COMPARISON OF PROLIFERATING CELL NUCLEAR ANTIGEN TO TRITIATED-THYMIDINE AS A MARKER OF PROLIFERATING HEPATOCYTES IN RATS

Proliferating cell nuclear antigen (PCNA), an endogenous nuclear protein, has recently been used to identify replicating cells. PCNA was compared to tritiated thymidine ([H-3].TdR), a reliable and accurate exogenous labeling agent, to ascertain if PCNA gives comparable results for quantitative cell proliferation. Male F344 rats were treated with a single dose of 500 mg/kg 4-acetylaminofluorene (4-AAF), a known liver mitogen. Rats (n = 5) were euthanized and necropsied at 6, 12, 18, 24, 36, 48, 96, or 192 hr after treatment. Two hours before necropsy, rats were pulse-dosed with [H-3]-TdR (2 mCi/kg body weight). Livers were sectioned, autoradiography performed, and labeling indexes (LI), a measurement of the percentage of S-phase hepatocytes, determined. One and a half years after the completion of this study, the archival paraffin blocks of the liver tissue were sectioned and stained for PCNA by an immunohistochemical procedure. Immunocytochemical staining patterns of proliferating cell nuclear antigen expression permitted the recognition of G(1), S, G(2),escent cells. PCNA LI, generated by scoring only cells exhibiting S-phase staining patterns, was compared to the pulse [H-3]-TdR LI for each animal. Similar periportal staining patterns of S-phase nuclei were detected by both markers. The [H-3]-TdR LI and the PCNA LI exhibited a peak at 24 hr of approximately the same magnitude. However, while the [H-3]-TdR LI had returned to near baseline at the 48-hr time point, the PCNA LI remained elevated until the 96-hr time point. This sustained elevation of the PCNA index cannot be explained at this time. Examination of all other time points revealed similar S-phase LI by either method. PCNA immunostaining allowed for the estimation of the growth fraction. A time-dependent alteration in the hepatic growth fraction curve was a consequence of 4-AAF treatment.