|Year : 2015 | Volume
| Issue : 4 | Page : 190-194
Differences between nucleophosmin isoforms in de-novo acute myeloid leukemia: possible implications in developing targeted therapy for acute myeloid leukemia with normal karyotype
Safaa A. A. Khaled MD 1, John Burthem2, El Badry E Abu-ElNoor3, Lobna F ELTony3, Sohier M Ahmed4, Hanan M Ahmed3
1 Hematology and BMT Unit, Department of Internal Medicine, Assiut University Hospital, Assiut, Egypt
2 Manchester Royal Infirmary, Manchester, United Kingdom
3 Department of Internal Medicine, Assiut University Hospital, Assiut, Egypt
4 Department of Clinical Pathology, Assiut University Hospital, Assiut, Egypt
|Date of Submission||03-Feb-2015|
|Date of Acceptance||16-Apr-2015|
|Date of Web Publication||23-Nov-2015|
Safaa A. A. Khaled
Hematology and BMT Unit, Department of Internal Medicine, Assiut University Hospital, Assiut 71111
Source of Support: None, Conflict of Interest: None
Background The current approach of treating de-novo acute myeloid leukemia (AML) is still ineffective. This is in part due to the clinical and molecular diversity of the disease. Accordingly, development of new effective targeted therapies for AML is mandatory. The current study investigated the differences between nucleophosmin (NPM) isoforms in de-novo AML. The work focused on AML with a normal karyotype as it constitutes 45% of AMLs and bears mutated nucleophosmin (mNPM). The main objective was to identify the active region in the NPM molecule to be targeted with a specific therapy.
Materials and methods In this study human leukemia cell lines HL60 and OCI-AML3 were used as models for AMLs bearing wild-type NPM and mNPM, respectively. The study was conducted through indirect immunofluorescence and immunoblotting techniques. The obtained results were presented and analyzed with appropriate computer software.
Results and conclusion The analyzed data proved, for the first time, that mNPM is phosphorylated at Thr199. As cdk2 is the main mediator of Thr199 phosphorylation, we speculated that a specific cdk2 inhibitor could be highly valuable as targeted therapy in de-novo AML with a normal karyotype. However, further experimental work in the presence of cdk2 inhibitors is needed.
Keywords: acute myeloid leukemia, isoforms, normal karyotype, nucleophosmin, targeted therapy
|How to cite this article:|
Khaled SA, Burthem J, Abu-ElNoor EE, ELTony LF, Ahmed SM, Ahmed HM. Differences between nucleophosmin isoforms in de-novo acute myeloid leukemia: possible implications in developing targeted therapy for acute myeloid leukemia with normal karyotype. Egypt J Haematol 2015;40:190-4
|How to cite this URL:|
Khaled SA, Burthem J, Abu-ElNoor EE, ELTony LF, Ahmed SM, Ahmed HM. Differences between nucleophosmin isoforms in de-novo acute myeloid leukemia: possible implications in developing targeted therapy for acute myeloid leukemia with normal karyotype. Egypt J Haematol [serial online] 2015 [cited 2020 Jan 18];40:190-4. Available from: http://www.ehj.eg.net/text.asp?2015/40/4/190/170220
| Introduction|| |
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults  . Unfortunately, it carries a very poor prognosis under conventional chemotherapy. Over the past 10 years, improvement in AML prognosis has been achieved, in part due to the marvelous progress in cytogenetic and molecular studies and the application of risk-dependent management protocols , . Nucleophosmin (NPM) gene mutations were identified in 65% of de-novo AMLs with a normal karyotype  .
NPM is a nucleocytoplasmic shuttling protein  that carries many intracellular functions ,, . Mutated nucleophosmin (mNPM) plays an important role in AML leukemogenesis and development  . Accordingly, NPM was suggested by many researchers as a molecular target in the development of new targeted therapies for AML  . Most NPM functions are dependent on its subcellular localization and phosphorylation  . The cdk2/cyclin E-dependent and A-dependent phosphorylation of NPM at Thr199 is crucial for centrosome duplication and possibly for other cell cycle events  .
In this study we investigated for NPM phosphorylation in OCI-AML3, which is an AML cell line that expresses mNPM in its cytoplasm  , compared with HL60, which carries wild-type nucleophosmin (wtNPM) only. Differences in localization, expression, and cell cycle kinetic changes between phosphorylated nucleophosmin (pNPM) and unphosphorylated NPM in both cell lines were estimated. The main objective was to study the possibility of targeting mNPM with an effective therapy.
| Materials and methods|| |
Both OCI-AML3 and HL60 purchased from DSMZ-German Collection of Cell Cultures (ACC 582, 583) were grown in RPMI media 1640, with GlutaMAX (Invitrogen, California, USA) supplemented with 10% FCS and 1% Pen/Strep (61870-044, 10108-165, and 15070-063; Gibco, UK), at optimum gas and temperature conditions (37°C, 5% CO 2 ). A 1 μmol/l concentration of colchicine was added to the media for synchronization.
Antibodies for immunoblotting
Primary antibodies were mouse anti-NPM (Oxford Laboratories, Oxford, UK) and pNPM (Thr199) (3541; Cell Signaling, Netherlands). Secondary antibodies were ECL anti-mouse and anti-rabbit (NA931, NA934; GE Healthcare, Buckinghamshire, UK).
Cells were pelleted, washed twice, and lysed by 30-min incubation with RIPA buffer containing 50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, 1% Triton-x, 10% sodium deoxycholate, and 1 mmol/l EDTA, with intermittent vortexing. Lysates were centrifuged; the supernatant was collected, diluted, and boiled for 5 min, recollected by brief centrifugation, and then loaded into 12% SDS-PAGE gels and electroblotted following the same protocol described by Towbin et al.  , with some modifications. Finally, images of the obtained blot were captured using a Kodak X-Ray film and cassette (Kodak Biomax XAR Film size 18/24 cm; Sigma, Aldrich, UK). The films were developed using a Kodak Developer/Fixer Kit (Kodak). The obtained protein bands were then marked compared with the Bio-Rad (UK) Precision Protein Standards.
Antibodies for immunostaining
Primary antibodies used were mouse anti-NPM (Zymed, FC-61991, California, USA,), antifibrillarin (ab5821, Abcam, Cambridge, UK), pNPM (Thr199, Cell Signaling), and a polyclonal to NPM (15440; Abcam, Cambridge, UK).
Secondary antibodies used were goat anti-rabbit (ab5999; Abcam) conjugated with fluorescein isothiocyanate and goat anti-mouse (ab6003; Abcam) conjugated with Texas Red.
Cellular cytospins were prepared from living cells. We followed the manufacturer's recommended protocol for immunostaining cytospins for wtNPM and pNPM Thr199. Next, the slides were viewed under a Nikon Eclipse 80I Fluorescence Microscope (ICCT Technologies, Sheppard, Canada), equipped with three fluorescence filter cubes. The obtained images were captured with a Hamamatsu ORCA HR (C4742-95-12HR, Hamamatsu, Japan) high-resolution digital camera, with full remote control from a PC. Another fast high-sensitivity black and white camera was used for low-intensity fluorescence slides.
Data analysis and computer technology
Results of the immunoblot analysis were scanned and saved with an Epson Photoscanner V5.0 (Digital ICE technology, UK) and subsequently edited and illustrated with the Adobe Photoshop V5.0 LE program (Adobe Systems Incorporated, USA).
Immunofluorescence images were captured, analyzed, and saved with the NIS-Elements imaging software (Nikon, UK). Also, creation of overlay images - for example, DAPI/NPM overlay - was carried out with the same program. The saved images were edited with Adobe Photoshop or Picasa programs (Google.com).
| Results|| |
Wild-type nucleophosmin and mutated nucleophosmin subcellular localization in HL60 and OCI-AML3
First, we carried out coimmunostaining of HL60 and OCI-AML3 cells with wtNPM and fibrillarin, a known marker for the dense nucleolar-fibrillar component, to detail wtNPM localization. The obtained results showed an interesting distribution pattern of wtNPM within the nucleolus of these cells, localizing at the nucleolar periphery and sparing the central area. This central nucleolar region showed a fibrillarin immunostaining pattern. These findings were similar in the interphase nuclei of both HL60 and OCI-AML3 cells, as depicted in [Figure 1].
|Figure 1 Immunofluorescence images of interphase HL60 and OCI-AML3 coimmunostained with fib (green) and wild-type nucleophosmin (wtNPM) (red); nuclei were stained with DAPI (blue). Black and white images of fib/NPM coimmunostaining were captured with a Nikon Eclipse black and white camera.|
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When colchicine-synchronized HL60 and OCI-AML3 were immunostained for wtNPM using FC-61991, wtNPM was translocated to the cytoplasm in actively dividing cells of either type ([Figure 2]). Using western blot analysis, it was found that the cytoplasmic translocation of NPM was associated with an overall reduction in the expression of total NPM ([Figure 3], upper panel compare lanes A and B).
|Figure 2 Immunocytofluorescent staining of cytospin preparations of HL60 and OCI-AML3 cells using primary antibodies against NPM (red) (a & b resting cells, C & D dividing cells and the arrows show the cytoplasmic translocation of NPM) or pNPM(green) (E & F) as indicated in text on the left of the figure, nuclei were counterstained with DAPI (blue).|
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|Figure 3 Western blot analysis of HL60 cell lysates from nondividing or colchicine-treated cultures as indicated. The upper panel shows total nucleophosmin (NPM); the lower panel shows only phosphorylated NPM (pNPM) (Thr 199).|
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Immunostaining with anti-panNPM (detects wtNPM and mNPM) showed both nucleolar and nucleocytoplasmic localization in OCI-AML3 ([Figure 4]). This appearance was not seen in HL60 cells and was not seen using antibodies that did not detect mNPM.
|Figure 4 Immunocytofluorescent staining of cytospin preparations of OCI-AML3 cells using primary antibodies against wtNPM only (red) or wt and mNPM (green)as indicated in text at the bottom of the figure, nuclei were counterstained with DAPI(blue).|
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The cytoplasmic-located and nucleoplasmic-located nucleophosmin in OCI-AML3 is phosphorylated at Thr199
Immunostaining of HL60 and OCI-AML3 with pNPM (Thr199) showed that only actively dividing HL60 cells expressed pNPM Thr199 in their cytoplasm, whereas interphase cells did not express pNPM, neither in their nuclei nor in their cytoplasm. In contrast to HL60, OCI-AML3 expressed pNPM in the nucleoplasm of dividing as well as resting cells. Furthermore, pNPM Thr199 staining was detected at the spindle asters of dividing cells. This finding was much more apparent in OCI-AML3 rather than in HL60, as shown in [Figure 2]. Comparing this pattern with the immunostaining results of mNPM in OCI-AML3 ([Figure 4]), it was concluded that the cytoplasmic NPM is phosphorylated at Thr199 even in resting cells. This finding was confirmed by immunoblotting of pNPM in HL60 and OCI-AML3 whole cellular lysates (WCLs). The obtained blot showed pNPM protein bands in cellular lysates of proliferating HL60 and OCI-AML3 cells. In contrast, pNPM was expressed in lysates of resting OCI-AML3 only ([Figure 5] and [Figure 6]).
|Figure 5 Immunoblotting of pNPM Thr199 in protein extracts of resting & proliferating OCI-AML3 in lanes a, b and in extracts of resting & proliferating HL60 in lane c.|
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|Figure 6 Immunoblotting of phosphorylated nucleophosmin (pNPM) Thr199 in protein extracts of serial dilutions of resting OCI-AML3 (lanes 1, 2, 3, and 4), in extracts of proliferating OCI-AML3 (lane 5), in proliferating HL60 (lane 6), and in resting HL60 (lane 7).|
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| Discussion|| |
AML is a hematopoietic neoplasm with diverse molecular features  . Research is ongoing in an attempt to develop a new effective targeted therapy for AML. mNPM is very common in AML and is implicated in disease pathogenesis , . However, many researchers focused on FLT3 and JAK2 inhibitors as a possible targeted therapy in AML , . Schlenk et al.  found that, in the absence of FLT3-ITD mutation, AML with mNPM is sensitive to the addition of all transretinioc acid (ATRA) to the chemotherapeutic regimen. However, this observation was not confirmed further  . As NPM functions were found to be related to its Thr199 phosphorylation, this study was conducted to find out whether mNPM is phosphorylated at Thr199. We followed other researchers and used OCI-AML3 cells as a model for AML with mNPM and HL60 cells as a comparative cell line , .
First, immunostaining of both cell types for wtNPM and then for panNPM was done. The obtained immunofluorescence images showed strict nucleolar and nucleocytoplasmic localization of wtNPM in resting and dividing cells of both cell types, respectively. This finding was consistent with that of other researchers who concluded that NPM, although a nucleolar protein, has a cell cycle-dependent chaperone activity , . Consistent with the reported behavior of mNPM by Burnett and colleagues , , we confirmed that immunostaining of OCI-AML3 with anti-panNPM showed both nucleolar and nucleocytoplasmic localization, strongly suggesting that the nucleoplasmic and cytoplasmic NPM was of mNPM type. Nishimura et al.  explained this localization of mNPM by reporting that any deletions of the protein sequence immediately before Trp288, or mutation to alanine of both Trp residues, was sufficient to dislocate the protein in the nucleoplasm.
Second, immunostaining of both Hl60 and OCI-AML3 with Thr199 phosphospecific antibody showed that mNPM is phosphorylated at Thr199. This novel finding was further confirmed by the immunoblotting results of HL60 cellular lysates with pNPM Thr199 where the protein band was detected in lysates of dividing cells only. Also, our immunofluorescence pattern confirmed the finding of Tarapore et al.  , who found that Thr199 phosphorylation targets NPM to nuclear speckles.
In conclusion, our results revealed that OCI-AML3 expressed both wtNPM and mNPM. Also, we demonstrated that wtNPM has a typical nucleolar distribution in stationary phase cells and is translocated to the cytoplasm and phosphorylated during cell division.
mNPM in OCI-AML3 is expressed in the nucleoplasm and cytoplasm, but not in nucleoli. Furthermore, by immunocytofluorescence, mNPM is phosphorylated at Thr199 in nondividing cells. This was further confirmed semiquantitatively with the immunoblotting analysis of pNPM Thr199 in both HL60 and OCI-AML3 lysates.
OCI-AML3 is phenotypically similar to cases of AML that express mNPM. Therefore, our finding that the mNPM in this cell line is phosphorylated at Thr199 in resting cells may have implications in suggesting cdk2 (the mediator of phosphorylation) inhibitors as a possible effective targeted therapy in de-novo AML with mNPM. We proposed that clinical trials of cdk2 inhibitor in AML should be started in patients with NPMc+ AML. This proposition is supported by the work of Okuda et al.  who reported that activation of cdk2/cyclin E occurs simultaneously with NPM phosphorylation. Cdk2/cyclin E activation was found previously to be important for DNA replication-initiation factor function.
Furthermore, we speculated that molecular targeting of mNPM will be a less toxic type of therapy for AML, referring to what has been reported by others  that NPM is highly expressed in tumor cells than in normal cells. However, repeating the same experiments of this work in the presence of cdk2 inhibitors is highly recommended.
| Acknowledgements|| |
The authors thank Dr Karen-Rees Unwin and Dr Suzanne Johnson, at Manchester School of Medicine, UK, for their continuous help throughout the study.
This work was supported by the Egyptian Cultural Bureau Educational grant.
Conflicts of interest
There are no conflicts of interest.
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