DNA intercalators, actinomycin D (Act D) and Adriamycin (Doxo) possess anti-cancer activity through the inhibition of DNA topoisomerase II. It is reported that Doxo induces histone eviction from open chromatin. In this study, we assessed the effects of evicted histones on gene expression in an artificial wound healing system using cultured human keratinocyte cells HaCaT. Act D or Doxo was added to the medium from 5 minutes prior to the artificial wounding until 25 minutes after, and cells were cultured for a total of 6 hours after the wounding. Total RNAs were isolated both from whole cells and polysomal fractions (TLN) and used in differential gene expression analysis by DNA microarray. The DNA microarray analysis revealed that Act D and Doxo regulated the ribosome loading to mRNAs of histones, centromere-related proteins and other chromatin associated proteins. In those proteins, the subcellular distribution of histones H1, H2A.1, H2B, phospho-H2A.X and acetyl-H3 were assessed by western blot analysis. Thirty minute treatment of Act D or Doxo induced an increase of histones H2B in nuclear plasm fractions and heavy TLN (over 6 ribosomes), although 6 hour treatment induced accumulation of H2A.1 and phospho-H2A.X in mRNP fractions. Immunocytochemical analysis revealed that H2A.1, H2B and phospho-H2A.X existed both in nuclei and in cytosolic granules after 6 hour treatment of Act D or Doxo. Stem loop binding protein which binds to the stem loop structure of histone mRNA, and centromere-related kinesin family member 11 and 14 were co-distributed with H2A.1, H2B and phospho-H2A.X. These results suggest that Act D and Doxo are able to exert a coordinated translational control of the expression of histones and centromere-related genes.
