FLOW CYTOMETRIC ANALYSIS OF THE EXPRESSION OF PCNA DURING THE CELL-CYCLE IN HELA-CELLS AND EFFECTS OF THE INHIBITION OF DNA-SYNTHESIS ON IT

Flow cytometric bivariate DNA/PCNA analysis was performed to investiga te the expression of PCNA during the cell cycle and the implication in DNA replication in HeLa cells, using a monoclonal antibody (PC10) to PCNA. The expression of PCNA was evident in almost all cells growing e xponentially, when cells were fixed in methanol. The total amount of P CNA altered a little during the cell cycle. However, the treatment wit h Triton X-100 extracted 80-89% of total PCNA from the cells, resultin g in the dramatic change of bivariate DNA/PCNA distribution pattern. P CNA was completely removed from nuclei in both GI and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S p hase nuclei. After the treatment of cells with Triton X-100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribu tion pattern in cells treated with Triton X-100 was strikingly so simi lar to the DNA/BrdUrd distribution pattern that it was unable to diffe rentiate one from the other. It is concluded that the detergent treatm ent of cells allows the rapid analysis of the cell cycle. The inhibiti on of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA cont ent mainly due to the increase in the fraction of the detergent extrac table PCNA. It was apparent, however, that in cells incubated with Tri ton X-100, the pattern of the bivariate DNA/PCNA distribution was not basically different from that in cells without HU treatment. The level of PCNA bound to nuclear structures (PCNA not extracted with detergen t) increased in cells arrested at the G1/S boundary with the time of h ydroxy-urea treatment. Eventually, it became difficult to differentiat e between cells in the Gl/S boundary and the early S phase on the basi s of DNA and PCNA contents. However, the increase in bound PCNA was mi nimal in S phase cells. The observation suggests that PCNA is bound to the potential initiation sites of DNA replication together with DNA r eplication associated enzymes in advance of the commencement of DNA re plication and that it is never associated with late replicating DNA wi thout the duplication of early replicating DNA. (C) 1993 Wiley-Liss, I nc.