Immunochemical detection of the proliferating cell nuclear antigen (PCNA/cyclin) in ocular tissues
Yamaguchi, K.; Stell, W.K.
Nippon Ganka Gakkai Zasshi 96(8): 954-958
1992
ISSN/ISBN: 0029-0203 PMID: 1355628 Document Number: 394267
The authors developed radioimmunoassay (RIA) for proliferating cell nuclear antigen (PCNA/cyclin), which a protein synthesizes in phase with DNA, as a marker for cell replication. We used it to detect the amounts of PCNA and estimated rates of cell replication in ocular tissues (cornea, lens epithelium, retina-plus-choroid and optic nerve) and in non-ocular tissues (skin, skeletal muscle, brain, thymus, small intestine and liver) in rats. Antisera were raised in rabbits to oligopeptide fragment of human PCNA (hPCNA): the C-terminal fragment (Tyr-254)-hPCNA (254 apprx 261). For RIA the synthetic peptides were used as standards and radioiodinated (125I-Tyr-254)-hPCNA (254 apprx 261) as radioligands. The IC-50 was 30 apprx 40 f mole/tube with a sensitivity 0.5 apprx 10 f mole/tube for this assay. Tissue extracts in 1 M acetic acid were digested with trypsin to release fragment (255 apprx 261) from whole PCNA and were assayed by this method. The highest amounts of immunoreactive PCNA were found in lens epithelium and skin, while the lowest amounts were found in optic nerve and muscle. The cornea showed a higher value than retina-plus-choroid or optic nerve. A high cell replication in the lens epithelium was revealed.