ANTISENSE INHIBITION OF VITAMIN-D-RECEPTOR EXPRESSION INDUCES APOPTOSIS IN MONOBLASTOID U937 CELLS

The active vitamin D-3 metabolite 1,25-dihydroxycholecalciferol (1,25( OH)(2)D-3) acts as an antiproliferative and differentiating agent for the monoblastoid cell line U937 and as an important immunologic mediat or implicated particularly in the function of cells belonging to the m onocyte/macrophage lineage. These effects are controlled by the vitami n D receptor (VDR), a member of the steroid hormone receptor family. T he objective of this study was to develop U937 transfectants expressin g antisense VDR mRNA, and to use these to examine the role of 1,25(OH) (2)D-3-VDR interaction in this lineage. A 2-kb VDR cDNA insert (includ ing the complete VDR coding region) was cloned in an antisense orienta tion into the EBV episomal vector pMEP4 under the control of an induci ble promoter and transfected into U937. The resultant cell line, DH42, was hygromycin resistant, contained VDR cDNA, expressed fewer VDRs th an controls, and showed a substantial decrease in antiproliferative re sponse to 1,25(OH)(2)D-3. However, 1,25(OH)(2)D-3 increased the number of cells expressing macrophage cell surface Ags, including CD14 and C D11b. A subpopulation of smaller cells did not express the differentia tion markers after cadmium stimulation. Cell cycle analysis showed shi fts in the distribution of cells from G1 to S phase, which were more p ronounced after cadmium treatment. A considerable proportion of cells were outside the cycle and DNA fragmentation confirmed apoptosis. Thus , the functional outcome of the VDR antisense transfection suggests th at in the myelomonocytic lineage, VDR expression may act as a protecti ve mechanism against programmed cell death.