Proliferating Cell Nuclear Antigen: A Marker for Hepatocellular Proliferation in Rodents

Abstract

Two different markers for quantitating cell proliferation were evaluated in livers of control and chemically treated mice and rats. Proliferating cell nuclear antigen (PCNA), an endogenous cell replication marker, and bromodeoxyuridine (BrdU), an exogenously administered DNA precursor label, were detected in formalin-fixed, paraffin-embedded tissues using immunohisto-chemical techniques. The percentage of cells in S phase (labeling indexes, LI) evaluated as PCNA-or BrdU-positive hepatocellular nuclei was compared in recut tissue sections from animals given BrdU by a single IP injection 2 hr before killing the animals. Ten-week-old male B6C3F, mice and F344 rats were exposed to known mitogenic hepatocarcinogens, Wy-14,643 (WY) in the diet at 0.1% for 2 days or 1,4-dichlorobenzene (DCB) in corn oil by gavage for 2 days (600 mg/kg/day in mice; 300 mg/kg/day in rats). In mice, PCNA and BrdU hepatocyte LI were similar in control, WY-treated, and DCB-treated animals. In rats, PCNA and BrdU gave similar LI in controls and Wy-treated animals. Although PCNA LI was statistically lower than BrdU LI in DCB-treated rats, both PCNA and BrdU LI for DCB-treated rats was increased over LI in control rats. Different patterns of PCNA immunohistochemical staining, interpreted to represent different subpopulations of cells at various phases of the cell cycle, were quantitated using PCNA immunohistochemistry. The proliferating index (PI), defined as the percentage of cells in the cell cycle (GI + S + G2 + M), was more sensitive than the LI (S phase only) in detecting a chemically induced cell proliferative response. Due to reports of adverse effects of BrdU on cell proliferation , PCNA immunohistochemical methods were used to determine the effect of duration of exogenously administered DNA precursor label (BrdU or [3H]-thymidine [3H-TdRl) on rodent hepatocyte proliferation measurements. PCNA LI were determined in control animals pulse labeled with BrdU or 3H-Tdr, or labeled continuously for 3 or 6 days. PCNA LI did not increase with duration of exposure to BrdU or 3H-Tdr, suggesting that these labeling conditions are not causing a hepatocellular proliferative response. These results demonstrate comparable hepato-cyte labeling of cells in S phase in control and chemically treated mice and rats with PCNA and pulse-BrdU labeling methods, supporting the use of PCNA as an alternative marker in either retrospective or prospective cell proliferation studies.

Comparison of Acute Hepatocellular Proliferating Cell Nuclear Antigen Labeling Indices and Growth Fractions, p34cdc2 Kinases, and Serum Enzymes in Carbon Tetrachloride-Treated Rats

“…3) (31). The increased sensitivity of PCNA-GF in our study was attributed to the detection of more cells in the cell cycle (G1 + S + G2 + M), as compared to PCNA-LI, in which only S-phase cells are quantified, thereby allowing a &dquo;larger window&dquo; of observation of the cell cycle ( 11 ). PCNA-GF may also be a more reliable and reproducible parameter than the PCNA-LI because all &dquo;labeled&dquo; cells are quantified, thereby avoiding subjective interpretations in defining cell cycle stage based on staining distribution and intensity.…”

“…An advantage of PCNA detection is that the immunohistochemical technique can be performed retrospectively on previously formalin-fixed, paraffin-embedded material (16). Moreover, PCNA tissue analysis has the potential to aide in the identification of cells in the different stages of the cell cycle (11,14).…”

“…PCNA-GF may also be a more reliable and reproducible parameter than the PCNA-LI because all &dquo;labeled&dquo; cells are quantified, thereby avoiding subjective interpretations in defining cell cycle stage based on staining distribution and intensity. Eldridge et al (11) also reported an increased sensitivity of hepatic PCNA-GF, as compared to PCNA-LI, in Fischer-344 rats exposed to WY14,643 ([4-chloro-6-(2-3-xylidino)-2-pyrimidinylthio] ace- (4). It has been hypothesized that hepatocellular division occurring after toxic insult is due to the induction of cells into G, and the cell cycle or, possibly, the activation of hepatocytes blocked in the G2 phase of the cell cycle (5).…”

p-Nitrobenzoic Acid α2u Nephropathy in 13-week Studies is not Associated with Renal Carcinogenesis in 2-year Feed Studies

“…Selected doses (0, 5,000, 10,000 ppm) in the 13-week study were chosen for PCNA evaluation; these included the high doses from the 13-week and 2-year studies. Positive staining for PCNA was categorized based on cellular distribution and intensity of the chromagen that correlates with the different phases of the cell cycle as reported by Foley et al (10) and Eldridge et al (9). Uniform dark nuclear staining was judged to be positive for S-phase cells.…”

Inhibitory Effects of S‐Methylcysteine and Cysteine on the Promoting Potential of Sodium Phenobarbital on Rat Liver Carcinogenesis

“…19,20) Immunohistochemical analysis of glutathione S-transferase placental form (GST-P)-positive foci, one of the widely accepted end-point markers of hepatocellular carcinoma, 19) was conducted. In addition, the effects of the compounds on proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, 21) and on ornithine decarboxylase (ODC), a rate-limiting enzyme of polyamine metabolism which is induced by promoters, 22,23) were examined.…”