Cytochalasin D-induced actin gene expression in murine erythroleukemia cells.
There is a dynamic equilibrium between monomeric G-actin and polymeric F-actin microfilaments (MFs) in eucaryotic cells. We have previously shown that disruption of MFs with cytochalasin D (CD) induced beta-actin gene transcription, resulting in elevated levels of beta-actin mRNA and protein synthesis. CD also inhibited cell growth by arresting progression through the S phase of the cell cycle. These CD-induced responses were reversible since recovering cells progressed through the G2 phase and resumed normal growth while beta-actin mRNA and protein synthesis rapidly returned to control levels. In the present study, we show that the response of beta- and gamma-actin genes is due to the synthesis of a protein(s) acting at a 5' regulatory element that may be independent of or require sequences in addition to the serum response element (SRE). CD induces beta- and gamma-actin mRNA in a dose-dependent manner, reaching a maximum of 20-fold over control mRNA levels at 30 microM. beta- and gamma-Actin gene expression was also induced 5-fold by serum stimulation of quiescent murine erythroleukemia (MEL) cells, while combined treatment with serum and CD had an additive effect. Two protein synthesis inhibitors, cycloheximide and puromycin, blocked the CD-induced increase in beta-actin mRNA, in contrast to the serum-induced increase which is insensitive to inhibitors of protein synthesis. The rapid return of beta-actin mRNA to basal levels following CD removal did not require protein synthesis nor did it require progression through the G2 phase of the cell cycle. A vector containing the 5' end of the beta-actin gene linked to a CAT reporter responded to CD when transfected into MEL cells, localizing the responsive element to the 5' portion of the beta-actin gene. By contrast, a minimal 99-bp actin promoter-CAT construct containing a functional SRE did not respond to CD.
Sympson
C J
CJ
Department of Biochemistry, University of Louisville School of Medicine, Kentucky 40292.
Singleton
D
D
Geoghegan
T E
TE
D000199:Actins / Q000235:genetics*; D000818:Animals; D015638:Cytochalasin D / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects; D066298:In Vitro Techniques; D004915:Leukemia, Erythroblastic, Acute / Q000473:pathology; D051379:Mice; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D016196:S Phase; D014407:Tumor Cells, Cultured
D000199:Actins; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D015638:Cytochalasin D