Granulocyte colony-stimulating factor potentiates differentiation induction by all-trans retinoic acid and arsenic trioxide and enhances arsenic uptake in the acute promyelocytic leukemia cell line HT93A

Abstract

Abstract. The effects of arsenic trioxide (ATO), all-trans retinoic acid (ATRA) and granulocyte colony-stimulating factor (G-CSF), alone or in combination, were investigated by focusing on differentiation, growth inhibition and arsenic uptake in the acute promyelocytic leukemia (APL) cell line HT93A. ATO induced differentiation at low concentrations (0.125 µM) and apoptosis at high concentrations (1-2 µM). Furthermore, ATRA induced greater differentiation than ATO. No synergistic effect of ATRA and ATO was found on differentiation. G-CSF promoted differentiation-inducing activities of both ATO and ATRA. The combination of ATRA and G-CSF showed maximum differentiation and ATO addition was not beneficial. Addition of 1 µM ATRA and/or 50 ng/ml G-CSF to ATO did not affect apoptosis compared to ATO treatment alone. ATRA induced expression of aquaporin-9 (AQP9), a transmembrane transporter recognized as a major pathway of arsenic uptake, in a time-and dose-dependent manner. However, treatment with 1 µM ATRA decreased arsenic uptake by 43.7% compared to control subject. Although G-CSF addition did not enhance AQP9 expression in the cells, the reduced arsenic uptake was recovered to the same level as that in controls. ATRA decreased cell viability and addition of 50 ng/ml G-CSF to ATRA significantly increased the number of viable cells compared with that in ATRA alone treated cells. G-CSF not only promotes differentiation-inducing activities of both ATRA and ATO, but also makes APL cells vulnerable to increased arsenic uptake. These observations provide new insights into combination therapy using these three agents for the treatment of APL.

Anti-leukemic activity of DNA methyltransferase inhibitor procaine targeted on human leukaemia cells

“…The induction of the granulocytic differentiation of leukemic cells by ATRA has been a major breakthrough in terms of survival for acute promyelocytic leukaemia (APL) patients [17]. In our study we reported that DNMTI procaine alone did not induce G-CSF expression, but in the combined treatment with ATRA enhanced G-CSF expression compared with ATRAtreatment.…”

Perspective on Therapeutic Strategies of Leukemia Treatment — Focus on Arsenic Compounds

“…Morphological changes such as condensation of chromatin, lobulation of nuclei, and the expression profiling of surface markers have been used as markers of the differentiation of APL cells [15,81,85]. Consistent with previous findings, we have recently demonstrated that 0.125 μM ATO also induced differentiation in HT93A cells, another t(15;17)-positive APL cell line established from the peripheral blood of a patient with APL [86], as evidenced by the appearance of jelly-bean-shaped nuclei in almost all cells, accompanied by a significant increase in CD11c and CD15 expression [87]. Furthermore, we demonstrated that As 2 S 2 induced granulocyte differentiation in HL-60 cells, as evidenced by the increment in CD11b expression, in which oxidative stress associated with GSH depletion and p38 mitogenactivated protein kinase (MAPK) activation was involved [77,84].…”

Enhancement of differentiation induction and upregulation of CCAAT/enhancer-binding proteins and PU.1 in NB4 cells treated with combination of ATRA and valproic acid

“…For morphological assessment, cytospin preparations of treated cells stained with Wright-Giemsa were evaluated by light microscopy as previously described (24). Furthermore, the numbers of cells with differentiation-associated morphological changes such as apparent lobulated nuclei, multi-lobulated nuclei were counted and presented as the percent of differentiated cells.…”

“…Furthermore, the numbers of cells with differentiation-associated morphological changes such as apparent lobulated nuclei, multi-lobulated nuclei were counted and presented as the percent of differentiated cells. Myeloid maturation with cell surface marker was analyzed by FACSCanto flow cytometer (BD Immunocytometry System) using antibodies for CD11b and CD10 as previously described with minor modifications (24). In brief, approximately 1x10 6 cells were washed with PBS containing 2.5% FBS and 0.5% NaN 3 (PBSF) and stained with PE-conjugated mouse anti-human CD11b IgG or FITC-conjugated mouse anti-human CD10 IgG for 30 min at 4˚C in the dark.…”

“…Expression profiles of transcription factors in NB4 cells. The expression levels of transcription factors, including C/EBPα, C/EBPβ, C/EBPε and PU.1 were evaluated by flow cytometry (Cyto ACE-150, Jasco) using antibody for each as previously described with minor modifications (24). In brief, approximately 1x10 6 cells were washed with PBS, and fixed with 4% formaldehyde for 10 min at 37˚C.…”