Effect of 5-bromodeoxyuridine on the transcriptional properties of the genome in WI-38 human diploid fibroblasts

Abstract

Growth of WI-38 diploid fibroblasts in a medium containing 5-bromodeoxyuridine (BrdU) resulted in an increased GMP and a decreased AMP incorporation into the RNA synthesised in vitro on a chromatin template. This effect was similar to that previously reported using 3T6 mouse fibroblasts-1. Substitution of thymidine by BrdU in DNA, also altered the characteristics of the DNA template itself, since the increased incorporation of guanine and decreased incorporation of adenine into RNA were evident also when purified, isolated DNA was used as template. The extent of replacement of AMP by GMP was proportional to the extent of replacement of thymidine by BrdU. Although there are variations in the base composition of RNA transcribed from BrdU-containing DNA templates, there are no significant difference in overall template activity or in the number of available chromatin binding sites for E. coli RNA polymerase. Confluent monolayers of BrdU-treated WI-38 fibroblasts are still able to respond with cell proliferation to a change of medium, as evidenced by an increased incorporation of (-3H)thymidine and an increase in chromatin template activity. The length of the prereplicative phase is similar in both BrdU-treated and untreated cells, although the magnitude of the increase of (-3H)thymidine incorporation is reduced by approximately 30% after BrdU treatment. The increase in chromatin template activity is associated with an increase in the number of chromatin binding sites for E. COLI RNA polymerase, suggesting that the presence of BrdU does not interfere with the availability of initiation sites or alter the actual rate of transcription.