Altered expression of serum N-glycoproteins in colorectal cancer patients detected by two-dimensional electrophoresis.

A.M. Rodríguez-Piñeiro, BSc , D. Ayude, PhD, M. Páez de la Cadena, PhD, M.F. Montenegro, BSc, M. Acuña, BSc, F.J. Fraíz, PhD, and F.J. Rodríguez-Berrocal, PhD.

Department of Biochemistry, Genetics and Immunology, University of Vigo, Vigo Pontevedra, Spain.

AIM: Colorectal cancer is one of the most common cancers in males and females, and represents the second cause of death in Western countries. Due to the almost completely lack of symptoms, this neoplasia is often diagnosed when metastasis has already spread. On the other hand, alterations in serum constituents have been used for many years as indicators of the presence of malignancy. Specifically, cell glycoproteins can be released into the circulation through increased turnover, secretion or shedding from the malignant cells and have been considered as potential tumour markers. In this work, serum N-glycoproteins from healthy donors and colorectal cancer patients have been detected and compared in order to analyse their expression. METHODS: N-glycoproteins from filtered sera were selectively eluted through a Concanavalin A-Sepharosa (Con A) column with 0.5 M alpha-methylmannopiranoside. These fractions were separated by charge in a protean IEF system using 17 cm strips with pH gradient 4-7. Then, they were transferred onto 9-16% gradient polyacrylamide gels to perform the second dimension. At the end of the SDS-PAGE, gels were stained either with silver nitrate or coomassie blue, scanned and analysed with suitable software. RESULTS AND CONCLUSIONS: A linear regression analysis has been done among two maps of the same sample in different days, in order to test the reproducibility of the technique, showing a correlation coefficient of 0.86. Then, we have compared 2D-maps from four control and four case samples. Due to the variability among healthy donors, we have normalised the intensity signals on the basis of some unvaried spots. Qualitative and semi quantitative differences could be observed when comparing the same areas of maps from colorectal cancer patients against controls. E.g., some high molecular spots appear in patients, whereas they are nearly absent in controls. We also have found a number of protein species that are, at least, 10 times increased or decreased in maps from patients in relation with donors.

For more information, contact

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Paris, France; February 9 - 12, 2002; in the section on Diagnostic Markers.