Abstract
Photoaffinity labelling (PAL) with [32P]8-azido-cAMP and polyacrylamide gel electrophoresis (PAGE) has been used to identify three specific cAMP-binding proteins (cAMP-BPs) within cytosols derived from the centre and periphery of 32 human colorectal cancers and from related adjacent (less than 5 cm from the tumour) and distant (more than 5 cm from the tumour) microscopically benign mucosa. By immunoprecipitation with specific anti-RI and anti-RII antibodies these proteins have subsequently been characterised as a single form of RI (48 kDa) and two forms of RII (50 and 52 kDa). The relative expression of isoforms in each specimen has been quantified by laser densitometry. There was significantly more RI expressed in both tumour centre and periphery than in either adjacent or distant mucosa (P < 0.008 by Wilcoxon signed-rank test). There was no significant difference in relative RI expression between tumour centre and periphery, or between adjacent and distant mucosa. There was no association between relative RI expression and Dukes' stage. Poorly differentiated tumours expressed significantly more RI than those that were either moderately or well differentiated (P = 0.016 by Mann-Whitney U-test). This study is the first to have characterised cAMP-BPs within human colorectal tissues and has demonstrated that colorectal cancers, and in particular those of poor histological grade, relatively overexpress RI when compared with related benign mucosa.
Highlights
Molecular weights of cAMP-binding proteins Several distinct cAMP-BPs were identified in cytosols derived from both tumours and normal mucosa (Figure 1)
In all tissues examined the predominant forms were R52, R50 and R48, with the other forms contributing less than 15% of the total cAMP-BPs present
In most tissues examined a protein of 65 kDa was found to bind [32P]8-azido-cAMP and was termed R65
Summary
Specimens were obtained from 32 patients undergoing elective surgery for colorectal cancer, from which sufficient material was available for study after histological confirmation of disease. Operative specimens were obtained fresh from theatre and kept on ice until processed. Samples were taken from tumour centre and periphery and from both adjacent and distant macroscopically normal mucosa. Attempts were made to avoid obviously necrotic or haemorrhagic areas. Operative specimens were routinely processed for diagnosis, stage and grade (Dukes, 1932). Individual tumour and mucosa samples were examined histologically to confirm their malignant or benign nature. Samples were stored at -70C until use
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