The Transmembrane Domain of CEACAM1-4S Is a Determinant of Anchorage Independent Growth and Tumorigenicity

CEACAM1 is a multifunctional Ig-like cell adhesion molecule expressed by epithelial cells in many organs. CEACAM1-4L and CEACAM1-4S, two isoforms produced by differential splicing, are predominant in rat liver. Previous work has shown that downregulation of both isoforms occurs in rat hepatocellular carcinomas. Here, we have isolated an anchorage dependent clone, designated 253T-NT that does not express detectable levels of CEACAM1. Stable transfection of 253-NT cells with a wild type CEACAM1-4S expression vector induced an anchorage independent growth in vitro and a tumorigenic phenotype in vivo. These phenotypes were used as quantifiable end points to examine the functionality of the CEACAM1-4S transmembrane domain. Examination of the CEACAM1 transmembrane domain showed N-terminal GXXXG dimerization sequences and C-terminal tyrosine residues shown in related studies to stabilize transmembrane domain helix-helix interactions. To examine the effects of transmembrane domain mutations, 253-NT cells were transfected with transmembrane domain mutants carrying glycine to leucine or tyrosine to valine substitutions. Results showed that mutation of transmembrane tyrosine residues greatly enhanced growth in vitro and in vivo. Mutation of transmembrane dimerization motifs, in contrast, significantly reduced anchorage independent growth and tumorigenicity. 253-NT cells expressing CEACAM1-4S with both glycine to leucine and tyrosine to valine mutations displayed the growth-enhanced phenotype of tyrosine mutants. The dramatic effect of transmembrane domain mutations constitutes strong evidence that the transmembrane domain is an important determinant of CEACAM1-4S functionality and most likely by other proteins with transmembrane domains containing dimerization sequences and/or C-terminal tyrosine residues

Regulation of RKIP Function by Helicobacter pylori in Gastric Cancer

Helicobacter pylori (H. pylori) is a gram-negative, spiral-shaped bacterium that infects more than half of the world’s population and is a major cause of gastric adenocarcinoma. The mechanisms that link H. pylori infection to gastric carcinogenesis are not well understood. In the present study, we report that the Raf-kinase inhibitor protein (RKIP) has a role in the induction of apoptosis by H. pylori in gastric epithelial cells. Western blot and luciferase transcription reporter assays demonstrate that the pathogenicity island of H. pylori rapidly phosphorylates RKIP, which then localizes to the nucleus where it activates its own transcription and induces apoptosis. Forced overexpression of RKIP enhances apoptosis in H. pylori-infected cells, whereas RKIP RNA inhibition suppresses the induction of apoptosis by H. pylori infection. While inducing the phosphorylation of RKIP, H. pylori simultaneously targets non-phosphorylated RKIP for proteasome-mediated degradation. The increase in RKIP transcription and phosphorylation is abrogated by mutating RKIP serine 153 to valine, demonstrating that regulation of RKIP activity by H. pylori is dependent upon RKIP’s S153 residue. In addition, H. pylori infection increases the expression of Snail, a transcriptional repressor of RKIP. Our results suggest that H. pylori utilizes a tumor suppressor protein, RKIP, to promote apoptosis in gastric cancer cells

Possible Role of Extracellular Vesicles in Hepatotoxicity of Acetaminophen

We examined proteomic profiles of rat liver extracellular vesicles (EVs) shed following treatment with a sub-toxic dose (500 mg/kg) of the pain reliever drug, acetaminophen (APAP). EVs representing the entire complement of hepatic cells were isolated after perfusion of the intact liver and analyzed with LC-MS/MS. The investigation was focused on revealing the function and cellular origin of identified EVs proteins shed by different parenchymal and non-parenchymal liver cells and their possible role in an early response of this organ to a toxic environment. Comparison of EV proteomic profiles from control and APAP-treated animals revealed significant differences. Alpha-1-macroglobulin and members of the cytochrome P450 superfamily were highly abundant proteins in EVs shed by the normal liver. In contrast, proteins like aminopeptidase N, metalloreductase STEAP4, different surface antigens like CD14 and CD45, and most members of the annexin family were detected only in EVs that were shed by livers of APAP-treated animals. In EVs from treated livers, there was almost a complete disappearance of members of the cytochrome P450 superfamily and a major decrease in other enzymes involved in the detoxification of xenobiotics. Additionally, there were proteins that predominated in non-parenchymal liver cells and in the extracellular matrix, like fibronectin, receptor-type tyrosine-protein phosphatase C, and endothelial type gp91. These differences indicate that even treatment with a sub-toxic concentration of APAP initiates dramatic perturbation in the function of this vital organ

Immunoblot Analysis Shows that Wild Type and Mutant CEACAM1-4S Constructs Have Identical Molecular Mass.

<p>Protein lysates prepared from wild type cells and each of the 253T-NT transfected cell lines were resolved on 7.5% SDS-polyacylamide gels, transferred onto nitrocellulose and labeled with MAb 9.2 specific to CEACAM1. Analysis of expressed wild type and mutant CEACAM1-4S protein shows that reactive band corresponding to CEACAM1-4S had the same apparent molecular mass (105 kDa).</p

Mobility of G to L Mutants of CEACAM1-4S Resolved by BN-PAGE.

<p>Protein lysates were prepared and resolved by BN-PAGE as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029606#s2" target="_blank">materials and methods</a>. Proteins were transferred onto PVDF membranes and probed with monoclonal antibody 9.2. When separated on native gels, wild type CEACAM1-4S and the single G mutants migrated with an apparent molecular mass that was approximately 100 kDa higher than the double glycine mutant.</p

CEACAM1-4s Transmembrane Domain.

<p>CEACAM1-4s Transmembrane Domain.</p

Cell Surface Expression of CEACAM1-4S.

<p>253T-NT cultures stably transfected with wild type or mutated forms of CEACAM1-4S were labeled by indirect immunofluorescence with MAb 9.2, a monoclonal antibody specific for CEACAM1. Cell nuclei were stained with propidium iodide. Confocal digital images constructed from 7–15 optical sections for each labeled subline are shown in panels A–L. (<b>A</b>) wild type CEACAM1-4S; (<b>B</b>) G424L mutant; (<b>C</b>) G432L mutant; (<b>D</b>) G424L and G432L double mutant; (<b>E</b>) Empty vector; (<b>F</b>) Y445V mutant; (<b>G</b>) Y448V mutant; (<b>H</b>) Y445V and Y448V double mutant; (<b>I</b>) G424L, G432L, Y445V, Y448V quadruple mutant; (<b>J</b>) untransfected 253T-NT cells; (<b>K</b>) G424L, G432L and Y445V triple mutant; (<b>L</b>) G424L, G432L and Y448V triple mutant. Scale bar represents 20 mm.</p

Tumorigenicity of 253T-NT Transfected with WT and Mutated CEACAM1-4S.

<p>Three nude mice were injected in the front flanks with each subline of 253T-NT (6 injection sites for each cell line). At three weeks after injection, tumor nodules were harvested and weighed. Columns marked with an asterisk indicate cell lines that according to P values (p<0.05) were significantly larger (Y448V and the quadruple mutant) or smaller (G424L) than 253T-NT cells expressing wild type CEACAM1-4S. Although the effects of mutations were not as clear-cut as those observed for growth in soft agar, the G424L mutation compromised and the Y448V or the quadruple mutation enhanced the ability of CEACAM1-4S to produce tumors in nude mice.</p

Rates of Cell Proliferation of CEACAM1-4S Transfectants.

<p>The bars show the fold-increase in cells at 96 hours after plating. The columns marked with an asterisk were sublines showing an increase in cell number between 0 and 96 hours that according to P values (p<0.05), was significantly higher or lower than 253T-NT cells transfected with wild type (WT) CEACAM1-4S. In general, the number of 253T-NT cells expressing G to L mutants increased at a slower rate and Y to V mutants at a higher rate than cells expressing wild type CEACAM1-4S. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029606#s3" target="_blank">Results</a> shown represent four separate assays.</p

Size Distribution of Soft Agar Colonies.

<p>Image ProPlus software was used to calculate the average area of soft agar colonies. The results represent three separate soft agar growth experiments. The columns marked with an asterisk indicate sublines that were either significantly smaller or larger than those formed by cells transfected with wild type (WT) CEACAM1-4S (p<0.05).</p