Limits...
Deregulation of the CEACAM expression pattern causes undifferentiated cell growth in human lung adenocarcinoma cells.

Singer BB, Scheffrahn I, Kammerer R, Suttorp N, Ergun S, Slevogt H - PLoS ONE (2010)

Bottom Line: Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable.Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth.Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, University Hospital Essen, Essen, Germany. BBSinger@gmx.de

ABSTRACT
CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

Show MeSH

Related in: MedlinePlus

CEACAM1-L but not CEACAM1-S secures cell contact-inhibition.A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in Materials and Methods using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in Materials and Methods. A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2807459&req=5

pone-0008747-g005: CEACAM1-L but not CEACAM1-S secures cell contact-inhibition.A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in Materials and Methods using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in Materials and Methods. A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.

Mentions: To analyze the functional impact of the different CEACAMs, we altered their expression patterns by stable transfection of parental A549 cells with CEACAM1-4L, CEACAM1-4S, the ITIM-mutant form of CEACAM1 [CEACAM1-4L(Y459F/Y486F)], CEACAM5 and CEACAM6, respectively. Since proliferating A549 cells did not express CEACAMs, the transfection success could be monitored by flow cytometry utilizing exponentially growing A549 transfectants. Empty vector transfected A549 cells served as a control and showed no expression of CEACAMs in the proliferative stage (Figure 5A, a). In contrast, A549-CEACAM1-4L (Figure 5A, b), A549-CEACAM1-4L(Y459F/Y486F) (Figure 5A, c) and A549-CEACAM5 (Figure 5A, d) cell lines revealed a strong exogenous CEACAM expression, respectively. The capability to produce A549-CEACAM5 cell lines demonstrated that in principle A549 cells are able to express GPI-anchored CEACAMs on their cell surface. Unexpectedly, we were not able to establish stable transfectants of A549-CEACAM1-4S and A549-CEACAM6, although the individual vectors transfected into HeLa cells resulted in CEACAM1-4S and CEACAM6 expression, respectively, proving the efficacy of these vectors (data not shown). Repeated transfections of A549 cells with CEACAM1-4S and CEACAM6 were carried out resulting in A549 cells, which were no longer able to exhibit normal proliferation. Analyzes by phase-contrast microscopy revealed that A549-CEACAM1-4S and A549-CEACAM6 cells, which initially survived the G418-selection procedure, detached from the culture vessel and subsequently died (data not shown). However, in contrast to A549-CEACAM6, several A549-CEACAM1-4S cells remained attached on the cell culture vessel and partially formed giant cells containing several nuclei (Figure 5B, e). This finding implied a malfunction of the regular cell division processes caused by over-expression of CEACAM1-4S. Compared with un-transfected and control-vector transfected A549 cells, A549-CEACAM1-4L exhibited a marked increase of the homogeneous, cobblestone-like epithelial cell morphology of a well spread monolayer, suggesting a potentiation of contact inhibition (Figure 5B, b). Importantly, none of the CEACAM1-4L over-expressing cell lines piled up from the confluent monolayer, and no spheroidal cell aggregates were formed. To determine, whether an intact cytoplasmic tail of CEACAM1-4L is essential for mediating the improved contact inhibition found in A549-CEACAM1-4L, un-separated A549 cells were stably transfected with an expression plasmid coding for a cytoplasmic ITIM mutant form of CEACAM1-4L[3], [30]. We found, that unlike CEACAM1-4L over-expressing cells, A549 cells carrying the mutant plasmid CEACAM1-4L (Y459F/Y486F) failed to grow as a homogenous monolayer. These cells barely established cell-matrix and cell-cell-contacts and appeared mainly as rounded cells. They grew as scattered, slightly attached cell-aggregates without forming solid monolayers (Figure 5B, c). Additionally, a number of A549-CEACAM1-4L (Y459F/Y486F) cells grew in suspension (data not shown), supporting an essential role of CEACAM1-4L in the mediation of contact inhibition of cell proliferation.


Deregulation of the CEACAM expression pattern causes undifferentiated cell growth in human lung adenocarcinoma cells.

Singer BB, Scheffrahn I, Kammerer R, Suttorp N, Ergun S, Slevogt H - PLoS ONE (2010)

CEACAM1-L but not CEACAM1-S secures cell contact-inhibition.A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in Materials and Methods using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in Materials and Methods. A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2807459&req=5

pone-0008747-g005: CEACAM1-L but not CEACAM1-S secures cell contact-inhibition.A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in Materials and Methods using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in Materials and Methods. A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.
Mentions: To analyze the functional impact of the different CEACAMs, we altered their expression patterns by stable transfection of parental A549 cells with CEACAM1-4L, CEACAM1-4S, the ITIM-mutant form of CEACAM1 [CEACAM1-4L(Y459F/Y486F)], CEACAM5 and CEACAM6, respectively. Since proliferating A549 cells did not express CEACAMs, the transfection success could be monitored by flow cytometry utilizing exponentially growing A549 transfectants. Empty vector transfected A549 cells served as a control and showed no expression of CEACAMs in the proliferative stage (Figure 5A, a). In contrast, A549-CEACAM1-4L (Figure 5A, b), A549-CEACAM1-4L(Y459F/Y486F) (Figure 5A, c) and A549-CEACAM5 (Figure 5A, d) cell lines revealed a strong exogenous CEACAM expression, respectively. The capability to produce A549-CEACAM5 cell lines demonstrated that in principle A549 cells are able to express GPI-anchored CEACAMs on their cell surface. Unexpectedly, we were not able to establish stable transfectants of A549-CEACAM1-4S and A549-CEACAM6, although the individual vectors transfected into HeLa cells resulted in CEACAM1-4S and CEACAM6 expression, respectively, proving the efficacy of these vectors (data not shown). Repeated transfections of A549 cells with CEACAM1-4S and CEACAM6 were carried out resulting in A549 cells, which were no longer able to exhibit normal proliferation. Analyzes by phase-contrast microscopy revealed that A549-CEACAM1-4S and A549-CEACAM6 cells, which initially survived the G418-selection procedure, detached from the culture vessel and subsequently died (data not shown). However, in contrast to A549-CEACAM6, several A549-CEACAM1-4S cells remained attached on the cell culture vessel and partially formed giant cells containing several nuclei (Figure 5B, e). This finding implied a malfunction of the regular cell division processes caused by over-expression of CEACAM1-4S. Compared with un-transfected and control-vector transfected A549 cells, A549-CEACAM1-4L exhibited a marked increase of the homogeneous, cobblestone-like epithelial cell morphology of a well spread monolayer, suggesting a potentiation of contact inhibition (Figure 5B, b). Importantly, none of the CEACAM1-4L over-expressing cell lines piled up from the confluent monolayer, and no spheroidal cell aggregates were formed. To determine, whether an intact cytoplasmic tail of CEACAM1-4L is essential for mediating the improved contact inhibition found in A549-CEACAM1-4L, un-separated A549 cells were stably transfected with an expression plasmid coding for a cytoplasmic ITIM mutant form of CEACAM1-4L[3], [30]. We found, that unlike CEACAM1-4L over-expressing cells, A549 cells carrying the mutant plasmid CEACAM1-4L (Y459F/Y486F) failed to grow as a homogenous monolayer. These cells barely established cell-matrix and cell-cell-contacts and appeared mainly as rounded cells. They grew as scattered, slightly attached cell-aggregates without forming solid monolayers (Figure 5B, c). Additionally, a number of A549-CEACAM1-4L (Y459F/Y486F) cells grew in suspension (data not shown), supporting an essential role of CEACAM1-4L in the mediation of contact inhibition of cell proliferation.

Bottom Line: Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable.Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth.Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

View Article: PubMed Central - PubMed

Affiliation: Institute of Anatomy, University Hospital Essen, Essen, Germany. BBSinger@gmx.de

ABSTRACT
CEACAM1, CEA/CEACAM5, and CEACAM6 are cell adhesion molecules (CAMs) of the carcinoembryonic antigen (CEA) family that have been shown to be deregulated in lung cancer and in up to 50% of all human cancers. However, little is known about the functional impact of these molecules on undifferentiated cell growth and tumor progression. Here we demonstrate that cell surface expression of CEACAM1 on confluent A549 human lung adenocarcinoma cells plays a critical role in differentiated, contact-inhibited cell growth. Interestingly, CEACAM1-L, but not CEACAM1-S, negatively regulates proliferation via its ITIM domain, while in proliferating cells no CEACAM expression is detectable. Furthermore, we show for the first time that CEACAM6 acts as an inducer of cellular proliferation in A549 cells, likely by interfering with the contact-inhibiting signal triggered by CEACAM1-4L, leading to undifferentiated anchorage-independent cell growth. We also found that A549 cells expressed significant amounts of non-membrane anchored variants of CEACAM5 and CEACAM6, representing a putative source for the increased CEACAM5/6 serum levels frequently found in lung cancer patients. Taken together, our data suggest that post-confluent contact inhibition is established and maintained by CEACAM1-4L, but disturbances of CEACAM1 signalling by CEACAM1-4S and other CEACAMs lead to undifferentiated cell growth and malignant transformation.

Show MeSH
Related in: MedlinePlus