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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.

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Characterization of the cell surface expression of different CEACAMs in A549-NT and A549-T cells.A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the Materials and Methods section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.
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pone-0008747-g002: Characterization of the cell surface expression of different CEACAMs in A549-NT and A549-T cells.A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the Materials and Methods section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.

Mentions: These observations concerning different growth properties of the A549 subpopulations led us to assume that different expression patterns of CEACAMs might be important in the loss of contact inhibition in these cells. Subsequently, the cell surface expression of CEACAM1, CEACAM5, CEACAM6, and CEACAM7 was measured on trypsin-dissociated A549-NT and A549-T cells harvested from non-confluent, confluent monolayers, or - in the case of A549-T cells, – cells growing as spheroids, by flow cytometry. As shown in Figure 2, neither proliferating cells of subpopulations cultivated in the log phase, nor A549-T cells growing as spheroids, expressed CEACAM1, CEACAM5, CEACAM6, or CEACAM7 on their cell surface. In contrast, in the tightly confluent monolayer of both the A549-T and A549-NT cell subpopulations, we found significant cell surface expression of CEACAM1, but not CEACAM5, in all A549 cells (Figure 2A). However, while cells of the A549-NT subpopulation also did not express cell surface bound CEACAM6, around 15% of the A549-T cells expressed significant levels of CEACAM6 on their cell surface (15%±6%, n = 7; Figure 2, lower panel). Western blot analyses confirmed that non-confluent A549 cells did not express CEACAM1, CEACAM5, or CEACAM6 (Figure 2B). Confluent A549 cells showed significant levels of CEACAM1, but in addition revealed a significant expression of CEACAM5 and CEACAM6 as well (Figure 2B). Thus, CEACAM5 and CEACAM6 proteins were expressed in confluent A549 cells, but apparently they did not appear at the cell surface except for some CEACAM6 in a minor fraction of cells (Figure 2A). To elucidate the discrepancy in our findings, we analyzed the cell culture supernatant of confluent A549 cells. We found that both CEACAM5, CEACAM6, and, to a minor extent, CEACAM1, were released into the cell culture supernatant of confluent A549-NT (Figure 2C) and A549-T cells (data not shown).


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)

Characterization of the cell surface expression of different CEACAMs in A549-NT and A549-T cells.A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the Materials and Methods section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2807459&req=5

pone-0008747-g002: Characterization of the cell surface expression of different CEACAMs in A549-NT and A549-T cells.A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the Materials and Methods section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.
Mentions: These observations concerning different growth properties of the A549 subpopulations led us to assume that different expression patterns of CEACAMs might be important in the loss of contact inhibition in these cells. Subsequently, the cell surface expression of CEACAM1, CEACAM5, CEACAM6, and CEACAM7 was measured on trypsin-dissociated A549-NT and A549-T cells harvested from non-confluent, confluent monolayers, or - in the case of A549-T cells, – cells growing as spheroids, by flow cytometry. As shown in Figure 2, neither proliferating cells of subpopulations cultivated in the log phase, nor A549-T cells growing as spheroids, expressed CEACAM1, CEACAM5, CEACAM6, or CEACAM7 on their cell surface. In contrast, in the tightly confluent monolayer of both the A549-T and A549-NT cell subpopulations, we found significant cell surface expression of CEACAM1, but not CEACAM5, in all A549 cells (Figure 2A). However, while cells of the A549-NT subpopulation also did not express cell surface bound CEACAM6, around 15% of the A549-T cells expressed significant levels of CEACAM6 on their cell surface (15%±6%, n = 7; Figure 2, lower panel). Western blot analyses confirmed that non-confluent A549 cells did not express CEACAM1, CEACAM5, or CEACAM6 (Figure 2B). Confluent A549 cells showed significant levels of CEACAM1, but in addition revealed a significant expression of CEACAM5 and CEACAM6 as well (Figure 2B). Thus, CEACAM5 and CEACAM6 proteins were expressed in confluent A549 cells, but apparently they did not appear at the cell surface except for some CEACAM6 in a minor fraction of cells (Figure 2A). To elucidate the discrepancy in our findings, we analyzed the cell culture supernatant of confluent A549 cells. We found that both CEACAM5, CEACAM6, and, to a minor extent, CEACAM1, were released into the cell culture supernatant of confluent A549-NT (Figure 2C) and A549-T cells (data not shown).

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