Effects of Histone Deacetylase Inhibitors, Sodium Phenyl Butyrate and Vitamin B3, in Combination with Retinoic Acid on Granulocytic Differentiation of Human Promyelocytic Leukemia HL-60 Cells

2006 ◽  
Vol 1091 (1) ◽  
pp. 356-367 ◽  
Author(s):  
RASA MERZVINSKYTE ◽  
GRAZINA TREIGYTE ◽  
JURATE SAVICKIENE ◽  
KARL-ERIC MAGNUSSON ◽  
RUTA NAVAKAUSKIENE
Keyword(s):  
Retinoic Acid ◽  
Histone Deacetylase ◽  
Histone Deacetylase Inhibitors ◽  
Promyelocytic Leukemia ◽  
Vitamin B3 ◽  
Granulocytic Differentiation

Histone Deacetylase Inhibitors and Filgrastim Do Not Synergize with ATRA in the Induction of Changes of Acute Promyelocytic Leukemia Cells Adhesive Properties.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2548-2548
Author(s):  
Gil C. De Santis ◽  
Suzana E. Moreno ◽  
Hamilton L.G. Teixera ◽  
Ana Silvia G. Lima ◽  
Aglair B. Garcia ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Retinoic Acid ◽  
Histone Deacetylase ◽  
Acute Promyelocytic Leukemia ◽  
Histone Deacetylase Inhibitors ◽  
Myeloid Cells ◽  
Promyelocytic Leukemia ◽  
Myeloperoxidase Activity ◽  
Nb4 Cells

Abstract All-trans retinoic acid (ATRA) in combination with anthracyclines induces long term complete remission in approximately 80% of patients with acute promyelocytic leukemia (APL). However, ATRA causes the retinoic acid syndrome (RAS) characterized by respiratory distress, pleural effusions, fever and weight gain. RAS is associated with changes in the expression of adhesion molecules (AMs) in the leukemic blasts. Nevertheless, which AMs are essential to RAS development is not clear. In addition, the effect on AMs expression of new therapeutic agents for APL such as histone deacetylase inhibitors (HDACis) or filgrastim is presently unknown. HDACis have been successfully used to treat ATRA-refractory cases and they potentiate ATRA-induced differentiation. The association of ATRA+ filgastrim induced remission in an APL patient harboring the t(11;17)/PLZF/RARα, which is resistant to ATRA. In order to determine the effect of ATRA, filgrastim, HDACis and their associations on cell adhesion, we analyzed the expression of the AMs: CD11a, CD11b, CD18, CD29, CD54, CD62L and CD162 on leukemic cells from 18 patients with APL and in NB4 cells treated ex vivo for 12 hours with DMSO (control), ATRA (1mM), filgrastrim (100ng/mL), trichostatin A (TSA, 0.1 mM), phenyl butirate (PB, 1 mM) (the latter two are bona fine HDACis), ATRA+TSA, ATRA+PB and ATRA+filgrastim (at the same doses). The number of positive cells for each of this markers and their respective fluorescence intensity was determined by flow cytometry. We detected a significant increase in the number of CD54+ and CD18+ cells, associated with an increase in the intensity of expression of CD54, CD11a, CD11b and CD18 in both NB4 and primary cells treated with ATRA alone or associated with PB or G-CSF. No difference was observed between samples treated exclusively with ATRA and those with the associations. We then analyzed if the changes in AMs expression were accompanied by changes in the adhesion to Matrigel or endothelial cells. ATRA and its associations, but not TSA, PB or filgrastim alone, increased significantly cell adhesion in vitro, an effect that was reversed by pre-incubating treated cells with anti-CD54 or anti-CD18 antibodies (Abs), or with dexametasone. ATRA induced cell adhesion was not dependent on myeloid maturation as it could be detected after short (12h) incubations. Finally, we analyzed the effects of ATRA, filgrastim and their association in a mouse model. NB4 cells were treated with ATRA, filgrastim, ATRA+filgrastim and injected IV through the tail vein. After 6h mice, the number of myeloid cells retained in the lungs was evaluated by measuring the myeloperoxidase activity. Compared to control groups (untreated cells or saline), the ATRA and ATRA+filgrastim but not the Filgrastim alone group presented a significant increase in the number of myeloid cells infiltrating the lungs. Similarly to the observed in vitro, pre incubation with anti-CD54, anti-CD18 Abs or with dexametasone reversed the increased cell adhesion in vivo. In conclusion, our results show that treatment with HDACis or filgrastim alone do not affect AM expression or cell adhesion and that there is no significant synergism between these agents and ATRA. In addition, our data suggest that SAR development is dependent on ATRA induced changes in CD54 and CD18 expression.

scholarly journals Reduced Retinoic Acid-Sensitivities of Nuclear Receptor Corepressor Binding to PML- and PLZF-RARα Underlie Molecular Pathogenesis and Treatment of Acute Promyelocytic Leukemia

Blood ◽  
1998 ◽  
Vol 91 (8) ◽  
pp. 2634-2642 ◽  
Author(s):  
Fabien Guidez ◽  
Sarah Ivins ◽  
Jun Zhu ◽  
Mats Söderström ◽  
Samuel Waxman ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Fusion Protein ◽  
Histone Deacetylase ◽  
Acute Promyelocytic Leukemia ◽  
Nuclear Receptor ◽  
Histone Deacetylase Inhibitors ◽  
Promyelocytic Leukemia ◽  
Molecular Pathogenesis ◽  
Wild Type ◽  
Nuclear Receptor Corepressor

Typical acute promyelocytic leukemia (APL) is associated with expression of the PML-RARα fusion protein and responsiveness to treatment with all-trans retinoic acid (ATRA). A rare, but recurrent, APL has been described that does not respond to ATRA treatment and is associated with a variant chromosomal translocation and expression of the PLZF-RARα fusion protein. Both PML- and PLZF-RARα possess identical RAR sequences and inhibit ATRA-induced gene transcription as well as cell differentiation. We now show that the above-mentioned oncogenic fusion proteins interact with the nuclear receptor corepressor N-CoR and, in comparison with the wild-type RARα protein, their interactions display reduced sensitivities to ATRA. Although pharmacologic concentration of ATRA could still induce dissociation of N-CoR from PML-RARα, it had a very little effect on its association with the PLZF-RARα fusion protein. This ATRA-insensitive interaction between N-CoR and PLZF-RARα was mediated by the N-terminal PLZF moiety of the chimera. It appears that N-CoR/histone deacetylase corepressor complex interacts directly in an ATRA-insensitive manner with the BTB/POZ-domain of the wild-type PLZF protein and is required, at least in part, for its function as a transcriptional repressor. As the above-noted results predict, histone deacetylase inhibitors antagonize oncogenic activities of the PML-RARα fusion protein and partially relieve transcriptional repression by PLZF as well as inhibitory effect of PLZF-RARα on ATRA response. Taken together, our results demonstrate involvement of nuclear receptor corepressor/histone deacetylase complex in the molecular pathogenesis of APL and provide an explanation for differential sensitivities of PML- and PLZF-RARα–associated leukemias to ATRA.

Targeted removal of PML-RARα protein is required prior to inhibition of histone deacetylase for overcoming all-trans retinoic acid differentiation resistance in acute promyelocytic leukemia

Blood ◽  
2002 ◽  
Vol 100 (3) ◽  
pp. 1008-1013 ◽  
Author(s):  
Yongkui Jing ◽  
Lijuan Xia ◽  
Samuel Waxman
Keyword(s):  
Retinoic Acid ◽  
Histone Deacetylase ◽  
Acute Promyelocytic Leukemia ◽  
Promyelocytic Leukemia ◽  
Dominant Negative ◽  
Gene Expressions ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Nitroblue Tetrazolium Reduction ◽  
A Cell

Abstract All-trans retinoic acid (tRA)–induced differentiation in NB4 cells, a cell line derived from an acute promyelocytic leukemia patient with t(15;17) translocation, is markedly facilitated by sodium butyrate (NaB), a histone deacetylase inhibitor (HDACI), or by hexamethylene bisacetamide (HMBA), a non–HDACI tRA-differentiation inducer, as determined by nitroblue tetrazolium reduction. The tRA-induced expression of RIG-G, Bfl-1/A1, and p21waf1 and, to a lesser extent, of CCAAT/enhancer binding protein–ε (C/EBPε) are also enhanced by such combined treatments. Both responses are associated with a facilitated diminution of the leukemogenic PML-RARα protein and retained ΔPML-RARα, a cleavage product. Treatment with tRA in tRA differentiation–resistant NB4 subclones R4 and MR-2 does not result in PML-RARα diminution and the tested gene expressions. Moreover, the addition of HMBA or NaB with tRA results in only minimal increase of differentiation in the tRA differentiation–resistant subclones. The increases in acetylated histone H3 (AcH3) and AcH4 in NaB-treated NB4, R4, and MR-2 cells are similar and do not correlate with the extent of differentiation induction when NaB and HMBA are given in combination with tRA. Arsenic trioxide (As2O3) treatment results in the total degradation of PML-RARα without increasing AcH3 or AcH4 or inducing differentiation in R4 cells. As2O3 in combination with tRA induces gene (Bfl-1/A1 and C/EBPε) expression and partial differentiation. Both NaB and HMBA addition to As2O3-plus-tRA–treated R4 cells further enhances differentiation. These results suggest that elimination of the dominant negative PML-RARα protein is required prior to inhibition of histone deacetylase to fully overcome tRA-differentiation resistance in APL cells.

A Case of Relapsed Variant Acute Promyelcytic Leukemia with t(11;17)(q23q21), PLZF/RARa Fusion Gene, with Durable Molecular Remission Following an Induction and Consolidation Regimen That Included ATRA with Low Dose G-CSF and Valproic Acid as a Histone Deacetylase Inhibitor.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4584-4584
Author(s):  
Daniel N. Efiom-Ekaha ◽  
Jasotha Sanmugarajah ◽  
William B. Solomon
Keyword(s):  
Bone Marrow ◽  
Valproic Acid ◽  
Retinoic Acid ◽  
Histone Deacetylase ◽  
Peripheral Blood ◽  
Fusion Gene ◽  
Flow Cytometric Analysis ◽  
Promyelocytic Leukemia ◽  
Blood Analysis ◽  
Fusion Product

Abstract Background: Acute promyelocytic leukemia (APL) accounts for 10% of acute myeloid leukemias. Greater than 95% of cases are associated with the t(15;17)(q22;q21) translocation with the PML-RARa fusion product which is associated with increased affinity for nuclear co-repressor protein complex and recruitment of histone deacetylase. This leads to an alteration in chromatin conformation and inhibition of transcription and differentiation in promyelocytes in the presence of physiologic levels of retinoic acid. Pharmacologic doses of retinoic acid however are usually required to overcome this repression of transcription and leads to promyelocyte differentiaton and improvement in clinical outcomes in classic APL. A variant translocation in APL, t(11;17) (q23;q21) where the 3′ end of the RARa gene is fused to the 5′ end of a gene called PLZF (promyelocytic leukemia zinc finger), accounts for about 1% of cases. This disease phenotype is associated with refractoriness to pharmacologic doses of retinoic acid. Case Report; 52 year-old male presented with fever and rapidly progressive leukocytosis. His HCT was 24%, WBC count was 75,000/mcl and platelet count was 109,000/mcl. He required endotracheal intubation and mechanical ventilation with leukapharesis on his second day of admission due to leukostasis. He had been diagnosed with APL variant with t(11;17) (q23;q21) two years prior to this admission and had induction with Ara-c + daunorubicin (7+3), with an additional cycle for consolidation. He was then placed on maintenance with 6-MP + MTX for 24 months. Bone marrow and peripheral blood analysis confirmed disease relapse. Flow cytometric analysis revealed immature myeloid population CD34−, CD15−, CD13+, CD56+, HLA-DR-, with partial expression of CD33. FISH analyses on peripheral blood was negative for t(15;17) and positive for the variant t(11;17)(q23q21). He was started on induction therapy with Idarubicin 12mg/m2/day for three consecutive days with ATRA 45mg/m2/day (via NGT) + G-CSF 5mcg/kg/day. His Day 21 bone marrow examination revealed complete hematologic response. Consolidation/ maintanance with sodium valproate 500mg po BID (to maintain serum levels of 50–100mcg/ml), ATRA 45mg/m2/day (via NGT) + G-CSF 5mcg/kg/day (with G-CSF dose reductions for leukocytosis with a final stable dose of 30mcg/day). He subsequently achieved a complete cytogenetic remission and relapsed after 18 months on this regimen. Conclusion: Induction of promyelocyte differentiation and reversal of retinoic acid refractoriness in variant APL with t(11;17) (q23;q21) can be achieved with G-CSF priming and the addition of valproic acid s a HDAC inhibitor.

APX3330 inhibition of the redox function of ape-1/ref-1 (Ref-1) in promyelocytic leukemia cells enhances retinoic acid (ATRA) induced myeloid differentiation and limits cell proliferation as an approach to the prevention of the retinoic acid syndrome (RAS)

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. e14613-e14613
Author(s):  
K. A. Robertson ◽  
E. S. Colvin ◽  
M. R. Kelley ◽  
M. L. Fishel
Keyword(s):  
Flow Cytometry ◽  
Retinoic Acid ◽  
Cell Growth ◽  
Real Time ◽  
Real Time Pcr ◽  
Mapk Pathway ◽  
Promyelocytic Leukemia ◽  
Leukemia Cells ◽  
Myeloid Differentiation ◽  
Granulocytic Differentiation

e14613 Background: ATRA + chemotherapy has improved the treatment of promyelocytic leukemia(APL). However, 25% of ATRA treated APL patients experience toxicities that comprise the RAS (life-threatening respiratory distress, edema, renal failure, hypotension, coagulopathy and rising blast count). One approach to prevent RAS is to limit blast proliferation and enhance myeloid differentiation. Ref-1 is a DNA repair protein that functions in redox regulation of cellular proteins, such as Fos, Jun, p53, and NFkB. HL60 myeloid leukemia cells are promyeloblasts that respond to ATRA with granulocytic differentiation/growth arrest. Prior studies suggest Ref-1 redox control is integral to ATRA-induced differentiation. To define the role of the redox function of Ref-1, we used the Ref-1 specific drug, APX3330, to block Ref-1 redox function and examined the response of HL60 cells to ATRA. Methods: Cell growth assessed using trypan blue. Differentiation was evaluated by morphology and expression of CD11b by flow cytometry. Apoptosis was assayed by annexin-PI staining on flow cytometry and cell cycle analysis assayed with propidium iodide flow cytometry. To assess activation of the MAPK pathway, BLR-1 expression was determined by real time PCR. Results: 1) APX3330 blockade of Ref-1 redox function resulted in limited cell growth yet a profound increase in differentiation and a moderate increase in apoptosis. 2) dose dependent studies with ATRA showed a similar degree of differentiation in cells treated with 10 μM ATRA to cells treated with APX3330 + 0.01 μM ATRA; allowing HL60 cells + APX3330 to give a similar response to a 1000 fold lower dose of ATRA. APX3330 alone did not induce differentiation and induced only minimal apoptosis but in combination with ATRA, increased the number of cells in G1/G0 phase significantly. 3) APX3330 + ATRA increased BLR-1 expression significantly by real time PCR suggesting enhanced activation of the MAPK pathway. Conclusions: APX3330 + ATRA limits HL60 growth and dramatically enhances terminal granulocytic differentiation. These finding may provide a therapeutic approach for prevention of the RAS. No significant financial relationships to disclose.

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