We have analyzed the differentiation program of a U937 promonocytic leukemia clone transduced with the acute promyelocytic leukemia specific PML/RARα fusion gene, the expression of which is under the control of the inducible metallothionine (MT) I promoter (MTPR9 clone). MTPR9 cells treated with Zn²⁺ hence exhibit levels of PML-RARα protein as high as fresh acute promyelocytic leukemia blasts.

In the absence of Zn²⁺, i.e., upon low level PML/RARα expression, 1,25-dihydroxyvitamin D₃ (D3) and particularly D3 plus transforming growth factor β₁ (TGF-β₁) 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 the presence of Zn²⁺, D3 and D3 plus TGF-β₁ failed to induce terminal differentiation, as evaluated by the above parameters. Interestingly, retinoic acid (RA) treatment suppresses the differentiation blockade induced by high level PML-RARα protein; indeed, Zn²⁺-treated MTPR9 cells incubated with RA plus D3 exhibited significant terminal monocytic maturation, comparable to that of cells treated with D3 alone or combined with RA in absence of Zn²⁺.

Similar observations were made in NB4, a PML-RAR⁺ human acute leukemic line. As expected RA treatment of NB4 cells causes granulocytic differentiation. Interestingly, the cell line is only scarcely induced to mature monocytic cells by D3 or D3 plus TGF-β₁ 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 or D3 plus TGF-β₁ 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/RARα expression inhibits the monocytic terminal differentiation program triggered by D3 or D3 plus TGF-β₁, whereas RA treatment effectively antagonizes this inhibitory PML-RARα action and restores the D3 differentiative effect.