Genome-wide examination of myoblast cell cycle withdrawal during differentiation.
Skeletal and cardiac myocytes cease division within weeks of birth. Although skeletal muscle retains limited capacity for regeneration through recruitment of satellite cells, resident populations of adult myocardial stem cells have not been identified. Because cell cycle withdrawal accompanies myocyte differentiation, we hypothesized that C2C12 cells, a mouse myoblast cell line previously used to characterize myocyte differentiation, also would provide a model for studying cell cycle withdrawal during differentiation. C2C12 cells were differentiated in culture medium containing horse serum and harvested at various time points to characterize the expression profiles of known cell cycle and myogenic regulatory factors by immunoblot analysis. BrdU incorporation decreased dramatically in confluent cultures 48 hr after addition of horse serum, as cells started to form myotubes. This finding was preceded by up-regulation of MyoD, followed by myogenin, and activation of Bcl-2. Cyclin D1 was expressed in proliferating cultures and became undetectable in cultures containing 40% fused myotubes, as levels of p21(WAF1/Cip1) increased and alpha-actin became detectable. Because C2C12 myoblasts withdraw from the cell cycle during myocyte differentiation following a course that recapitulates this process in vivo, we performed a genome-wide screen to identify other gene products involved in this process. Using microarrays containing approximately 10,000 minimally redundant mouse sequences that map to the UniGene database of the National Center for Biotechnology Information, we compared gene expression profiles between proliferating, differentiating, and differentiated C2C12 cells and verified candidate genes demonstrating differential expression by RT-PCR. Cluster analysis of differentially expressed genes revealed groups of gene products involved in cell cycle withdrawal, muscle differentiation, and apoptosis. In addition, we identified several genes, including DDAH2 and Ly-6A, whose expression specifically was up-regulated during cell cycle withdrawal coincident with early myoblast differentiation.
Shen
Xun
X
Cardiovascular Research Institute, University of California, San Francisco, California, USA.
Collier
J Michael
JM
Hlaing
Myint
M
Zhang
Leanne
L
Delshad
Elizabeth H
EH
Bristow
James
J
Bernstein
Harold S
HS
D000818:Animals; D017209:Apoptosis; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002453:Cell Cycle; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D019938:Cyclin D1 / Q000096:biosynthesis; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000096:biosynthesis; D019992:Databases as Topic; D016678:Genome; D015151:Immunoblotting; D051379:Mice; D008954:Models, Biological; D018482:Muscle, Skeletal / Q000196:embryology* / Q000378:metabolism; D017570:MyoD Protein / Q000096:biosynthesis; D032446:Myoblasts / Q000378:metabolism* / Q000502:physiology*; D018008:Myogenin / Q000096:biosynthesis; D020411:Oligonucleotide Array Sequence Analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D015854:Up-Regulation
C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D017570:MyoD Protein; C503801:Myog protein, mouse; D018008:Myogenin; D019253:Proto-Oncogene Proteins c-bcl-2; D019938:Cyclin D1; D001973:Bromodeoxyuridine