title: Loss of glucocorticoid-dependent growth inhibition in transformed mouse lung cells.
abstract: Transformed A5 mouse lung cells were examined for mechanisms that may explain their loss of glucocorticoid-induced growth inhibition. These cells were compared to nontransformed C10 mouse lung cells, which retain this response. Southern blot analysis revealed no major differences in the amount or pattern of restriction fragments for the glucocorticoid receptor (GR) gene between the responsive and nonresponsive cells. Northern blot analysis demonstrated that both cell lines expressed GR mRNA at similar levels and that these mRNAs had similar relative stabilities. The mRNA from both cell lines was used for reverse transcription-polymerase chain reaction amplification and direct sequencing with primers for different regions of the GR cDNA. A conservative mutation previously shown not to affect receptor function was detected within the DNA-binding domain region of the GR from both cell lines. Because of the ability of the transcription factors for activator protein-1 to antagonize GR function, c-jun and c-fos mRNA levels were examined. A5 cells were found to have higher levels of c-jun mRNA than C10 cells both during active cell growth and after serum starvation. Stable transfection of the nonresponsive A5 cells with a rat GR expression vector (A5GR7) resulted in strong glucocorticoid-induced growth inhibition, demonstrating that these cells retain the ability to be growth inhibited by these steroids. The A5GR7 transfectants also had higher mouse mammary tumor virus (MMTV)-chloramphenicol acetyltransferase (CAT) activity than the parental A5 cells and lower levels of c-jun during active cell growth. Transient transfection of the C10 cells with c-jun expression vector strongly reduced glucocorticoid-inducible MMTV-CAT activity. These results suggest that the transformed A5 cells apparently contain functional GR but that the high level of c-jun mRNA expression (probably resulting from the activated Ki-ras allele in these cells) may antagonize their ability to respond to the growth-inhibitory signaling of glucocorticoids.
authors.affiliation: Department of Surgery, University of Michigan Medical School, Ann Arbor 48109, USA.
authors.forename: J
authors.initials: J
authors.lastname: Li
authors.forename: T A
authors.initials: TA
authors.lastname: Johnson
authors.forename: L A
authors.initials: LA
authors.lastname: Hanson
authors.forename: D G
authors.initials: DG
authors.lastname: Beer
mesh_terms: D000818:Animals; D001483:Base Sequence; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D002471:Cell Transformation, Neoplastic; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation; D011905:Genes, ras; D008168:Lung / Q000166:cytology* / Q000187:drug effects* / Q000648:ultrastructure; D051379:Mice; D008969:Molecular Sequence Data; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000502:physiology*; D018808:Transcription Factor AP-1 / Q000378:metabolism
chemical_list: D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011965:Receptors, Glucocorticoid; D018808:Transcription Factor AP-1; D003907:Dexamethasone