Validation of growth condition-associated subcellular localization of ADRBK1.

<p>ADRBK1-YFP fusion constructs (green signals) were transfected into cultured Panc-1 cells cultured in the absence (A, C) or presence (B, D) of 10% serum. After 24h or 48h of incubation, cells were fixed and counterstained with DAPI (blue signals). After 24h, ADRBK1-YFP signals were detectable in both, cytoplasm and nuclei, of cells cultured without serum (A), while nuclei of cells cultured with serum were devoid of YFP fluorescence (B). In contrast, after 48h of incubation, cells grown under either condition showed prominent staining of nuclei (C, D). Optical sections of fluorescent images were obtained using the ApoTome technology. Original magnifications are indicated in the images.</p

A Multistep High-Content Screening Approach to Identify Novel Functionally Relevant Target Genes in Pancreatic Cancer

<div><p>In order to foster the systematic identification of novel genes with important functional roles in pancreatic cancer, we have devised a multi-stage screening strategy to provide a rational basis for the selection of highly relevant novel candidate genes based on the results of functional high-content analyses. The workflow comprised three consecutive stages: 1) serial gene expression profiling analyses of primary human pancreatic tissues as well as a number of <i>in vivo</i> and <i>in vitro</i> models of tumor-relevant characteristics in order to identify genes with conspicuous expression patterns; 2) use of ‘reverse transfection array’ technology for large-scale parallelized functional analyses of potential candidate genes in cell-based assays; and 3) selection of individual candidate genes for further in-depth examination of their cellular roles. A total of 14 genes, among them 8 from “druggable” gene families, were classified as high priority candidates for individual functional characterization. As an example to demonstrate the validity of the approach, comprehensive functional data on candidate gene ADRBK1/GRK2, which has previously not been implicated in pancreatic cancer, is presented.</p></div

Schematic representation of the workflow in the different stages of the screening and functional characterization process.

<p>Schematic representation of the workflow in the different stages of the screening and functional characterization process.</p

ADRBK1 overexpression promotes growth in human PDAC.

<p>A: Box-and-whisker plot showing ADRBK1 mRNA expression in primary human pancreatic tumor tissue samples, chronic pancreatitis and normal pancreas as analyzed by quantitative realtime reverse transcription PCR (qRT-PCR). Expression was normalized to ribosomal protein, large, P0 (RPLP0) mRNA levels. Data in Fig. represent median and 2nd and 3rd quartiles (boxes) as well as minimum and maximum values (whiskers). CP = Chronic Pancreatitis. ** p<0.01, *** p<0.001 (Student’s t-test). B: Immunohistological analyses of ADRBK1 expression using human pancreatic tissue microarrays (TMAs) comprising 33 donor, 28 chronic pancreatitis and 65 tumor tissues. Micrographs are representative of typical staining results for donor samples (left panel), which were devoid of ADRBK1 staining, or ADRBK1-positive neoplastic tissues (right panel) (original magnification 100x). Note that in positive PDAC cases, neoplastic ducts as well as individual infiltrating immune cells (arrows) stained postitve for ADRBK1. Lower panel: Distribution of staining intensities across different tumor grades (G1: n = 23; G2: n = 19; G3: n = 23). C: TTK mRNA expression levels in a variety of different pancreatic cancer and control cell lines (qRT-PCR; expression was normalized to ribosomal protein, large, P0 (RPLP0) mRNA levels). D, E: MTT assays demonstrated increased numbers of viable S2-007 (C) and Panc-1 (D) cells 72h after transfection of fluorescence-tagged ADRBK1 expression constructs (CFP: N-terminal CFP-ADRBK1 fusion; YFP: C-terminal ADRBK1-YFP fusion) compared to GFP controls in the presence (dark bars) or absence (light bars) of 10% serum in the culture medium.</p

Examples of distinct subcellular localization of fluorescence-tagged candidate gene peoducts after reverse transfection.

<p>Shown are CFP (A) or YFP (B-C) fusion constructs. Following transfection and fixation of cells on reverse transfection microarrays, coverslips were applied using DAPI-containing mounting medium. For CFP fusion proteins, DAPI signals are shown in brown (false color) to provide suitable contrast (A). Shown are examples of purely cytoplasmic (A), uniform nuclear-cytoplasmic (B) and purely nuclear localization (C) as well as localization to endosome-like structures (D). Original magnifications are indicated in the images.</p

Examples of the different antibody stainings for functional analyses.

<p>A: Caspase-3 staining (Cy-3, red), HEK-293 cells transfected with H1F0-YFP (green); B: Ki-67 staining (Cy-3, red), HEK-293 transfected with NKX2-5-YFP (green); C: Cyclin B staining (Cy-3, red), PANC-1 transfected with PRKCZ-YFP (green); D: Vimentin staining (Cy-3, red), PANC-1 transfected with FASTK-YFP (green); E: E-Cadherin staining (Cy-3, red), PANC-1 transfected with PPP2R1A-YFP (green). Original magnifications are indicated in the images.</p

Inhibition of endogeneous ADRBK1 expression impairs cell growth.

<p>A: Transfection of three individual siRNAs against ADRBK1 resulted in at least 70% reduction of ADRBK1 mRNA levels in two pancreatic cancer cell lines with high (S2-28) and intermediate (PaTu-8988t) levels of endogeneous ADRBK1 expression, as well as the non-transformed HEK293 cell line. mRNA levels were determined by qRT-PCR and normalized to non-silencing control siRNA (“siControl”). B: MTT assays showed significantly reduced numbers of viable cells 72h after transfection of ADRBK1-specific siRNAs as compared to non-silencing control siRNA. C: PI-staining and flow cytometry analyses were performed 48h after siRNA transfection into PaTu 8988t cells. The results demonstrate strongly increased proportions of cells in G1 phase and strongly decreased proportions of cells in S phase after ADRBK1 knockdown, while G2 phase remained essentially unchanged. Shown is one representative example of three independent experiments.E, F: PARP cleavage was analyzed by Western Blot analyses 72h after transfection of ADRBK1-specific and control siRNAs, respectively. The results showed slightly elevated levels of cleaved PARP protein in S2-028 cells (E, right panel), but this was not apparent in PaRu-8988t (E, left panel) or HEK293 (F) cells. * p<0.05, ** p<0.01 (Student’s t-test).</p