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The level of claudin-7 is reduced as an early event in colorectal carcinogenesis.

Bornholdt J, Friis S, Godiksen S, Poulsen SS, Santoni-Rugiu E, Bisgaard HC, Lothe IM, Ikdahl T, Tveit KM, Johnson E, Kure EH, Vogel LK - BMC Cancer (2011)

Bottom Line: We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression.A 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001).The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Science, University of Copenhagen, Denmark.

ABSTRACT

Background: Compromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both in vivo and in vitro. We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression. We have previously shown that the matriptase expression level decreases during colorectal carcinogenesis. In the present study we investigated whether claudin-7 expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue.

Methods: The mRNA level of claudin-7 (CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings.

Results: A 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the claudin-7 mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.

Conclusions: Our results show that the claudin-7 mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.

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Immunohistochemical staining for claudin-7 in colorectal adenomas, carcinomas and adjacent normal tissue. The top four pictures are taken from an individual with dysplasia but no record of carcinoma and the bottom four pictures are from a patient with colorectal cancer. A) Tissue section including both normal mucosa and dysplastic tissue, stained with PAS/Alcian. B) Neighbouring tissue section stained for claudin-7. The white arrow indicates mucosa of normal appearance and the black arrow indicates dysplastic tissue. C) High magnification of the normal mucosa from B, showing staining mainly at the basolateral cell membranes of the epithelial cells. D) High magnification of the dysplastic tissue from B showing a patchy staining pattern with areas of low and normal staining. E) Tissue section with cancerous tissue to the right and normal mucosa to the left, stained with Hematoxylin and Eosin. F) Neighbouring tissue section stained for claudin-7. The white arrow indicates the mucosa with normal appearance. The black arrow points out carcinomatous tissue. G) High magnification of histologically normally appearing mucosa from F, showing staining mainly localised to the basolateral cell membranes of the surface epithelial cells. H) High magnification of carcinomatous tissue showing faint, patchy staining of the epithelial strands of tumour tissue. Scale bars: 500 μm (A + B + E + F), 100 μm (C + D + G + H).
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Figure 4: Immunohistochemical staining for claudin-7 in colorectal adenomas, carcinomas and adjacent normal tissue. The top four pictures are taken from an individual with dysplasia but no record of carcinoma and the bottom four pictures are from a patient with colorectal cancer. A) Tissue section including both normal mucosa and dysplastic tissue, stained with PAS/Alcian. B) Neighbouring tissue section stained for claudin-7. The white arrow indicates mucosa of normal appearance and the black arrow indicates dysplastic tissue. C) High magnification of the normal mucosa from B, showing staining mainly at the basolateral cell membranes of the epithelial cells. D) High magnification of the dysplastic tissue from B showing a patchy staining pattern with areas of low and normal staining. E) Tissue section with cancerous tissue to the right and normal mucosa to the left, stained with Hematoxylin and Eosin. F) Neighbouring tissue section stained for claudin-7. The white arrow indicates the mucosa with normal appearance. The black arrow points out carcinomatous tissue. G) High magnification of histologically normally appearing mucosa from F, showing staining mainly localised to the basolateral cell membranes of the surface epithelial cells. H) High magnification of carcinomatous tissue showing faint, patchy staining of the epithelial strands of tumour tissue. Scale bars: 500 μm (A + B + E + F), 100 μm (C + D + G + H).

Mentions: Carcinoma tissue and normal mucosa from five CRC patients were extracted and the proteins were analysed on Western blots (Figure 3). Only one distinct band of approximately 22 kDa was detected using an antibody against claudin-7. This is in accordance with the expected size of claudin-7 [29]. Immunohistochemical stainings were performed on adenomas from four individuals without any history of carcinomas and on CRC tissue and normal mucosa from five CRC patients (Figure 4 and additional files 1). All stainings of adenomas showed essentially the same, and a representative example is shown in Figure 4. An intense claudin-7 reaction was seen in histologically normal appearing mucosa (Figure 4B and 4C) whereas dysplastic tissue ranged from almost no staining to staining similar to the normal mucosa (Figure 4B and 4D). Stainings of the five colorectal cancer samples also showed essentially the same and is shown in figure 4 and additional file 2. Histologically normal mucosa from cancer patients also displayed an intense claudin-7 reaction that was detected from the transitional zone (Figure 4F and 4G). In the cancerous tissue, the staining was reduced or almost absent (Figure 4F and 4H). These observations are consistent with the claudin-7 mRNA levels obtained in this study. The normal appearing mucosa from the transitional zone showed, at high magnification, staining for claudin-7. The staining was mainly localised at the basolateral plasma membranes of the surface epithelial cells (Figure 4C and 4G), whereas the ademomatous and carcinomatous tissue showed faint, patchy staining of the epithelial strands of tumor tissue (Figure 4B and 4F).


The level of claudin-7 is reduced as an early event in colorectal carcinogenesis.

Bornholdt J, Friis S, Godiksen S, Poulsen SS, Santoni-Rugiu E, Bisgaard HC, Lothe IM, Ikdahl T, Tveit KM, Johnson E, Kure EH, Vogel LK - BMC Cancer (2011)

Immunohistochemical staining for claudin-7 in colorectal adenomas, carcinomas and adjacent normal tissue. The top four pictures are taken from an individual with dysplasia but no record of carcinoma and the bottom four pictures are from a patient with colorectal cancer. A) Tissue section including both normal mucosa and dysplastic tissue, stained with PAS/Alcian. B) Neighbouring tissue section stained for claudin-7. The white arrow indicates mucosa of normal appearance and the black arrow indicates dysplastic tissue. C) High magnification of the normal mucosa from B, showing staining mainly at the basolateral cell membranes of the epithelial cells. D) High magnification of the dysplastic tissue from B showing a patchy staining pattern with areas of low and normal staining. E) Tissue section with cancerous tissue to the right and normal mucosa to the left, stained with Hematoxylin and Eosin. F) Neighbouring tissue section stained for claudin-7. The white arrow indicates the mucosa with normal appearance. The black arrow points out carcinomatous tissue. G) High magnification of histologically normally appearing mucosa from F, showing staining mainly localised to the basolateral cell membranes of the surface epithelial cells. H) High magnification of carcinomatous tissue showing faint, patchy staining of the epithelial strands of tumour tissue. Scale bars: 500 μm (A + B + E + F), 100 μm (C + D + G + H).
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Related In: Results  -  Collection

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Figure 4: Immunohistochemical staining for claudin-7 in colorectal adenomas, carcinomas and adjacent normal tissue. The top four pictures are taken from an individual with dysplasia but no record of carcinoma and the bottom four pictures are from a patient with colorectal cancer. A) Tissue section including both normal mucosa and dysplastic tissue, stained with PAS/Alcian. B) Neighbouring tissue section stained for claudin-7. The white arrow indicates mucosa of normal appearance and the black arrow indicates dysplastic tissue. C) High magnification of the normal mucosa from B, showing staining mainly at the basolateral cell membranes of the epithelial cells. D) High magnification of the dysplastic tissue from B showing a patchy staining pattern with areas of low and normal staining. E) Tissue section with cancerous tissue to the right and normal mucosa to the left, stained with Hematoxylin and Eosin. F) Neighbouring tissue section stained for claudin-7. The white arrow indicates the mucosa with normal appearance. The black arrow points out carcinomatous tissue. G) High magnification of histologically normally appearing mucosa from F, showing staining mainly localised to the basolateral cell membranes of the surface epithelial cells. H) High magnification of carcinomatous tissue showing faint, patchy staining of the epithelial strands of tumour tissue. Scale bars: 500 μm (A + B + E + F), 100 μm (C + D + G + H).
Mentions: Carcinoma tissue and normal mucosa from five CRC patients were extracted and the proteins were analysed on Western blots (Figure 3). Only one distinct band of approximately 22 kDa was detected using an antibody against claudin-7. This is in accordance with the expected size of claudin-7 [29]. Immunohistochemical stainings were performed on adenomas from four individuals without any history of carcinomas and on CRC tissue and normal mucosa from five CRC patients (Figure 4 and additional files 1). All stainings of adenomas showed essentially the same, and a representative example is shown in Figure 4. An intense claudin-7 reaction was seen in histologically normal appearing mucosa (Figure 4B and 4C) whereas dysplastic tissue ranged from almost no staining to staining similar to the normal mucosa (Figure 4B and 4D). Stainings of the five colorectal cancer samples also showed essentially the same and is shown in figure 4 and additional file 2. Histologically normal mucosa from cancer patients also displayed an intense claudin-7 reaction that was detected from the transitional zone (Figure 4F and 4G). In the cancerous tissue, the staining was reduced or almost absent (Figure 4F and 4H). These observations are consistent with the claudin-7 mRNA levels obtained in this study. The normal appearing mucosa from the transitional zone showed, at high magnification, staining for claudin-7. The staining was mainly localised at the basolateral plasma membranes of the surface epithelial cells (Figure 4C and 4G), whereas the ademomatous and carcinomatous tissue showed faint, patchy staining of the epithelial strands of tumor tissue (Figure 4B and 4F).

Bottom Line: We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression.A 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001).The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Health Science, University of Copenhagen, Denmark.

ABSTRACT

Background: Compromised epithelial barriers are found in dysplastic tissue of the gastrointestinal tract. Claudins are transmembrane proteins important for tight junctions. Claudins regulate the paracellular transport and are crucial for maintaining a functional epithelial barrier. Down-regulation of the oncogenic serine protease, matriptase, induces leakiness in epithelial barriers both in vivo and in vitro. We found in an in-silico search tight co-regulation between matriptase and claudin-7 expression. We have previously shown that the matriptase expression level decreases during colorectal carcinogenesis. In the present study we investigated whether claudin-7 expression is likewise decreased during colorectal carcinogenesis, thereby causing or contributing to the compromised epithelial leakiness of dysplastic tissue.

Methods: The mRNA level of claudin-7 (CLDN7) was determined in samples from 18 healthy individuals, 100 individuals with dysplasia and 121 colorectal cancer patients using quantitative real time RT-PCR. In addition, immunohistochemical stainings were performed on colorectal adenomas and carcinomas, to confirm the mRNA findings.

Results: A 2.7-fold reduction in the claudin-7 mRNA level was found when comparing the biopsies from healthy individuals with the biopsies of carcinomas (p < 0.001). Reductions in the claudin-7 mRNA levels were also detected in mild/moderate dysplasia (p < 0.001), severe dysplasia (p < 0.01) and carcinomas (p < 0.01), compared to a control sample from the same individual. The decrease at mRNA level was confirmed at the protein level by immunohistochemical stainings.

Conclusions: Our results show that the claudin-7 mRNA level is decreased already as an early event in colorectal carcinogenesis, probably contributing to the compromised epithelial barrier in adenomas.

Show MeSH
Related in: MedlinePlus