Image analysis of in situ cell cycle related changes of PCNA and Ki‐67 proliferating antigen expression

. This study reports on the proliferating cell nuclear antigen (PCNA) and Ki‐67 cell cycle related expression and distribution pattern analysed in the same cells. MCF‐7 cells were synchronized by mitotic detachment and triple stained for DNA, PCNA and Ki‐67. The major cell type was identified on each time sample as a function of the PCNA/Ki‐67 pattern, and both antigens as well as DNA were quantified. During G 1 phase, the expression of PCNA greatly increased whereas Ki‐67 content decreased. During S phase, nuclear Ki‐67 content continuously increased especially in the second half of this phase, mainly due to the accumulation of the antigen in the nucleoli. During G 2 phase, the antigen significantly passed into the nucleoplasm, its content continued to increase and reached its maximum in mitotic cells. Nuclear PCNA content mostly increased in the first part of S phase and sharply declined in mitotic cells as the antigen shifted to the cytoplasm. Cells showing PCNA positive Ki‐67 negative labelling were observed in all time samples from the beginning of the experiment. Their nuclear size, DNA content (of G 1 cells), PCNA content (equivalent to the content of some late G, cells) and time occurrence (their percentage increased after the last late G 1 cells had disappeared) tend to indicate that these cells have left the cycle by the end of G 1 phase to enter a quiescent state. Cells coming out of mitosis split into two groups according to their Ki‐67/PCNA content. The biggest fraction was PCNA negative and Ki‐67 positive while the smallest showed positive staining for both antibodies. Cells of this second cohort slowly lost their 1–67 while their PCNA content increased as they moved through G 1 . Concurrently, most of the cells of the first cohort (here called Q2 and Q3 cell types) lost their Ki‐67 without increasing their PCNA content; then they joined cells of the second cohort by increasing their PCNA content at the end of G, phase. Some cells of this first cohort can also increase their PCNA and thus reach cells of the first cohort before the end of G 1 phase. The existence of these two main cell cohorts suggests that cells after mitosis differ in some way that make them progress dlfferently through G 1 . Some cells seem to go through early G 1 (G 1a and late G 1 (G lb ) while others may come out of mitosis committed to go through the following cycle by directly entering late G 1 compartment.