Application of laser capture microdissection combined with two-dimensional electrophoresis for the discovery of differentially regulated proteins in pancreatic ductal adenocarcinoma

被引:140
作者
Shekouh, AR
Thompson, CC
Prime, W
Campbell, F
Hamlett, J
Herrington, CS
Lemoine, NR
Crnogorac-Jurcevic, T
Buechler, MW
Friess, H
Neoptolemos, JP
Pennington, SR
Costello, E
机构
[1] Royal Liverpool Univ Hosp, Dept Surg, Liverpool, Merseyside, England
[2] Univ Liverpool, Canc Tissue Bank, Res Ctr, Dept Pathol, Liverpool L69 3BX, Merseyside, England
[3] Univ Liverpool, Dept Pathol, Liverpool L69 3BX, Merseyside, England
[4] Univ Liverpool, Dept Human Anat & Cell Biol, Liverpool L69 3BX, Merseyside, England
[5] Univ London Imperial Coll Sci Technol & Med, Fac Med, Dept Canc Med, Cancer Res UK Mol Oncol Unit, London, England
[6] Heidelberg Univ, Dept Gen Surg, Heidelberg, Germany
关键词
differential protein expression; laser capture microdissection; pancreatic cancer;
D O I
10.1002/pmic.200300466
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Pancreatic ductal adenocarcinoma (PDAC) is the most lethal of all the common malignancies and markers for early detection or targets for treatment of this disease are urgently required. The disease is characterised by a strong stromal response, with cancer cells usually representing a relatively small proportion of the cells in the tumor mass. We therefore performed laser capture microdissection (LCM) to enrich for both normal and malignant pancreatic ductal epithelial cells. Proteins extracted from these cells were then separated by two-dimensional gel electrophoresis (2-DE). The limited amounts of protein in the LCM procured samples necessitated the detection of 2-DE resolved proteins by silver staining. Consequently, loading equivalent amounts of protein onto gels was essential. However, we found that conventional means of measuring total protein in the samples were not sufficiently accurate. We therefore adopted a strategy in which the samples were first separated by one-dimensional sodium dodecyl sulphate-polyacryl-amide gel electrophoresis, stained with silver stain and subjected to densitometry. Evaluation of the staining intensity was then used to normalise the samples. We found that the protein profiles from undissected normal pancreas and LCM-acquired nonmalignant ductal epithelial cells from the same tissue block were different, underpinning the value of LCM in our analysis. The comparisons of protein profiles from nonmalignant and malignant ductal epithelial cells revealed nine protein spots that were consistently differentially regulated. Five of these proteins showed increased expression in tumor cells while four showed diminished expression in these cells. One of the proteins displaying enhanced expression in tumor cells was identified as the calcium-binding protein, S100A6. To determine the incidence of S100A6 overexpression in pancreatic cancer, we carried out immunohistochemical analysis on sections from a pancreas cancer tissue array containing 174 duplicate normal and malignant pancreatic tissue samples, from 46 pancreas cancer patients. Normal pancreatic ductal epithelia were either devoid of detectable S100A6 or showed weak expression only. Moderately or poorly differentiated tumors, by contrast, showed a higher incidence and a higher level of S100A6 expression. These observations indicate that the combination of LCM with 2-DE provides an effective strategy to discover proteins that are differentially expressed in PDAC.
引用
收藏
页码:1988 / 2001
页数:14
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