Abstract
Ectopic expression of DNA methyltransferase transforms vertebrate cells, and inhibition of DNA methyltransferase reverses the transformed phenotype by an unknown mechanism. We tested the hypothesis that the presence of an active DNA methyltransferase is required for DNA replication in human non-small cell lung carcinoma A549 cells. We show that the inhibition of DNA methyltransferase by two novel mechanisms negatively affects DNA synthesis and progression through the cell cycle. Competitive polymerase chain reaction of newly synthesized DNA shows decreased origin activity at three previously characterized origins of replication following DNA methyltransferase inhibition. We suggest that the requirement of an active DNA methyltransferase for the functioning of the replication machinery has evolved to coordinate DNA replication and inheritance of the DNA methylation pattern.
Highlights
Aberrant patterns of DNA methylation are observed in many cancer cells, and these changes occur in parallel with hyperactivation of DNA methyltransferase (DNMT-1)1 [1, 2]
Antisense oligonucleotides and an adenovirus expressing DNMT-1 antisense mRNA were use to test the hypothesis that the inhibition of DNMT-1 directly affects the growth of A549 cells by inhibiting DNA replication
Inhibitors of DNMT-1 Slow Cell Growth, the Progression through the Cell Cycle, and the Rate of DNA Replication—We have previously demonstrated that the treatment of A549 cells with direct inhibitors of DNMT-1 results in an inhibition of their anchorage-independent growth [15]
Summary
Aberrant patterns of DNA methylation are observed in many cancer cells, and these changes occur in parallel with hyperactivation of DNA methyltransferase (DNMT-1)1 [1, 2]. To investigate how the inhibition of DNMT-1 results in the inhibition of tumorigenesis, we have developed phosphorothioate-modified hemimethylated oligonucleotides that, in the presence of a lipophilic carrier, can enter into the nucleus of cancer cells in culture, form a stable complex with DNMT-1, and inhibit its activity with an EC50 of approximately 60 nM [15, 16]. We have developed an inactive analog of this phosphorothioate hemimethylated inhibitor of the same sequence, which does not form a stable complex with DNMT-1 and does not inhibit its activity, that can serve as an experimental control [15, 16]. Antisense oligonucleotides and an adenovirus expressing DNMT-1 antisense mRNA were use to test the hypothesis that the inhibition of DNMT-1 directly affects the growth of A549 cells by inhibiting DNA replication
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