Cancer Procoagulant and Tissue Factor Are Differently Modulated by All-trans-Retinoic Acid in Acute Promyelocytic Leukemia Cells

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Abstract
All-trans-retinoic acid (ATRA) downregulates the expression of two cellular procoagulants, tissue factor (TF) and cancer procoagulant (CP), in human promyelocytic leukemia cells. To evaluate whether or not changes of the procoagulant activities (PCAs) may share mechanisms with the ATRA-induced cyto-differentiation process, we have characterized the effect of ATRA on the TF and CP expression by NB4 cells, an ATRA maturation-inducible cell line, and two NB4-derived cell lines resistant to ATRA-induced maturation, the NB4.306 and NB4.007/6 cells. Next, we evaluated the effect on the PCAs of the NB4 parental cells of three synthetic retinoid analogues, ie: AM580 (selective for the retinoic acid receptor [RAR] α), capable to induce the granulocytic differentiation of NB4 cells; and CD2019 (selective for RARβ) and CD437 (selective for RARγ), both lacking this capability. Cells were treated with either ATRA or the analogues (10−6 to 10−8 mol/L) for 96 hours. The effect on cell differentiation was evaluated by morphologic changes, cell proliferation, nitro blue tetrazolium reduction assay, and flow cytometry analysis of the CD33 and CD11b surface-antigen expression. PCA was first measured in 20 mmol/L Veronal Buffer cell extracts by the one-stage clotting assay of normal and FVII-deficient plasmas. Further TF and CP have been characterized and quantified in cell-sample preparations by chromogenic and immunological assays. In the first series of experiments, ATRA downregulates both TF and CP in NB4 parental cells, as expected. However, in the differentiation-resistant cell lines, it induced a significant loss of TF but had little or no effect on CP. In a second series of experiments, in the NB4 parental cells, the RARα agonist (AM580) induced cell maturation and reduced 91% CP expression, whereas CD437 and CD2019 had no cyto-differentiating effects and did not affect CP levels. On the other hand, in the same cells the TF expression was reduced by ATRA and AM580, but also by the RARβ agonist CD2019, which did not induce cell maturation. These data indicate that in NB4 cells, ATRA modulation of CP occurs in parallel with signs of cell differentiation, while the regulation of TF appears to be at least in part independent from these processes, and involves both α and β nuclear retinoid receptors.