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Colorectal hyperplasia and dysplasia due to human carcinoembryonic antigen (CEA) family member expression in transgenic mice.

Chan CH, Camacho-Leal P, Stanners CP - PLoS ONE (2007)

Bottom Line: The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6.Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis.All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology.

View Article: PubMed Central - PubMed

Affiliation: McGill Cancer Centre, Department of Biochemistry, McGill University, Montréal, Québec, Canada. carlos.chan@mcgill.ca

ABSTRACT
CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer.

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Inhibition of apoptosis/anoikis in CEABAC mice.A) TUNEL assays on formaldehyde-fixed frozen sections of WT and CEABAC20 colons. Cyan (red arrow): apoptotic nuclei; Blue (yellow arrow): non-apoptotic nuclei. Note the presence of non-apoptotic anchorless cells, which are CEA/CEACAM6-positive (data not shown), in the crypt lumens of CEABAC20 colons (yellow arrow). Magnification: 400×. Staining: DAPI. B) Immunoblots of colon protein extracts for PARP show reduced cleavage product of PARP (ΔPARP) indicating an overall reduction of apoptosis in CEABAC20 mouse colons. C) TUNEL assays on purified colonocytes show a marked inhibition of anoikis of CEABAC colonocytes compared to WT colonocytes after 3 hours in single cell suspension. Negative control (freshly purified WT colonocytes) is completely TUNEL negative. Positive control (freshly purified WT colonocytes treated with DNAseI) is TUNEL positive. D) Apoptotic indices estimated from 3 independent experiments shown in C. E) Immunoblots of colonocyte lysates from anoikis experiment for caspase-3 show a baseline cleavage of caspase-3 (ΔCaspase3) at time 0 (freshly purified colonocytes) and a marked reduction of caspase-3 cleavage at time 1 hr (after 1 hour in single cell suspensions) for CEABAC mice, indicating inhibition of anoikis in the CEABAC colonocytes.
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pone-0001353-g008: Inhibition of apoptosis/anoikis in CEABAC mice.A) TUNEL assays on formaldehyde-fixed frozen sections of WT and CEABAC20 colons. Cyan (red arrow): apoptotic nuclei; Blue (yellow arrow): non-apoptotic nuclei. Note the presence of non-apoptotic anchorless cells, which are CEA/CEACAM6-positive (data not shown), in the crypt lumens of CEABAC20 colons (yellow arrow). Magnification: 400×. Staining: DAPI. B) Immunoblots of colon protein extracts for PARP show reduced cleavage product of PARP (ΔPARP) indicating an overall reduction of apoptosis in CEABAC20 mouse colons. C) TUNEL assays on purified colonocytes show a marked inhibition of anoikis of CEABAC colonocytes compared to WT colonocytes after 3 hours in single cell suspension. Negative control (freshly purified WT colonocytes) is completely TUNEL negative. Positive control (freshly purified WT colonocytes treated with DNAseI) is TUNEL positive. D) Apoptotic indices estimated from 3 independent experiments shown in C. E) Immunoblots of colonocyte lysates from anoikis experiment for caspase-3 show a baseline cleavage of caspase-3 (ΔCaspase3) at time 0 (freshly purified colonocytes) and a marked reduction of caspase-3 cleavage at time 1 hr (after 1 hour in single cell suspensions) for CEABAC mice, indicating inhibition of anoikis in the CEABAC colonocytes.

Mentions: A further change, characteristic of neoplasia, was the inhibition of anoikis, the apoptotic process that normally removes cells that are not properly anchored to the extra-cellular membrane and which therefore acts as a quality control mechanism for tissue architecture [24]. If the CEA/CEACAM6-mediated disruption of tissue architecture is to persist, this process needs to be inhibited. In fact, crypts from 3 month-old CEABAC20 mice often showed CEA/CEACAM6-positive anchorless cells in their lumens that were negative for staining by the TUNEL assay; such cells were not seen in WT crypts (Figure 8A). Moreover, protein extracts from CEABAC20 colonic mucosa showed less PARP cleavage than from WT mucosa (Figure 8B), suggesting that the proportion of cells undergoing apoptosis was less in the CEABAC20 mice than in WT mice. In addition, colonocytes purified from isolated crypts of even the reduced CEABAC dose CEABAC2 and CEABAC10 mice, when maintained in suspension, showed a dramatic reduction in apoptosis (Figures 8C and 8D). At the molecular level, the cleavage of caspase-3, which represents a hallmark of apoptosis [32], was also reduced in the suspended CEABAC2 and CEABAC10 colonocytes (Figure 8E).


Colorectal hyperplasia and dysplasia due to human carcinoembryonic antigen (CEA) family member expression in transgenic mice.

Chan CH, Camacho-Leal P, Stanners CP - PLoS ONE (2007)

Inhibition of apoptosis/anoikis in CEABAC mice.A) TUNEL assays on formaldehyde-fixed frozen sections of WT and CEABAC20 colons. Cyan (red arrow): apoptotic nuclei; Blue (yellow arrow): non-apoptotic nuclei. Note the presence of non-apoptotic anchorless cells, which are CEA/CEACAM6-positive (data not shown), in the crypt lumens of CEABAC20 colons (yellow arrow). Magnification: 400×. Staining: DAPI. B) Immunoblots of colon protein extracts for PARP show reduced cleavage product of PARP (ΔPARP) indicating an overall reduction of apoptosis in CEABAC20 mouse colons. C) TUNEL assays on purified colonocytes show a marked inhibition of anoikis of CEABAC colonocytes compared to WT colonocytes after 3 hours in single cell suspension. Negative control (freshly purified WT colonocytes) is completely TUNEL negative. Positive control (freshly purified WT colonocytes treated with DNAseI) is TUNEL positive. D) Apoptotic indices estimated from 3 independent experiments shown in C. E) Immunoblots of colonocyte lysates from anoikis experiment for caspase-3 show a baseline cleavage of caspase-3 (ΔCaspase3) at time 0 (freshly purified colonocytes) and a marked reduction of caspase-3 cleavage at time 1 hr (after 1 hour in single cell suspensions) for CEABAC mice, indicating inhibition of anoikis in the CEABAC colonocytes.
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Related In: Results  -  Collection

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pone-0001353-g008: Inhibition of apoptosis/anoikis in CEABAC mice.A) TUNEL assays on formaldehyde-fixed frozen sections of WT and CEABAC20 colons. Cyan (red arrow): apoptotic nuclei; Blue (yellow arrow): non-apoptotic nuclei. Note the presence of non-apoptotic anchorless cells, which are CEA/CEACAM6-positive (data not shown), in the crypt lumens of CEABAC20 colons (yellow arrow). Magnification: 400×. Staining: DAPI. B) Immunoblots of colon protein extracts for PARP show reduced cleavage product of PARP (ΔPARP) indicating an overall reduction of apoptosis in CEABAC20 mouse colons. C) TUNEL assays on purified colonocytes show a marked inhibition of anoikis of CEABAC colonocytes compared to WT colonocytes after 3 hours in single cell suspension. Negative control (freshly purified WT colonocytes) is completely TUNEL negative. Positive control (freshly purified WT colonocytes treated with DNAseI) is TUNEL positive. D) Apoptotic indices estimated from 3 independent experiments shown in C. E) Immunoblots of colonocyte lysates from anoikis experiment for caspase-3 show a baseline cleavage of caspase-3 (ΔCaspase3) at time 0 (freshly purified colonocytes) and a marked reduction of caspase-3 cleavage at time 1 hr (after 1 hour in single cell suspensions) for CEABAC mice, indicating inhibition of anoikis in the CEABAC colonocytes.
Mentions: A further change, characteristic of neoplasia, was the inhibition of anoikis, the apoptotic process that normally removes cells that are not properly anchored to the extra-cellular membrane and which therefore acts as a quality control mechanism for tissue architecture [24]. If the CEA/CEACAM6-mediated disruption of tissue architecture is to persist, this process needs to be inhibited. In fact, crypts from 3 month-old CEABAC20 mice often showed CEA/CEACAM6-positive anchorless cells in their lumens that were negative for staining by the TUNEL assay; such cells were not seen in WT crypts (Figure 8A). Moreover, protein extracts from CEABAC20 colonic mucosa showed less PARP cleavage than from WT mucosa (Figure 8B), suggesting that the proportion of cells undergoing apoptosis was less in the CEABAC20 mice than in WT mice. In addition, colonocytes purified from isolated crypts of even the reduced CEABAC dose CEABAC2 and CEABAC10 mice, when maintained in suspension, showed a dramatic reduction in apoptosis (Figures 8C and 8D). At the molecular level, the cleavage of caspase-3, which represents a hallmark of apoptosis [32], was also reduced in the suspended CEABAC2 and CEABAC10 colonocytes (Figure 8E).

Bottom Line: The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6.Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis.All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology.

View Article: PubMed Central - PubMed

Affiliation: McGill Cancer Centre, Department of Biochemistry, McGill University, Montréal, Québec, Canada. carlos.chan@mcgill.ca

ABSTRACT
CEA and CEACAM6 are immunoglobulin family intercellular adhesion molecules that are up-regulated without structural mutations in approximately 70% of human cancers. Results in in vitro systems showing tumorigenic effects for these molecules suggest that this correlation could indicate an instrumental role in tumorigenesis. To test whether this applies in vivo, transgenic mice harboring 187 kb of the human genome containing four CEA family member genes including the CEA and CEACAM6 genes were created and their copy numbers increased by mating until colonocyte expression levels reached levels seen in human colorectal carcinomas. The colonocyte surface level of integrin alpha5 and the activation of AKT increased progressively with the expression levels of CEA/CEACAM6. Colonic crypts showed a progressive increase in colonocyte proliferation, an increase in crypt fission, and a strong inhibition of both differentiation and anoikis/apoptosis. All transgenic mice showed massively enlarged colons comprising a continuous mosaic of severe hyperplasia, dysplasia and serrated adenomatous morphology. These results suggest that up-regulated non-mutated adhesion molecules could have a significant instrumental role in human cancer.

Show MeSH
Related in: MedlinePlus