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Cell Growth & Differentiation Vol. 13, 173-183, April 2002
© 2002 American Association for Cancer Research

The Retinoic Acid Receptor {alpha} (RAR{alpha}) Chimeric Proteins PML-, PLZF-, NPM-, and NuMA-RAR{alpha} Have Distinct Intracellular Localization Patterns1

Jeff L. Hummel, Tong Zhang, Richard A. Wells and Suzanne Kamel-Reid2

The Institute of Medical Sciences [J. L. H., R. A. W., S. K-R.] and Department of Laboratory Medicine and Pathobiology [S. K-R.], University of Toronto, Toronto, Ontario, M5S 1A8; The Ontario Cancer Institute, The Department of Pathology, The University Health Network, Toronto, Ontario, M5G 2M9 [J. L. H., T. Z., S. K-R.], Canada


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoic acid receptor {alpha} (RAR{alpha}) gene rearrangement by reciprocal chromosome translocation is the molecular signature of acutepromyelocytic leukemia (APL). Disruption of RAR{alpha} function appears to be the likely cause of aberrant myelopoiesis observed in APL, because PML-RAR{alpha} expression has been shown to deregulate the transcription of genes that control myelopoiesis. To target RAR{alpha} chimeric proteins, we engineered epitope-tagged versions of PML-RAR{alpha}, PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} (X-RAR{alpha}V5) and generated a panel of stable COS cell lines expressing X-RAR{alpha}V5. Protein fractionation and Western analysis of these COS lines reveal that X-RAR{alpha} proteins localize to both the cytoplasm and nucleus. NPM-RAR{alpha} is predominantly nuclear whereas NuMA-RAR{alpha} is predominantly cytoplasmic. Confocal immunofluorescent microscopy reveals that PML-RAR{alpha} and PLZF-RAR{alpha} share a primarily diffuse nuclear pattern that excludes the nucleolus. NPM-RAR{alpha} is also diffuse in the nucleus but, in contrast to PML-RAR{alpha} and PLZF-RAR{alpha}, is strongly associated with the nucleolus. Strikingly, NuMA-RAR{alpha} predominantly localizes throughout the cytoplasm in a microspeckled pattern. We further demonstrate that NPM and NuMA interact with NPM-RAR{alpha} and NuMA-RAR{alpha}, respectively. The distinct intracellular localization patterns and the shared ability of X-RAR{alpha} to interact with their respective RAR{alpha} partner proteins (X) further support the hypothesis that deregulation of these partners may play a role in APL pathogenesis.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
APL3 is characterized by the accumulation of abnormal promyelocytes in the bone marrow, attributable to a block in the myeloid differentiation program. Nearly 100% of APL patients achieve remission after differentiation therapy with RA (1) . This is the direct result of the abnormal promyelocytes reentering the differentiation program. APL is associated with a balanced t(15;17)(q22;q21) translocation (2) that fuses the RAR{alpha} (17q21) gene (3 , 4) to the PML (15q22) gene (5, 6, 7, 8) and results in the expression of a PML-RAR{alpha} chimeric protein. Several rare variant APL translocations have been identified involving the PLZF, (11q23; Ref. 9 ), NuMA (11q13; Refs. 10 , 11 ), NPM (5q35; Refs. 12 , 13 ), and STAT5b genes (17q21; Ref. 14 ) fused to the essential functional domains of RAR{alpha}. RAR{alpha}, a member of the steroid/hormone nuclear receptor family that specifically binds to 9-cis RA and ATRA (3 , 15 , 16) , helps orchestrate myeloid development by acting as a transcription factor at specific DNA sequences called RAREs (17, 18, 19, 20, 21, 22) . Similar to other steroid/hormone nuclear receptors (TR, VD3R, and PPAR), RAR{alpha} functions as a heterodimer with the RXR{alpha} (19 , 21 , 23) . Because RAREs have been identified in the upstream regions of several genes believed to control myelopoiesis (2 , 20 , 23) the disruption of RAR{alpha} function is believed to be the likely cause of the aberrant myelopoiesis observed in APL. In addition, all APL fusion genes result in the expression of RAR{alpha} chimeric proteins (X-RAR{alpha}), which have been directly implicated in APL pathogenesis. PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha} have been shown to possess altered transcriptional activity at RAREs, with a tendency to repress transcription in the presence of physiological levels of RA. Pharmacological levels of ATRA (10-6 M) can reestablish transcriptional activation at RAREs in cells expressing PML-RAR{alpha} and NPM-RAR{alpha}; however, these levels have little effect on cells expressing PLZF-RAR{alpha} (7 , 24, 25, 26, 27, 28) . Consistent with this, APL patients bearing the t(11;17)(q23;q21) respond poorly to ATRA treatment (29) .

Although the RAR{alpha} partner proteins (X) appear to have different functions, each contains a multimerization domain that potentiates the formation of X-RAR{alpha} homodimers. It has recently been determined by several groups that RAREs appear to be occupied by X-RAR{alpha} homodimers in conditions where PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha} are overexpressed (30, 31, 32, 33, 34, 35) . In fact, increased repression at RAREs by PML-RAR{alpha} can be attributed to an increased affinity that the homodimers possess for corepressors, because PML-RAR{alpha} is capable of binding two SMRT complexes instead of one (34) . PLZF itself has the capacity to bind SMRT through its POZ domain (9 , 30 , 36) , and in vitro studies have recently revealed that the unresponsiveness of PLZF-RAR{alpha}-expressing cells to pharmacological doses of ATRA is caused, in part, by PLZF-RAR{alpha} homodimers interacting with as many as four corepressor complexes; two of which are at the PLZF moiety and are ATRA unresponsive (31, 32, 33 , 37) . Although the stoichiometry of corepressor binding and ATRA-mediated corepressor release, as well as the subsequent effects on RAR{alpha} and X-RAR{alpha} transcriptional regulation at RAREs, are incompletely understood, these studies describe a molecular mechanism that helps explain the differential response to ATRA observed in APL. Physiological levels of ATRA are sufficient to dissociate one corepressor complex from RXR{alpha}/RAR{alpha} and activate transcription, whereas pharmacological levels are required to dissociate two corepressor complexes from PML-RAR{alpha} homodimers. Pharmacological levels of ATRA appear insufficient to dissociate four corepressor complexes from PLZF-RAR{alpha} homodimers. Whether or not STAT5b-RAR{alpha}, associated with an APL that did not respond to ATRA (14) , functions like PLZF-RAR{alpha} by recruiting four corepressor complexes to RAREs, remains to be determined.

Proper protein function is highly dependent on intracellular localization. For example, the transforming ability of the ABL tyrosine kinase is related to its activity as well as to its cellular localization. It has been found that deletion of the NH2 terminus of ABL results in the translocation of ABL from the nucleus to the cytoplasm, and to the transformation of mouse fibroblasts (38, 39, 40) . In CML, the translocation that gives rise to the Philadelphia chromosome (41) fuses the BCR and ABL genes and results in the expression of BCR-ABL that is also transforming and found predominantly in the cytoplasm (42 , 43) . All of the RAR{alpha} partner proteins associated with APL typically interact with the nuclear matrix architecture. Immunofluorescent studies show that PML and PLZF are largely found in the nucleus associated within 10–30 nuclear bodies (26 , 44, 45, 46, 47, 48) . It has more recently been observed that PLZF tends to be more diffuse when compared with PML (49) , and that PML and PLZF proteins localize to adjacent, but functionally distinct, nuclear bodies (50) . NPM primarily localizes to the nucleoli of cells, in two to five discreet aggregates (51, 52, 53) . Our group and others have studied the intracellular localization of PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha} in APL cells, as well as hematopoietic and nonhematopoietic cell lines, and have shown that these RAR{alpha} chimeras share the ability to colocalize and interact with their respective partners, as well as with RXR{alpha}. Specifically, PML-RAR{alpha} disrupts PML localization within the nuclear bodies and delocalizes PML in a microspeckled, nuclear pattern (45, 46, 47) . PML-RAR{alpha} interacts with RXR{alpha} as well, potentially deregulating retinoid signaling by sequestering this critical factor (54) . In support of this hypothesis, PML-RAR{alpha} has been observed in large cytoplasmic aggregates (44 , 46) . Interestingly, the treatment of cells that express PML-RAR{alpha} with RA results in the reformation of PML nuclear bodies (46 , 47 , 55) , the degradation of PML-RAR{alpha} (44) , and the restoration of RAR{alpha} signaling (7) . In contrast, APL cells and cells engineered to express PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} show normal PML localization within nuclear bodies (11 , 13 , 56) . Like PML-RAR{alpha}, PLZF-RAR{alpha} localizes in a diffuse nuclear pattern and is completely excluded from the nucleolus (26) . We have previously shown that NPM-RAR{alpha} is localized in a diffuse nuclear pattern in t(5;17) APL cells (13) . Like PML-RAR{alpha}, PLZF-RAR{alpha} and NPM-RAR{alpha} interact with their respective partner proteins and RXR{alpha} (13 , 25) . Although PML nuclear body disruption may not be critical in all APL cases, X-RAR{alpha} chimeras likely contribute to APL pathogenesis by delocalizing and thereby altering normal intracellular trafficking of RAR{alpha} partner proteins as well as critical retinoid factors.

Having identified and characterized single APL cases expressing each of NPM-RAR{alpha} and NuMA-RAR{alpha}, our laboratory has focused on understanding the roles of NPM-RAR{alpha} and NuMA-RAR{alpha} in APL pathogenesis. Whereas engineering cell types to express PML-RAR{alpha} has contributed significantly to a better understanding of the role of PML-RAR{alpha} in APL pathogenesis, these studies have been complicated by an apparent toxicity of this chimeric protein. As a result, comparison of the intracellular localization patterns of X-RAR{alpha} among different studies is limited. Here, we examined the intracellular localization of PML-RAR{alpha}, PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} in a single-cell system to better understand the functional consequences of RAR{alpha} chimeric proteins. We report that X-RAR{alpha} chimeric proteins are localized to both the cytoplasm and nucleus, and in addition have distinct intracellular localization patterns. We show that, unlike PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha}, the NuMA-RAR{alpha} chimera is localized predominantly in the cytoplasm. In addition, NPM-RAR{alpha} is the only X-RAR{alpha} protein detected in the nucleolus. Consistent with our previous patient reports (11 , 13) , NPM-RAR{alpha} and NuMA-RAR{alpha} colocalize with NPM and NuMA, respectively, in this system.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Tagging X-RAR{alpha}.
Lacking specific antibodies capable of directly targeting X-RAR{alpha} proteins, we subcloned full-length PML-RAR{alpha}, PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} cDNA cassettes into pcDNA3.1V5/HIS (pV5). X-RAR{alpha} cDNAs were subcloned such that the V5 epitope was in frame at the COOH termini. The same restriction enzyme/PCR strategy was applied to all cDNAs to remove a short RAR{alpha} region including the stop codon (see "Materials and Methods"). We were able to generate full length X-RAR{alpha} cDNAs lacking the RAR{alpha} stop codon in pBluescript, and subclone each into EcoRI/EcoRV-prepared pV5, in frame. Three versions of pV5, representing all possible reading frames, were created for each fusion cDNA to accommodate the variability of Klenow-polished DNA ends. Intact pX-RAR{alpha}V5 constructs were identified through sequence analysis of the cDNA-V5 junctions, and full-length protein expression was verified in transiently transfected COS-7 cells by Western blot analysis. As well as specific X-RAR{alpha}V5 proteins, high molecular weight signals were observed (Fig. 1A)Citation . These signals could be reduced through increased boiling of the protein extracts, and through the addition of higher concentrations of ß-mercaptoethanol in the loading buffer.



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Fig. 1. APL fusion proteins expressed in stable COS lines. A, Western blot analysis of COS-7 cells transiently transfected with pX-RAR{alpha}V5 constructs. Arrows, the expression of PML-RAR{alpha} (PRA), PLZF-RAR{alpha} (PLRA), NPM-RAR{alpha} (NRA), and NuMA-RAR{alpha} (NuRA) directly detected by the V5 monoclonal antibody. A pLacZV5 construct was used as a positive transfection and immunoblot control. The molecular weights observed for X-RAR{alpha}V5 were consistent with the expected sizes calculated from the primary amino acid sequence of PRA (~95,000), PLRA (~100,000), NRA (~60,000) and NuRA (~230,000). kDa, molecular weight in thousands; (*), high-molecular-weight complexes above 210 kDa for PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha}. B, Western blot analysis of COS-7 cells stably transfected with pX-RAR{alpha}V5 constructs. Representative COS lines are shown for PML-RAR{alpha} (PRA-2), PLZF-RAR{alpha} (PLRA-5), NPM-RAR{alpha} (NRA-3), and NuMA-RAR{alpha} (NuRA-10). Arrows, the V5 monoclonal antibody directly detected the expression of the four X-RAR{alpha}V5 chimeric proteins; *, high-molecular-weight complexes were observed, as well as degradation products below the major bands. kDa, molecular weight in thousands.

 
COS Cells Support Stable Expression of X-RAR{alpha}.
To study X-RAR{alpha} in a single-cell system, we created COS cells that stably expressed the epitope-tagged versions of X-RAR{alpha}. G418-selected colonies from each of PML-RAR{alpha}V5, PLZF-RAR{alpha}V5, NPM-RAR{alpha}V5, and NuMA-RAR{alpha}V5 COS lines were successfully expanded into cell lines expressing high levels of X-RAR{alpha}V5. COS lines containing pLacZV5 and pcDNA plasmids were also generated as unrelated gene and vector controls, respectively. Established COS X-RAR{alpha}V5 and control lines were routinely maintained under G418 selection pressure, and the presence of X-RAR{alpha}V5 was confirmed by Western analysis for up to 3 months. Representative COS X-RAR{alpha}V5 lines, based on the expression of appropriately sized X-RAR{alpha}V5, were chosen for further analysis (Fig. 1B)Citation . In culture, the COS PML-RAR{alpha}V5 line grew more slowly, taking approximately twice as much time to reach confluence.

X-RAR{alpha} Proteins Are Cytoplasmic and Nuclear.
By targeting the V5 epitope, we were able to characterize and compare PML-RAR{alpha}, PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} localization patterns in COS X-RAR{alpha}V5 lines. Intracellular protein fractions, analyzed by Western blot, revealed that the LacZ control protein was cytoplasmic. COS PML-RAR{alpha}V5 and PLZF-RAR{alpha}V5 lines represented additional controls because their localization patterns in COS cells have been previously described (26 , 44) . By this technique, PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha} were predominantly nuclear, although these proteins were also detectable in the cytoplasmic fraction. In contrast, NuMA-RAR{alpha} appeared primarily within the cytoplasm (Fig. 2)Citation . To address the possibility that the NuMA-RAR{alpha} observation was the result of cloning or transfection artifact, we examined U937 cells expressing NuMA-RAR{alpha} that were established in our laboratory through retroviral transduction, as well as COS cells transiently transfected with two different vectors driving NuMA-RAR{alpha} expression. Although intracellular protein fractions revealed detectable NuMA-RAR{alpha} in the nucleus, the vast majority of NuMA-RAR{alpha} still appeared in the cytoplasm despite the use of different cell lines and different infection/transfection methods (Fig. 3)Citation .



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Fig. 2. Cytoplasmic and nuclear fractionation of X-RAR{alpha}. Western blot analysis of cytoplasmic and nuclear lysates extracted from COS X-RAR{alpha}V5 lines. Blots were immunoprobed with the V5 antibody to determine the intracellular fractionation of control and X-RAR{alpha} proteins. The control COS lines harbor the pcDNA empty vector and pLacZV5, whereas the COS X-RAR{alpha}V5 lines express PML-RAR{alpha} (PRA), PLZF-RAR{alpha} (PLRA), NPM-RAR{alpha} (NRA) and NuMA-RAR{alpha} (NuRA). Top panel, the cytoplasmic fraction; bottom panel, the nuclear fraction from the same extracts. All of the proteins examined were observed in the cytoplasm, but only PML-RAR{alpha}, PLZF-RAR{alpha} and NPM-RAR{alpha} were detected in the nuclear fraction. kDa, molecular weight in thousands.

 


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Fig. 3. NuMA-RAR{alpha} is predominantly cytoplasmic. Western blot analysis of cytoplasmic and nuclear lysates extracted from different cell lines. Blots were dually immunoprobed with V5 and RAR{alpha} antibodies to determine the intracellular fractionation of tagged and untagged NuMA-RAR{alpha}. U937 MIEV cells included an empty vector/GFP control and a U937 line expressing untagged NuMA-RAR{alpha}. COS X-RAR{alpha}V5 cells included the NPM-RAR{alpha} line as a nuclear fractionation control, and the NuMA-RAR{alpha} line. COS cells transiently transfected with pV5 empty vector, pNPM-RAR{alpha}V5, pNuMA-RAR{alpha}V5, and a pSG5 version of NuMA-RAR{alpha} were also tested. Top panel, the cytoplasmic fraction; bottom panel, the nuclear fraction from the same lysates. NPM-RAR{alpha} was observed in both the cytoplasm and the nucleus with a bias toward the nucleus. In contrast, NuMA-RAR{alpha}, expressed in two different cell types and from three different expression constructs, appeared largely in the cytoplasm. *, NuMA-RAR{alpha} was weakly detected in the nuclear fraction of all samples. Duplicate blots were also immunoprobed with a dynein antibody to demonstrate cytoplasmic fractionation. kDa, molecular weight in thousands.

 
X-RAR{alpha} Proteins Display Distinct Intracellular Patterns.
Next, we examined parental COS, COS pcDNA (empty vector control) and the COS X-RAR{alpha}V5 cells by immunofluorescent confocal microscopy to generate detailed images of X-RAR{alpha} intracellular localization patterns for direct comparison. Although these cell lines were generated from single colonies, we observed that not all of the cells within a given COS X-RAR{alpha}V5 line overexpressed X-RAR{alpha}. Immunofluorescent confocal microscopy revealed that ~30–50% of the cells in a given field express X-RAR{alpha} to a high degree. Consistent with previous reports, PML-RAR{alpha} and PLZF-RAR{alpha} shared a diffuse nuclear pattern that completely excluded the nucleolus. Whereas cytoplasmic PLZF-RAR{alpha} was weakly detected, PML-RAR{alpha} was detected in large perinuclear and cytoplasmic aggregates (Fig. 4, A and B)Citation . Similar to PLZF-RAR{alpha}, NPM-RAR{alpha} primarily localized in the nucleus; however, in contrast, NPM-RAR{alpha} appeared to be strongly associated with the nucleoli (Fig. 4C)Citation . Strikingly, NuMA-RAR{alpha} was found to primarily localize throughout the cytoplasm in a microspeckled pattern ((Figs. 4DCitation and 5BCitation ). Nuclear NuMA-RAR{alpha} was also detected, albeit weakly. These data reveal not only similarities, but also the unique differences between the intracellular localization of X-RAR{alpha}, and are consistent with the results of protein fractionation experiments. Nonspecific V5 antibody staining was not observed in COS or COS pcDNA control cells (Fig. 4E)Citation , or in COS X-RAR{alpha}V5 cells that appeared to lack X-RAR{alpha} expression (Fig. 5)Citation . Table 1Citation provides a summary of the X-RAR{alpha} localization patterns observed in this study.



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Fig. 4. Intracellular localization of X-RAR{alpha}. Images of COS X-RAR{alpha}V5 and pcDNA cell lines analyzed by immunofluorescent confocal microscopy. Cells were immunostained with primary V5 antibody, then FITC-conjugated secondary antibody (green) to visualize the intracellular localization patterns of PML-RAR{alpha} (A), PLZF-RAR{alpha} (B), NPM-RAR{alpha} (C), and NuMA-RAR{alpha} (D). Here, specific cytoplasmic and nuclear patterns can be seen for each of the X-RAR{alpha} chimeric proteins. PML-RAR{alpha}, PLZF-RAR{alpha}, and NPM-RAR{alpha} were predominantly localized in a diffuse nuclear pattern. PML-RAR{alpha} also localized to large cytoplasmic aggregates. NuMA-RAR{alpha} localized strongly in the cytoplasm and weakly in the nucleus. NPM-RAR{alpha} was the only chimeric protein observed in the nucleoli. No significant anti-V5 signal was observed in COS pcDNA cells (E). Bars, 10 µm.

 


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Fig. 5. NPM and NuMA colocalize with NPM-RAR{alpha} and NuMA-RAR{alpha}, respectively. Images of COS NPM-RAR{alpha}V5 and NuMA-RAR{alpha}V5 cell lines analyzed by immunofluorescent confocal microscopy. A, COS NPM-RAR{alpha}V5 cells dually immunostained with NPM polyclonal (Rhodamine) and V5 monoclonal (FITC) antibodies. In cells highly expressing NPM-RAR{alpha} (arrows), NPM was microspeckled in the nucleus and concentrated in large nucleolar bodies. NPM overlapped and colocalized with NPM-RAR{alpha} (screen). B, COS NuMA-RAR{alpha}V5 cells dually immunostained with NuMA polyclonal (Rhodamine) and V5 monoclonal (FITC) antibodies. In cells highly expressing NuMA-RAR{alpha} (arrow), NuMA strongly colocalized with cytoplasmic NuMA-RAR{alpha} (screen). C, COS NuMA-RAR{alpha}V5 cells immunostained with monoclonal V5 or monoclonal Nu1 antibody (FITC) confirmed that endogenous NuMA is delocalized to the cytoplasm (arrows). In COS pcDNA control, cells immunostained with monoclonal Nu1 antibody (right panel), NuMA was observed in the nucleus. Bars, 10 µm.

 

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Table 1 Summary of X-RAR{alpha} intracellular localization

A general comparison of the X-RAR{alpha} intracellular localization patterns observed. X-RAR{alpha} chimeric proteins were characterized by their presence in the cytoplasm and nucleus/nucleolus, and by whether the localization patterns were diffuse (+) or aggregated (described). The predominant intracellular localization of each X-RAR{alpha} chimeric protein was noted (C, cytoplasmic; N, nuclear; No, nucleolar). Patterns not observed were also recorded (—).

 
NPM-RAR{alpha} and NuMA-RAR{alpha} Interact with Wild-Type NPM and NuMA, Respectively.
We used the COS NPM-RAR{alpha}V5 and NuMA-RAR{alpha}V5 lines and immunofluorescent confocal microscopy to show that, as observed in patient APL cells bearing the variant t(5;17) and t(11;17) translocations, wild-type NPM and NuMA colocalize with NPM-RAR{alpha} and NuMA-RAR{alpha}, respectively. In COS NPM-RAR{alpha}V5 cells, dually immunostained with polyclonal NPM and monoclonal V5 antibodies, endogenous NPM was found to be microspeckled in the nucleus as well as being strongly associated with 1–2 large nucleoli (Fig. 5A)Citation . In adjacent cells, in which NPM-RAR{alpha} expression was not detectable, NPM localized normally to three to five nucleoli. In addition, we observed that NPM overlapped with NPM-RAR{alpha} in a microspeckled nuclear pattern, as well as colocalized with NPM-RAR{alpha} throughout the nucleoli. In COS NuMA-RAR{alpha}V5 cells, dually immunostained with polyclonal NuMA and monoclonal V5 antibodies, NuMA was largely cytoplasmic and exhibited strong colocalization with NuMA-RAR{alpha} (Fig. 5B)Citation . In adjacent cells in which NuMA-RAR{alpha} expression was not detectable, NuMA localized normally to the spindle poles, or in a diffuse nuclear pattern that excluded the nucleoli. Although Western analyses performed in our laboratory suggest the polyclonal NuMA antibody targets the COOH-terminal portion of NuMA, we examined endogenous NuMA localization in COS NuMA-RAR{alpha}V5 cells using a monoclonal Nu1 antibody that specifically targets the COOH-terminal portion of NuMA. Consistent with Fig. 5BCitation , strong cytoplasmic NuMA staining was observed in cells expressing NuMA-RAR{alpha} (Fig. 5C)Citation .

To determine whether NuMA-RAR{alpha} interacted directly with NuMA, immunoprecipitation reactions were performed with V5 antibodies in COS NuMA-RAR{alpha}V5 cell extracts. Western blot analysis of the A/G bead fractions immunoprobed with Nu1 antibody showed that NuMA coimmunoprecipitated with NuMA-RAR{alpha}. NuMA did not coimmunoprecipitate with the LacZ negative-control protein (Fig. 6A)Citation . We also tested whether NPM-RAR{alpha} interacted directly with NPM. Bacterial lysates containing GST-fusion proteins GST-NPMwt and the multimerization deficient mutant GST-NPM{Delta}md were combined in a binding reaction with NPM-RAR{alpha} or the negative control LacZ protein, both generated using a transcription/translation in vitro kit (see "Materials and Methods"). Western blots immunoprobed with V5 antibody demonstrated that unlike LacZV5, NPM-RAR{alpha}V5 interacts with GST-NPM. In contrast, we observed a weak interaction between NPM-RAR{alpha}V5 and GST-NPM{Delta}md (Fig. 6B)Citation .



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Fig. 6. NPM and NuMA interact with NPM-RAR{alpha} and NuMA-RAR{alpha}, respectively. A, Western blot analysis of V5 immunoprecipitation assays performed on protein lysates extracted from COS LacZV5 (LacZ) and NuMA-RAR{alpha}V5 (NuRA) cell lines. Supernatant (sup) and A/G agarose (bead) fractions were blotted and immunoprobed with Nu1 antibody that specifically targets wild-type NuMA (COOH terminus). The first NuRA lane (1) represents a COS NuMA-RAR{alpha} bead control because no anti-V5 antibody was added. Interaction between endogenous NuMA and NuMA-RAR{alpha} was demonstrated by the presence of NuMA in the bead fraction. LacZ, the negative binding control, did not interact with NuMA-RAR{alpha}. The immunoglobulin heavy and light chains are marked in the bead fraction (IgHc and IgLc). B, Western blot analysis of GST pull-downs from binding assays containing GST-NPM fusion proteins and in vitro TNT NPM-RAR{alpha}V5. In vitro TNT LacZV5 protein was used as a negative binding control. Supernatant (sup) and 4B Sepharose (bead) fractions were blotted and immunoprobed with the V5 antibody. Interaction between full length NPM (GST-NPM-wt) and NPM-RAR{alpha}V5 (NRA) was confirmed by the presence of NRA in the bead fraction. The multimerization domain-deleted mutant (GST-NPM-{Delta}md) showed a weak interaction with NRA. The LacZ negative control protein did not interact with either of the GST-NPM fusion proteins. kDa, molecular weight in thousands.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Having identified and characterized two variant APL cases, our laboratory has focused on understanding the roles of X-RAR{alpha} in APL pathogenesis. In particular, we are interested in examining the effects of X-RAR{alpha} on the function of the RAR{alpha} partner proteins PML, PLZF, NPM, and NuMA, as well as on retinoid signaling factors like RXR{alpha}. In this study, we describe the generation of COS cell lines that retain the ability to express V5 epitope-tagged versions of PML-RAR{alpha}, PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} (X-RAR{alpha}). By applying a PCR- and restriction endonuclease-based approach, pX-RAR{alpha}V5 constructs were made to allow for direct immunotargeting, as well as to eliminate the possibility of detecting basal levels of RAR{alpha} in subsequent immunochemical analyses (31 , 57) . X-RAR{alpha}V5 chimeric proteins were highly expressed and specifically detected by V5 antibody in both transiently transfected COS cells and stably expressing COS X-RAR{alpha}V5 lines (Fig. 1)Citation . Frequently, we observed high-molecular-weight species that we reasoned were stable X-RAR{alpha} complexes. PML-RAR{alpha} and PLZF-RAR{alpha} can form multimeric complexes through multimerization motifs retained in the partner portion of these chimeric proteins (34 , 49 , 54 , 57 , 58) . Because the high-molecular-weight X-RAR{alpha} species that we observed were dissociated by treating protein lysates with higher ß-mercaptoethanol concentrations and increased boiling times before SDS-PAGE (59) , we concluded that these signals likely represent X-RAR{alpha} multimers.

Even although COS cells are not of the myeloid lineage, we found that they can be used as a valuable model for the study of X-RAR{alpha} intracellular localization patterns and potential protein/protein interactions. In contrast to hematopoietic cell lines such as U937 and HL60, COS cells can be efficiently transfected for both transient and stable analyses. In addition, COS cells are adherent and have a low nuclear:cytoplasmic ratio that provides ideal topography for intracellular localization and protein translocation studies. In this study, the G418 selection of COS cells, transfected with pX-RAR{alpha}V5 constructs, resulted in the outgrowth of G418-resistant colonies that were individually isolated and expanded into COS X-RAR{alpha}V5 lines (Fig. 1B)Citation . COS X-RAR{alpha}V5 lines were examined by Western analyses after 3 months of continuous culture and were shown to have maintained X-RAR{alpha} expression (Fig. 2)Citation . Several groups have reported that PML-RAR{alpha} has a potent growth-inhibitory or toxic effect on mammalian nonhematopoietic cells, as well as on several hematopoietic cells tested (60, 61, 62) , particularly when PML-RAR{alpha} is delivered by retroviral transduction. An avian retrovirus engineered to carry PML-RAR{alpha}, however, is capable of stably transducing early chick hematopoietic progenitors in vitro and in vivo (63 , 64) . In our study, we were able to transfect, expand, and stably culture COS PML-RAR{alpha}V5 cells. Consistent with the observations of others, our COS PML-RAR{alpha}V5 line had a reduced growth rate in culture when compared with the other COS X-RAR{alpha}V5 lines (data not shown). Although PML-RAR{alpha} expression had a growth-inhibitory effect in our COS lines, toxicity was not observed in these studies.

Because of the many different hematopoietic and nonhematopoietic cell types, and different expression constructs used to characterize PML-RAR{alpha} and PLZF-RAR{alpha} intracellular localization, the comparison of X-RAR{alpha} is difficult and current data are disparate. For example, in vitro studies of COS and NB4 cells expressing PML-RAR{alpha} have demonstrated three possible intracellular patterns: predominantly nuclear (45 , 59) ; both nuclear and cytoplasmic (44, 45, 46, 47 , 54) ; and predominantly cytoplasmic (46 , 55) . Observations for PLZF-RAR{alpha} have been more consistent, showing predominantly nuclear localization (26 , 31 , 37 , 49 , 50 , 65 , 66) . In this study, we used protein fractionation and immunofluorescent confocal microscopy of COS X-RAR{alpha}V5 cells to examine the intracellular localization patterns for PML-RAR{alpha}, PLZF-RAR{alpha}, NPM-RAR{alpha}, and NuMA-RAR{alpha} in a single-cell system. Protein fractionation demonstrated that PML-RAR{alpha} was detected in both the nucleus and the cytoplasm, but was predominantly nuclear. The immunofluorescent image of PML-RAR{alpha} revealed a diffuse nuclear pattern that excluded the nucleolus, and a heavily aggregated cytoplasmic pattern (Fig. 4A)Citation . These data are consistent with the original reports of in vitro PML-RAR{alpha} expression in COS cells (44 , 46) .

By protein fractionation, comparison of PLZF-RAR{alpha} and NPM-RAR{alpha} to PML-RAR{alpha} showed that all three RAR{alpha} chimeric proteins were detectable in the nucleus and the cytoplasm, with a bias toward the nucleus. In contrast to the other three, NuMA-RAR{alpha} was found to be predominantly cytoplasmic (Fig. 2)Citation . To be certain that this unique NuMA-RAR{alpha} pattern was not the result of transfection or cloning artifact, we extended the protein fractionation analysis to include a number of cell types expressing NuMA-RAR{alpha} from different constructs. In U937 cells stably expressing a retrovirally transduced NuMA-RAR{alpha} gene, and in COS cells transiently transfected with pV5- and pSG5 NuMA-RAR{alpha} constructs, NuMA-RAR{alpha} was still found to be predominantly cytoplasmic (Fig. 3)Citation . Because it is widely held that disruption of RARE transcriptional activation by X-RAR{alpha} is the primary cause of aberrant myelopoiesis in APL, it has been suggested that cytoplasmic localization of X-RAR{alpha} is an artifact of overexpression in nonhematopoietic cell lines (45 , 47) . Our data, combined with the fact that PML-RAR{alpha} is typically overexpressed in APL patients, and the findings that PML-RAR{alpha} can be detected in cytoplasmic fractions of APL patient cells as well as in hematopoietic cell lines (44 , 58 , 67) , do not support this contention.

To obtain a more detailed view of X-RAR{alpha} intracellular localization, we compared COS PLZF-RAR{alpha}V5, NPM-RAR{alpha}V5, and NuMA-RAR{alpha}V5 with COS PML-RAR{alpha}V5 cells by immunofluorescent confocal microscopy. In keeping with current data, PLZF-RAR{alpha} shared a diffuse nuclear pattern with PML-RAR{alpha}, which excluded the nucleolus (Ref. 26 ; Figs. 4, A and BCitation ). PLZF-RAR{alpha} was weakly detected in the cytoplasm. NPM-RAR{alpha} was also localized in a diffuse nuclear pattern (Fig. 4C)Citation but, in contrast to PML-RAR{alpha} and PLZF-RAR{alpha}, was strongly associated with the nucleoli (Fig. 4C)Citation . Despite the apparent lack of nucleolar NPM-RAR{alpha} staining in t(5;17) APL patient cells (13) , the presence of NPM-RAR{alpha} in nucleoli of COS cells is not surprising because the multimerization domain of NPM, retained in NPM-RAR{alpha}, facilitates the formation of functional NPM hexamers typically localized to the nucleolus (68, 69, 70) . Moreover, two other NPM chimeric proteins, NPM-ALK and NPM-MLF1, which are associated with anaplastic large cell lymphoma and AML/myelodysplastic syndrome, respectively, are predominantly localized in the nucleus and nucleoli (71 , 72) . Consistent with the results of our protein fractionation experiments, immunofluorescent images of NuMA-RAR{alpha} localization revealed a predominantly microspeckled, cytoplasmic pattern (Fig. 4D)Citation . These data clearly demonstrate that X-RAR{alpha} proteins have distinct intracellular localization patterns and, importantly, that NuMA-RAR{alpha} differs from the other RAR{alpha} chimeric proteins. Although the specific cytoplasmic or perinuclear structures with which NuMA-RAR{alpha} and PML-RAR{alpha} interact remain unidentified, our data suggest that X-RAR{alpha} chimeric proteins associate with distinct intracellular compartments. These data also represent the first description of NPM-RAR{alpha} and NuMA-RAR{alpha} intracellular localization in vitro.

To study the effects of NPM-RAR{alpha} and NuMA-RAR{alpha} on the normal patterns of wild-type NPM and NuMA, respectively, we examined NPM and NuMA immunofluorescent patterns in COS NPM-RAR{alpha}V5 and NuMA-RAR{alpha}V5 cells. Cells dually stained with NPM and V5 antibodies revealed partial overlap between NPM and NPM-RAR{alpha} in the nucleus, and colocalization in the nucleolus (Fig. 5A)Citation . NuMA and V5 antibodies showed that NuMA strongly colocalizes with NuMA-RAR{alpha} in the cytoplasm (Fig. 5B)Citation . Through GST pull-down and coimmunoprecipitation assays, we further demonstrated that this colocalization was the result of direct interaction of NPM-RAR{alpha} and NuMA-RAR{alpha} with NPM and NuMA, respectively (Fig. 6)Citation . Moreover, we demonstrated that the NPM multimerization domain mediates the interaction between NPM-RAR{alpha} and NPM, consistent with previous reports showing that PML interacts with PML-RAR{alpha} and PLZF interacts with PLZF-RAR{alpha} (9 , 36 , 44 , 54 , 58 , 73 , 74) . This is the first demonstration of endogenous NuMA interacting with, and being translocated to the cytoplasm by, NuMA-RAR{alpha}. Taken together with the finding that STAT5b is delocalized by STAT5b-RAR{alpha} (14) , our data show that all RAR{alpha} partners (X) interact with their respective X-RAR{alpha} chimeric protein.

NPM-RAR{alpha} and NuMA-RAR{alpha} displayed intracellular localization patterns unique to each protein. Unlike the other X-RAR{alpha} proteins, which are predominantly nuclear, NuMA-RAR{alpha} is found predominantly in the cytoplasm. NPM-RAR{alpha} appears to be the only RAR{alpha} chimeric protein localized at the nucleoli. It has been shown that cytoplasmic PML-RAR{alpha} requires PML heterodimerization to shuttle to the nucleus, where it affects retinoid signaling (46 , 62) . Whereas our data show that NPM-RAR{alpha} is shuttled effectively to the nucleus and nucleolus, most likely by NPM (53) , NuMA-RAR{alpha} nuclear translocation appears to be hindered. Because NuMA is shuttled back to the nucleus by ß-importin after mitosis (75) , there is the possibility that expression of NuMA-RAR{alpha} impairs the ß-importin shuttling pathway or the interaction between NuMA and ß-importin. Wild-type NuMA is normally found in a dispersed nuclear pattern in the interphase nucleus and is associated with the polar region of the mitotic apparatus (Fig. 5BCitation ; Ref. 76 ). NuMA is critical for the completion of mitosis (77) . NuMA gene sequence predicts a large protein containing two terminal globular domains, separated by a large coiled-coil domain (78 , 79) . The functional importance of the tail domain has been partially determined through the use of tailless NuMA mutants (77 , 80) . NuMA, lacking the tail domain, fails to target correctly and results in the formation of micronuclei but does not otherwise interfere with mitosis. The predominantly cytoplasmic localization of NuMA-RAR{alpha} in COS cells stably expressing this protein suggests that NuMA-RAR{alpha} functions as a tailless version of NuMA. In further support of this hypothesis, it has been shown that, in cells engineered to express NuMA in the cytoplasm, NuMA is assembled in extensive filamentous structures similar to the cytoplasmic, microspeckled pattern that we observed in COS NuMA-RAR{alpha}V5 cells (Fig. 5BCitation ; Ref. 81 ).

The distinct intracellular localization patterns, and potential deregulation of RAR{alpha} partner protein function, may play a role in generating subtle differences in APL phenotypes. When the clinical presentations of APL patients bearing variant RAR{alpha} translocations are closely studied, subtle differences can be discerned. For example, the patient carrying the NuMA-RAR{alpha} gene fusion was initially diagnosed with CML, the patient carrying the STAT5b-RAR{alpha} gene fusion had acute myeloid leukemia M1, whereas the PLZF-RAR{alpha} and NPM-RAR{alpha} gene fusion cases had APL M3v morphology (2) . These observations also parallel those found in the study of APL transgenic mice models. Transgenic mice expressing PML-RAR{alpha} develop an APL-like syndrome after a preleukemic phase characterized by the accumulation of myeloid precursors in the bone marrow (82 , 83) . In contrast, transgenic mice expressing PLZF-RAR{alpha} develop leukemia with symptoms resembling CML (37 , 84) , whereas mice expressing NPM-RAR{alpha} develop leukemia with features ranging from APL-like to CML-like (84) . Together with the observation that X-RAR{alpha} proteins have distinct intracellular localization patterns, we hypothesize that the deregulation of individual RAR{alpha} partner genes may play a role in the variation observed in the clinical presentation of APL patients.

In conclusion, we have demonstrated that X-RAR{alpha} can all be detected in the nucleus of COS cells: a critical event that supports the widespread view that X-RAR{alpha} regulate the expression of RA-responsive genes in a dominant negative, ligand-dependent manner. The COS X-RAR{alpha}V5 lines provide a valuable model by which to study intracellular localization patterns and protein/protein interactions. Specifically, we observed that X-RAR{alpha} localize in distinct intracellular patterns. NPM-RAR{alpha} is predominantly nuclear, like PML-RAR{alpha} and PLZF-RAR{alpha}, and is the only X-RAR{alpha} protein associated with the nucleolus. In contrast, NuMA-RAR{alpha} is predominantly cytoplasmic. Like PML-RAR{alpha} and PLZF-RAR{alpha}, NPM-RAR{alpha} and NuMA-RAR{alpha} can potentially deregulate the function of their wild-type partners through sequestration to novel nuclear and or cytoplasmic compartments. These data also suggest that the deregulation of RAR{alpha} partner proteins may play a role in APL pathogenesis, and contribute to the subtle phenotypic differences observed in variant APL cases.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cloning and Epitope Tagging of X-RAR{alpha}.
PCR was performed on full length RAR{alpha} cDNA with a primer 5' of the RAR{alpha} RsrII restriction site (5'-GGAGGTGCCCAAGCCCGAGT-3') and a 3' primer containing a mutated stop codon (5'-GGGGAGTGGGTGGCCGGG-3'). This PCR generated a 3' {Delta}stop RAR{alpha} fragment. Four pBluescript X-RAR{alpha} constructs were digested with BamHI, polished with Klenow fragment, then digested with RsrII to remove the wild-type RAR{alpha}-3' fragment containing the stop codon. Next, PCR fragments were polished with Klenow fragment, digested with RsrII, then ligated into the RsrII/blunt-prepared pBluescript X-RAR{alpha} constructs. Finally, EcoRI/blunt, full-length X-RAR{alpha} cDNAs lacking the RAR{alpha} stop codon were subcloned from their pBluescript background into EcoRI/EcoRV prepared pV5 (pcDNA3.1V5/HIS; Invitrogen). By this technique, X-RAR{alpha} cDNAs, containing an in-frame COOH-terminal V5 epitope tag, were created.

COS-7 Transfections.
Using Effectene (Qiagen), a non-liposomal DNA carrier, COS-7 cells were transfected with linearized pX-RAR{alpha}V5 constructs. The manufacturer’s suggested protocol was followed for a maximum of 500 µg of plasmid DNA for each transfection. For transient transfections, ~2 x 105 COS cells/well were seeded in 6-well plates and cultured in {alpha}MEM supplemented with 5% fetal bovine serum, 0.05 units/ml penicillin, 0.05 µg/ml streptomycin, 0.5 µg/ml Fungizone, and 2 mM L-glutamine) for 16 h at 32°C and 5% CO2. Transiently transfected COS cells were harvested for X-RAR{alpha}V5 expression analysis (see below). In addition to the transient analyses, transfected COS cells were cultured in the presence of G418 (Life Technologies, Inc.) to generate PML-RAR{alpha}V5, PLZF-RAR{alpha}V5, NPM-RAR{alpha}V5, and NuMA-RAR{alpha}V5 expressing colonies. To generate these lines, ~5 x 105 COS cells/dish were seeded in 100-mm culture dishes and cultured for 16 h at 32°C and 5% CO2. After the transfection period, the COS cells were cultured in 500 µg/ml G418 supplemented medium, sufficient to eliminate cells not expressing the neor gene carried on the pV5 plasmid within 10–14 days. Individual COS X-RAR{alpha}V5, G418-resistant colonies were isolated by trypsinization within glass cloning rings (Bellco). These colonies were expanded under G418 selection pressure, through 24- and 6-well plates and then up to T25 culture flasks (Nunc) for X-RAR{alpha}V5 expression analysis and cryopreservation.

Protein Extraction, Intracellular Fractionation, and Western Blotting.
When protein was required, COS cells were washed twice in PBS, trypsinized from culture flasks, resuspended in fresh medium, and pelleted. After a final wash in PBS, cells were pelleted once more. Native-state proteins were extracted either as whole cell lysates [Native buffer: 50 mM Tris-HCl (pH 8), 150 mM NaCl, 10 mM EDTA, and 0.5% Triton X] or as intracellular cytoplasmic (buffer A: 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, and 1 mM DTT) and nuclear fractions (buffer C: 20 mM HEPES, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT). Protein samples were separated by SDS-PAGE in 8% gels and transferred to polyvinylidene difluoride membrane (Bio-Rad). Blots were blocked in PBS + 4% skim milk + 0.1% Tween + 1:500 preimmune goat serum, then immunoprobed with V5 monoclonal antibody (Invitrogen) in PBS + 3% FCS at 1:1500 dilution. Electrochemical luminescent detection (NEN Life Sciences) of LacZV5 (Mr ~130,000), PML-RAR{alpha}V5 (Mr ~95,000), PLZF-RAR{alpha}V5 (Mr ~100,000), NPM-RAR{alpha}V5 (Mr ~60,000), and NuMA-RAR{alpha}V5 (Mr ~230,000), from transiently transfected COS cells as well as from COS X-RAR{alpha}V5 lines, was carried out with an antimouse-HRP-conjugated secondary antibody at 1:7000 dilution (Santa Cruz Biotechnology). A polyclonal RAR{alpha} antibody (Santa Cruz Biotechnology) at 1:500 dilutions, in conjunction with an antirabbit-HRP-conjugated secondary at 1:7000 dilutions (Santa Cruz Biotechnology), was also used to detect X-RAR{alpha} proteins.

Immunofluorescent Confocal Microscopy.
LAB-TEK chambered glass microscope slides (Nunc) were used to grow COS parental and COS pcDNA empty vector control cells, as well as the COS X-RAR{alpha}V5 lines. Cells were seeded at medium density and paraformaldehyde fixed at ~70% confluency. Fixed cells were washed in Tris-buffered saline (TBS) + 3% BSA + 0.5% Triton X for 5 min, and then washed in TBS + BSA. Slides were blocked with 1% preimmune goat serum in PN buffer (0.1 M Na2HPO4/NaH2PO4 and 0.1% Tween 20) for 1 h at 37°C. After two PN buffer washes, individual chambers were incubated for 2 h at 37°C with single monoclonal V5 (1:2000) or monoclonal NuMA (1:500 COOH-terminal Nu1 antibody; Oncogene Science) antibodies, or combinations of V5 with polyclonal NPM (1:1000) antibody or V5 with polyclonal NuMA (1:2000) antibody. The polyclonal NPM (kind gift M. O. J. Olson, University of Mississippi Medical Center, Jackson, MS) was raised against recombinant NPM containing the COOH-terminal portion (85 , 86) . The polyclonal NuMA (kind gift of D. Compton, Dartmouth Medical School, Hanover, NH, and D. Cleveland, Ludwig Institute for Cancer Research, Baltimore, MD) antibody was able to detect endogenous NuMA, but not NuMA-RAR{alpha}V5 in Western blot analyses performed in our laboratory. For red and green immunofluorescent detection, rhodamine-conjugated goat antirabbit (Calbiochem) and FITC-conjugated goat antimouse (Oncogene Science) were added and incubated for 1 h at 37°C. After two PN buffer washes, the chamber covers were removed and slides were mounted in vectashield antifade (Vector). Immunofluorescent confocal microscopy was performed under a ZEISS laser scanning confocal microscope.

Immunoprecipitation and GST Pull-Downs.
For immunoprecipitation and coimmunoprecipitation analyses, native-state protein from COS X-RAR{alpha}V5 cells was extracted as described above. A typical immunoprecipitation reaction contained 10 µl of V5 monoclonal antibody (Invitrogen) added to 500 µl of protein extract. After a 2-h rotating incubation at room temperature, 20 µl of A/G PLUS-Agarose (Santa Cruz Biotechnology) was added, and the mixture was further incubated for 1 h. The agarose beads were spun down at 2500 rpm at 4°C for 5 min, and the supernatant was collected. The bead fraction was washed three times in 500 µl of Native buffer, and then resuspended in 50 µl of SDS loading dye for Western analysis. Blots were immunoprobed with a monoclonal antibody that specifically targets the COOH terminus of NuMA (Nu1; Oncogene Sciences) to test for coimmunoprecipitation.

For GST pull-down reactions, GST fusion proteins were generated from a full-length, wild-type NPM construct (GST-NPMwt), and a mutated NPM construct with ~75% of the multimerization domain deleted (GST-NPM{Delta}md; kind gift K. Fukasawa, University of Cincinnati College of Medicine, Cincinnati, OH), as recommended by the manufacturer (Amersham Pharmacia Biotech). 4B Sepharose-conjugated GST fusion proteins were resuspended in GST protein/protein binding buffer [GBB: 20 mM Tris-HCl (pH 8), 150 mM NaCl, 1 mM EDTA, and 0.5% NP40] and quantitated against BSA standards by SDS-PAGE and Coomassie Blue staining. LacZV5 and NPM-RAR{alpha}V5 were generated in the cell-free Quick Coupled Transcription/Translation system as recommended by the manufacturer (TNT-Promega). Binding reactions were carried out in 300 µl of GBB in which 1 µl from a 50-µl in vitro TNT reaction was added to equimolar amounts of GST fusion protein.


    Acknowledgments
 
We thank Dr. K. Fukasawa and Yukari Tokuyama (University of Cincinnati College of Medicine) for supplying the GST-NPM constructs; Drs. D. Compton (Hanover, NH) and D. Cleveland (Baltimore, MD) for the NuMA antibody; Dr. M. O. J. Olson for the NPM antibody; Dr. D. Cleveland (Baltimore, MD) and Dr. S. Morris (St. Jude Children’s Research Hospital, Memphis, TN) for NuMA and NPM cDNA constructs, respectively, which facilitated the cloning of full-length NuMA-RAR{alpha} and NPM-RAR{alpha} cDNAs; and Soheila A. Hamadanizadeh (Institute of Medical Sciences, Toronto, Ontario, Canada) for supplying the U937 NuMA-RAR{alpha} cell line.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from the National Cancer Institute of Canada (to S. K-R.), the Canadian Institute of Health Research (to S. K-R.), and the Leukemia Research Fund of Canada (to S. K-R.), and by the Ontario Graduate Scholarship Program (to J. L. H.). Back

2 To whom requests for reprints should be addressed, at Princess Margaret Hospital, Ontario Cancer Institute, Room 9-622, 610 University Avenue, Toronto, Ontario, M5G 2M9. Phone: (416) 946-2806; E-mail: s.kamel.reid{at}utoronto.ca Back

3 The abbreviations used are: APL, acute PML; PML, promyelocytic leukemia; RA, retinoic acid; RAR{alpha}, RA receptor {alpha}; PLZF, promyelocytic zinc finger; NPM, nucleophosmin; NuMA, nuclear mitotic apparatus; STAT5b, signal transducer and transcriptional activator 5b; ATRA, all-trans RA; RARE, RA response element; RXR{alpha}, retinoid X receptor {alpha}; CML, chronic myeloid leukemia; GST, glutathione S-transferase; TNT, transcribed/translated. Back

Received for publication 11/15/01. Revision received 2/19/02. Accepted for publication 2/20/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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