Bromodeoxyuridine Effects on the RNA Sequence Complexity and Phenotype in a Neuroblastoma Clone

Abstract

The effects on morphology, activities of cholinergic enzymes, and RNA sequence complexity were measured in a subclone of the cholinergic mouse neuroblastoma line NS20 after treatment of the cells with 5-bromo-2′-deoxyuridine or with other conditions which have been reported to cause ‘differentiation’ in neuroblastoma cells. Treatment with bromodeoxyuridine caused a flattening of the cells so that they resembled fibroblasts, a marked increase in the activity of choline acetyltransferase, and little change in cholinesterase activity. In medium without 5-bromo-2′-deoxyuridine but with a low content (0.1%) of serum, cells attained a more neuronal morphology, although choline acetyltransferase activity was unaffected and cholinesterase activity was decreased. Treatment with dibutyryl-cyclic-3’: 5′-AMP affected neither the cellular morphology nor the activities of the cholinergic enzymes. When neuroblastoma RNA preparations were hybridized to single-copy mouse DNA, there were no measurable differences in total RNA sequence complexities between control and 5-bromo-2′-deoxyuridine-treated cells. For whole cell and nuclear RNAs, the reaction kinetics demonstrated the presence of single abundance classes of RNA which hybridized with approximately 6% of the total single-copy DNA. Poly(A)-containing mRNAs of 5-bromo-2′-deoxyuridine-treated and control cells both hybridized to approximately 2.1% of the single-copy DNA, with kinetics which were consistent with the presence of two major RNA abundance classes. The single-copy DNA sequences expressed in the nuclear RNA of 5-bromo-2′-deoxyuridine-treated cells were partially purified and then re-reacted with RNAs from treated and control cells. Approximately one-third of the nuclear RNA sequences expressed in 5-bromo-2′-deoxyuridine-treated cells were absent from, or at a much lower concentration in, control cells. Since the RNA complexities of control and 5-bromo-2′-deoxyuridine-treated cells were equivalent, the changes produced by 5-bromo-2′-deoxyuridine must have involved the activation of transcription of some sequences in the genome and repression of a similar number of sequences.