|Deregulation of NPM and PLZF in a variant t(5;17) case of acute promyelocytic leukemia|
|Jeff L Hummel1, Richard A Wells4, Ian D Dubé1,3, Jonathan D Licht5 and Suzanne Kamel-Reid1,2,a|
1University of Toronto and the Institute of Medical Sciences, 7213 Medical Sciences Building, M5S 1A8, Toronto, Ontario, Canada
2The Ontario Cancer Institute and The Toronto Hospitals Cancer Cytogenetics and Molecular Oncology Program, The Toronto Hospital/Princess Margaret Hospital, 610 University Avenue, M5G 2M9, Toronto, Ontario, Canada
3Laboratory Medicine, Sunnybrook Health Sciences Center, 2075 Bayview Avenue, M4N 3M5, Toronto, Ontario, Canada
4Children's Hospital and Dana Farber Cancer Institute, Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115, USA
5Derald H Ruttenberg Cancer Center and Departments of Medicine and Molecular Biology, Mount Sinai School of Medicine, New York 10029, USA
aAuthor for correspondence: The Ontario Cancer Institute and The Toronto Hospitals Cancer Cytogenetics and Molecular Oncology Program, The Toronto Hospital/Princess Margaret Hospital, 610 University Avenue, M5G 2M9
Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RAR. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXR), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RAR to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identified in a pediatric case of APL. RT - PCR, cloning, and sequence studies identified NPM as the RAR partner on chromosome 5, and both NPM-RAR and RAR-NPM fusion mRNAs were expressed in this patient. We further explored the effects of the NPM-RAR chimeric protein on the subcellular localization of PML, RXR, NPM, and PLZF using immunofluorescent confocal microscopy. While PML remained localized to its normal 10 - 20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXR, and NPM-RAR in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RAR partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.
APL; NPM-RAR; PLZF; subcellular localization
For over 40 years acute promyelocytic leukemia (APL) has been recognized as a subtype of acute leukemia because of its morphology and clinical association with fatal coagulopathy (Hillestad, 1957), as well as its unique association with translocations involving the retinoic acid receptor alpha gene (RAR) (Giguère et al., 1987). Nearly 100% of APL cases are associated with a t(15;17)(q22;q21) translocation that fuses RAR to the promyelocytic leukemia gene (PML) at chromosome 15q22 (de Thé et al., 1990, 1991; Alcalay et al., 1991; Goddard et al., 1991; Kakizuka et al., 1991). The resulting PML-RAR gene is invariably expressed in t(15;17) APL (Castaigne et al., 1992; Grignani et al., 1994) in comparison to the reciprocal RAR-PML gene which is expressed in approximately 80% of cases (Alcalay et al., 1992; Borrow et al., 1992; Grimwade et al., 1996; Li et al., 1997).
Although the molecular mechanisms of action of PML-RAR are still unclear, it has been shown to exert its transforming effects by sequestering hematopoietic factors like RXR, PML, and PLZF (Dyck et al., 1994; Weis et al., 1994; Koken et al., 1997; He et al., 1998); or by acquiring altered transactivating properties that affect retinoic acid (RA) and PML responsive genes (de Thé et al., 1991; Kakizuka et al., 1991). Much like RAR (Chen and Evans, 1995; Nagy et al., 1997), PML-RAR retains the ability to recruit the transcriptional co-repressors N-CoR and SMRT to RA responsive genes (Hong et al., 1997). In contrast to RAR, PML-RAR appears to interact with N-CoR and SMRT to form stable transcriptional repressor complexes under physiological concentrations of RA (Grignani et al., 1998; Guidez et al., 1998; He et al., 1998; Lin et al., 1998).
PML is a nuclear matrix protein (Chang et al., 1995) that localizes in the nucleus to 10 - 20 discreet nuclear bodies or PML oncogenic domains (PODs) (Daniel et al., 1993; Dyck et al., 1994). In t(15;17) APL cells it has been shown that PML is delocalized, and closely associated with PML-RAR in a dispersed, subcellular pattern (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). The finding that t(15;17) cells treated with all trans retinoic acid (atRA) reform PML nuclear bodies resulted in the hypothesis that the disruption of PML nuclear bodies is a key molecular event in APL pathogenesis (Dyck et al., 1994). Although the cellular mechanisms of PML are poorly understood, PML has been shown to exhibit growth suppressor function (Mu et al., 1994; Koken et al., 1995; Liu et al., 1995; Le et al., 1996) and induce apoptosis through its RING domain (Borden et al., 1997).
Molecular genetic techniques identify the PML-RAR fusion gene in nearly 100% of APL cases, although recently, rare cases lacking the t(15;17) have been characterized. Three variant APL translocations have been described, all of which involve translocations fusing RAR to partner genes other than PML. The t(11;17)(q23;q21) translocation, in which RAR is fused to the promyelocytic leukemia zinc finger gene (PLZF) (Chen et al., 1993), appears to be the most commonly occurring variant (Licht et al., 1995). It has been observed that ectopically expressed PLZF-RAR inhibits normal RAR function in a trans dominant fashion (Chen et al., 1994; Licht et al., 1996). Like PML-RAR, PLZF-RAR also appears to behave as a transcriptional repressor, capable of interacting with N-CoR and SMRT (Hong et al., 1997). However, PLZF-RAR forms co-repressor complexes that are insensitive to pharmacological concentrations of RA (Grignani et al., 1998; Guidez et al., 1998; He et al., 1998; Lin et al., 1998). Taken together, these data implicate PLZF-RAR in the pathogenesis of APL as well as characterize a molecular mechanism for the differential response of APLs to RA. Recently, our group characterized a novel t(11;17)(q13;q21) variant APL translocation (Wells et al., 1996, 1997) which resulted in the fusion of RAR to the nuclear mitotic apparatus gene, NuMA (Lydersen and Pettijohn, 1980; Sparks et al., 1993). A variant t(5;17)(q35;q21) APL translocation, which fuses RAR to the nucleophosmin gene (NPM) (Kang et al., 1974; Chan et al., 1989) has also been recently identified (Redner et al., 1996). Like PML-RAR and PLZF-RAR chimeric proteins, it appears that NPM-RAR shares the ability to act as a ligand-dependent transcriptional activator of retinoic acid responsive genes, in vitro (Redner et al., 1996).
NPM is a nuclear matrix phosphoprotein that is ubiquitously expressed, and primarily localizes to the dense fibrillar and granular components of the nucleolus where it is believed to participate in ribosome assembly (Borer et al., 1989; Chan et al., 1990). NPM also appears to interact specifically with proteins bearing nuclear/nucleolar localization signals (Li et al., 1996), which is consistent with the proposal that NPM shuttles proteins destined for the nucleolus (Borer et al., 1989). The NPM protein has a modular structure with four main functional motifs: a ribonuclease domain, a multimerization domain, a dimerization domain, and a DNA/RNA binding domain (Wang et al., 1994; Herrera et al., 1995, 1996; Li et al., 1996). Interestingly, NPM rearrangements have been reported in specific translocations associated with hematological malignancies other than APL. Approximately one third of anaplastic large cell lymphoma (ALCL) tumors have a t(2;5)(p23;q35) translocation that fuses NPM to a novel transmembrane tyrosine-specific protein kinase, anaplastic lymphoma kinase (ALK) (Morris et al., 1994; Downing et al., 1995). Similarly, cases of myelodysplastic syndrome and a subset of acute myeloid leukemia have been shown to be associated with a t(3;5)(q25;q34) translocation that fuses NPM to a novel myelodysplasia/myeloid leukemia factor (MLF1) (Yoneda-Kato et al., 1996).
We describe the molecular characterization of a t(5;17) variant APL translocation involving NPM, and explore the effects NPM-RAR has on the subcellular localization of PML, RXR, NPM, and PLZF in the bone marrow cells of a t(5;17) bearing patient.
A variant APL translocation
All cells examined by cytogenetics revealed a complex karyotype that included a variant APL translocation involving chromosomes 5 and 17. The karyotype was 47, XY, t(5;17)(q35;q21), der (8)(p23), der(10)(q26), del(12)(q13q22), del(13)(q12q14), -16, -18, +21, +22 and +M. The t(15;17)(q22;q21) translocation was not detected in any of the metaphases examined. Southern blot analysis was performed to confirm the molecular involvement of RAR, as well as to detect any occult rearrangement of PML. In patient DNA, additional restriction fragments were detected using the RAR specific LCN4A3B probe when compared to non-leukemic cells (Figure 1a). No evidence of PML rearrangement was detected using overlapping PML cDNA probes.
The t(5;17) translocation fuses nucleophosmin to RAR
When study of the t(5;17) APL translocation began, the identity of the RAR fusion partner was unknown. Due to limited patient material, somatic cell hybrid lines were established and screened for the retention of one of the derivative chromosomes. Through cytogenetic and Southern analyses, we identified a somatic cell hybrid that had retained the derivative 17 chromosome (data not shown). Northern blot analysis of this cell line, named A6, further revealed the presence of an aberrant 1.8 kb RAR mRNA (Figure 1b). Following a report that demonstrated an NPM-RAR fusion event in a similar t(5;17) APL case (Redner et al., 1996), the presence of a reciprocal RAR-NPM mRNA in somatic cell hybrid A6 was confirmed by RT - PCR analysis (data not shown).
t(5;17) APL blasts express NPM-RAR and RAR-NPM mRNA
The above results prompted us to examine patient RNA through a semi-nested RT - PCR approach (Figure 2a) designed to detect NPM-RAR and RAR-NPM fusion mRNA. Independent reactions containing RAR primers R231 and R361, and NPM primers N96 and N1180 routinely detected normal RAR (130 bp) and NPM (1084 bp) mRNA. The NPM-RAR primer N96 with R361 and R724 produced a 429 bp fragment, while the RAR-NPM primers R231 and R254 with N1180 produced a 733 bp fragment. In addition, smaller 150 bp and 250 bp fragments were amplified in both NPM-RAR and RAR-NPM reactions, respectively (Figure 2b). Sequence analysis indicated that the 429 bp NPM-RAR fragment was the product of a fusion event between NPM nucleotide 445 and RAR nucleotide 282 (Figure 3a). In addition, we found that the 733 bp RAR-NPM fragment was identical to that derived from the somatic cell hybrid A6 (Figure 3b). Sequences from both of the smaller RT - PCR products revealed that they were PCR artifacts.
NPM-RAR and RAR-NPM sequences predict modular proteins
The NPM breakpoint in this patient occurred between nucleotides 445 - 446 of the NPM cDNA (amino acids 118 - 119), while RAR was disrupted between nucleotides 280 and 281 (amino acids 59 - 60). cDNA sequence analysis of the NPM-RAR and RAR-NPM breakpoint junctions revealed that the NPM junction was identical to previously reported NPM-ALK, and the short isoform of NPM-RAR (NPMs-RAR) (Morris et al., 1994; Redner et al., 1996) (Figure 3a and b). The long isoform of NPM-RAR (NPML-RAR) observed by Redner and colleagues was not expressed in our patient. We also noted that the NPM-RAR and reciprocal RAR-NPM cDNA sequences maintained intact opening reading frames capable of encoding 52 and 24 kd proteins, respectively. From these sequence data we were able to determine that the NPM-RAR protein would consist of the ribonuclease and multimerization domains of NPM joined to the DNA-binding, ligand-binding and dimerization domains of RAR. Conversely, RAR-NPM would consist of the cell/promoter specific transactivating domain of RAR joined to the dimerization and nucleic acid-binding domains of NPM (Figure 4).
PML localizes to intact nuclear bodies
Immunofluorescent confocal microscopy of U937 and NB4 control cells, treated with PML antibody, resulted in the expected localization patterns. Figure 5 shows that in U937 cells, PML localized to the nuclear matrix in 10 - 20 distinct nuclear bodies or PODS. In NB4 cells, distinct nuclear bodies were absent and PML was found in a dispersed, microspeckled pattern. In contrast to the pattern seen in NB4, PML localized to the nuclear matrix in distinct nuclear bodies in bone marrow cells analysed from the t(5;17) patient.
Nucleolar localization of NPM is disrupted
Immunofluorescent confocal microscopy of U937 control cells, treated with the NPM antibody, showed that NPM localized specifically to the nucleolus. While it was shown that NPM may not be exclusively nucleolar, we noted that in these control cells the majority of NPM was localized to the nucleolus in 1 - 5 discreet dots (Figure 6). Nucleolar NPM localization was also confirmed in normal, human bone marrow (data not shown). In contrast, the nucleolar localization of NPM was disrupted in t(5;17) cells analysed from the patient. In these cells, NPM was delocalized in a dispersed nuclear pattern (Figure 6).
PLZF expression is upregulated in t(5;17) APL cells
Expression and nuclear localization patterns for PLZF in U937 and HL60 control cells were determined by immunofluorescent confocal microscopy. As shown in Figure 7, PLZF antibody detected the normal microspeckled and dispersed nuclear pattern in HL60 cells, whereas in the U937 negative control, only background signals were observed. While normal cells present in the patient's marrow showed no PLZF expression, leukemic bone marrow cells analysed from the t(5;17) patient exhibited significant expression of PLZF in a microspeckled and diffuse pattern (Figure 7).
NPM, NPM-RAR, and RXR co-localize in t(5;17) APL cells
As shown in Figure 6, immunofluorescent confocal microscopy of U937 control cells treated with RAR antibody confirmed a dispersed, nuclear localization pattern. Similar results were observed for RXR. When t(5;17) patient cells were examined, no discernable change in RAR and RXR localization was observed. However, slides containing t(5;17) patient cells dually treated with NPM and RAR, or NPM and RXR antibodies revealed that NPM, RAR and RXR signals co-localized. Since the C-terminal RAR antibody is capable of detecting NPM-RAR, these data are consistent with co-localization of NPM, NPM-RAR, and RXR in a dispersed nuclear manner.
We report the molecular characterization of a variant t(5;17) translocation observed in a pediatric case of APL. RT - PCR analysis using NPM and RAR specific primers revealed that a fusion event between NPM and RAR had occurred in the t(5;17) patient. The NPM junctions were found to be identical in sequence to the previously reported NPM-ALK junction sequences observed in ALCL (Morris et al., 1994; Downing et al., 1995). In contrast to the Redner report, we identified only one NPM-RAR isoform with an NPM junction sequence consistent with NPMs-RAR. Similar to the Redner case, we observed the expression of the reciprocal RAR-NPM mRNA which was found to contain the N-terminal RAR transactivating domain juxtaposed to the NPM dimerization and DNA/RNA binding domains. It is of interest that RAR-PLZF appears to be expressed in all t(11;17)(q23;q21) patients, and that it also contains the N-terminal RAR transactivating domain juxtaposed to a DNA binding domain. Since RAR-PLZF seems to play a role in APL pathogenesis by acting as a trans dominant activator of PLZF responsive genes (Li et al., 1997); Sitterlin et al., 1997), it is possible that RAR-NPM might also act as a second, dominant interfering gene in t(5;17)-associated APL.
In this study, as well as in the previous t(5;17) APL report, the N-terminal portion of NPM is joined to the essential functional domains of RAR. The same RAR domains are invariably joined to the N-terminal portions of PML, PLZF, and NuMA. In vitro studies have demonstrated that PML-RAR and PLZF-RAR disrupt the transactivating effects of RAR at the RARE2 promoter, and that the dominant negative effects of PML-RAR and PLZF-RAR are reversed in the presence of ectopically expressed RXR (Chen et al., 1994; Licht et al., 1996). PML-RAR and PLZF-RAR heterodimerize with RXR through the RAR dimerization domain retained in the RAR portion of the fusions (Jansen et al., 1995; Licht et al., 1996). Recently it has been shown that both PML-RAR and PLZF-RAR silence the transcription of RA responsive genes under physiological concentrations of RA (Grignani et al., 1998; He et al., 1998; Lin et al., 1998). In light of these studies, as well as the fact that RAR/RXR heterodimerization is required for RAR function (Pandolfi et al., 1991; Bugge et al., 1992; Zhang et al., 1992), it is surprising that PML-RAR/RXR heterodimers do not appear essential for PML-RAR's effect on differentiation (Grignani et al., 1996). Taken together, however, it appears that PML-RAR, PLZF-RAR, and possibly NPM-RAR disrupt wildtype RAR function by sequestering RXR to co-repressor complexes. Our observations of co-localization of NPM-RAR and RXR in t(5;17) blasts demonstrate that NPM-RAR may share the ability to sequester RXR, and potentially disrupt RAR function.
In addition to the disruption of the retinoid signaling pathway, APL fusion genes may contribute to the disease phenotype by disrupting the normal functions of the partner genes. Mutation analysis of the PML portion of PML-RAR has shown that the PML multimerization domain is critical in promoting altered differentiation in APL cells (Grignani et al., 1996). The POZ domain of PLZF, which is also a functional multimerization domain (Bardwell and Treisman, 1994), appears to be an essential component of PLZF-RAR that confers altered response to differentiation signals (Dong et al., 1996). In addition, it has been demonstrated that PML and PLZF form heterodimers with PML-RAR and PLZF-RAR, respectively, through these multimerization domains (Perez et al., 1993; Licht et al., 1996; Dong et al., 1996). Structural analysis of NPM-RAR indicates that the multimerization domain of NPM is retained in the chimeric protein. Our immunofluorescent confocal microscopy studies demonstrate that the nucleolar localization of NPM is disrupted in t(5;17) APL cells, and that NPM co-localizes with NPM-RAR in a dispersed nuclear pattern. These data suggest that NPM-RAR affects wildtype NPM in t(5;17) APL, and support the notion that chimeric RAR proteins in APL deregulate the wildtype function of the partner through a mechanism similar to that of RXR sequestration.
In normal hematopoietic cells, PML localizes to the nuclear matrix in 10 - 20 discreet nuclear bodies or PODS (Daniel et al., 1993; Dyck et al., 1994). In t(15;17) APL cells, several groups have observed that the expression of PML-RAR delocalizes PML from these nuclear bodies in an RA dependent fashion (Dyck et al., 1994; Koken et al., 1994; Weis et al., 1994). This has led to the proposal that PML nuclear body formation plays a role in the differentiation response of myeloid progenitors to growth factors (Dyck et al., 1994). Evidence that disruption of PML nuclear bodies may be an epiphenomenon associated specifically with t(15;17) APL is seen in t(11;17) APL cells expressing PLZF-RAR (Grimwade et al., 1997) or NuMA-RAR (Wells et al., 1997). In these cells immunofluorescent confocal microscopy reveals that PML localizes normally to discreet nuclear bodies. We confirm these findings in t(5;17) APL cells expressing NPM-RAR, where PML nuclear bodies are also intact. Thus, chimeric RAR proteins derived from variant APL translocations do not appear to affect PML localization, suggesting that APL pathogenesis is not dependent on delocalization of PML from the nuclear body structure.
While the existence of a functional link between the RAR partner proteins has yet to be described, all four are nuclear matrix phosphoproteins with the capacity to form dimers or multimers. Recent data have demonstrated that PLZF expression is upregulated in t(11;17)(q23;q21) and t(15;17) APL cells, and that the nuclear localization of PLZF is disrupted in these forms of APL as a direct result of PLZF/PLZF-RAR and PLZF/PML-RAR heterodimers, respectively (Koken et al., 1997). Consistent with the above data we show that PLZF expression appears to be similarly upregulated in a microspeckled pattern in the nucleus of t(5;17) cells. Although specific interactions between NPM and PLZF remain to be proven, these data suggest that PLZF may play a more universal role in the pathogenesis of all forms of APL.
Even though the t(5;17) variant has only been identified in a minority of APL patients, it may represent a distinct subtype of APL and a model system that can aid in the molecular study of APL pathogenesis. While it does not interfere with PML nuclear body formation, the expression of NPM-RAR in t(5;17) APL results in the co-localization of NPM, RXR, and NPM-RAR. Moreover, PLZF expression is also affected, as it is observed in a microspeckled pattern in the nucleus of patient leukemic cells but not in patient normal bone marrow cells. In summary, our data provide further evidence that the molecular consequences of APL translocations are the deregulation of both the retinoid signaling pathway and endogenous translocation partner proteins. Furthermore, our data confirm that APL pathogenesis is not dependent on the delocalization of PML from its nuclear bodies. Further characterization of the mechanisms of action of NPM-RAR should lead to a better understanding of normal hematopoiesis and its deregulation in APL.
Materials and methods
The patient, a 12 year-old male, presented with disseminated intravascular coagulation, and bilateral lower limb hematomas. Bone marrow examination revealed a hypercellular specimen that contained >70% promyelocytes. Auer rods were not detected. Few mature cells of the granulocytic series were present and cells of the erythroid lineage appeared dysplastic. Cells surface immunophenotype of the abnormal cells was determined by flow cytometry to be HLA-DR-, CD13+, CD14-, CD33+. A diagnosis of acute myelogenous leukemia subtype M3v was made, and remission induction chemotherapy (cytosine arabinoside [AraC] 100 mg/m2/day for 7 days and daunorubicin [DNR] 45 mg/m2/day for 3 days) was followed by consolidation chemotherapy that combined DNR, AraC, 6-thioguanine, etoposide, and dexamethasone. A bone marrow sample taken after the first consolidation cycle revealed morphological and cytogenetic remission, however, a follow-up specimen (5 months post-diagnosis) showed evidence of relapse. Re-induction therapy consisted of AraC and atRA (25 mg/m2/day). Cytogenetic remission was documented 5 weeks post-induction, and maintenance therapy with atRA (45 mg/m2/day) was administered for 2 months leading up to bone marrow transplantation. The patient underwent allogeneic bone marrow transplantation from a matched sibling donor 13 weeks after re-induction. While patient response to atRA alone is difficult to assess since AraC was part of the re-induction treatment, the differentiation effects of atRA probably contributed to this patient's remission status prior to transplantation. Consistent with this, Redner and colleagues have recently demonstrated that in short term culture systems, cells bearing the t(5;17) translocation terminally differentiate in response to retinoic acid (Redner et al., 1997).
Somatic cell hybrids
A panel of somatic cell hybrids was produced using PEG fusion (Davidson et al., 1976; Norwood et al., 1976), and screened to identify patient/CHO hybrid clones bearing derivative t(5;17) chromosomes. The parental cells used to construct these hybrids were patient marrow cells, and the MEV-1, ADE-B mutant CHO cell lines. 25 MEV-1 hybrids (M1 - M25) were selected for the presence of the short arm of chromosome 5 (3-hydroxy-3-methylglutaryl-coenzyme A synthase), hence, the patient derivative 5 chromosome. Ten ADE-B hybrids (A1 - A10) were selected for the presence of the short arm of human chromosome 17 (5-bromodeoxyuridine), hence, the patient derivative 17 chromosome. Thirty-four hybrid lines were successfully maintained in Ham's F12 media and harvested for cytogenetic, Southern, RT - PCR and Northern analyses.
Standard phenol-chloroform techniques were applied to genomic DNA from 1´107 fresh/cryopreserved patient mononuclear cells, and somatic cell hybrid lines. Restriction enzymes BamHI, PstI, and EcoRI were used independently and in combination to digest 10 g samples of DNA. Standard Southern blotting techniques were used in these studies (Southern, 1975). RAR involvement was determined by using a previously described genomic fragment LCN4A3B (Borrow et al., 1990) as a hybridization probe. Two overlapping PML cDNA probes, pAGH7 and pAGU3 (E Solomon, London, UK), were used to screen for occult PML rearrangements.
RT - PCR
cDNA was reversed transcribed from sample RNA extracts for the purposes of PCR analyses. Total RNA (1 g) was heated for 2 min at 65°C to denature any secondary mRNA structure, and was reverse transcribed in 20 l cDNA reactions containing 5´ first strand buffer (50 mM Tris-HCl, pH=8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 mM dNTP final concentration), 100 ng of random hexamer, 20 units of RNA guard (Pharmacia, Baie d'Urfé, QC), and 10 units of DNase I (Boehringer-Mannheim, Laval, QC). Primers were generated from RAR and NPM cDNA sequences retrieved from GENBANK to allow the amplification of RAR (130 bp), NPM (1084 bp), as well as NPM-RAR (429 bp) and RAR-NPM (733 bp) mRNA species. 50 l reactions (PCR buffer made up of 2.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl pH=8.8, 0.01% gelatin, and 0.1% Triton X-100) were amplified in a TRIO-Thermoblock thermocycler (Biometra, Tampa, FL, USA). The location of the following primers is shown in Figure 2a:
Cloning and sequencing
RT - PCR products were purified from low melting point agarose gels, concentrated in a microconcentrator (Amicon, Beverly, MA, USA), and cloned directly into the SrfI restriction site of the pCR-SCRIPTTM vector (Stratagene, La Jolla, CA, USA). Highly concentrated, pure plasmid DNA was isolated from positive clones using standard alkaline-lysis mini-preparation techniques and sequenced using a Sequenase DNA Sequencing kit (USB-Cleveland, OH, USA) according to the manufacturer's protocol for double stranded plasmid DNA templates. Sequenced samples were analysed on a 6% urea-polyacrylamide gel prepared using Long Ranger Gel Solution Concentrate (JT Baker, Phillipsburg, NJ, USA), and screened against GENBANK for high percentage sequence matches.
Immunofluorescent confocal microscopy
APES-coated, glass microscope slides were prepared for the t(15;17) APL cell line NB4 (M Lanotte, INSERM, Paris, France), the myeloid progenitor cell line U937 (ATCC, Rockville, MD, USA), the promyelocytic cell line HL60 (ATCC, Rockville, MD, USA), and t(5;17) APL patient bone marrow cells. Samples were spread at medium density, fixed for 2 min at -10°C in 1 : 1 methanol : acetone and allowed to air-dry for 20 min. Slides were blocked with 3% pre-immune goat serum in PN buffer (0.1 M Na2HPO4/NaH2PO4, Tween 20) for 1 h at 37°C. After two PN buffer washes, slides were incubated for 2 h at 37°C with dilutions of RAR, RXR, PML (Oncogene Science, Cambridge, MA, USA), NPM (S Morris, Memphis, TN, USA), and PLZF antibodies (J Licht, New York, NY, USA) in PN buffer. Polyclonal antibodies were used against RAR and RXR, whereas monoclonal antibodies were used against PML, NPM, and PLZF. Texas red-conjugated goat anti-rabbit (Santa Cruz Biotech, Santa Cruz, CA, USA) or FITC-conjugated goat anti-mouse (Oncogene Science) secondary antibodies were then added and incubated for 1 h at 37°C. After a final rinse in PN buffer, slides were mounted in vectashield antifade (Vector, Burlingame, CA, USA), and cells were viewed on a laser scanning confocal microscope (Zeiss, Hospital for Sick Children, Toronto, Canada).
We are indebted to M Freedman, W Vanek and T Grunberger (Hospital for Sick Children, Toronto, Canada) for providing patient material for our studies and for help with the interpretation of clinical information; C Jones and D Geyer of the Eleanor Roosevelt Institute in Denver, Colorado for their expertise in creating the panel of somatic cell hybrids: and to M Starr for assistance with confocal microscopy (Hospital for Sick Children, Toronto, Canada). This work was partially funded by the Leukemia Research Fund (S Kamel-Reid), the National Cancer Institute of Canada (S Kamel-Reid), and NIH R01 CA 59936 (JD Licht). JD Licht is a Scholar of the Leukemia Society of America.
Alcalay M, Zangrilli D, Pandolfi PP, Longo L, Mencarelli A, Giacomucci A, Rocchi M, Biondi A, Rambaldi A, Lo Coco F, Diverio D, Donti E, Grignani F and Pelicci PG. (1991). Proc. Natl. Acad. Sci. 88, 1977-1981. MEDLINE
Alcalay M, Zangrilli D, Fagiolo M, Pandolfi PP, Mencarelli A, Lo Coco F, Biondi A, Grignani F and Pelicci PG. (1992). Proc. Natl. Acad. Sci. USA 89, 4840-4844. MEDLINE
Bardwell VJ and Treisman R. (1994). Genes Dev. 8, 1664-1677. MEDLINE
Borden KLB, CampbellDwyer EJ and Salvato MS. (1997). FEBS Letters 418, 30-34. MEDLINE
Borer RA, Lehner CF, Eppenberger HM and Nigg EA. (1989). Cell 56, 379-390. MEDLINE
Borrow J, Goddard AD, Sheer D and Solomon E. (1990). Science 249, 1577-1580. MEDLINE
Borrow J, Goddard AD, Gibbons B, Katz F, Swirsky D, Fioretos T, Dubé I, Winfield DA, Kingston J, Hagemeijer A, Rees JKH, Lister A and Solomon E. (1992). Br. J. Haematol. 82, 529-540. MEDLINE
Bugge TH, Pohl J, Lonnoy O and Stunnenberg H. (1992). EMBO 11, 1409-1418.
Castaigne S, Balitrand N, de Thé H, Dejean A, Degos L and Chomienne C. (1992). Blood 79, 3110-3115. MEDLINE
Chan PK, Liu Q-R and Durban E. (1990). Biochem. J. 270, 549-552. MEDLINE
Chan W-Y, Liu Q-R, Borjigin J, Busch H, Rennert OM, Tease LA and Chan P-K. (1989). Biochemistry 28, 1033-1039. MEDLINE
Chang K, Fan Y-H, Andreeff M, Liu J and Mu Z-M. (1995). Blood 85, 3646-3653. MEDLINE
Chen JD and Evans RM. (1995). Nature 377, 454-457. MEDLINE
Chen Z, Brand NJ, Chen A, Chen S-J, Tong J-H, Wang Z-Y, Waxman S and Zelent A. (1993). EMBO 12, 1161-1167.
Chen Z, Fabien G, Rousselot P, Agadir A, Chen S-J, Wang Z-Y, Degos L, Zelent A, Waxman S and Chomienne C. (1994). Proc. Natl. Acad. Sci. 91, 1178-1182. MEDLINE
Daniel MT, Koken M, Romagné O, Barbey S, Bazarbachi A, Stadler M, Guillemin MC, Degos L, Chomienne C and de Thé H. (1993). Blood 82, 1858-1867. MEDLINE
Davidson RL, O'Malley KA and Wheeler TB. (1976). Som. Cell Genet. 2, 271-280.
de Thé H, Chomienne C, Lanotte M, Degos L and Dejean A. (1990). Nature 347, 558-561. MEDLINE
de Thé H, Lavau C, Marchio A, Chomienne C, Degos L and Dejean A. (1991). Cell 66, 675-684. MEDLINE
Dong S, Zhu J, Reid A, Strutt P, Guidez F, Zhong H-J, Wang Z-Y, Licht J, Waxman S, Chomienne C, Chen Z, Zelent A and Chen S-J. (1996). Proc. Natl. Acad. Sci. 93, 3624-3629. MEDLINE
Downing JR, Shurtleff SA, Zielenska M, Curcio-Brint AM, Behm FG, Head DR, Sandlund JT, Weisenburger DD, Kossakowska AE, Thorner P, Lorenzana A, Ladanyi M and Morris SW. (1995). Blood 85, 3416-3422. MEDLINE
Dyck JA, Maul GG, Miller Jr. WH, Chen JD, Kakizuka A and Evans RM. (1994). Cell 76, 333-343. MEDLINE
Giguère V, Ong E, Segui P and Evans R. (1987). Nature 330, 624-629. MEDLINE
Goddard AD, Borrow J, Freemont PS and Solomon E. (1991). Science 254, 1371-1374. MEDLINE
Grignani F, Fagioli M, Alcalay M, Longo L, Pandolfi PP, Donti E, Biondi A, Lo Coco F, Grignani F and Pelicci PG. (1994). Blood 83, 10-25. MEDLINE
Grignani F, Testa U, Rogaia D, Ferrucci PF, Samoggia P, Pinto A, Aldinucci D, Gelmetti V, Fagioli M, Alcalay M, Seeler J, Grignani F, Nicoletti I, Peschle C and Pelicci PG. (1996). EMBO 15, 4949-4958.
Grignani F, Matteis SD, Nervi C, Tomassoni L, Gelmetti V, Cioce M, Fanelli M, Ruthardt M, Ferrara FF, Zamir I, Seiser C, Grignani F, Lazar MA, Minucci S and Pelicci PG. (1998). Nature 391, 815-818. Article MEDLINE
Grimwade D, Howe K, Langabeer S, Davies L, Oliver F, Walker H, Swirsky D, Wheatley K, Goldstone A, Burnett A and Solomon E. (1996). Br. J. Haematol. 94, 557-573. MEDLINE
Grimwade D, Gorman P, Duprez E, Howe K, Langabeer S, Oliver F, Walker H, Culligan D, Waters J, Pomfret M, Goldstone A, Burnett A, Freemont P, Sheer D and Solomon E. (1997). Blood 90, 4876-4885. MEDLINE
Guidez F, Ivins S, Zhu J, Söderström M, Waxman S and Zelent A. (1998). Blood 91, 2634-2642. MEDLINE
He L-Z, Guidez F, Tribioli C, Peruzzi D, Ruthardt M, Zelent A and Pandolfi PP. (1998). Nature Genet. 18, 126-135. MEDLINE
Herrera JE, Savkur R and Olson MOJ. (1995). Nuc. Acids Res. 23, 3974-3979.
Herrera JE, Correia JJ, Jones AE and Olson MOJ. (1996). Biochemistry 35, 2668-2673. MEDLINE
Hillestad L. (1957). Acta Med. Scand. 69, 189-198.
Hong S-H, David G, Wong C-W, Dejean A and Privalsky ML. (1997). Proc. Natl. Acad. Sci. USA 94, 9028-9033. Article MEDLINE
Jansen JH, Mahfoudi A, Rambaud S, Lavau C, Wahli W and Dejean A. (1995). Proc. Natl. Acad. Sci. 92, 7401-7405. MEDLINE
Kakizuka A, Miller Jr WH, Umesono K, Warrell Jr RP, Frankel SR, Murty VVVS, Dmitrovsky E and Evans RM. (1991). Cell 66, 663-674. MEDLINE
Kang YJ, Olson MOJ and Busch H. (1974). J. Biol. Chem. 249, 5580-5585. MEDLINE
Koken M, Puvion-Dutilleul F, Guillemin M, Viron A, Linares-Cruz G, Stuurman N, de Jong L, Szostecki C, Calvo F, Chomienne C, Degos L, Puvion E and de Thé H. (1994). EMBO 13, 1073-1083. .
Koken MHM, Linares-Cruz G, Quignon F, Viron A, Chelbi-Alix MK, Sobczak-Thépot J, Juhlin L, Degos L, Calvo F and de Thé H. (1995). Oncogene 10, 1315-1324. MEDLINE
Koken MHM, Reid A, Quignon F, Chelbi-Alix, Davies JM, Kabarowski JHS, Zhu J, Dong S, Chen S-J, Chen Z, Tan CC, Licht J, Waxman S, de Thé H and Zelent A. (1997). Proc. Natl. Acad. Sci. USA 94, 10255-10260. MEDLINE
Le XF, Yang P and Chang KS. (1996). J. Biol. Chem. 271, 130-135. MEDLINE
Li Y-P, Busch RK, Valdez BC and Busch H. (1996). Eur. J. Biochem. 237, 153-158. MEDLINE
Li J-Y, English MA, Ball HJ, Yeyati PL, Waxman S and Licht JD. (1997). J. Biol. Chem. 272, 22447-22455. Article MEDLINE
Licht JD, Chomienne C, Goy A, Chen A, Scott AA, Head DR, Michaux JL, Wu Y, DeBlasio A, Miller WH, Zelenetz AD, Willman CL, Chen Z, Chen S, Zelent A, Macintyre E, Veil A, Cortes J, Kantarjian H and Waxman S. (1995). Blood 85, 1083-1094. MEDLINE
Licht JD, Shaknovitch R, English MA, Melnick A, Li J, Reddy JC, Dong S, Chen S, Zelent A and Waxman S. (1996). Oncogene 12, 323-336. MEDLINE
Lin RJ, Nagy L, Inoue S, Shao W, Miller Jr WH and Evans RM. (1998). Nature 391, 811-814. Article MEDLINE
Liu J-H, Mu Z-M and Chang K-S. (1995). J. Exp. Med. 181, 1965-1973. MEDLINE
Lydersen BK and Pettijohn DE. (1980). Cell 22, 489-499. MEDLINE
Morris SW, Kirstein MN, Valentine MB, Dittmer KG, Shapiro DN, Saltman DL and Look AT. (1994). Science 263, 1281-1284. MEDLINE
Mu Z-M, Chin K-V, Liu J-H, Lozano G and Chang K-S. (1994). Mol. Cell. Biol. 14, 6858-6867. MEDLINE
Nagy L, Kao H-Y, Chakravarti D, Lin RJ, Hassig CA, Ayer DE, Schreiber SL and Evans RM. (1997). Cell 89, 373-380. MEDLINE
Norwood TH, Zeigler CJ and Martin GM. (1976). Som. Cell Genet. 2, 263-270.
Pandolfi PP, Grignani F and Alcalay M. (1991). Oncogene 6, 1285. MEDLINE
Perez A, Kastner P, Sethi S, Lutz Y, Reibel C and Chambon P. (1993). EMBO 12, 3171-3182.
Redner RL, Rush EA, Faas S, Rudert WA and Corey SJ. (1996). Blood 87, 882-886. MEDLINE
Redner RL, Corey SJ and Rush EA. (1997). Leukemia 11, 1014-1016. MEDLINE
Sitterlin D, Tiollasis P and Transy C. (1997). Oncogene 14, 1067-1074. MEDLINE
Southern EM. (1975). J. Mol. Biol. 98, 503-517. MEDLINE
Sparks CA, Bangs PL, McNeil GP, Lawrence JB and Fey EG. (1993). Genomics 17, 222-224. MEDLINE
Wang D, Baumann A, Szebeni A and Olson MOJ. (1994). J. Biol. Chem. 269, 30994-30998. MEDLINE
Weis K, Rambaud S, Lavau C, Jansen J, Carvalho T, Carmo-Fonseca M, Lamond A and Dejean A. (1994). Cell 76, 345-356. MEDLINE
Wells RA, Hummel JL, DeKoven A, Zipursky A, Kirby M, Dubé ID and Kamel-Reid S. (1996). Leukemia 10, 735-740. MEDLINE
Wells RA, Catzavelos C and Kamel-Reid S. (1997). Nature Genet. 17, 109-113. MEDLINE
Yoneda-Kato N, Look AT, Kirstein MK, Valentine MB, Raimondi SC, Cohen KJ, Carroll AJ and Morris SW. (1996). Oncogene 12, 265-275. MEDLINE
Zhang X-K, Hoffmann B, Tran PB-V, Graupner G and Pfahl M. (1992). Nature 355, 441-446. MEDLINE
Figure 1 (a) Southern blot analysis of patient (J700) bone marrow DNA and normal placental control DNA using the LCN4A3B genomic probe. Size in kilobases is indicated on the right, and the control and patient samples are labeled. Note the aberrantly sized fragments in the patient samples confirming RAR involvement. Restriction sites: B=BamHI, P=PstI, E=EcoRI. (b) Northern blot analysis of the somatic cell hybrid A6 which bears the patient derivative 17 chromosome. Note the expression of an aberrant RAR mRNA. The two major RAR isoforms are also evident. Normal bone marrow (BM) and the U937 cell line were included as controls along with somatic cell hybrids M20 and A5 lacking the derivative 17 chromosome
Figure 2 (a) Schematic of the specific intron/exon structure of RAR and NPM. The NPM-RAR and RAR-NPM fusion genes are also shown. Coding regions are represented by shaded boxes while the white boxes denote untranslated regions. RT - PCR primers (arrows) are labeled and are positioned under the exons from which they were generated. Dashed lines indicate alternative splicing patterns. (b) RT - PCR analysis of cDNA prepared from patient bone marrow cells demonstrating the expression of NPM-RAR and RAR-NPM mRNA. Reagent (R) and reverse transcriptase minus (RT-) controls containing all primer pairs tested are also shown. The expression of NPM and RAR is shown in lanes 1 and 2, respectively. The expression of NPM-RAR and RAR-NPM is shown in lanes 3 and 4, respectively
Figure 3 (a) cDNA sequence analysis of the 429 bp NPM-RAR RT - PCR product. Single letter amino acid sequence is shown above the second letter of each codon. Stop codons are indicated (*), and the breakpoint junctions are marked with arrows. Note that the NPM-RAR breakpoint junction maintains the alanine (A) at amino acid 118 of NPM. (b) cDNA sequence analysis of the 733 bp RAR-NPM RT - PCR product. Note that the RAR-NPM breakpoint junction maintains the threonine (T) at amino acid 59 of RAR
Figure 4 Schematic of RAR, NPM, NPM-RAR, and RAR-NPM protein sequences. The major breakpoint cluster regions (bcr) for RAR and NPM are shown (arrows), as well as essential functional motifs: TA=transactivating, DNA=DNA binding, D=dimerization, L=ligand binding, R=ribonuclease activity, M=multimerization
Figure 5 Immunofluorescent confocal microscopy of control cell lines U937 and NB4 treated with a monoclonal PML antibody (FITC, green). Note that in U937, PML is localized to 10 - 20 nuclear bodies. t(15;17) bearing NB4 cells confirmed that PML is dispersed throughout the nucleus. Analysis of bone marrow cells from the t(5;17) patient (J700) demonstrates that PML is localized to 10 - 20 nuclear bodies, as in U937. White bars represent 10 m
Figure 6 Immunofluorescent confocal microscopy of control cells U937 dually treated with a monoclonal NPM antibody (FITC) plus a polyclonal RAR antibody (TRITC, red), or dually treated with NPM (as above) plus a polyclonal RXR antibody (TRITC). Note that in U937, NPM is localized to 1 - 5 nucleolar dots, whereas RAR and RXR are both dispersed throughout the nucleus. Analysis of bone marrow cells from the t(5;17) patient (J700) dually treated with NPM plus RAR/RXR antibodies demonstrates that NPM localization is completely disrupted. Note that NPM, NPM-RAR and RXR co-localize in a dispersed nuclear manner
Figure 7 Immunofluorescent confocal microscopy of U937 and HL60 control cells, and bone marrow cells from the t(5;17) patient (J700) treated with a monoclonal PLZF antibody (FITC). U937 cells show no PLZF expression (background), whereas in HL60, PLZF is localized both in a microspeckled and dispersed nuclear pattern. Analysis of patient leukemic cells demonstrates that PLZF is expressed in a microspeckled pattern in the nucleus
|Received 12 March 1998; revised 12 August 1998; accepted 12 August 1998|
|21 January 1999, Volume 18, Number 3, Pages 633-641|
|Table of contents Previous Article Next [PDF]