Validity of PCNA immunostaining in normal and tumor cells: Comparison with Ki-67 labeling

We analyzed the relationship between tumor proliferation and expression of PCNA, whose role as a proliferative marker remains controversial. Bivariate DNA/PCNA flow cytometric analysis of both clonogenic cell lines and several human tumor and normal samples were compared to Ki-67 immunostaining. Because there are 2 populations of PCNA in cells, a replicon-bound and a free nucleoplasmic form, PCNA labeling was strongly affected by the cell fixation procedure used poor with paraformaldehyde, strong and uniform with methanol, and variable with acetone/methanol. Only the acetone/methanol fixation demonstrated good correlation between S-phase specificity and cell proliferation marker. Cells blocked in specific cell cycle phases were distinguished by different staining levels; PCNA expression was detectable from early G1, increased in late G1, reached a maximum in S, and remained high in G2M. Because of its long half-life, residual levels of PCNA were still detectable in G0, whereas expression of the proliferative marker Ki-67, expressed later in the cell cycle than PCNA, was very weak or even undetectable in G0 cells and in early G1 cells. Staining levels of PCNA in tumor cells were always higher than in normal cells whatever their origin. Similarly, resting normal lymphocytes displayed lower PCNA levels than those observed in leukemia lymphoblasts. Fewer normal cells stained with Ki-67: PCNA labeling tended to give an overestimation of the growth fraction. Comparison between PCNA and Ki-67 labeling showed a linear correlation; but when compared in S-phase fraction, Ki-67 performed better than PCNA. PCNA may be used with caution and attention to fixation. It may be difficult to distinguish between proliferative and newly quiescent cells, because of its residual prolonged expression.