Retinoic acid resistance of the variant embryonal carcinoma cell line RAC65 is caused by expression of a truncated RAR alpha.
P19 embryonal carcinoma (EC) cells differentiate when treated with retinoic acid (RA). The P19 EC-derived mutant cell line RAC65 is resistant to the differentiation-inducing activity of RA. We show that these cells express a truncated retinoic acid receptor alpha(mRAR alpha-RAC65), probably due to the integration of a transposon-like element in the RAR alpha gene. This receptor lacks 71 C-terminal amino acids and terminates in the ligand-binding domain. In CAT assays in RAC65 cells, mRAR alpha-RAC65 fails to trans-activate the RAR beta promoter, which contains a RA-response element. In wild-type P19 EC cells mRAR alpha-RAC65 functions as a dominant-negative repressor of RA-induced RAR beta activation. Gel retardation assays demonstrate that mRAR alpha-RAC65 is still able to bind to the RA-response element of the RAR beta promoter, indicating that competition with functional RARs for the same binding site leads to the observed dominant-negative effect. In addition, in two RAC65 clones in which wild-type hRAR alpha was stably transfected RA-sensitivity was restored and in one RAR beta expression could be induced by RA. Taken together, these data show that the primary cause of RA-resistance of RAC65 cells is the expression of a defective RAR alpha, which prevents the trans-activation of RA-responsive genes and results in a loss of the ability to differentiate.
Kruyt
F A
FA
Hubrecht Laboratory, Netherlands Institute for Development Biology, Utrecht.
van der Veer
L J
LJ
Mader
S
S
van den Brink
C E
CE
Feijen
A
A
Jonk
L J
LJ
Kruijer
W
W
van der Saag
P T
PT
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002352:Carrier Proteins / Q000502:physiology*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis; D004351:Drug Resistance; D005786:Gene Expression Regulation; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D018168:Receptors, Retinoic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013724:Teratoma / Q000235:genetics*; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*
D002352:Carrier Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D004247:DNA