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Desmoglein 2 is a substrate of kallikrein 7 in pancreatic cancer.

Ramani VC, Hennings L, Haun RS - BMC Cancer (2008)

Bottom Line: BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues.When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA. vpramani@uab.edu

ABSTRACT

Background: In a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (KLK7/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells.

Methods: The expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using in vitro degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.

Results: The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.

Conclusion: A reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using in vitro degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion.

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Expression of desmogleins in human pancreatic adenocarcinoma cell lines. (A) Equal amounts of total protein from whole cell lysates of human pancreatic cancer cell lines BxPC-3 (lanes 1 and 3) and Panc-1 (lanes 2 and 4) were separated by SDS-PAGE and visualized by western blot using anti-Dsg1 and anti-Dsg2 antibodies, as indicated. GAPDH levels were monitored as a loading control (arrowhead). Sizes of protein markers are indicated on the left. Appreciable levels of Dsg2 were detected in BxPC-3 cells, but not in Panc-1 cells. Dsg1 was not detected in either of the cell lines. (B) Immunocytochemistry of Dsg2 in BxPC-3 cells using a FITC-conjugated secondary antibody showed specific cell membrane localization. Original magnification x400.
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Figure 4: Expression of desmogleins in human pancreatic adenocarcinoma cell lines. (A) Equal amounts of total protein from whole cell lysates of human pancreatic cancer cell lines BxPC-3 (lanes 1 and 3) and Panc-1 (lanes 2 and 4) were separated by SDS-PAGE and visualized by western blot using anti-Dsg1 and anti-Dsg2 antibodies, as indicated. GAPDH levels were monitored as a loading control (arrowhead). Sizes of protein markers are indicated on the left. Appreciable levels of Dsg2 were detected in BxPC-3 cells, but not in Panc-1 cells. Dsg1 was not detected in either of the cell lines. (B) Immunocytochemistry of Dsg2 in BxPC-3 cells using a FITC-conjugated secondary antibody showed specific cell membrane localization. Original magnification x400.

Mentions: To identify pancreatic cancer cell lines that could be used to examine hK7-dependent cleavage of desmosomal proteins in a cell-based system, cell lysates were prepared from Panc-1 and BxPC-3 cell lines and assessed for the levels of Dsg1 and Dsg2 by western blot analysis. Dsg1 was not detected in either of the cell lines (Fig. 4A, lanes 1 and 2), whereas Dsg2 was readily detected in BxPC-3 but not Panc-1 cell lysates (Fig. 4A, lanes 3 and 4, respectively). Due to the significant expression of Dsg2 in BxPC-3 cell lysates, immunocytochemical staining was performed on intact cells to determine its cellular localization and distribution. The staining revealed a robust cell-cell border distribution similar to the pattern seen in epithelial tissues (Fig. 4B).


Desmoglein 2 is a substrate of kallikrein 7 in pancreatic cancer.

Ramani VC, Hennings L, Haun RS - BMC Cancer (2008)

Expression of desmogleins in human pancreatic adenocarcinoma cell lines. (A) Equal amounts of total protein from whole cell lysates of human pancreatic cancer cell lines BxPC-3 (lanes 1 and 3) and Panc-1 (lanes 2 and 4) were separated by SDS-PAGE and visualized by western blot using anti-Dsg1 and anti-Dsg2 antibodies, as indicated. GAPDH levels were monitored as a loading control (arrowhead). Sizes of protein markers are indicated on the left. Appreciable levels of Dsg2 were detected in BxPC-3 cells, but not in Panc-1 cells. Dsg1 was not detected in either of the cell lines. (B) Immunocytochemistry of Dsg2 in BxPC-3 cells using a FITC-conjugated secondary antibody showed specific cell membrane localization. Original magnification x400.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2628383&req=5

Figure 4: Expression of desmogleins in human pancreatic adenocarcinoma cell lines. (A) Equal amounts of total protein from whole cell lysates of human pancreatic cancer cell lines BxPC-3 (lanes 1 and 3) and Panc-1 (lanes 2 and 4) were separated by SDS-PAGE and visualized by western blot using anti-Dsg1 and anti-Dsg2 antibodies, as indicated. GAPDH levels were monitored as a loading control (arrowhead). Sizes of protein markers are indicated on the left. Appreciable levels of Dsg2 were detected in BxPC-3 cells, but not in Panc-1 cells. Dsg1 was not detected in either of the cell lines. (B) Immunocytochemistry of Dsg2 in BxPC-3 cells using a FITC-conjugated secondary antibody showed specific cell membrane localization. Original magnification x400.
Mentions: To identify pancreatic cancer cell lines that could be used to examine hK7-dependent cleavage of desmosomal proteins in a cell-based system, cell lysates were prepared from Panc-1 and BxPC-3 cell lines and assessed for the levels of Dsg1 and Dsg2 by western blot analysis. Dsg1 was not detected in either of the cell lines (Fig. 4A, lanes 1 and 2), whereas Dsg2 was readily detected in BxPC-3 but not Panc-1 cell lysates (Fig. 4A, lanes 3 and 4, respectively). Due to the significant expression of Dsg2 in BxPC-3 cell lysates, immunocytochemical staining was performed on intact cells to determine its cellular localization and distribution. The staining revealed a robust cell-cell border distribution similar to the pattern seen in epithelial tissues (Fig. 4B).

Bottom Line: BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues.When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA. vpramani@uab.edu

ABSTRACT

Background: In a previous report we have demonstrated that the chymotryptic-like serine protease kallikrein 7 (KLK7/hK7) is overexpressed in pancreatic cancer. In normal skin, hK7 is thought to participate in skin desquamation by contributing in the degradation of desmosomal components, such as desmogleins. Thus, the ability of hK7 to degrade desmogleins was assessed and the effect of hK7 expression on desmoglein 2 was examined in cultured pancreatic cancer cells.

Methods: The expression of Dsg1, Dsg2, and Dsg3 in pancreatic tissues was examined by immunohistochemistry and their expression in two pancreatic cancer cell lines, BxPC-3 and Panc-1, was determined by western blot analysis. The ability of hK7 to degrade Dsg1 and Dsg2 was investigated using in vitro degradation assays. BxPC-3 cells stably transfected to overexpress hK7 were used to examine the effect of hK7 on cell-surface resident Dsg2.

Results: The levels of immunoreactive Dsg1 and Dsg2 were reduced in pancreatic adenocarcinomas compared with both normal pancreatic and chronic pancreatitis tissues. Among the desmosomal proteins examined, Dsg2 exhibited robust expression on the surface of BxPC-3 cells. When hK7 was overexpressed in this cell line, there was a significant increase in the amount of soluble Dsg2 released into the culture medium compared with vector-transfected control cells.

Conclusion: A reduction in the amount of the cell adhesion components Dsg1 and Dsg2 in pancreatic tumors suggests that loss of these desmosomal proteins may play a role in pancreatic cancer invasion. Using in vitro degradation assays, both Dsg1 and Dsg2 could be readily proteolyzed by hK7, which is overexpressed in pancreatic adenocarcinomas. The enforced expression of hK7 in BxPC-3 cells that express significant amounts of Dsg2 resulted in a marked increase in the shedding of soluble Dsg2, which is consistent with the notion that aberrant expression of hK7 in pancreatic tumors may result in diminished cell-cell adhesion and facilitate tumor cell invasion.

Show MeSH
Related in: MedlinePlus