Ultraviolet exposure of melanoma cells induces fibroblast activation protein-α in fibroblasts: Implications for melanoma invasion.

Fibroblast activation protein-a (FAP-a) promotes tumor growth and cell invasiveness through extracellular matrix degradation. How ultraviolet radiation (UVR), the major risk factor for malignant melanoma, influences the expression of FAP-a is unknown. We examined the effect of UVR on FAP-a expression in melanocytes, keratinocytes and fibroblasts from the skin and in melanoma cells. UVR induces upregulation of FAP-a in fibroblasts, melanocytes and primary melanoma cells (PM) whereas keratinocytes and metastatic melanoma cells remained FAP-a negative. UVA and UVB stimulated FAP-a-driven migration and invasion in fibroblasts, melanocytes and PM. In co-culture systems UVR of melanocytes, PM and cells from regional metastases upregulated FAP-a in fibroblasts but only supernatants from non-irradiated PM were able to induce FAP-a in fibroblasts. Further, UV-radiated melanocytes and PM significantly increased FAP-a expression in fibroblasts through secretory crosstalk via Wnt5a, PDGF-BB and TGF-ß1. Moreover, UV radiated melanocytes and PM increased collagen I invasion and migration of fibroblasts. The FAP-a/DPPIV inhibitor Gly-ProP(OPh)2 significantly decreased this response implicating FAP-a/DPPIV as an important protein complex in cell migration and invasion. These experiments suggest a functional association between UVR and FAP-a expression in fibroblasts, melanocytes and melanoma cells implicating that UVR of malignant melanoma converts fibroblasts into FAP-a expressing and ECM degrading fibroblasts thus facilitating invasion and migration. The secretory crosstalk between melanoma and tumor surrounding fibroblasts is mediated via PDGF-BB, TGF-ß1 and Wnt5a and these factors should be evaluated as targets to reduce FAP-a activity and prevent early melanoma dissemination.

Cytotoxicity of 1-alkylquinolinium bromide ionic liquids in murine fibroblast NIH 3T3 cells

Minimal toxicity data are available for 1-alkylquinolinium bromide ionic liquids. Here, their toxicity to NIH 3T3 murine fibroblast cells, of relevance to their potential antimicrobial application, is presented. Toxicity data, presented by time-point analysis with a particular focus on the immediate toxicity upon short term cellular exposure, indicate a link between the length of the alkyl chain substituent and resultant biological toxicity. 1-Tetradecylquinolinium bromide was found to exhibit cellular toxicity comparable to benzalkonium chloride over all time points tested. By comparison, 1-octylquinolinium bromide initially exerted significantly lower cytotoxicity at one hour; however, toxicity was found to have a cumulative effect over time-course analysis up to three days. This illustrates that alkyl chain components may govern not only overall toxicity, but also the rate of toxicity. Fluorescence microscopy was utilised to examine destabilisation of the plasma membrane by 1 tetradecylquinolinium bromide and benzalkonium chloride after one hour, with membrane destabilisation not observed for 1-octylquinolinium bromide, or the base constituent quinoline.

Gingival fibroblast response to cyclosporin A and transforming growth factor beta(1)

This study investigates a potential role for TGF beta(1), in the pathogenesis of cyclosporin A-induced gingival overgrowth (CsA-OG). TGF beta(1) was localized immunohistochemically in the connective tissue of both normal gingiva and CsA-OG. Intense staining for TGF beta(1) was detected at the tips of the dermal papillae of the overgrown gingiva. In addition, fibroblasts derived from healthy gingiva and fibroblasts derived from CsA-OG were cultured both as monolayers or embedded in a 3D-collagen gel. Fibroblast activity was monitored in terms of protein and collagen production in the presence of (i) 1 ng/ml TGF beta(1), (ii) 500 ng/ml CsA, or (iii) 500 ng/ml CsA and 1 ng/ml TGF beta(1). In monolayer culture TGF beta(1) significantly increased protein and collagen production in all cell strains (p

Calcium fluxes modulate the radiation-induced bystander responses in targeted glioma and fibroblast cells

Bystander responses have been reported to be a major determinant of the response of cells to radiation exposure at low doses, including those of relevance to therapy. This study investigated the role of changes in calcium levels in bystander responses leading to chromosomal damage in nonirradiated T98G glioma cells and AG01522 fibroblasts that had been either exposed to conditioned medium from irradiated cells or co-cultured with a population where a fraction of cells were individually targeted through the nucleus or cytoplasm with a precise number of microbeam helium-3 particles. After the recipient cells were treated with conditioned medium from T98G or AG01522 cells that had been irradiated through either nucleus or cytoplasm, rapid calcium fluxes were monitored in the nonirradiated recipient cells. Their characteristics were dependent on the source of the conditioned medium but had no dependence on radiation dose. When recipient cells were co-cultured with an irradiated population of either T98G or AG01522 cells, micronuclei were induced in the nonirradiated cells, but this response was eliminated by treating the cells with calcicludine (CaC), a potent blocker of Ca2+ channels. Moreover, both the calcium fluxes and the bystander effect were inhibited when the irradiated T98G cells were treated with aminoguanidine, an inhibitor of nitric oxide synthase (NOS), and when the irradiated AG01522 cells were treated with DMSO, a scavenger of reactive oxygen species (ROS), which indicates that NO and ROS were involved in the bystander responses generated from irradiated T98G and AG01522 cells, respectively. Our findings indicate that calcium signaling may be an early response in radiation-induced bystander effects leading to chromosome damage. (c) 2006 by Radiation Research Society.

Contributions of two UDP-glucose dehydrogenases to viability and polymyxin B resistance of Burkholderia cenocepacia

Burkholderia cenocepacia is highly resistant to antimicrobial peptides and we hypothesized that the conversion of UDP-glucose to UDP-glucuronic acid, a reaction catalysed by the enzyme UDP-glucose dehydrogenase (Ugd) would be important for this resistance. The genome of B. cenocepacia contains three predicted ugd genes: ugd(BCAL2946), ugd(BCAM0855) and ugd(BCAM2034), all of which were individually inactivated. Only inactivation of ugd(BCAL2946) resulted in increased sensitivity to polymyxin B and this sensitivity could be overcome when either ugd(BCAL2946) or ugd(BCAM0855) but not ugd(BCAM2034) was expressed from plasmids. The growth of a conditional ugd(BCAL2946) mutant, created in the Deltaugd(BCAM0855) background, was significantly impaired under non-permissive conditions. Growth could be rescued by either ugd(BCAL2946) or ugd(BCAM0855) expressed in trans, but not by ugd(BCAM2034). Biochemical analysis of the purified, recombinant forms of Ugd(BCAL2946) and Ugd(BCAM0855) revealed that they are soluble homodimers with similar in vitro Ugd activity and comparable kinetic constants for their substrates UDP-glucose and NAD(+). Purified Ugd(BCAM2034) showed no in vitro Ugd activity. Real-time PCR analysis showed that the expression of ugd(BCAL2946) was 5.4- and 135-fold greater than that of ugd(BCAM0855) and ugd(BCAM2034), respectively. Together, these data indicate that the combined activity of Ugd(BCAL2946) and Ugd(BCAM0855) is essential for the survival of B. cenocepacia but only the most highly expressed ugd gene, ugd(BCAL2946), is required for polymyxin B resistance.

A putative gene cluster for aminoarabinose biosynthesis is essential for Burkholderia cenocepacia viability

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.

Chymotrypsin-like serine proteinases are involved in the maintenance of cell viability

An increasing number of studies have implicated serine proteinases in the development of apoptosis. In this study, we assessed the ability of a set of highly specific irreversible inhibitors (activity probes), incorporating an a-amino alkane diphenyl phosphonate moiety, to modulate cell death. In an initial assessment of the cellular toxicity of these activity probes, we discovered that one example, N-a-tetramethylrhodamine phenylalanine diphenylphosphonate {TMR-Phe^P(OPh)₂} caused a concentration-dependent decrease in the viability of HeLa and U251 mg cells. This reduced cell viability was associated with a time-dependent increase in caspase-3 activity, PARP cleavage and phosphatidylserine translocation, establishing apoptosis as the mechanism of cell death. SDS-PAGE analysis of cell lysates prepared from the HeLa cells treated with TMR-Phe^P(OPh)₂, revealed the presence of a fluorescent band of molecular weight 58 kDa. Given that we have previously reported on the use of this type of activity probe to reveal active proteolytic species, we believe that we have identified a chymotrypsin-like serine proteinase activity integral to the maintenance of cell viability.

In vitro viability of choroidal melanomata following pre-enucleation irradiation

In a group of eighteen patients with uveal melanomas, seven underwent low-dose pre-enucleation irradiation of approximately 2000 cGy. All the tumours were propagated in tissue culture and the growth characteristics of tumour cells from irradiated eyes were compared with tumour cells from non-irradiated eyes. Cultures were observed with phase-contrast microscopy, and radioactive thymidine labelling was used to study cell turnover. Although tissue samples from peripheral areas of irradiated tumours produced a mixture of viable and non-viable cells, with reduced ability to attach to substrate, central regions of irradiated tumours contained viable cells which propagated freely in tissue culture.

Effect of laser treatment on the attachment and viability of mesenchymal stem cell responses on shape memory NiTi alloy

The objectives of this study were to investigate the effect of laser-induced surface features on the morphology, attachment and viability of mesenchymal stem cells (MSCs) at different periods of time, and to evaluate the biocompatibility of different zones: laser-melted zone (MZ), heat-affected zone (HAZ) and base metal (BM) in laser-treated NiTi alloy. The surface morphology and composition were studied by scanning electron microscope (SEM) and X-ray photoemission spectroscopy (XPS), respectively. The cell morphology was examined by SEM while the cell counting and viability measurements were done by haemocytometer and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. The results indicated that the laser-induced surface features, such as surface roughening, presence of anisotropic dendritic pattern and complete surface Ni oxidation were beneficial to improve the biocompatibility of NiTi as evidenced by the highest cell attachment (4 days of culture) and viability (7 days of culture) found in the MZ. The biocompatibility of the MZ was the best, followed by the BM with the HAZ being the worst. The defective and porous oxide layer as well as the coarse grained structure might attribute to the inferior cell attachment (4 days of culture) and viability (7 days of culture) on the HAZ compared with the BM which has similar surface morphology.