Cyclic AMP binding proteins and ras p21 oncogene expression in human colorectal cancer and mucosa

Abstract

A competitive cyclic AMP binding assay was developed and verified to allow reliable and reproducible measurement of cyclic AMP binding levels within benign and malignant human colorectal tissues. Cyclic AMP binding was determined in cytosols derived from the centre and periphery of 69 human colorectal cancers and adjacent and distant mucosa gathered from a series of patients undergoing elective surgery for colorectal cancer. Cyclic AMP binding levels were significantly higher in the periphery of tumour than in either adjacent or distant mucosa. Binding levels in tumour centre were the same as those in adjacent mucosa but significantly lower than those in distant mucosa. Within both the periphery and centre of tumour low levels of cAMP binding correlated with advanced disease in terms of stage and grade. Binding levels in all four specimens were also highly related within individuals.

Cyclic AMP binding protein isotype (RI and RII) expression was determined in cytosols derived from the centre and periphery of 32 human colorectal cancers and from related adjacent and distant mucosa by means of [32p]-8-azido-cAMP photoaffinity labelling, polyacrylamide gel electrophoresis, autoradiography and laser densitometry. A single form of RI was identified with a molecular weight of 48kDa (RI-48). There were two forms of RII with molecular weights of 52 and 54kDa (RII- 52 and RII-54). Both the centre and periphery of colorectal cancers expressed significantly more RI than either adjacent or distant mucosa. Tumours of poor histological grade expressed significantly more RI than tumours that were either well or moderately differentiated. High levels of RI were associated with a virtual absence of RII-52. R subunits in membrane enriched preparations of colorectal cancers and mucosa were also characterised in terms of molecular weight and found to be similar to those within cytosol preparations. There was a significant negative correlation between total cAMP binding and RI expression in both the centre and periphery of tumour but not in either adjacent or distant mucosa.

By means of Western blotting using Y13-259 antibody, ras p21 levels were determined in membrane preparations derived from the periphery of 21 human colorectal cancers and related adjacent and distant mucosa. Levels of p21 were lower in tumour than in adjacent mucosa but did not relate to stage or grade of disease. There was a significant negative correlation between p21 levels and RI expression in tumour but not mucosal preparations. In conclusion, total cAMP binding levels and RI expression are related to stage and histological grade of human colorectal cancer; and are related to the level of ras p21 expression within tumours but not within benign colorectal mucosa.