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Leukemia (2010) 24, 729–739 & 2010 Macmillan Publishers Limited All rights reserved 0887-6924/10 $32.00 www.nature.com/leu ORIGINAL ARTICLE Targeting PKCd-mediated topoisomerase IIb overexpression subverts the differentiation block in a retinoic acid-resistant APL cell line S McNamara1,3, JN Nichol1,3, H Wang2 and WH Miller Jr1 1Division of Experimental Medicine, Department of Oncology, Segal Cancer Comprehensive Centre, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, McGill University, Montre´al, Quebec, Canada and 2Segal Cancer Comprehensive Centre, Lady Davis Institute for Medical Research, Sir Mortimer B Davis Jewish General Hospital, Montre´al, Quebec, Canada Retinoic acid (RA) relieves the maturation block in t(15:17) therapeutic doses of all-trans RA, a vitamin A derivative that acute promyelocytic leukemia (APL), leading to granulocytic activates RARA and circumvents the differentiation block.4 differentiation. However, RA treatment alone invariably results in RA resistance, both in vivo and in vitro. RA-resistant cell Unfortunately, although treatment with RA alone results in a lines have been shown to serve as useful models for elucida- complete remission, the reprieve is not long-lasting, as RA 5 tion of mechanisms of resistance. Previously, we identified resistance develops in vivo, a phenomenon that can be topoisomerase II beta (TOP2B) as a novel mediator of modeled in vitro.6 RA-resistance in APL cell lines. In this study, we show that Acquired mutation in the PML-RARA oncoprotein is one both TOP2B protein stability and activity are regulated by a source of RA-resistant APL. Studies on the PML-RARA protein in member of the protein kinase C (PRKC) family, PRKC delta (PRKCD). Co-treatment with a pharmacologic inhibitor of RA-resistant patient cells or cell lines established that roughly PRKCD and RA resulted in the induction of an RA responsive 33% possessed a mutation in the ligand-binding domain of the reporter construct, as well as the endogenous RA target genes, RARA portion of the fusion protein, resulting in a dysfunctional CEBPE, CYP26A1 and RIG-I. Furthermore, the co-treatment PML-RARA that is unable to respond to pharmacological doses overcame the differentiation block in RA-resistant cells, as of RA.7–10 However, the mechanisms of acquired resistance assessed by morphological analysis, restoration of promyelo- in the remaining 67% are undefined. The cells continue to cytic leukemia nuclear bodies, induction of CD11c cell surface expression and an increase in nitro-blue-tetrazolium reduction. express wild-type PML-RARA, yet are resistant to RA-induced Cumulatively, our data suggest a model whereby inhibition of differentiation. PRKCD decreases TOP2B protein levels, leading to a loss of In vitro-derived RA-resistant cell lines are useful experimental TOP2B-mediated repressive effects on RA-induced transcrip- models for the study of mechanisms of RA resistance in APL. tion and granulocytic differentiation. Our lab has previously isolated three RA-resistant subclones Leukemia (2010) 24, 729–739; doi:10.1038/leu.2010.27; from the parental RA-sensitive cell line NB4, denoted NB4- published online 4 March 2010 11,12 Keywords: acute promyelocytic leukemia; topoisomerase II beta; MR2, NB4-MR4 and NB4-MR6. Consistent with the pattern retinoic acid; resistance; protein kinase C delta in other models of RA-resistance, one of these resistant subclones, NB4-MR4, contains a mutation in the ligand-binding domain of PML-RARA, whereas the other two retain wild-type PML-RARA expression.12 We have reported that resistance to RA-mediated transcription and differentiation in these cell lines Introduction is associated with enhanced binding of corepressor complexes to PML-RARA.11 We furthered this observation by identifying Acute promyelocytic leukemia (APL) is a subtype of acute topoisomerase II beta (TOP2B) as a novel member of these myelogenous leukemia, representing 5–8% of acute myelo- complexes. In addition, we showed that TOP2B inhibits RA- genous leukemia cases in adults. At the genetic level, APL is induced gene expression and granulocytic differentiation by characterized by a specific chromosomal rearrangement be- negatively modulating RARA transcriptional activity.13 Type II tween the retinoic acid (RA) receptor-a (RARA) and the topoisomerases catalyze changes in the topological state of promyelocytic leukemia (PML) genes.1–3 The resulting chimeric DNA by generating transient breaks in double-strand DNA.14,15 protein, PML-RARA, acts as a dominant negative inhibitor of Owing to their DNA cleavage properties, TOP2s serve as target normal retinoid receptor function. It locates to promoters of antineoplastic agents, including DNA damaging agents.16 normally regulated by RARA, aberrantly recruits co-repressor Two isoforms of TOP2 (A and B) exist in mammals. TOP2B proteins, and thereby inhibits the RARA-mediated gene expres- is a phosphoprotein in which most of the phosphorylation sites sion. On the cellular level, the result is a block in granulocytic have been mapped to the C-terminal domain and are thought differentiation and an accumulation of myeloid progenitors to be mostly serine or threonine residues.17 Sequence analysis arrested at the promyelocyte stage. APL patients are treated with of TOP2 phosphorylation sites revealed 30 possible protein kinase C (PRKC) sites and 40 casein kinase II sites.14 However, Correspondence: Dr WH Miller Jr, Department of Oncology, Segal the functions of TOP2B’s phosphorylation sites have not yet Cancer Comprehensive Center, Lady Davis Institute for Medical been characterized and the identification of possible phosphory- Research, Sir Mortimer B. Davis Jewish General Hospital, McGill lation sites responsible for TOP2B stability has remained University, 3755 Coˆte Ste-Catherine Road, Montre´al, Quebec, Canada elusive. H3T 1E2. RA treatment leads to an increase of TOP2B protein levels in E-mail: [email protected] 18 3These authors contributed equally to this work. hematopoietic cells during differentiation. Interestingly, this Received 2 November 2009; revised 22 December 2009; accepted correlates with an increase in TOP2B protein stability and 4 January 2010; published online 4 March 2010 phosphorylation levels,18 although the exact mechanism by PKCd regulation of topoisomerase IIb in APL S McNamara et al 730 which RA upregulates and stabilizes TOP2B protein levels in chloramphenicol acetyltransferase, with or without the pTB114 differentiating cells has not been determined. Previously, we plasmid, which contains the full-length TOP2B isoform fused to demonstrated that increased TOP2B levels mediate RA resis- green fluorescent protein in the pEGFP-C3 vector as previously tance by inhibiting RA-induced gene expression and subsequent described.19 Following electroporation, cells were replenished maturation toward granulocytes in APL cell lines. Consistent in media, and grown for 48 h in the absence or presence of RA. with this finding, the RA-resistant NB4-MR2 subline has Chloramphenicol acetyltransferase activity was measured by comparatively higher basal TOP2B protein levels.13 Interest- means of a modified protocol of the organic diffusion method.20 ingly, RA leads to increased levels of TOP2B protein in the RA- The chloramphenicol acetyltransferase counts were normalized sensitive NB4 cell line, suggesting the possibility of a negative with protein concentration to obtain the relative chloramphe- feedback loop. We speculated that RA may activate specific nicol acetyltransferase activity. kinases that lead to increased phosphorylation levels of TOP2B, thereby increasing its stability and decreasing its rate of degradation. In this study, we investigate the mechanism by which TOP2B protein levels are regulated in APL cells. RNA extraction and analysis Total mRNA was isolated using the TRIzol method (Invitrogen, Carlsbad, CA, USA). cDNA was generated from 5 mg total RNA Materials and methods using random primers and SuperScript II reverse transcriptase (Invitrogen). CEBPE and CYP26A1 mRNA levels were assessed Materials by quantitative real-time PCR analysis using Power SYBR green RPMI 1640 and fetal bovine serum were purchased from master mix (Applied Biosystems, FosterCity, CA, USA) with the Wisent (St Bruno, QC, Canada). All-trans RA and 4a-phorbol following primer sets: CEBPE: sense 50-CGGCTGGCCCCTTAC 12-myristate 13-acetate (PMA) were obtained from Sigma- AC-30, antisense 50-AGCCGGTACTCAAGGCTATCTTT-30; and Aldrich (St Louis, MO, USA). Rottlerin was purchased from CYP26A1: sense 50-GACATGCAGGCACTAAAGCAAT-30, anti- Biomol (Plymouth Meeting, PA, USA). The PRKCD pSUPER sense 50-CACTGGCCGTGGTTTCGT-30. cDNA was amplified short hairpin RNA-producing plasmid (Oligoengine, Seattle, for RIGI using a Taqman hybridization probe and Taqman Fast WA, USA) was directed against the following target sequence: Master Mix (Applied Biosystems). DDCt values were normalized 50-AAACTCATGGTTCTTGATGTAGTGG-30. The PRKCD kinase with those obtained from the amplification of endogenous dead (PRKCDKD) construct was kindly provided by Dr Trevor J glyceraldehyde-3-phosphate dehydrogenase mRNA. Quantita- Biden (Garvan Institute of Medical Research, Sydney, Australia). tive real-time PCR was performed on the 7500 Fast Real-time PCR system (Applied Biosystems) using standard parameters and analyzed using relative quantification with dimethylsulfoxide- Cell culture treated NB4 cells as the calibrator. Derivation of the RA-resistant cell line NB4-MR2 from the parental APL
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