It has been shown recently in China that arsenic trioxide (As2O3) is a very effective treatment for acute promyelocytic leukemia (APL). APL patients resistant to all-trans retinoic acid (ATRA) and conventional chemotherapy can still respond to AS2O3. In this study, we addressed the possible cellular and molecular mechanisms of this treatment by using NB4 cells as a model. The results show that: (1) As2O3 triggers relatively specific NB4 cell apoptosis at micromolar concentration, as proved by morphology, histogramic related nuclear DNA contents, and DNA gel eletrophoresis. (2) As2O3 does not influence bax, bcl-x, c-myc, and p53 gene expression, but downregulates bcl-2 gene expression at both mRNA and protein levels. (3) As2O3 induces a significant modulation of the PML staining pattern in NB4 cells and HL-60 cells. The micropunctates characteristic of PML-RAR alpha in NB4 cells dissappear after treatment with As2O3, whereas a diffuse PML staining occurs in the perinuclear cytoplasmic region. In addition, a low percentage of untreated NB4 cells exhibits an accumulation of PML positive particles in a compartment of cytoplasm. The percentage of these cells can be significantly increased after As2O3 treatment. A similar PML staining pattern is observed in apoptotic cells. (4) ATRA pretreatment does not influence As2O3-induced apoptosis. These results suggest that induction of cell apoptosis can be one of the mechanisms of the therapeutic effect of As2O3. Moreover, this apoptosis induction occurs independently of the retinoid pathway and may be mediated, at least partly, through the modulation of bcl-2, as well as PML-RAR alpha and/ or PML proteins.

Cellular and systemic O 2 concentrations are tightly regulated to maintain delicate oxygen homeostasis. Although the roles of hypoxia in solid tumors have been widely studied, few studies were reported regarding the possible effects of hypoxia on leukemic cells. Here, we showed for the first time that low concentrations of cobalt chloride (CoCl 2 ), a hypoxia-mimicking agent, and 2-3% O 2 triggered differentiation of various subtypes of human acute myeloid leukemic (AML) cell lines, including NB4, U937 and Kasumi-1 cells, respectively, from M3, M5 and M2b-type AML, but CoCl 2 did not modulate AML subtype-specific fusion proteins promyelocytic leukemia-retinoic acid receptor alpha (PML-RARa) and AML1-ETO. Treatment with CoCl 2 also induced primary leukemic cells from some AML patients to undergo differentiation. Similar to what occurs in solid tumor cells, CoCl 2 -mimicked hypoxia also increased the level of hypoxia-inducible factor (HIF)-1a protein and its DNAbinding activity in leukemic cells. The CoCl 2 induction of HIF-1a protein and its DNA-binding activity were inhibited by 3-morpholinosydnonimine, which also blocked CoCl 2 -induced cell differentiation in leukemic cells. These results provide an insight into a possible link of hypoxia or HIF-1a and leukemic cell differentiation, and are possibly of significance to explore clinical potentials of hypoxia or hypoxia-mimicking agents and novel target-based drugs for differentiation therapy of leukemia

The second messenger cyclic adenosine monophosphate (cAMP) plays an important role in cell proliferation, differentiation and apoptosis. In the present work, we evaluated the cAMP signaling in acute promyelocytic leukemia (APL) cells in the context of differentiation induced by all-trans retinoic acid (ATRA). There was a marked increase in the intracellular cAMP level within a few minutes after treatment with ATRA in APL cell line NB4 and fresh APL cells, whereas no such phenomenon was observed in NB4-R1 cells that are resistant to ATRAinduced maturation. In addition, the basal level of intracellular cAMP was lower in NB4-R1 than in NB4 cells. Mechanistic study showed that this induction of cAMP was mediated through the activation of adenylate cyclase. Moreover, we found that cAMPdependent protein kinase (PKA) activity was quickly upregulated in parallel in ATRA-treated NB4 cells, and the phosphorylation of RARa by PKA could increase its transactivation effect. Use of H-89, an inhibitor of PKA, could partially suppress the transcriptional expression of ATRA target genes and ATRAinduced differentiation of APL cells. Taken together, we suggested a crosstalk between ATRA-induced cytosolic pathway and nuclear pathway in APL cell differentiation.