Atonal homolog 1 Is a Tumor Suppressor Gene

Colon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from Drosophila and previous in vitro studies of mammalian Atonal homolog 1 (Atoh1, also called Math1 or Hath1) suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation

The Flavonoid Luteolin Increases the Resistance of Normal, but Not Malignant Keratinocytes, Against UVB-Induced Apoptosis

Adequate protection of skin against the carcinogenic effects of UVB irradiation is essential. Flavonoids may have a conspicuous role in cancer prevention because of their antioxidant, anti-inflammatory, and growth-inhibitory effects. Therefore, we tested the effects of the flavone luteolin (LUT) on selected parameters of the sunburn response in normal human keratinocytes, exposed to physiological doses of UVB. LUT attenuated UVB-induced cell death through delay and inhibition of intrinsic apoptotic signaling. Moreover, LUT not only predominantly affected the mitochondrial apoptosis pathway through its antioxidant capacity, but also changed the balance of Bcl2 (B-cell leukemia/lymphoma 2)-family members. Furthermore, LUT had inhibitory effects on the UVB-induced release of the inflammatory mediators, IL-1α and prostaglandin-E2. Using different cell lines derived from squamous cell carcinomas, we showed that LUT did not increase the resistance of malignant keratinocytes to UVB. Our data suggest that LUT inhibits different aspects of the sunburn response, which results ultimately in an increased survival of normal keratinocytes, whereas the sensitivity of malignant cells to UVB remain unchanged. Hence, LUT might have value in new photoprotective applications or improve existing ones

Enhanced visualization of blood and pigment in multispectral skin dermoscopy

Background and objectives Dermoscopy has proven its value in the diagnosis of skin cancer and, therefore, is well established in daily dermatology practice. Up until now, analogue white light dermoscopy is the standard. Multispectral dermoscopy is based on illumination of the skin with narrowband light sources with different wavelengths. Each of these wavelengths is differently absorbed by skin chromophores, such as pigment or (de)oxygenated blood. Multispectral dermoscopy could be a way to enhance the visualization of vasculature and pigment. We illustrate possible additional information by such "skin parameter maps" in some cases of basal cell carcinoma and Bowen's disease. Methods Using a new digital multispectral dermatoscope, skin images at multiple wavelengths are collected from different types of skin lesions. These particular images together with the knowledge on skin absorption properties, result in so called "skin parameter maps". Results A "pigment contrast map," which shows the relative concentration of primarily pigment, and a "blood contrast map" which shows the relative concentration of primarily blood were created. Especially, the latter is of importance in diagnosing keratinocyte skin cancer hence vascular structures are a characteristic feature, as further illustrated in the study. Conclusions Skin parameter maps based on multispectral images can give better insight in the inner structures of lesions, especially in lesions with characteristic blood vessels such as Bowen's disease and basal cell carcinoma. Skin parameter maps can be used complementary to regular dermoscopy and could potentially facilitate diagnosing skin lesions

Conditional Mutagenesis in Drosophila

Most genes function at multiple stages of metazoan development, in dividing and nondividing cells. Generating mouse conditional knock-outs (cKO), where a gene can be eliminated in a temporally and spatially controlled manner, is a valuable technique because it allows study of gene function at any stage of life. In contrast and despite the development of many other powerful genetic tools, cKO has thus far been lacking in Drosophila. We combined several recent molecular and genetic technical advances in an approach termed integrase-mediated approach for gene knock-out (IMAGO). IMAGO allows the replacement of any genomic sequence, such as a gene, with another desired sequence, including cKO alleles that can be used to create positively marked mutant cells. IMAGO should also be applicable to other genetic model organisms.status: publishe

Autophagy inhibitor chloroquine enhanced the cell death inducing effect of the flavonoid luteolin in metastatic squamous cell carcinoma cells

Flavonoids are widely proposed as very interesting compounds with possible chemopreventive and therapeutic capacities.In this study, we showed that in vitro treatment with the flavonoid Luteolin induced caspase-dependent cell death in a model of human cutaneous squamous cell carcinoma (SCC) derived cells, representing a matched pair of primary tumor and its metastasis. Notably, no cytotoxic effects were observed in normal human keratinocytes when treated with similar doses of Luteolin. Luteolin-induced apoptosis was accompanied by inhibition of AKT signaling, and sensitivity decreased with tumor progression, as the primary MET1 SCC cells were considerably more sensitive to Luteolin than the isogenic metastatic MET4 cells. Extensive intracellular vacuolization was observed in Luteolin-treated MET4 cells, which were characterized as acidic lysosomal vacuoles, suggesting the involvement of autophagy. Transmission electron microscopy, mRFP-GFP-LC3 assay and p62 protein degradation, confirmed that Luteolin stimulated the autophagic process in the metastatic MET4 cells. Blocking autophagy using chloroquine magnified Luteolin-induced apoptosis in the metastatic SCC cells.Together, these results suggest that Luteolin has the capacity to induce selectively apoptotic cell death both in primary cutaneous SCC cells and in metastatic SCC cells in combination with chloroquine, an inhibitor of autophagosomal degradation. Hence, Luteolin might be a promising agent for the treatment of cutaneous SCC

Inhibition of late phase autophagy increases LUT induced cell death in metastatic SCC.

<p><b>A.</b> Cells were pre-treated with CQ (0 or 50 µM) and after 1 hour LUT (0, 20 or 50 µM) was added for 24 hours. The amount of apoptotic DNA was determined by the Cell death detection ELISA. Enrichment factor relative to untreated controls (UNTR/-) was calculated. Experiment was performed twice in duplicate. <b>B.</b> Western blot of cell lysates of MET4 cells pre-treated for 1 hour with CQ (50 µM) and/or 3-MA (10 mM) before the addition of LUT (50 µM) for 24 hours. Densitometric analysis of PARP% (cleaved Parp/(total Parp + cleaved Parp) and of cleaved caspase 3 relative to actin level is shown. Representative blot of three independent experiments is shown. <b>C.</b> Trypan blue exclusion assay of MET4 cells treated as indicated and described in <b>B.</b> (Experiment performed twice, n = 4).</p

Expanded lysosomal compartment following LUT-treatment in metastatic SCC cells.

<p><b>A. </b><i>First row</i>: Bright field microscopic pictures of MET4 treated or not with LUT (50 μM) for 24 hours. <i>Second and third row</i>: Fluorescent microscopic images of AO stained MET4 cells treated with LUT (50 µM) or left untreated. Red  =  AVO; Green: non-acidic cell compartment (scale bar  = 20 µm). <b>B.</b> Representive flow cytometric quantification of AVO formation (increase in red fluorescence in MET1, MET4 and NHKs following treatment with indicated concentrations of LUT <b>C.</b> Confocal images of LAMP-1 and LAMP-2 stained <i>(green)</i> MET4 cells treated with LUT (50 µM) for 16 h. DAPI <i>(blue)</i> was used for nuclear staining.</p

Involvement of autophagy in the response of SCC cells on LUT.

<p><b>A.</b> Electron micrographs showing the ultrastructure of MET4-cells treated with LUT (20 µM) or not. Arrows indicate autophagosomes (scale bar  = 1 µm). <b>B. </b><i>(left panel)</i> Fluorescent microscopy analysis of MET4 cells transiently overexpressing mRFP-GFP-LC3, treated or not with LUT (10 or 50 µM) for 24 hours (scale bar  = 20 µm). (right panel) Quantification of red and green fluorescent punctae of at least 10 cells per condition is shown. <b>C.</b> Protein levels of p62 and LC3-II upon LUT treatment in MET4 cells. One representative blot of at least three independent experiments is shown. Values are the ratio's p62 and LC3-II to actin level of densitometric analysis. <b>D.</b> Cells were pre-treated (1 hour) with an autophagy inhibitor (INH), CQ (50 µM) or apoptosis inhibitor (INH), ZVAD-fmk (50 µM), before LUT treatment (50 µM). Lysates were made after 24 hours and analysed by western blot. A representative blot of at least three independent experiments is shown. Numbers are the ratios of the densitometric analysis for p62/actin and LC3II/actin.</p

LUT induced apoptosis involves modulation of AKT signaling.

<p><b>A.</b> Time dependent effect of LUT on AKT phosphorylation (AKT-P; Ser473) and on apoptosis markers (Parp-cleavage and caspase 3 activation) was studied using western blot in MET1 and MET4 cells <b>B.</b> Protein and phosphorylation levels of AKT and its downstream targets in MET1 and MET4 cells treated with LUT (20 and 50 µM) for 24 hours and 48 h obtained using western blot analysis. Numbers, obtained by densitometric analysis of the western blot, indicate the ratio of phosphorylated to total AKT and p70S6 protein. <b>C.</b> MET1 and MET4 cells were pre-treated or not with AI (10 µM) for 1 hour and subsequently treated with LUT as indicated. Densitometric analysis of cleaved caspase 3 relative to actin level is shown. A representative blot of at least 3 independent experiments is shown. <b>D.</b> Cells were treated with AI and LUT for 24 hours as indicated and the amount of apoptotic DNA was determined by the Cell death detection ELISA as described in Materials and Methods. Experiment was performed twice in duplicate <b>E.</b> MET1 and MET4 cells transiently transfected with a construct expressing Myr-AKT-HA, were treated or not with LUT (50 µM) for 24 hours. Fugene HD  = transfectant only; Myr-AKT =  constitutively active AKT). Densitometric analysis of cleaved caspase 3 relative to actin level is shown. A representative blot of at least 3 independent experiments is shown.</p

LUT induces apoptotic cell death specifically in malignant keratinocytes.

<p><b>A.</b> Detection of metabolic activity by MTT assay, 24 hours after treatment with LUT (10–100 µM) or DMSO (equal amount as highest LUT concentration) in different SCC cell lines. (Representive experiment out of 3, n = 16) <b>B.</b> Flow cytometric analysis of propidium iodide (PI) stained cells, 24 hours after treatment with LUT (50 µM). PI-positive cells = dead cells. (Representive experiment is shown, n = 3) <b>C.</b> Detection of caspase signaling by western blot analysis 24 hours after different concentrations of LUT (10–100 µM). UVB (120 mJ/cm²) was used as a positive control for cell death in NHK. <b>D.</b> Luminescent detection of caspase 8 and caspase 9 activity 24 hours after treatment with 50 µM LUT. Fold increase values against untreated control are shown. (Experiment performed three times in duplicate) <b>E.</b> Measurement of viability by trypan blue exclusion assay, 24 hours after treatment with LUT (50 µM) and zVAD-fmk (50 µM).</p