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The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas.

St Hill CA, Farooqui M, Mitcheltree G, Gulbahce HE, Jessurun J, Cao Q, Walcheck B - BMC Cancer (2009)

Bottom Line: The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR.Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Clinical Sciences, University of Minnesota, Room C339, Veterinary Medical Center, 1352 Boyd Avenue, St, Paul, MN 55108, USA. sthil001@umn.edu

ABSTRACT

Background: The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).

Results: We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.

Conclusion: C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.

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Human metastatic liver tumors of primary colorectal adenocarcinomas expressed C2-O-sLex. (A) Photomicrograph of a tissue section of metastatic colorectal adenocarcinoma tumors in liver stained with hematoxylin and eosin, scale bar = 150 μm. (B) A serial section of the same tissue as shown in (A), stained with CHO-131 mAb (15 μg/ml). Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 150 μm. (C) A different tissue section of metastatic colorectal adenocarcinoma in liver stained with CHO-131 mAb, scale bar = 100 μm. (D) A higher magnification of the same tissue section as in (C), stained with CHO-131 mAb (15 μg/ml), scale bar = 50 μm. (E) A serial section of the same tissue as shown in (C) and (D), stained with CEA mAb (1.6 μg/ml) as a positive control. Brown stained cells are positive for CEA mAb, scale bar = 100 μm. (F) Normal liver stained with CHO-131 mAb (15 μg/ml). Note the absence of red color, indicating a lack of reactivity with CHO-131 mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figures (A) and (B) 100× magnification, (C) 200× magnification, (D) 400× magnification, (E) and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.
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Figure 6: Human metastatic liver tumors of primary colorectal adenocarcinomas expressed C2-O-sLex. (A) Photomicrograph of a tissue section of metastatic colorectal adenocarcinoma tumors in liver stained with hematoxylin and eosin, scale bar = 150 μm. (B) A serial section of the same tissue as shown in (A), stained with CHO-131 mAb (15 μg/ml). Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 150 μm. (C) A different tissue section of metastatic colorectal adenocarcinoma in liver stained with CHO-131 mAb, scale bar = 100 μm. (D) A higher magnification of the same tissue section as in (C), stained with CHO-131 mAb (15 μg/ml), scale bar = 50 μm. (E) A serial section of the same tissue as shown in (C) and (D), stained with CEA mAb (1.6 μg/ml) as a positive control. Brown stained cells are positive for CEA mAb, scale bar = 100 μm. (F) Normal liver stained with CHO-131 mAb (15 μg/ml). Note the absence of red color, indicating a lack of reactivity with CHO-131 mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figures (A) and (B) 100× magnification, (C) 200× magnification, (D) 400× magnification, (E) and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.

Mentions: We assessed whether the reactivity of CHO-131 mAb and CSLEX1 mAb overlapped. All primary colorectal adenocarcinomas and metastatic liver tumors examined reacted positively with CSLEX1 mAb. The percentage of colorectal adenocarcinomas that were highly reactive with CHO-131 mAb (2+ to 3+ staining intensity) are shown in Figure 4B. The cytoplasm of glandular structures in primary colorectal adenocarcinomas and metastatic tumors was positively stained with CSLEX1 mAb and reactivity was not related to differentiation status of the tumor. We observed strong, highly positive 2+ to 3+ reactivity with CSLEX1 mAb in similar tissue locations of colorectal adenocarcinomas and metastatic liver tumors to those areas that positively reacted with CHO-131 mAb (Figure 5A, B, C, D and Table 3). However, positive staining with CSLEX1 mAb was more widely distributed in tissues than that with CHO-131 mAb. Normal colonic mucosa and hepatocytes in normal liver lacked reactivity with CSLEX1 mAb (Figure 5E, F). Representative sections of metastatic liver tumors that were positively stained with CHO-131 mAb are shown in Figure 6.


The high affinity selectin glycan ligand C2-O-sLex and mRNA transcripts of the core 2 beta-1,6-N-acetylglucosaminyltransferase (C2GnT1) gene are highly expressed in human colorectal adenocarcinomas.

St Hill CA, Farooqui M, Mitcheltree G, Gulbahce HE, Jessurun J, Cao Q, Walcheck B - BMC Cancer (2009)

Human metastatic liver tumors of primary colorectal adenocarcinomas expressed C2-O-sLex. (A) Photomicrograph of a tissue section of metastatic colorectal adenocarcinoma tumors in liver stained with hematoxylin and eosin, scale bar = 150 μm. (B) A serial section of the same tissue as shown in (A), stained with CHO-131 mAb (15 μg/ml). Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 150 μm. (C) A different tissue section of metastatic colorectal adenocarcinoma in liver stained with CHO-131 mAb, scale bar = 100 μm. (D) A higher magnification of the same tissue section as in (C), stained with CHO-131 mAb (15 μg/ml), scale bar = 50 μm. (E) A serial section of the same tissue as shown in (C) and (D), stained with CEA mAb (1.6 μg/ml) as a positive control. Brown stained cells are positive for CEA mAb, scale bar = 100 μm. (F) Normal liver stained with CHO-131 mAb (15 μg/ml). Note the absence of red color, indicating a lack of reactivity with CHO-131 mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figures (A) and (B) 100× magnification, (C) 200× magnification, (D) 400× magnification, (E) and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.
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Figure 6: Human metastatic liver tumors of primary colorectal adenocarcinomas expressed C2-O-sLex. (A) Photomicrograph of a tissue section of metastatic colorectal adenocarcinoma tumors in liver stained with hematoxylin and eosin, scale bar = 150 μm. (B) A serial section of the same tissue as shown in (A), stained with CHO-131 mAb (15 μg/ml). Red color indicates positive reactivity with the CHO-131 mAb, scale bar = 150 μm. (C) A different tissue section of metastatic colorectal adenocarcinoma in liver stained with CHO-131 mAb, scale bar = 100 μm. (D) A higher magnification of the same tissue section as in (C), stained with CHO-131 mAb (15 μg/ml), scale bar = 50 μm. (E) A serial section of the same tissue as shown in (C) and (D), stained with CEA mAb (1.6 μg/ml) as a positive control. Brown stained cells are positive for CEA mAb, scale bar = 100 μm. (F) Normal liver stained with CHO-131 mAb (15 μg/ml). Note the absence of red color, indicating a lack of reactivity with CHO-131 mAb, scale bar = 100 μm. All tissue sections were 4 μm thickness. Figures (A) and (B) 100× magnification, (C) 200× magnification, (D) 400× magnification, (E) and (F) 200× magnification. Mayer's hematoxylin was used as a counterstain for tissues stained with CHO-131 and CEA mAbs. Representative sections from multiple stained tissues are shown.
Mentions: We assessed whether the reactivity of CHO-131 mAb and CSLEX1 mAb overlapped. All primary colorectal adenocarcinomas and metastatic liver tumors examined reacted positively with CSLEX1 mAb. The percentage of colorectal adenocarcinomas that were highly reactive with CHO-131 mAb (2+ to 3+ staining intensity) are shown in Figure 4B. The cytoplasm of glandular structures in primary colorectal adenocarcinomas and metastatic tumors was positively stained with CSLEX1 mAb and reactivity was not related to differentiation status of the tumor. We observed strong, highly positive 2+ to 3+ reactivity with CSLEX1 mAb in similar tissue locations of colorectal adenocarcinomas and metastatic liver tumors to those areas that positively reacted with CHO-131 mAb (Figure 5A, B, C, D and Table 3). However, positive staining with CSLEX1 mAb was more widely distributed in tissues than that with CHO-131 mAb. Normal colonic mucosa and hepatocytes in normal liver lacked reactivity with CSLEX1 mAb (Figure 5E, F). Representative sections of metastatic liver tumors that were positively stained with CHO-131 mAb are shown in Figure 6.

Bottom Line: The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR.Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Veterinary Clinical Sciences, University of Minnesota, Room C339, Veterinary Medical Center, 1352 Boyd Avenue, St, Paul, MN 55108, USA. sthil001@umn.edu

ABSTRACT

Background: The metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. Specialized carbohydrates modified with sialyl Lewis x (sLex) antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. The activity of the enzyme core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT1) in leukocytes greatly increases their ability to bind to endothelial selectins. C2GnT1 is essential for the synthesis of core 2-branched O-linked carbohydrates terminated with sLex (C2-O-sLex). Our goal was to determine the expression profiles of C2-O-sLex in the malignant progression and metastasis of colorectal adenocarcinomas. The well characterized CHO-131 monoclonal antibody (mAb) specifically recognizes C2-O-sLex present in human leukocytes and carcinoma cells. Using CHO-131 mAb, we investigated whether C2-O-sLex was present in 113 human primary colorectal adenocarcinomas, 10 colorectal adenomas, 46 metastatic liver tumors, 28 normal colorectal tissues, and 5 normal liver tissues by immunohistochemistry. We also examined mRNA levels of the enzyme core 2 beta1,6-N-acetylglucosaminyltransferase (C2GnT1) in 20 well, 15 moderately, and 2 poorly differentiated colorectal adenocarcinomas, and in 5 normal colorectal tissues by using quantitative real-time polymerase chain reactions (RT-PCR).

Results: We observed high reactivity with CHO-131 mAb in approximately 70% of colorectal carcinomas and 87% of metastatic liver tumors but a lack of reactivity in colorectal adenomas and normal colonic and liver tissues. Positive reactivity with CHO-131 mAb was very prominent in neoplastic colorectal glands of well to moderately differentiated adenocarcinomas. The most intense staining with CHO-131 mAb was observed at the advancing edge of tumors with the deepest invasive components.Finally, we analyzed C2GnT1 mRNA levels in 37 colorectal adenocarcinomas and 5 normal colorectal tissues by RT-PCR. Significantly, we observed a greater than 15-fold increase in C2GnT1 mRNA levels in colorectal adenocarcinomas compared to normal colorectal tissues.

Conclusion: C2-O-sLex, detected by the CHO-131 mAb, is a tumor associated antigen whose expression is highly upregulated in colorectal adenocarcinomas and metastatic liver tumors compared to normal tissues. C2-O-sLex is a potentially useful early predictor of metastasis.

Show MeSH
Related in: MedlinePlus