DISRUPTION OF VITAMIN-D RECEPTOR-RETINOID-X RECEPTOR HETERODIMER FORMATION FOLLOWING RAS TRANSFORMATION OF HUMAN KERATINOCYTES

A partial resistance to the growth inhibitory influence of 1,25-dihydr oxyvitamin D-3 is apparent when immortalized keratinocytes are transfo rmed by the ras oncogene. The vitamin D receptor (VDR) was isolated, a nalyzed, and found to be identical in normal, immortalized, and ras-tr ansformed keratinocytes. Subsequently, nuclear extracts from immortali zed and ras-transformed keratinocytes were analyzed in gel mobility sh ift assays utilizing labeled vitamin D response elements or thyroid ho rmone response elements. A specific protein DNA complex that was shown to contain VDR using an anti-VDR antibody was identified in both type s of extracts; however, the addition of an anti-retinoid X receptor (R XR) antibody identified RXR in the complex of both normal and immortal ized keratinocyte cell extracts, but not in ras-transformed keratinocy tes. Furthermore, transfection of ras-transformed keratinocytes with w ild-type human RXR alpha rescued VDR.RXR and thyroid hormone receptor. RXR complexes as demonstrated by a supershift in the presence of the a nti-RXR antibody. Both cell lines were found to express RXR alpha mess age in equal amounts. Western blot analysis revealed that RXR alpha pr otein from ras-transformed keratinocytes was indistinguishable from th at from immortalized keratinocytes and from control cells. These resul ts suggest a causal relationship between resistance to the growth inhi bitory influences of 1,25-dihydroxyvitamin D-3 and disruption of the V DR.RXR complex in malignant keratinocytes.