To the Editor:

We recently found that a subtype of retinoic acid receptors (RARs), RARα, mediates upregulation of TM gene expression in human promyelocytic leukemia cells (NB4), acute monocytic leukemia cells, and human umbilical vein endothelial cells and that the cooperation of RARα and RARβ exerts downregulation of TF gene expression in NB4 cells.1 We introduce here new data to support our previous findings using RARα, RARβ, and RARγ selective agonists, Ro40-6055, Ro48-2249, and Ro44-4753, respectively. These synthetic retinoids were kindly provided by F. Hoffman-La Roche, Ltd (Basel, Switzerland). Our experiments also showed that the incubation of NB4 cells with the RARα selective agonist, Ro40-6055, induced cell differentiation into neutrophils and apoptosis. These effects were not exerted by Ro48-2249 and Ro44-4753.

When NB4 cells were incubated with 0.01 μmol/L of Ro40-6055, Ro48-2249, or Ro44-4753 for 24 hours, Ro40-6055 caused induction of TM antigen and reduction of TF antigen (Fig 1A and B), which was similar to the results induced by a synthetic retinoid Am80, which is an agonist to RARα and RARβ.1 The TM upregulation by the incubation with Ro40-6055 was more effective than that by the stimulation of the combination with Ro40-6055 and Ro48-2249 (Fig 1A). Ro40-6055 and Ro48-2249 showed downregulation of TF in NB4 cells, and the TF downregulation by the combined incubation of Ro40-6055 and Ro48-2249 was more significant than that by each retinoid (Fig 1B). Interestingly, the incubation of NB4 cells with Ro44-4753 induced upregulation of TF antigen level, although its mechanism is not yet known.

Fig. 1.

Effects of RARα, RARβ, and RARγ selective retinoids on the expression of the toal TM an TF antigens in NB4 cells. NB4 cells were incubated with 0.01 μmol/L of Ro40-6055 (lane 2), Ro48-2249 (lane 3), Ro44-4753 (lane 4), or a combination of 0.005 μmol/L each of Ro40-6055 and Ro48-2249 (lane 5) for 24 hours. Total TM (A) and TF (B) antigen levels in cell lysates were measured by enzyme-linked immunosorbent assay, as previously reported.1 Results are shown as the means ± SD of three different experiments.

Fig. 1.

Effects of RARα, RARβ, and RARγ selective retinoids on the expression of the toal TM an TF antigens in NB4 cells. NB4 cells were incubated with 0.01 μmol/L of Ro40-6055 (lane 2), Ro48-2249 (lane 3), Ro44-4753 (lane 4), or a combination of 0.005 μmol/L each of Ro40-6055 and Ro48-2249 (lane 5) for 24 hours. Total TM (A) and TF (B) antigen levels in cell lysates were measured by enzyme-linked immunosorbent assay, as previously reported.1 Results are shown as the means ± SD of three different experiments.

NB4 cells were further incubated with 0.01 μmol/L of Ro40-6055 for 7 days and were evaluated for changes in the morphology and surface markers of the leukemic cells. NB4 cells with frequent mitotic figures and morphologic features characteristic of acute promyelocytic leukemia cells were shown to have significant morphologic changes, such as condensed chromatin, smaller nuclei, decreased nuclei/cytoplasm ratio, and appearance of neutrophilic granules in the cytoplasm when treated with Ro40-6055. Differentiation of NB4 cells was also assessed by flow cytometric analysis for several membrane-bound differentiation markers. Expression of the cell surface antigens CD11b (a marker of β-integrin subunit expressed by both granulocytes and monocytes), CD11c (granulocytic lineage), and CD14 (late monocytic cell) is gradually detected during the normal development of mature monocytes from hematopoietic stem cells.2 The expression levels of CD11b and CD11c antigens (19.1% and 43.2%, respectively) were significantly increased after incubation with 0.01 μmol/L of Ro40-6055 to 91.6% and 90.8%, respectively. CD14 expression level was only slightly increased from 6.5% to 8.4%. DNA fragmentation assay of NB4 cells showed the occurrence of apoptosis by Ro40-6055 stimulation (0.01 μmol/L for 7 days).

These results support the idea that RARα mediates TM gene expression and that RARα and also RARβ are relevant to the retinoid-induced TF downregulation. A selective RARα agonist, Ro40-6055, is a potent inducer of differentiation and apoptosis with anticoagulant effect on acute promyelocytic leukemia cells.

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