PML RAR-ALPHA(+) U937 MUTANT AND NB4 CELL-LINES - RETINOIC ACID RESTORES THE MONOCYTIC DIFFERENTIATION RESPONSE TO VITAMIN-D-3/

We have analyzed the differentiation program of a U937 promonocytic le ukemia clone transduced with the acute promyelocytic leukemia specific PML/RAR alpha fusion gene, the expression of which is under the contr ol of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTP R9 cells treated with Zn2+ hence exhibit levels of PML-RAR alpha prote in as high as fresh acute promyelocytic leukemia blasts. In the absenc e of Zn2+, i.e., upon low level PML/RAR alpha expression, 1,25-dihydro xyvitamin D-3 (D3) and particularly D3 plus transforming growth factor beta(1) (TGF-beta(1)) induced terminal differentiation of MTPR9 cells (as observed in ''wild-type'' U937 cells), on the basis of morphology , membrane antigen pattern, and functional criteria. Conversely, in th e presence of Zn2+, D3 and D3 plus TGF-beta(1) failed to induce termin al differentiation, as evaluated by the above parameters. Interestingl y, retinoic acid (RA) treatment suppresses the differentiation blockad e induced by high level PML-RAR alpha protein; indeed, Zn2+-treated MT PR9 cells incubated with RA plus D3 exhibited significant terminal mon ocytic maturation, comparable to that of cells treated with D3 alone o r combined with RA in absence of Zn2+. Similar observations were made in NB4, a PML-RAR(+) human acute leukemic line. As expected RA treatme nt of NB4 cells causes granulocytic differentiation. Interestingly, th e cell line is only scarcely induced to mature monocytic cells by D3 o r D3 plus TGF-beta(1) treatment, whereas it is effectively induced to monocytic maturation by combined treatment with D3 and RA. Accordingly , the rate of NB4 cell proliferation is only slightly affected by D3 o r D3 plus TGF-beta(1) treatment, mildly inhibited by RA, and markedly decreased by D3 plus RA. These results indicate that in both U937 and NB4 cells high level PML/RAR alpha expression inhibits the monocytic t erminal differentiation program triggered by D3 or D3 plus TGF-beta(1) , whereas RA treatment effectively antagonizes this inhibitory PML-RAR alpha action and restores the D3 differentiative effect.