Regulation of smooth muscle alpha-actin promoter in ras-transformed cells: usefulness for setting up reporter gene-based assay system for drug screening.
Oncogenic activation of ras results in changes in the transcription of several genes leading to uncontrolled cell growth. In this paper, we demonstrate that transformation of fibroblast cells by the ras oncogene leads to transcriptional repression of the smooth muscle alpha-actin promoter. Transient transfection analysis of plasmids containing the 5' upstream region of the human alpha-actin gene fused to human growth hormone or bacterial chloramphenicol acetyltransferase coding sequences into Rat-2 and ras-transformed Rat-2 (HO6) cells indicates that alpha-actin promoter is repressed in ras-transformed cells. In addition, stable rat fibroblast cell lines expressing human growth hormone or beta-galactosidase under the control of alpha-actin promoter exhibit repressed reporter gene activity following transformation by the ras oncogene. alpha-Actin promoter-driven beta-galactosidase activity is derepressed in revertants of ras-transformed stable cell lines. This revertant cell line expresses elevated levels of ras p21 protein and is resistant to retransformation by Ki and Ha-ras oncogenes. The revertant may have either a defective target protein whose activity is essential for the transforming activity of ras or an activated tumor suppressor gene which can suppress the activity of ras. These results indicate that smooth muscle alpha-actin promoter activity is a sensitive marker to follow phenotypic changes following transformation by ras and subsequent reversion. The advantages of this alpha-actin promoter-reporter gene assay system to screen for drugs that inhibit the transforming activity of ras, either directly or indirectly, are discussed.
Kumar
C C
CC
Department of Tumor Biology, Schering-Plough Research Institute, Bloomfield, New Jersey 07003.
Bushel
P
P
Mohan-Peterson
S
S
Ramirez
F
F
D000199:Actins / Q000235:genetics*; D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000187:drug effects*; D004354:Drug Screening Assays, Antitumor / Q000379:methods*; D011905:Genes, ras; D009130:Muscle, Smooth / Q000737:chemistry*; D011401:Promoter Regions, Genetic; D051381:Rats
D000199:Actins