MODULATION OF HUMAN DNA METHYLTRANSFERASE ACTIVITY AND MESSENGER-RNA LEVELS IN THE MONOBLAST CELL-LINE U937 INDUCED TO DIFFERENTIATE WITH DIBUTYRYL-CYCLIC-AMP AND PHORBOL ESTER

The regulation of DNA (cytosine-5) methyltransferase (DNA MeTase) enzy me activity and gene expression was examined in the monoblastoid U937 cell line induced to differentiate with either dibutyryl cyclic AMP (d bcAMP) or phorbol ester. dbcAMP treatment was found to cause the rapid (<4 h) suppression of DNA MeTase specific activity, with no DNA MeTas e activity detectable after 10 h. Equally, no DNA MeTase activity was detectable in nuclear extracts of fresh peripheral blood monocytes. Us ing both a U937 DNA MeTase cDNA and a mouse DNA MeTase cDNA as probes, steady-state levels of DNA MeTase mRNA were found to decline sharply between 4 and 15 h after dbcAMP treatment. No DNA MeTase mRNA was dete ctable after 20 h of dbcAMP treatment. Nuclear run-on analysis showed there to be only a small (40%) suppression of DNA MeTase gene transcri ption in cells treated with dbcAMP for 24 h, implying a role for post- transcriptional processes in the regulation of DNA MeTase mRNA levels. The observed decline in DNA MeTase activity/mRNA levels appeared to p recede the dbcAMP-induced arrest in DNA replication, as judged by the incorporation of tritiated thymidine into DNA. In contrast to the effe ct of dbcAMP, treatment of U937 cells with the phorbol ester 12-O-tetr adecanoyl phorbol-13-acetate (TPA) led to an overall stimulation of DN A MeTase specific activity. The TPA response was found to be complex a nd broadly consisted of an early (0-15 h) burst of DNA MeTase activity followed by a more gradual sustained increase in DNA MeTase activity after prolonged (16-40 h) TPA treatment. The early phase of high DNA M eTase activity was not mirrored by an increase in steady-state levels of DNA MeTase mRNA, as judged by Northern blot analysis. However, a su bstantial induction of DNA MeTase mRNA levels was observed after 20-24 h of TPA treatment. Nuclear run-on analysis showed this not to be due to any significant increase in DNA MeTase gene transcription. The obs erved increases in DNA MeTase activity/mRNA levels were observed whils t cells were undergoing deproliferation. Interestingly, the addition o f TPA and more physiological protein kinase C (PKC) activators, such a s diacylglycerol and phosphatidylserine, to DNA MeTase-enriched nuclea r extracts generated a 4.5-fold and a 1.5-fold increase in DNA MeTase specific activity respectively. The TPA-induced stimulation of DNA MeT ase activity could be inhibited by the PKC inhibitor H-9, implicating a role for PKC in the regulation of DNA MeTase activity in vivo.