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Antineoplastic agent doxorubicin inhibits myogenic differentiation of C2 myoblasts
M Kurabayashi1, R Jeyaseelan, L Kedes
1Institute for Genetic Medicine, University of Southern California School of Medicine, Los Angeles 90033.
Abstract:
Doxorubicin (Dox, adriamycin), an antineoplastic agent that can cause dilated cardiomyopathy, selectively inhibits muscle-specific gene expression in rodent cardiac muscle cells. This study shows that Dox treatment of proliferating C2 myoblasts, an established cell line from mouse skeletal muscle, completely prevents both fusion and accumulation of muscle-specific gene transcripts without significantly altering non-muscle gene transcripts. When added to high density cultures, Dox only blocked myotube formation but did not inhibit induction of muscle-specific genes. Transient transfection into C2 myoblasts showed that the transcriptional expression of chloramphenicol acetyltransferase reporter plasmids regulated by either the cardiac alpha-actin promoter or the muscle creatine kinase enhancer, but not with a viral or beta-actin promoter, was significantly diminished by Dox in a dose-dependent manner. Moreover, exposure of C2 myoblasts to Dox had a profound effect on the expression of regulatory genes critical to the myogenic differentiation program; mRNAs for MyoD and myogenin were dramatically reduced and Id mRNA was concomitantly increased. In addition, there was diminished DNA binding activity of the muscle-specific transcription factor, MEF-2. These results suggest that Dox inhibits myogenesis by preventing muscle-specific gene expression, possibly through affecting the myogenic programs controlled by muscle-specific transcription factors.
Insights
Doxorubicin (Dox) prevents muscle cell development by inhibiting muscle-specific gene expression. This impacts key regulatory genes and transcription factors essential for myogenesis.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Doxorubicin (Dox) is an antineoplastic agent known to cause dilated cardiomyopathy.
- Dox selectively inhibits muscle-specific gene expression in rodent cardiac muscle cells.
Purpose of the Study:
- To investigate the effect of Dox on myogenesis in C2 myoblasts.
- To determine if Dox affects muscle-specific gene expression and the underlying regulatory mechanisms.
Main Methods:
- Treatment of proliferating C2 myoblasts with Dox.
- Analysis of muscle-specific gene transcripts and non-muscle gene transcripts.
- Transient transfection assays using reporter plasmids with muscle-specific promoters.
- Measurement of mRNA levels for myogenic regulatory factors (MyoD, myogenin, Id).
- Assessment of DNA binding activity of the muscle-specific transcription factor MEF-2.
Main Results:
- Dox treatment completely prevented C2 myoblast fusion and accumulation of muscle-specific gene transcripts.
- Dox blocked myotube formation but did not inhibit induction of muscle-specific genes in high-density cultures.
- Transcriptional expression from cardiac alpha-actin and muscle creatine kinase promoters was dose-dependently diminished by Dox.
- Dox significantly reduced MyoD and myogenin mRNA levels while increasing Id mRNA.
- DNA binding activity of MEF-2 was diminished following Dox exposure.
Conclusions:
- Doxorubicin inhibits myogenesis by preventing muscle-specific gene expression.
- Dox likely affects myogenic programs controlled by muscle-specific transcription factors.
- These findings provide insight into the cardiotoxic mechanisms of Doxorubicin at the molecular level.