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 C and protein kinase A.
We have recently shown that doxorubicin (Dox), an antineoplastic drug and an inhibitor of terminal differentiation of myogenic and adipogenic cells, induces expression of Id, a gene encoding a helix-loop-helix transcriptional inhibitor. In this study we have investigated the molecular mechanisms underlying Dox-induced Id2A expression. We have also attempted to determine whether the genetic responses to Dox are related to the UV response, a well-characterized set of reactions to UV and DNA-damaging compounds that is partly mediated by AP-1. Transient transfection of a series of deletions and point mutation derivatives of the human Id2A promoter sequence shows that two closely spaced and inverted short elements similar to an activating transcription factor (ATF) binding site or a cyclic AMP response element (CRE) are necessary and sufficient for a full response to Dox. We refer to this element as the IdATF site. Sequences containing an IdATF site conferred Dox inducibility on a minimal heterologous promoter. An electrophoretic mobility shift assay showed nuclear proteins specifically interacting with the IdATF sequence. While oligonucleotides containing either legitimate ATF/CRE or AP-1 binding sequences competed for binding, antibody supershift experiments suggested that neither CREB/ATF-1 nor AP-1 are major factors binding to IdATF. Several independent criteria suggest that Dox inducibility was independent of Ca2+/phospholipid-dependent protein kinase (protein kinase C), cyclic AMP-dependent protein kinase (protein kinase A), and tyrosine kinase. Moreover, we found that Dox also induces transcription from promoters of immediate-early genes through an AP-1-independent pathway. Taken together, our results suggest that Dox elicits a novel genetic response distinct from the classical UV response.
Kurabayashi
M
M
Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles 90033, USA.
Dutta
S
S
Jeyaseelan
R
R
Kedes
L
L
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001483:Base Sequence; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D004317:Doxorubicin / Q000494:pharmacology*; D017781:Genes, Immediate-Early; D006321:Heart / Q000187:drug effects*; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D051797:Inhibitor of Differentiation Protein 2; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000502:physiology*; D012097:Repressor Proteins / Q000235:genetics*; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects
D000903:Antibiotics, Antineoplastic; D017362:Cyclic AMP Response Element-Binding Protein; D017931:DNA Primers; D004268:DNA-Binding Proteins; C497450:ID2 protein, human; D051797:Inhibitor of Differentiation Protein 2; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D004317:Doxorubicin; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases