engleski

The effects of phosphoinositide 3-kinase/Akt inhibitors on two retinoid-responsive leukemia cell lines

Introduction: All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) patients is the first example of therapy by differentiation. The classical view of the action of ATRA in APL holds that ATRA releases corepressors from the chimeric PML-RARa protein and thus allows the growth inhibition, differentiation and apoptosis in t(15 ; 17) APL cells. The pharmacological inhibitors of phosphoinositide 3-kinase(PI3K)/Akt pathway have been proposed in the treatment of leukemia based on their antiproliferative effects. However, recent studies (Matkovic et al, Leukemia, 20:941-951, 2006) demonstrated Akt-activation in nuclei of ATRA-treated HL-60 cells raising the possibility that PI3K/Akt– inhibitors may block antitumor properties of retinoids. The aim of this study is to compare the sensitivity of two different leukemia cell lines to commercially available PI3K/Akt-inhibitors. NB4 cell line, established from an APL patient, is a typical AML-M3 carrying t(15 ; 17). HL-60 cells, isolated from AML-M2 patient, do not display specific t(15, 17) translocation but share the responsiveness to ATRA and are widely used to demonstrate the differentiative activity of ATRA and to predict its efficiency in vivo. Materials and Methods: HL-60 cells and NB4 cells were maintained in exponential growth and differentiated in the presence of ATRA (1microM). The Akt activity in different cell fractions was determined by Western blot analysis and kinase assays using Crosstide. The following inhibitors were tested: Akt inhibitor I (1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate, Calbiochem), Akt inhibitor II (SH-5, Calbiochem), LY 294002 (Calbiochem) and MEK-inhibitor PD 98059 (Calbiochem). The number of viable cells was quantified using a hemocytometer and trypan blue exclusion. The expression of CD11b and the cell cycle distribution of propidium iodide-labeled cells were determined by FACS analyses. Results: Both HL-60 and NB4 cells show an increase in the activity of Akt in nuclei and lysates after ATRA-mediated differentiation. PI3K inhibitor LY 294002 and MEK-inhibitor PD98059 reduced the number of viable cells and the expression of differentiation marker in both cell lines. The presence of Akt inhibitors inhibited the growth of both control and ATRA-treated NB4 and HL-60 cells and reduced the expression of CD11b in ATRA-treated NB4 cells. In contrast, Akt-inhibitors had no inhibitory effects on the expression of CD11b in ATRA-treated HL-60 cells, but increased the percentage of control cells expressing CD11b. The inhibition of Akt altered the life span of differentiated HL-60 cells as the number of events in sub-G1 measured at 7 days after the addition of ATRA was higher in samples treated with the combination of Akt inhibitors and ATRA that in the cells treated with ATRA alone. Conclusion: Two retinoid-responsive leukemia cell lines respond differently to the presence of PI3K/Akt inhibitors ; the expression of differentiation marker is reduced in ATRA-treated NB4 cells, there are no inhibitory effects on ATRA-induced differentiation of HL-60 cells and the percentage of control HL-60 cells expressing CD11b is increased. These results suggest that the combination of retinoids with Akt-inhibitors could be exploited to improve retinoid therapy in selected cases. In addition, data further suggest that ATRA may mediate a number of nontranscriptional changes in cell signaling by mechanisms apart from the direct effects of the nuclear receptor on transcription.

leukemia; phosphoinositide 3-kinase; Akt; inhibitor; NB4; HL-60

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