Identification of differentially expressed genes in cutaneous squamous cell carcinoma by microarray expression profiling.

BACKGROUND: Carcinogenesis is a multi-step process indicated by several genes up- or down-regulated during tumor progression. This study examined and identified differentially expressed genes in cutaneous squamous cell carcinoma (SCC). RESULTS: Three different biopsies of 5 immunosuppressed organ-transplanted recipients each normal skin (all were pooled), actinic keratosis (AK) (two were pooled), and invasive SCC and additionally 5 normal skin tissues from immunocompetent patients were analyzed. Thus, total RNA of 15 specimens were used for hybridization with Affymetrix HG-U133A microarray technology containing 22,283 genes. Data analyses were performed by prediction analysis of microarrays using nearest shrunken centroids with the threshold 3.5 and ANOVA analysis was independently performed in order to identify differentially expressed genes (p < 0.05). Verification of 13 up- or down-regulated genes was performed by quantitative real-time reverse transcription (RT)-PCR and genes were additionally confirmed by sequencing. Broad coherent patterns in normal skin vs. AK and SCC were observed for 118 genes. CONCLUSION: The majority of identified differentially expressed genes in cutaneous SCC were previously not described.

Expression regulation of MAO isoforms in monocytic cells in response to Th2 cytokines.

BACKGROUND: Th2-cytokines, such as interleukins-4 and -13 (IL-4, IL-13), have been identified as alternative stimuli of monocytes/macrophages. We have recently profiled the gene-expression pattern of IL-4-treated human peripheral monocytes and found that 15-lipoxygenase-1 (15-LOX1) and monoamine oxidase A (MAO-A) are among the five most strongly upregulated gene products in IL-4-treated cells. Transfection of monocytic cells (U937) with 15-LOX1 also induced MAO-A expression. These data suggested that 15-LOX1 products might play a role in the IL4-induced signaling cascade leading to expression of MAO-A in human monocytes. MATERIAL/METHODS: To test this hypothesis we incubated wild-type and 15-LOX1-transfected U937 cells with different concentrations of either IL-4 or 15-LOX-products [13S-H(p)ODE, 15S-H(p)ETE] and quantified the expression of 15-LOX1, MAO-A, and MAO-B by activity assays and real-time RT-PCR. RESULTS: Wild-type U937 cells express neither MAO-A nor MAO-B, but after three days of IL4 treatment, MAO-A mRNA was detected. A similar isoform-specific expression of MAO-A mRNA was observed when U937 cells were transfected with 15-LOX1 or when the cells were incubated with primary 15-LOX1 products (hydroperoxy fatty acids) or H2O2. In contrast, the corresponding hydroxy fatty acids were ineffective. CONCLUSIONS: These data indicate that increased intracellular peroxide concentrations (oxidative stress) induce MAO-A expression in monocytes/macrophages, which normally do not express the enzyme. Our findings also suggest that IL-4-induced upregulation of MAO-A expression in human peripheral monocytes may proceed via 15-LOX1-dependent and 15-LOX1-independent pathways. The biological role of MAO-A expression for monocyte function is discussed.

Gene expression alterations of human peripheral blood monocytes induced by medium-term treatment with the TH2-cytokines interleukin-4 and -13.

The TH2-cytokines interleukins-4 and -13 severely alter gene expression of monocytic cells. We quantified the impact of interleukins-4 and -13 on the gene expression pattern of human peripheral blood monocytes applying a strategy that involved microarray hybridization, RT-PCR, immunohistochemistry and activity assays. After 3 days of continuous cytokine exposure the six most strongly upregulated gene products (15-lipoxygenase-1, fibronectin, monoamine oxidase-A, CD1c, CD23A, coagulation factor XIII) included four proteins with potential anti-inflammatory properties: (i) 15-lipoxygenase-1 (290-fold upregulation), (ii) fibronectin (180-fold upregulation), (iii) monoamine oxidase-A (56-fold upregulation) and (iv) coagulation factor XIII (35-fold upregulation). In addition, a number of other gene products, the expression of which is consistent with inflammatory resolution (annexin 1, collagen 1alpha2, laminin alpha5, TIMP3, heme oxygenase-1, CCL22, heat shock protein A8), were upregulated to a lower extent. In contrast, expression of classical pro-inflammatory gene products, such as tumor necrosis factor alpha, monocyte chemotactic protein-1, interleukins-1, -6, -8, -18, cyclooxygenase-2, as well as enzymes and receptors of the leukotriene cascade (5-lipoxygenase, 5-lipoxygenase activating protein, leukotriene B(4) receptor, cysteinyl leukotriene receptor 2) were significantly downregulated. These data suggest that medium-term treatment of human peripheral blood monocytes with interleukins-4/13 alters the gene expression pattern so that the cells might adopt a resolving phenotype.

Expression of 12/15-lipoxygenase attenuates intracellular lipid deposition during in vitro foam cell formation.

OBJECTIVE: Lipoxygenases with different positional specificity have been implicated in atherogenesis, but the precise roles of the various isoforms remain unclear. Because of its capability of oxidizing low-density lipoprotein (LDL) to an atherogenic form, 12/15-lipoxygenases have been suggested to initiate LDL oxidation in vivo; thus, these enzymes may exhibit pro-atherogenic activities. However, in several rabbit atherosclerosis models, the enzyme appears to act atheroprotective. METHODS AND RESULTS: To test the impact of 12/15-lipoxygenase expression on early atherogenesis, we established an in vitro foam cell model, which is based on the uptake of acetylated LDL by murine macrophages. In this system, we found that 12/15-lipoxygenase expression protects the cells from intracellular lipid deposition. This effect was related to an attenuated uptake of modified LDL, as indicated by impaired expression of scavenger receptor A and to accelerated intracellular lipid metabolism. CONCLUSIONS: Our results indicate that the role of 12/15-lipoxygenase in atherogenesis may not be restricted to oxidative LDL modification. Expression of this lipid-peroxidizing enzyme may impact both lipid uptake and intracellular lipid turnover. These data provide a plausible explanation for the antiatherogenic effect of 12/15-LOX in rabbit atherosclerosis models.

Th2 response of human peripheral monocytes involves isoform-specific induction of monoamine oxidase-A.

Monocyte/macrophage function is critically regulated by specific cytokines and growth factors that they are exposed to at inflammatory sites. IL-4 and IL-13 are multifunctional cytokines generated mainly by Th2 lymphocytes that have important biological activities in allergy and inflammation. The Th2 response of human peripheral monocytes is characterized by complex alterations in the gene expression pattern, which involves dominant expression of CD23 cell surface Ag and lipid-peroxidizing 15-lipoxygenase-1 (15-LOX1). In this study, we report that the classical Th2 cytokines IL-4 and IL-13 strongly up-regulate expression of monoamine oxidase A (MAO-A) with no induction of the closely related isozyme, MAO-B. Real-time PCR indicated a >2000-fold up-regulation of the MAO-A transcripts, and immunohistochemistry revealed coexpression of the enzyme with 15-LOX1 in a major subpopulation of monocytes. MAO-A was also induced in lung carcinoma A549 cells by IL-4 in parallel with 15-LOX1. In promyelomonocytic U937 cells, which neither express 15-LOX1 nor MAO-A in response to IL-4 stimulation, expression of MAO-A was up-regulated following transfection with 15-LOX1. This is the first report indicating expression of MAO-A in human monocytes. Its isoform-specific up-regulation in response to Th2 cytokines suggests involvement of the enzyme in modulation of innate and/or acquired immune system.