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Altered expression of the klf4 in colorectal cancers

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avinash tiwari

it is about the transcription factor family KLF4 and their expression in the colon cancer at different stage.which is help full in diagnosis of such type of cancer

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Altered expression of the KLF4 in colorectal cancers Byung Joon Choi et. al.,2006 By Avinash tiwari M. Tech 1st year 201710902010002
Introduction: The mammalian gastrointestinal epithelium is a dynamic system in which cell proliferation is coupled to differentiation. Aberrations in this process frequently lead to colorectal cancer. colorectal cancer is one of the leading primary cancers
cause Minor reason (inherited predisposition) Hereditary non-polyposis c.c. familial adenomatous polyposis Major reason Somatic mutation or Altered expression of tumor- associated protein
KLF-4 in colorectal cancer: Kru即ppel-like factor 4 (KLF-4) is family of zinc finger containing transcription factor Highly expressed in epithelial of gastrointestinal tract, skin and vascular endothelial cells. Play a important roll during the differentiation of gut epithelial cell. KLF4 activates the promoter of the negative cell-cycle-regulatory cyclin-dependent kinase inhibitor p21WAF/Cip1 gene in a p53- dependent manner
Induces cell cycle arrest at the transition from G1 to S phase Altered expression of KLF4 may be involved in the tumorigenesis of several kinds of human cancers, including colon, breast, gastric, esophageal, and bladder cancers. the level of KLF4 and the KLF4 mRNA expression is significantly decreased in colonic adenomas & in sporadic colonic adenomas and carcinoma patients with familial adenomatous polyposis when compared with normal colonic tissues
Fig.- different function of KLF4 (form research gate)
Material and method: Method & material Tissue samples Immunohistochemistry for the KLF4:
A. Tissue samples 1. Formalin-fixed and paraffin-embedded samples were obtained from surgical resections of 123 colorectal cancers 2. Classification of tumor according Dukes criteria. 3. There were 12, 47, 56, and 8 cases with stage A, B, C, and D, respectively.
Tissue samples cont. 4. Screened the histological sections and selected areas of the representative tumor cells. 5. Tissue cores were taken from each tumor sample and placed in a paraffin block using a commercially available microarray instrument 6. One cylinder of normal colonic mucosa adjacent to each tumor was also transferred to the recipient block.
B. Immunohistochemistry for the KLF4: 1. 2 mm sections were cut the day before use and stained according to standard protocols. 2. To maximize the signal on immunohistochemistry, two strategies were used = antigen retrieval in citrate buffer Signal amplification with biotinylated tyramide
3. heat-induced epitope retrieval was conducted in citrate buffer (pH 6.0) and boiling the buffer for 30 min and cool 4. rinsing with PBS, the slides were treated with 1% H2O2 in PBS for 15 min at room temperature to abolish endogenous peroxidase activity. 5. washing with TNT buffer (0.1 mol/L Tris-HCl, pH for 20 min the slides were treated with TNB buffer. 6. incubated overnight at 4 degree C with the antibody (1/100 dilution) for KLF4 protein
7. detection was carried out using biotinylated goat anti-rabbit antibody, followed by incubation with peroxidase-linked avidinbiotin complex. 8. Diaminobenzidine was used as chromogen, and the slide was counterstained with Mayers hematoxylin. 9. As negative controls, the slide was treated by replacement of primary antibody with non-immune serum.
Results: Immunohistochemistry for the KLF4: In immunohistochemistry, normal colonic mucosa showed moderate to strong expression of KLF4 protein mainly in the nucleus of the glandular epithelial cells. Inflammatory cells, including lymphocytes in lamina propria of colonic mucosa, also Demonstrated focal, weakly positive staining Colorectal normal cells
Expression of KLF4 in tumor cells shows Moderate to strong immunopositivity for KLF4 protein was clearly marked on the nucleus of colorectal tumor cells compared with normal colonic mucosa. Colorectal cancer, well differentiated adenocarcinoma, displayed moderate to strong immunostaining in nucleus of cancer cells. Colorectal cancer stage B
Another cancer showed immunonegativity for the KLF4 protein in the nucleus of the cancer cells KLF4 protein was detected in 30 (24.4%) of 123 colorectal cancers Colorectal cancer stage C
KLF4 protein was detected in 30 (24.4%) of 123 colorectal cancers KLF4 expression was detected in 100% (6/6),73.2% (82/112), and 100% (5/5) of well, moderately,and poorly differentiated colorectal cancers In this classification, the expression of the KLF4 protein was not associated with the differentiation of tumor cells (w2 test, P40.05). Loss of KLF4 expression was seen in 4 (33.3%) of 12 cases corresponding to stage A, 9 (19.1%) of 47 to stage B, 16 (28.6%) of 56 to stage C, and 1 (12.5%) of 8 to stage D. In addition, KLF4 expression was lost in 16 (27.1%) of 59 cases with lymph node metastasis. There was a tendency that loss of KLF4 expression was more common in left-sided colon cancer (27.5%) than in right-sided colon cancer (12%).
Altered expression of the klf4 in colorectal cancers

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Altered expression of the klf4 in colorectal cancers

  • 1. Altered expression of the KLF4 in colorectal cancers Byung Joon Choi et. al.,2006 By Avinash tiwari M. Tech 1st year 201710902010002
  • 2. Introduction: The mammalian gastrointestinal epithelium is a dynamic system in which cell proliferation is coupled to differentiation. Aberrations in this process frequently lead to colorectal cancer. colorectal cancer is one of the leading primary cancers
  • 3. cause Minor reason (inherited predisposition) Hereditary non-polyposis c.c. familial adenomatous polyposis Major reason Somatic mutation or Altered expression of tumor- associated protein
  • 4. KLF-4 in colorectal cancer: Kru即ppel-like factor 4 (KLF-4) is family of zinc finger containing transcription factor Highly expressed in epithelial of gastrointestinal tract, skin and vascular endothelial cells. Play a important roll during the differentiation of gut epithelial cell. KLF4 activates the promoter of the negative cell-cycle-regulatory cyclin-dependent kinase inhibitor p21WAF/Cip1 gene in a p53- dependent manner
  • 5. Induces cell cycle arrest at the transition from G1 to S phase Altered expression of KLF4 may be involved in the tumorigenesis of several kinds of human cancers, including colon, breast, gastric, esophageal, and bladder cancers. the level of KLF4 and the KLF4 mRNA expression is significantly decreased in colonic adenomas & in sporadic colonic adenomas and carcinoma patients with familial adenomatous polyposis when compared with normal colonic tissues
  • 6. Fig.- different function of KLF4 (form research gate)
  • 7. Material and method: Method & material Tissue samples Immunohistochemistry for the KLF4:
  • 8. A. Tissue samples 1. Formalin-fixed and paraffin-embedded samples were obtained from surgical resections of 123 colorectal cancers 2. Classification of tumor according Dukes criteria. 3. There were 12, 47, 56, and 8 cases with stage A, B, C, and D, respectively.
  • 9. Tissue samples cont. 4. Screened the histological sections and selected areas of the representative tumor cells. 5. Tissue cores were taken from each tumor sample and placed in a paraffin block using a commercially available microarray instrument 6. One cylinder of normal colonic mucosa adjacent to each tumor was also transferred to the recipient block.
  • 10. B. Immunohistochemistry for the KLF4: 1. 2 mm sections were cut the day before use and stained according to standard protocols. 2. To maximize the signal on immunohistochemistry, two strategies were used = antigen retrieval in citrate buffer Signal amplification with biotinylated tyramide
  • 11. 3. heat-induced epitope retrieval was conducted in citrate buffer (pH 6.0) and boiling the buffer for 30 min and cool 4. rinsing with PBS, the slides were treated with 1% H2O2 in PBS for 15 min at room temperature to abolish endogenous peroxidase activity. 5. washing with TNT buffer (0.1 mol/L Tris-HCl, pH for 20 min the slides were treated with TNB buffer. 6. incubated overnight at 4 degree C with the antibody (1/100 dilution) for KLF4 protein
  • 12. 7. detection was carried out using biotinylated goat anti-rabbit antibody, followed by incubation with peroxidase-linked avidinbiotin complex. 8. Diaminobenzidine was used as chromogen, and the slide was counterstained with Mayers hematoxylin. 9. As negative controls, the slide was treated by replacement of primary antibody with non-immune serum.
  • 13. Results: Immunohistochemistry for the KLF4: In immunohistochemistry, normal colonic mucosa showed moderate to strong expression of KLF4 protein mainly in the nucleus of the glandular epithelial cells. Inflammatory cells, including lymphocytes in lamina propria of colonic mucosa, also Demonstrated focal, weakly positive staining Colorectal normal cells
  • 14. Expression of KLF4 in tumor cells shows Moderate to strong immunopositivity for KLF4 protein was clearly marked on the nucleus of colorectal tumor cells compared with normal colonic mucosa. Colorectal cancer, well differentiated adenocarcinoma, displayed moderate to strong immunostaining in nucleus of cancer cells. Colorectal cancer stage B
  • 15. Another cancer showed immunonegativity for the KLF4 protein in the nucleus of the cancer cells KLF4 protein was detected in 30 (24.4%) of 123 colorectal cancers Colorectal cancer stage C
  • 16. KLF4 protein was detected in 30 (24.4%) of 123 colorectal cancers KLF4 expression was detected in 100% (6/6),73.2% (82/112), and 100% (5/5) of well, moderately,and poorly differentiated colorectal cancers In this classification, the expression of the KLF4 protein was not associated with the differentiation of tumor cells (w2 test, P40.05). Loss of KLF4 expression was seen in 4 (33.3%) of 12 cases corresponding to stage A, 9 (19.1%) of 47 to stage B, 16 (28.6%) of 56 to stage C, and 1 (12.5%) of 8 to stage D. In addition, KLF4 expression was lost in 16 (27.1%) of 59 cases with lymph node metastasis. There was a tendency that loss of KLF4 expression was more common in left-sided colon cancer (27.5%) than in right-sided colon cancer (12%).

Editor's Notes

  • #5: KLF 4 is also known as the gut enriched KLF of epithelial zinc finger Post mitotic terminally differentiated cells..reported in middle layer to upper region of the crypt