Validity of PCNA immunostaining in normal and tumor cells: Comparison withKi-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 severa l human tumor and normal samples were compared to Ki-67 immunostaining. Bec ause there are 2 populations of PCNA in cells, a replicon-bound and a free nucleoplasmic form, PCNA labeling was strongly affected by the cell fixatio n procedure used poor with paraformaldehyde, strong and uniform with methan ol, and variable with acetone/methanol. Only the acetone/methanol fixation demonstrated good correlation between S-phase specificity and cell prolifer ation marker. Cells blocked in specific cell cycle phases were distinguishe d by different staining levels; PCNA expression was detectable from early G 1, 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 detectabl e in G0, whereas expression of the proliferative marker Ki-67, expressed la ter in the cell cycle than PCNA, was very weak or even undetectable in G0 c ells and in early G1 cells. Staining levels of PCNA in tumor cells were alw ays higher than in normal cells whatever their origin. Similarly, resting n ormal lymphocytes displayed lower PCNA levels than those observed in leukem ia lymphoblasts. Fewer normal cells stained with Ki-67: PCNA labeling tende d to give an overestimation of the growth fraction. Comparison between PCNA and Ki-67 labeling showed a linear correlation; but when compared in S-pha se fraction, Ki-67 performed better than PCNA. PCNA may be used with cautio n and attention to fixation. It may be difficult to distinguish between pro liferative and newly quiescent cells, because of its residual prolonged exp ression.