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 correlated expression when mammalian cells are stimulated to proliferate from G0. This is the case for proliferating cell nuclear antigen (PCNA), a cofactor of DNA polymerase delta that is essential for the synthesis of the leading and lagging strands of DNA. Normal rat kidney cells re-entering the cell cycle from quiescence start DNA synthesis at 12 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 addition 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-ts13 cells transfected with a plasmid containing the thymidine kinase gene under the control of the human 2.8 kb PCNA promoter, we demonstrated that this promoter is not regulated by calmodulin. The half-life of PCNA mRNA during G1/S transition was not modified by the treatment with W13, indicating that the decrease in the mRNA found when calmodulin was inhibited is not due to changes in its stability. Run-on assays revealed that control cells produced predominantly complete PCNA transcripts during S phase, while short incomplete transcripts were generated in W13-treated cells at the same time. These results indicate that calmodulin participates in a more direct or indirect way during G1 in the activation of PCNA expression. From data presented here it can be suggested that calmodulin activates the release of a transcriptional block leading to an increase in the amount of PCNA during S phase.
López-Girona
A
A
Departament de Biologia Cellular, Facultat de Medicina, Universitat de Barcelona, Spain.
Bosch
M
M
Bachs
O
O
Agell
N
N
D000818:Animals; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D004247:DNA / Q000096:biosynthesis; D016193:G1 Phase / Q000187:drug effects* / Q000235:genetics; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008647:Mesocricetus; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics*; D011401:Promoter Regions, Genetic; D051381:Rats; D013449:Sulfonamides / Q000494:pharmacology
D002147:Calmodulin; D018809:Proliferating Cell Nuclear Antigen; D013449:Sulfonamides; C040184:W 12; C033772:N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; D004247:DNA