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. 2012;7(1):e29606.
doi: 10.1371/journal.pone.0029606. Epub 2012 Jan 3.

The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity

Erica L Lawson  1 , David R Mills, Kate E Brilliant, Douglas C Hixson
Affiliations

The transmembrane domain of CEACAM1-4S is a determinant of anchorage independent growth and tumorigenicity

Erica L Lawson et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Anchorage Independent Growth Induced by Expression of CEACAM1-4S.
(A) A representative anchorage independent colony formed by parental 253T cells after 3 weeks in soft agar. (B) Sparsely scattered single cells that typified soft agar cultures of 253T-NT cells. (C) Scattered cells remaining after 3 weeks in soft agar cultures of 253T-NT cells stably transfected with empty vector. (D) A representative colony formed by253T-NT cells stably transfected with a wild type CEACAM1-4S expression vector.
Figure 2
Figure 2. Cell Surface Expression of CEACAM1-4S.
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. (A) wild type CEACAM1-4S; (B) G424L mutant; (C) G432L mutant; (D) G424L and G432L double mutant; (E) Empty vector; (F) Y445V mutant; (G) Y448V mutant; (H) Y445V and Y448V double mutant; (I) G424L, G432L, Y445V, Y448V quadruple mutant; (J) untransfected 253T-NT cells; (K) G424L, G432L and Y445V triple mutant; (L) G424L, G432L and Y448V triple mutant. Scale bar represents 20 mm.
Figure 3
Figure 3. Immunoblot Analysis Shows that Wild Type and Mutant CEACAM1-4S Constructs Have Identical Molecular Mass.
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).
Figure 4
Figure 4. FACS Analysis of CEACAM1-4S Expression in Transfected and Untransfected 253T-NT Sublines.
FACS analysis was performed to determine the percentage of CEACAM1-4S positive cells in the various sublines. When 253T-NT cells were labeled with a human HLA specific monoclonal antibody (negative control), only 5% of the cells showed fluorescence above background levels (A). When stained with MAb 188-A2 specific for transferrin receptor (positive control), 95% of the cells showed positive reactivity (B). (C–M) the percentage of positive cells is indicated in the upper left corner of each histogram. Sublines analyzed in each panel were transfected with: (C) wild type CEACAM1-4S; (D) empty vector; (E) G424L; (F) G432L; (G) G424L and G432L; (H) Y445V; (I) Y448V; (J) Y445V and Y448V; (K) G424L, G432L and Y445V; (L) G424L, G432L and Y448V; (M) G424L, G432L, Y445V and Y448V.
Figure 5
Figure 5. Anchorage Independent Growth in Soft Agar.
(A–I) show the appearance of colonies formed by the various CEACAM1-4S sublines after 3 weeks in soft agar: (A) G424L; (B) G432L; (C) G424L and G432L; (D) Y445V; (E) Y448V; (F) Y445V and Y448V; (G) G424L, G432L and Y445V; (H) G424L, G432L and Y448V; (I) G424L, G432L, Y445V and Y448V.
Figure 6
Figure 6. Size Distribution of Soft Agar Colonies.
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).
Figure 7
Figure 7. Rates of Cell Proliferation of CEACAM1-4S Transfectants.
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. Results shown represent four separate assays.
Figure 8
Figure 8. Tumorigenicity of 253T-NT Transfected with WT and Mutated CEACAM1-4S.
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.
Figure 9
Figure 9. Expression of WT and Mutant CEACAM1-4S on Tumor Nodules.
Frozen sections of normal liver or 253T-NT transfectants were stained by indirect immunofluorescence with MAbs 9.2 and 5.4, monoclonal antibodies recognizing the N-terminal Ig domain of CEACAM1-4S. (A) Normal rat liver with MAb 5.4; (B) 253T-NT expressing wild type CEACAM1-4S; (C–F) tumors of 253-NT cells expressing the following mutants: (C) G424L with MAb 9.2; (D) G424L and G432L with MAb 5.4; (E) Y448V with MAb 5.4; (F) Y445V and Y448V with MAb 9.2.
Figure 10
Figure 10. Mobility of G to L Mutants of CEACAM1-4S Resolved by BN-PAGE.
Protein lysates were prepared and resolved by BN-PAGE as described under materials and methods. 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.
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