Moderate overexpression of <i>RAS</i> and <i>ERBB2</i> by retroviral transfections of prostate cancer cells.

<p>ERBB2 and RAS protein levels were assessed by Western blots using antibodies against H-RAS or ERBB2 for total cell lysates prepared from prostate cancer cells transfected with either control retroviruses (<i>PBP</i>), or retroviruses overexpressing <i>PBP</i>-<i>H-RAS</i> (<i>RAS</i>) or <i>PBP-ERBB2</i> (<i>ERBB2</i>). Blots with antibodies against actin served as loading controls. Numbers in white represent fold changes in ERBB2 or RAS protein levels in <i>ERBB2</i>- or <i>RAS</i>-overexpressing cells relative to those in their corresponding <i>PBP</i> control cells after the actin normalization.</p

Moderate levels of <i>RAS</i> overexpression did not promote cellular senescence in prostate cancer cells.

<p>(A) Senescence-associated β-galactosidase activities were assessed by X-gal staining for human prostate cancer cell lines transfected with either control retroviruses (<i>PBP</i>) or retroviruses overexpressing <i>PBP-H-RAS</i> (<i>RAS</i>). PC3 cells expressing an extremely high level of <i>RAS</i> (<i>Hi-RAS</i>) were included for comparisons. Senescent BJ human skin fibroblast cells were used as a positive control for X-gal staining. Representative images were shown with representative X-gal-positive cells being marked with red arrows. Inserts in each image showed magnified, representative X-gal-positive cells. (<b>B</b>) Quantifications of data collected from panel (<b>A</b>). Data were presented as means ± SD from three replicates. (<b>C</b>) RAS protein levels assessed by Western blot analysis with antibodies against H-RAS for parental PC3 cells (P), PC3 cells transfected with control retroviruses (<i>PBP</i>), or PC3 cells transfected with the <i>PBP-H-RAS</i> retroviruses overexpressing a moderate level of <i>RAS</i> (<i>RAS</i>) or a much higher level of <i>RAS</i> (<i>Hi-RAS</i>). A blot using antibodies against actin was used as a loading control. Numbers in white represent RAS protein levels in fold changes in <i>ERBB2</i>- or <i>RAS</i>-overexpressing cells relative to those in <i>PBP</i> control cells after the actin normalization. Scare bar: 100 µM.</p

Overexpression of <i>RAS</i> or <i>ERBB2</i> did not increase lateral cell migration rates.

<p>Cell migration rates were estimated by a wound healing assay for prostate cancer cells that were transfected with either control retroviruses (<i>PBP</i>), or retroviruses overexpressing <i>PBP</i>-<i>H-RAS</i> (<i>RAS</i>) or <i>PBP-ERBB2</i> (<i>ERBB2</i>). Left panels showed percentages of wounds remained at different time points. The percentages of wounds were estimated based on the average of 12 measurements on each plate reflecting measurements of four evenly distributed sections on each of the three wounds/scratches on each plate. Data were presented as means ± SD from three replicates. Right panels showed representative images taken at different time points. All images were taken at the same scale with a scale bar of 200 µM displayed in the first image.</p

Overexpression of <i>RAS</i>/<i>ERBB2</i> activates the MAPK pathway and/or the PI3K–AKT pathway in prostate cancer cells.

<p>Protein levels for various kinases and their phosphorylated forms were assessed by Western blot analyses for parental prostate cancer cells that were transfected either with control retroviruses (<i>PBP</i>), or with retroviruses overexpressing <i>PBP-H-RAS</i> (<i>RAS</i>) or <i>PBP-ERBB2</i> (<i>ERBB2</i>). Actin was used as a loading control. Numbers in white represent protein levels in fold changes relative to those in <i>PBP</i> control cells after the actin normalization.</p

Summary of the principal characteristics of the prostate cancer cell lines included in the study.

<p>Summary of the principal characteristics of the prostate cancer cell lines included in the study.</p

<i>ERBB2</i> overexpression increased the invasiveness of DU145 cells and PC3 cells.

<p>Cell invasiveness was assessed by a transwell-based invasion assay for prostate cancer cells that were transfected with either control retroviruses (<i>PBP</i>), or retroviruses overexpressing <i>PBP</i>-<i>H-RAS</i> (<i>RAS</i>) or <i>PBP-ERBB2</i> (<i>ERBB2</i>). Each bar graph showed the numbers of cells that have passed through the collagen matrix either 72 hours (for PC3 cells) or 96 hours (for DU145 cells) after plating. Transwell inserts were stained and invading cells were counted for the entire inserts. Data were presented as means ± SD from three replicates. Representative images were shown underneath each bar graph. All images were taken at the same scale with a scale bar of 200 µM displayed in the first image.</p