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

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Abstract
Flow cytometric bivariate DNA/PCNA analysis was performed to investigate 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 exponentially, when cells were fixed in methanol. The total amount of PCNA altered a little during the cell cycle. However, the treatment with Triton X-100 extracted 80–89% of total PCNA from the cells, resulting in the dramatic change of bivariate DNA/PCNA distribution pattern. PCNA was completely removed from nuclei in both G1 and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S phase nuclei. After the treatment of cells with Triton X-100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribution pattern in cells treated with Triton X-100 was strikingly so similar to the DNA/BrdUrd distribution pattern that it was unable to differentiate one from the other. It is concluded that the detergent treatment of cells allows the rapid analysis of the cell cycle. The inhibition of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA content mainly due to the increase in the fraction of the detergent extractable PCNA. It was apparent, however, that in cells incubated with Triton 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 detergent) increased in cells arrested at the G1/S boundary with the time of hydroxyurea treatment. Eventually, it became difficult to differentiate between cells in the G1/S boundary and the early S phase on the basis of DNA and PCNA contents. However, the increase in bound PCNA was minimal in S phase cells. The observation suggests that PCNA is bound to the potential initiation sites of DNA replication together with DNA replication associated enzymes in advance of the commencement of DNA replication and that it is never associated with late replicating DNA without the duplication of early replicating DNA.

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