated in SCC-25 cells but not in SCC-9 cells. Gene expression levels were confirmed for seven of these genes by RT-qPCR. Expression of three genes that showed threefold downregulation was evaluated in SCC-25 cells treated with atRA for 3, 5, and 7 days. Three different patterns of atRA-dependent gene expression were observed. ALX1 showed downregulation only on day 7. DLX3 showed reduced expression on day 3 and further reduced on clay 7. TLX1 showed downregulation only on days 5 and 7. Clearly the expression of homeobox genes is modulated by atRA in OSCC cell lines. However, the time course of this modulation suggests that these genes are not direct targets of atRA mediating OSCC growth suppression. Instead they appear to act as downstream effectors of atRA signaling. J. Cell. Biochem. 111: 1437-1444, 2010. (C) 2010 Wiley-Liss, Inc.

A role for COX2-derived PGE2 and PGE2-receptor subtypes in head and neck squamous carcinoma cell proliferation

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies. Published by Elsevier Ltd.

GAPD and tubulin are suitable internal controls for qPCR analysis of oral squamous cell carcinoma cell lines

The selection of housekeeping genes is critical for gene expression studies. To address this issue, four candidate housekeeping genes, including several commonly used ones, were investigated in oral squamous cell carcinoma cell lines. A simple quantitative RT-PCR approach was employed by comparing relative expression of the four candidate genes within two cancerous cell tines (HN6 and HN31) and one noncancerous cell tine (HaCaT) treated or not with EGF and TGF-beta 1. Data were analyzed using ANOVA followed by the NormFinder software program. On this basis, stability of the candidate housekeeping genes was ranked and non statistical differences were found using ANOVA test. On the other hand, the NormFinder was able to show that GAPD and TUBB presented the less variable results, representing appropriated housekeeping genes for the samples and conditions anatyzed. In conclusion, this study suggests that the GAPD and the TUBB represent adequate normalizers for gene profiting studies in OSCC cell. tines, covering, respectively, high and low expression levels genes. (C) 2008 Elsevier Ltd. All rights reserved.

Testing for Market Power in Multiple-Input, Multiple-Output Industries: The Australian Grains and Oilseeds Industries

Recent empirical studies have found significant evidence of departures from competition in the input side of the Australian bread, breakfast cereal and margarine end-product markets. For example, Griffith (2000) found that firms in some parts of the processing and marketing sector exerted market power when purchasing grains and oilseeds from farmers. As noted at the time, this result accorded well with the views of previous regulatory authorities (p.358). In the mid-1990s, the Prices Surveillence Authority (PSA 1994) determined that the markets for products contained in the Breakfast Cereals and Cooking Oils and Fats indexes were "not effectively competitive" (p.14). The PSA consequently maintained price surveillence on the major firms in this product group. The Griffith result is also consistent with the large number of legal judgements against firms in this sector over the past decade for price fixing or other types of non-competitive behaviour. For example, bread manufacturer George Weston was fined twice during 2000 for non-competitive conduct and the ACCC has also recently pursued and won cases against retailer Safeway in grains and oilseeds product lines.