Abstract

To elucidate the role of proliferating cell nuclear antigen (PCNA) in vivo, PCNA immunohistochemistry combined with 3H-thymidine autoradiography was performed on 2 types of regenerating rat livers. One group of rats underwent 2/3 partial hepatectomy (PH), and the other group 1/3 PH. After 3H-thymidine labeling, the livers were fixed with paraformaldehyde (PFA) and methanol, and paraffin-embedded. In methanol-fixed tissues, the serial changes of the PCNA-positive index (PCNA-PI) corresponded well with those of the 3H-thymidine-labeling index (3H-thymidine-LI) after each PH. In PFA-fixed tissues, however, the PCNA-PI was 26% before PH, when the 3H-thymidine-LI was below 1%. Furthermore, the 1/3 PH group, despite a continued low 3H-thymidine-LI, showed a rise in PCNA-PI to a level comparable to that in the 2/3 PH group. From these results, we propose 3 phases of PCNA expression. In PFA-fixed tissues, PCNA that was detectable before PH was considered pooled in G0-phase nucleoplasm (Phase I), and some or many of the PCNA-positive cells failed to enter the S-phase in the 1/3 PH group, reflecting the increased PCNA not bound to DNA replicons (Phase II). In methanol-fixed tissues, PCNA was detected only in the S-phase, indicating its direct involvement in DNA synthesis (Phase III). Therefore, determining PCNA-PI in methanol-fixed tissues is useful to evaluate proliferative activity, whereas that in PFA-fixed tissues must be assessed with great caution.

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