ADDITION OF CALMODULIN ANTAGONISTS TO NRK CELLS DURING G1 INHIBITS PROLIFERATING CELL NUCLEAR ANTIGEN EXPRESSION

The mRNAs of most proteins involved in DNA synthesis show an S phase c orrelated expression when mammalian cells are stimulated to proliferat e from G0. This is the case for proliferating cell nuclear antigen (PC NA), a cofactor of DNA polymerase delta that is essential for the synt hesis of the leading and tagging strands of DNA. Normal rat kidney cel ls re-entering the cell cycle from quiescence start DNA synthesis at 1 2 h and reach a maximum at 20 h. The expression of PCNA parallels the synthesis of DNA, Progression through the S phase was inhibited by add ition of the anticalmodulin drug W13 to the cells during G1, 5 h after activation. W13 also inhibited the increase in both PCNA protein and mRNA indicating that calmodulin regulates its expression, Using TK(-)t s13 cells transfected with a plasmid containing the thymidine kinase g ene under the control of the human 2.8 kb PCNA promoter, we demonstrat ed that this promoter is not regulated by calmodulin. The half-life of PCNA mRNA during G1/S transition was not modified by the treatment wi th W13, indicating that the decrease in the mRMA found when calmodulin was inhibited is not due to changes in its stability. Run-on assays r evealed that control cells produced predominantly complete PCNA transc ripts during S phase, while short incomplete transcripts were generate d in W13-treated cells at the same time. These results indicate that c almodulin participates in a more direct or indirect way during G1 in t he activation of PCNA expression, From data presented here it can be s uggested that calmodulin activates the release of a transcriptional bl ock reading to an increase in the amount of PCNA during S phase.