We recently identified a new acute myeloid leukemia (AML) subtype characterized by mutations at exon-12 of the nucleophosmin (NPM) gene and aberrant cytoplasmic expression of NPM protein (NPMc+). NPMc+ AML accounts for about 35% of adult AML and it is associated with normal karyotype, wide morphological spectrum, CD34-negativity, high frequency of FLT3-ITD mutations and good response to induction therapy. In an attempt to identify a human cell line to serve as a model for the in vitro study of NPMc+ AML, we screened 79 myeloid cell lines for mutations at exon-12 of NPM. One of these cell lines, OCI/AML3, showed a TCTG duplication at exon-12 of NPM. This mutation corresponds to the type A, the NPM mutation most frequently observed in primary NPMc+ AML. OCI/AML3 cells also displayed typical phenotypic features of NPMc+ AML, that is, expression of macrophage markers and lack of CD34, and the immunocytochemical hallmark of this leukemia subtype, that is, the aberrant cytoplasmic expression of NPM. The OCI/AML3 cell line easily engrafts in NOD/SCID mice and maintains in the animals the typical features of NPMc+ AML, such as the NPM cytoplasmic expression. For all these reasons, the OCI/AML3 cell line represents a remarkable tool for biomolecular studies of NPMc+ AML.
We identified mutations at exon-12 of the nucleophosmin (NPM) gene as the genetic lesion most specifically and frequently associated with normal karyotype in adult4 and pediatric5 AML. Named NPMc+ AML, this subgroup accounts for about 35% of adult AML (60% of all AML with normal karyotype), encompasses all FAB/WHO categories6 except M3, M4eo and M7 and is characterized by multilineage involvement, CD34-negativity, high frequency of FLT3 mutations and good response to induction therapy.4
Exon-12 NPM gene mutations are AML-specific since they are not detected in normal cells or other neoplasms. Frameshifts alter NPM protein at its C-terminus4 and the mutated NPM protein localizes aberrantly in leukemic cell cytoplasm,4 while wild-type NPM, a protein with multiple functional domains,7 shuttles between nucleus and cytoplasm,8 and is found mainly in the nucleolus.9
The mechanisms underlying abnormal cytoplasmic accumulation of mutant NPM protein and its interference with wild-type NPM, which is involved in ARF-p53 pathway regulation10, 11, 12 and centrosome duplication,13 remain to be clarified.
In an attempt to identify a human NPMc+ AML cell line, which would greatly help in addressing these issues and in investigating the activity of new drugs, we screened 79 myeloid cell lines for the presence of exon-12 NPM gene mutations and found one, OCI/AML3, exhibiting the typical molecular and biological features of NPMc+ AML.
Materials and methods
Mutational analysis of human myeloid cell lines
A total of 79 human myeloid cell lines were evaluated for NPM mutations (Table 1). Mutations at the exon-12 of the NPM gene are recurrent genetic aberrations in AML.4 The NPM mutated and wild-type status of the 79 cell lines was verified by determining the size of the products of an NPM polymerase chain reaction (PCR) by capillary gel electrophoresis. NPM PCR analysis was performed with a DNA thermal cycler (Perkin Elmer Cetus, Heidelberg, Germany) under the following conditions: 30 s at 94°C for denaturation, 30 s at 53°C for annealing and 2 min at 72°C for extension. Genomic PCR amplification was performed using primer NPM intron 11 forward: IndexTermIndexTerm5′-TTA ACT CTC TGG TGG TAG AAT GAA-3′ and reverse-transcriptase PCR with primer NPM exon 11 forward: IndexTermIndexTerm5′-CAT CAA TTA TGT GAA GAA TTG CTT-3′. Reverse primer for both PCR analyses was NPM exon 12 reverse: IndexTermIndexTerm5′-TGT TAC AGA AAT GAA ATA AGA CGG-3′. The reverse primer was either labeled with the fluorescent dye D4 (for capillary electrophoresis) or not-labeled (for cloning experiments). Oligonucleotides were obtained from Proligo (Paris, France). For fragment size determination, 1 μl of the PCR products was combined with 0.25 μl of an internal size standard (size standard kit 400, Beckman-Coulter, Krefeld, Germany) in a total volume of 30 μl sample loading solution (SLS, Beckman-Coulter). Electrophoresis was carried out on the capillary electrophoresis system CEQ 8000 (Beckman-Coulter). The following injection and electrophoretic running conditions were used: 0.5 μl sample injection, 5 min DNA strand denaturation at 93°C, separation at 6000 V at 50°C and signal detection with calibrated D4 emission spectra. Fragment size determinations were performed using default fragment analysis parameters of the CEQ 8000 fragment analysis software, allowing the precise determination of fluorescent DNA fragments resulting in an electropherogram and a fragment summary list. NPMwt PCR products were 215 bp (genomic PCR) and 139 bp (RT-PCR). NPMm primary AML cells, used as positive control, exhibited 4 bp larger signals in addition to the NPMwt signals. For the identification of NPM variants in cell lines, a second PCR analysis was performed, this time with unlabeled oligonucleotides. The PCR products were cloned into the pGEM-T Easy Vector System (Promega, Mannheim, Germany), NPMm and wt clones were identified with the described detection system and positively identified plasmids were sequenced (MWG Biotech, Ebersberg, Germany).
Karyotypic analysis and fluorescence in situ hybridization (FISH) were performed as described previously14
Monoclonal anti-NPM and anti-CD68 antibodies have been previously described.9, 15, 16 An affinity-purified rabbit polyclonal antibody (SIL-A) was generated against a synthetic 18mer peptide (NH2-QEAIQDLCLAVEEVSLRK-COOH) (Primm srl, Milan, Italy), which recognizes the NPM mutant A4 but not wild-type NPM protein. Monoclonal antibody against nucleolin (C23) was purchased from Dako Cytomation (Glostrup, Denmark).
Engraftment in NOD/SCID mice
NOD/SCID mice were purchased from Charles River Italia Spa (Calco, Italy) and maintained under sterile conditions in the animal facilities at the University of Perugia, Perugia, Italy. Six mice were injected subcutaneously with OCI/AML3 cells at dosage ranging from 20 × 106 to 50 × 106 OCI/AML3 cells.
Immunocytochemical detection of NPM
For immuno-alkaline phosphatase (APAAP) detection of wild-type and mutant NPM proteins, we used cytocentrifuge preparations of acetone- or 4% paraformaldeyde-fixed (10 min) OCI/AML3 cells, and paraffin sections (3 μm) from B5-fixed cell line pellets (each containing 107 cells). Paraffin sections were subjected to antigen retrieval by microwaving in 0.1 mM EDTA pH 8.0.4 The immuno-alkaline phosphatase APAAP technique was used for immunostaining.17
For immunofluorescence, paraffin sections of OCI/AML3 cells were incubated with the anti-NPM monoclonal antibody9, 15 followed by a secondary goat anti-mouse antibody conjugated with Alexa 488 (Molecular Probes, OR, USA), counterstained with propidium iodide and analyzed with a confocal microscope (Zeiss, LSM 510). Images were collected with a Zeiss LSM 510 confocal microscope Carl Zeiss, Jena, DE, Germany) using the 488 nm (for Alexa 488) and the 543 nm (for propidium iodide) laser lines for excitation. AOTF-controlled tuning of laser lines, diameters of pinholes and light collection configuration were optimized to obtain the best signal-to-noise ratio and to avoid any fluorescence crossover.
The LSM 510 software was used for microscope regulation and collection of images. The images were transferred to an SGI Octane workstation (Silicon Graphics, Mountain View, CA, USA) for further processing. Three-dimensional reconstruction of the slices was performed with the shadow technique, using the Imaris (Bitplane, Zurich, CH) software.
Nuclear and cytoplasmic extracts were prepared using a slightly modified Schreiber method.18 Briefly, 5 × 106 cells were collected, washed with phosphate-buffered saline and used either fresh or after being snap frozen in liquid nitrogen. Either cells or frozen cell pellets were resuspended in 150 μl of buffer A containing 10 mmol/l HEPES (pH 7.9), 1.5 mmol/l MgCl2, 10 mmol/l KCl, 0.2 mmol/l EDTA, 1 mmol/l dithiothreitol and proteases inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 mM phenylmethane sulfonyl fluoride, 1 mM sodium orthovanadate) and incubated for 15 min on ice, after which 7.5 μl of a 10% solution of Nonidet P-40 (0.5%, final) was added and the tube was vigorously vortexed for 10 s. The homogenate was centrifuged at 12 000 r.p.m for 30 s. The cytoplasmic supernatant fraction was spun once more to ensure complete removal of nuclei. The nuclear pellet was washed again with buffer A with centrifugation as above. The nuclear pellet was then resuspended in 50 μl of lysis buffer C containing 10 mmol/l HEPES (pH 7.9), 0.42 mol/l NaCl, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 0.5 mmol/l dithiothreitol, 25% glycerol and proteases inhibitors. After incubation on ice for 15 min (with occasional vortexing) the samples were centrifuged at 12 000 r.p.m for 15 min at 4°C. The supernatant was recovered as nuclear extracts. Nuclear and cytoplasmic extracts were snap frozen in liquid nitrogen and stored at −80°C until use.
Whole cell lysates (WCL) were prepared by addition of ice-cold lysis buffer (1% NP-40, 150 mmol/l NaCl, 25 mmol/l Tris pH 7.5, 1 mmol/l EDTA, 1 mmol/l Na3VO4, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 mmol/l phenylmethylsulfonyl fluoride) on ice for 20 min. Cell lysates were clarified by centrifugation at 14 000 g for 10 min at 4°C. Proteins were boiled in SDS sample buffer, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF; Millipore, Bedford, MA, USA) and probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies. Polypeptides recognized in the Western blot were detected using the enhanced chemiluminescence (ECL) methods according to the manufacturer's instructions (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA).
OCI/AML3 cell line bears mutation A at exon-12 of the NPM gene
OCI/AML3 was the only myeloid cell line among the 79 tested that expressed a signal in addition to and 4 bp larger than the wild-type NPM signal (Figure 1a). Sequencing of cloned NPM-mutated PCR product showed TCTG duplication at positions 956–959 of exon-12 of the reference sequence (GenBank accession number NPM-002520). This mutation was heterozygous and corresponded to the type A we previously described in 77% of primary NPMc+ AMLs.4 Exon-12 NPM mutation of type A causes a frameshift in the region encoding the C-terminal of the NPM protein. As a consequence, the last seven amino acids (WQWRKSL) of NPM are replaced by 11 different residues (CLAVEEVSLRK). Important changes at the C-terminus of NPM mutant A include mutations of both tryptophans at position 288 and 290 which are important for nucleolar localization19 and the creation of an NES motif.20
Phenotypic and cytogenetic/FISH features of the OCI/AML3 cell line
The OCI/AML3 cell line was established from the peripheral blood taken from a 57-year-old male with AML FAB-M4.21 The OCI/AML3 cells show a myeloid morphology (Figure 1b, left-hand side) and the immunophenotype: CD3−, CD4+, CD8−, CD10−, CD13+, CD14−, CD15+, CD19−, CD30−, CD34−, CD41+, CD42b−, CD68+ (Figure 1b, right-hand side), CD117− and CD235a+.
Karyotype of the AML at diagnosis from which OCI/AML3 was established was not available. The consensus karyotype was 48(45-50)<2n>X/XY,+1,+5,+8, der(1)t(1;18) (p11;q11),i(5p), del(13)(q13q21),dup(17)(q21q25);sdl with r(Y)x1-2;hemizygous for RB1 (Figure 2).
FISH analysis of OCI/AML3 revealed no structural rearrangements involving MLL, MOZ, or CBFB. FLT3 internal tandem duplication (ITD) is a recurrent aberration in AML,22 but quite rare in cell lines.23 Although FTL3 ITD are twice as frequent in NPMc+ than in NPMc− AML with normal karyotype,4 the cell line OCI/AML3 exhibits neither FLT3 ITD nor FLT3 D835 point mutation.23
OCI/AML3 cell line exhibits aberrant cytoplasmic NPM expression
Immunostaining with specific monoclonal antibodies9 detected NPM both in the cytoplasm and nucleus (nucleoli) of OCI/AML3 cells (Figure 3a, top left, middle left). This contrasts with the nucleus-restricted expression of NPM observed in AMLs bearing wild-type NPM, such as the cell line HL-60 (Figure 3a, top right). Therefore, the NPM staining pattern in the OCI/AML3 cell line is identical to that observed in primary NPMc+ AML samples.4
However, anti-NPM monoclonal antibodies do not distinguish between wild-type and mutated NPM which are both present in leukemic cells due to the fact that exon-12 NPM mutations are consistently heterozygous.4 In order to investigate the subcellular expression of the mutant A without the interference of the NPM wild-type protein, we stained the OCI/AML3 cells with an antibody specific for mutant A and found that mutated NPM is expressed exclusively in the cytoplasm (Figure 3a, middle right); pre-incubation with the immunogen peptide abolished the reactivity of the anti-NPM-mutated antibody (Figure 3a, bottom left). In contrast, the anti-nucleolin antibody only labeled nucleoli (Figure 3a, bottom right). Immunohistochemical findings were confirmed by Western blotting analysis (Figure 3b).
Alltogether, these findings demonstrate that OCI/AML3 cells bear the hallmark of primary NPMc+ AML, that is mislocalization of the mutant NPM protein in the cytoplasm.
Engraftment in NOD/SCID mice
Subcutaneous injection of OCI/AML3 cells in NOD/SCID produced palpable tumors in all six injected animals by days 25–30. At post-mortem examination, mice showed disseminated disease that was confirmed by morphological and immunohistochemical studies showing the typical aberrant expression of NPM in the cytoplasm of the leukemic cells (Figure 4). Since the anti-NPM monoclonal antibodies recognize both human and mouse NPM, nucleus-restricted expression of murine NPM served as a control.
Human cell lines derived from AML bearing recurrent chromosomal abnormalities represent remarkable tools for biological and molecular studies of these diseases.14
We recently identified a new AML subtype characterized by mutations at exon-12 of the NPM gene and aberrant cytoplasmic expression of the NPM protein (NPMc+).4 NPMc+ AML accounts for about 35% of adult AML and it is characteristically associated with a normal karyotype, wide morphological spectrum, CD34 negativity, high frequency of FLT3-ITD mutations and a relatively good response to induction therapy.4 Our numerous attempts to establish a cell line from these leukemic patients were unsuccessful. This prompted us to carry out a mutational screening for exon-12 NPM mutations in a large number of available human myeloid cell lines.
Here, we report that the OCI/AML3 cell line bears the characteristic molecular and biological features of the NPMc+ AML. The OCI/AML3 cell line was the only human myeloid cell line, among the 79 tested, to bear mutations at the exon-12 of the NPM gene. As NPM mutations occur in about 35% of adult AML patients,4 their low frequency in myeloid cell lines is somewhat surprising, but may indicate this leukemia subtype is difficult to grow in culture, possibly because of its dependence on host growth factors. Notably, the OCI/AML3 cell line bears mutation A, which is the most frequent type of mutations occurring at exon-12 of the NPM gene in AML (about 80% of cases).4 In this regard, OCI/AML3 is a model representative of most patients with NPMc+ AML.
The immunophenotype of the OCI/AML3 cell line is also consistent with a derivation from a patient with NPMc+ AML. The OCI/AML3 cells express macrophage markers such as CD68 (PG-M1)16 and lack of CD34. This is in keeping with the finding that primary NPMc+ AML are most frequently of the M4 and M5 type and show absence of CD34 both at immunohistochemistry4 and gene expression profiling.24
Mutations at exon-12 of the NPM gene are the genetic lesions most specifically and frequently associated with normal karyotype in adult AML.4 NPMc+ AML account for about 60% of all adult AML with a normal karyotype. Karyotype of patient primary AML cells from which the OCI/AML3 was established was not known. The results of our cytogenetic investigations deserve some comments. Trisomy 8 occurs in 10–15% of AML in all FAB subgroups, alone or together with simple karyopypic changes.22 Interestingly, +8 partners i(5p) in a subset of AML-M5 patients, possibly associated with 1q trisomy25 as found in OCI/AML3. This is in keeping with the finding that NPMc+ AML are more frequent in M5 than other FAB subtypes.4 Deletion of proximal 13q (including the FLT3 locus at 13q13) as present in OCI/AML3 may also partner +8 in AML patients possibly targeting loss of heterozygosity affecting the RB1 locus at 13q14.26 The association in OCI/AML3 of the above chromosomal abnormalities and NPM gene mutation is not surprising since we previously reported that about 14% of NPMc+ AML bear nonmajor chromosomal abnormalities.4
The OCI/AML3 displays one of the most remarkable features of NPMc+ AML, that is, the dislocation of the NPM protein in the cytoplasm. According to our results, this finding is certainly due to the presence in the cytoplasm of the NPM mutant A and, possibly, of the wild-type NPM protein that could be aberrantly recruited in the cytoplasm through formation of heterodimers with the mutant.27 In contrast, we regard nuclear labeling of OCI/AML3 for NPM to be exclusively due to the presence in that site of the wild-type NPM protein, as proved by the finding that an antibody specifically directed against the mutant only detects the abnormal NPM protein in the cytoplasm. Mechanism of altered-nucleo-cytoplasmic traffic of NPM in leukemic cells is unknown and the OCI/AML3 represents a good in vitro model for addressing this issue and analyzing the drug's ability to interfere with this process. Moreover, OCI/AML3 cells easily engraft in NOD/SCID mice and this may serve as a pre-clinical model for testing new therapeutic modalities.
In conclusion, the OCI/AML3 represents the only permanent cell line sharing all molecular and biological features of primary NPMc+ AML and emerges as a remarkable tool for studying this new leukemia subtype.
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This work was supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC). We thank Claudia Tibidò for the assistance in the preparation of the manuscript. We are indebted to Roberta Pacini and Alessia Tabarrini for performing the immunohistochemical studies.
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