DOXORUBICIN-INDUCED ID2A GENE-TRANSCRIPTION IS TARGETED AT AN ACTIVATING TRANSCRIPTION FACTOR CYCLIC-AMP RESPONSE ELEMENT MOTIF THROUGH NOVEL MECHANISMS INVOLVING PROTEIN-KINASES DISTINCT FROM PROTEIN-KINASE-CAND PROTEIN-KINASE-A

We have recently shown that doxorubicin (Dor;), an antineoplastic drug and an inhibitor of terminal differentiation of myogenic and adipogen ic cells, induces expression of Id, a gene encoding a helix-loop-helix transcriptional inhibitor, In this study we have investigated the mol ecular mechanisms underlying Dox-induced Id2A expression. We have also attempted to determine whether the genetic responses to Dox are relat ed to the UV response, a well-characterized set of reactions to UV and DNA-damaging compounds that is partly mediated by AP-1. Transient tra nsfection of a series of deletions and point mutation derivatives of t he human Id2A promoter sequence shows that two closely spaced and inve rted short elements similar to an activating transcription factor (ATF ) binding site or a cyclic AMP response element (CRE) are necessary an d sufficient for a full response to Dox. We refer to this element as t he IdATF site, Sequences containing an IdATF site conferred Dox induci bility on a minimal heterologous promoter, tin electrophoretic mobilit y shift assay showed nuclear proteins specifically interacting with th e IdATF sequence, While oligonucleotides containing either legitimate ATF/CRE or AP-1 binding sequences competed for binding, antibody super shift experiments suggested that neither CREB/ATF-1 nor AP-1 are major factors binding to IdATF, Several independent criteria suggest that D ox inducibility was independent of Ca2+/phospholipid-dependent protein kinase (protein kinase C), cyclic AMP-dependent protein kinase (prote in kinase A), and tyrosine kinase. Moreover, we found that Dox also in duces transcription from promoters of immediate-early genes through an AP-l-independent pathway, Taken together, our results suggest that Do x elicits a novel genetic response distinct from the classical UV resp onse.