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Molecular and Cellular Biology, July 1999, p. 5170-5178, Vol. 19, No. 7
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

PIC-1/SUMO-1-Modified PML-Retinoic Acid Receptor alpha  Mediates Arsenic Trioxide-Induced Apoptosis in Acute Promyelocytic Leukemia

Thomas Sternsdorf,1 Elena Puccetti,2 Kirsten Jensen,1 Dieter Hoelzer,2 Hans Will,1 Oliver Gerhard Ottmann,2 and Martin Ruthardt2,*

Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität, D-20251 Hamburg,1 and Med. Klinik III/Abtl. Hämatologie, Johann Wolfgang Goethe-Universität, D-60590 Frankfurt,2 Germany

Received 30 November 1998/Returned for modification 12 February 1999/Accepted 14 April 1999


    ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Fusion proteins involving the retinoic acid receptor alpha  (RARalpha ) and PML or PLZF nuclear protein are the genetic markers of acute promyelocytic leukemia (APL). APLs with PML-RARalpha or PLZF-RARalpha fusion protein differ only in their response to retinoic acid (RA) treatment: the t(15;17) (PML-RARalpha -positive) APL blasts are sensitive to RA in vitro, and patients enter disease remission after RA treatment, while those with t(11;17) (PLZF-RARalpha -positive) APLs do not. Recently it has been shown that complete remission can be achieved upon treatment with arsenic trioxide (As2O3) in PML-RARalpha -positive APL, even when the patient has relapsed and the disease is RA resistant. This appears to be due to apoptosis induced by As2O3 in the APL blasts by poorly defined mechanisms. Here we report that (i) As2O3 induces apoptosis only in cells expressing the PML-RARalpha , not the PLZF-RARalpha , fusion protein; (ii) PML-RARalpha is partially modified by covalent linkage with a PIC-1/SUMO-1-like protein prior to As2O3 treatment, whereas PLZF-RARalpha is not; (iii) As2O3 treatment induces a change in the modification pattern of PML-RARalpha toward highly modified forms; (iv) redistribution of PML nuclear bodies (PML-NBs) upon As2O3 treatment is accompanied by recruitment of PIC-1/SUMO-1 into PML-NBs, probably due to hypermodification of both PML and PML-RARalpha ; (v) As2O3-induced apoptosis is independent of the DNA binding activity located in the RARalpha portion of the PML-RARalpha fusion protein; and (vi) the apoptotic process is bcl-2 and caspase 3 independent and is blocked only partially by a global caspase inhibitor. Taken together, these data provide novel insights into the mechanisms involved in As2O3-induced apoptosis in APL and predict that treatment of t(11;17) (PLZF-RARalpha -positive) APLs with As2O3 will not be successful.


    INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Acute promyelocytic leukemia (APL) is characterized by translocations that always involve chromosome 17, with the breakpoint in the locus that codes for the retinoic acid receptor alpha  (RARalpha ), and predominantly one of two partner chromosomes, chromosome 15 and, less frequently, chromosome 11, with breakpoints in the PML and PLZF loci, respectively (18, 52). The results of these translocations are fusion genes encoding the PML-RARalpha and the PLZF-RARalpha fusion proteins, respectively. The two fusion proteins retain the same portion of RARalpha , including the DNA-binding, transactivating, and ligand-binding domains (7, 25, 27, 40, 41). PML-RARalpha - and PLZF-RARalpha -positive APLs differ only in their response to retinoic acid (RA) and are otherwise clinically indistinguishable. PML-RARalpha APL blasts are highly sensitive to differentiation-inducing activity of RA (10, 24, 32, 53). In contrast, PLZF-RARalpha -expressing APLs are not sensitive to RA treatment (21, 23, 31, 44).

Recently it has been reported that arsenic trioxide (As2O3) is able to induce complete remission in t(15;17)-positive APLs independent of their sensitivity to RA (5, 6, 48). Whereas RA induces terminal differentiation, As2O3 seems to trigger apoptosis in t(15;17) APLs (5, 6). The mechanism of As2O3-induced apoptosis has not been elucidated. In the APL-derived NB4 cell line (30), As2O3 treatment is accompanied by bcl-2 down-regulation at late time points after apoptosis induction (5, 6, 16). Similar to what is known for RA treatment (56), it has been reported that As2O3 exposure of NB4 leads to rapid degradation of PML-RARalpha (5, 37, 57). Currently nothing is known about the effect of As2O3 on t(11;17)-positive APLs.

One of the RARalpha translocation partners, PML, is localized to specific nuclear matrix-associated subdomains, often referred to as PML nuclear bodies (PML-NBs), PML oncogenic domains, ND10 (nuclear domain 10), or Kr bodies (2, 14, 15, 28, 54). These structures can be visualized as specific "speckles" by immunostaining. In PML-RARalpha -expressing cells, PML-NBs are disrupted into a finely granular, so-called "microspeckled" immunostaining pattern (14, 15, 28, 54). Remarkably, treatment with both RA and As2O3 results in a redistribution of the microspeckled pattern and a reconstitution of the normal PML-NB pattern (9, 16, 57). Therefore, it has been hypothesized that the disruption of PML-NBs could play an important role in the pathogenesis of APL (14, 28, 54). Several proteins have been shown to colocalize with PML within the NBs, such as the Sp 100 protein, originally identified as an autoantigen in patients with primary biliary cirrhosis (51), LYSP100/Sp140 (3, 12), ISG20 (17), the retinoblastoma protein (Rb) (1), and Int-6 (13).

Recently it has been shown that PML is covalently modified by the PIC-1/SUMO-1 protein. PIC-1/SUMO-1 was first identified as interaction partner of PML by using the yeast two-hybrid assay (4). PIC-1/SUMO-1 is also referred as GAP modifying protein 1 (GMP1) (35), sentrin (39), and ubiquitin-like 1 (UBL1) (47). It has considerable sequence homology with ubiquitin and is covalently linked to the nuclear pore complex-associated protein RanGAP1 (33, 35). Furthermore, it is involved in apoptotic signalling (39) and DNA recombination and repair processes (47). It has been shown that PIC-1/SUMO-1 also binds to Sp100, another component of the PML-NBs (26, 37, 50).

PLZF, the translocation partner of RARalpha in t(11;17), has also been reported to localize in nuclear regions that are morphologically similar to the PML-NBs (42), the so-called PLZF-NBs (43). The PML-NBs and PLZF-NBs are in some cases adjacent, but functionally distinct, because PLZF-NBs, different from PML-NBs, are not affected by adenovirus E4-ORF3 expression and exposure to interferon (43). Coexpression experiments showed that PML-RARalpha and PLZF-RARalpha can colocalize perfectly into the microspeckles (43).

In the present work, we have investigated the molecular mechanisms of apoptosis induction and compared the effects of As2O3 on PML-RARalpha - and PLZF-RARalpha -expressing cells. Our data show that the presence of PML in the fusion protein is essential for efficient induction of apoptosis by As2O3 and that neither bcl-2 nor caspase 3-like activity is involved. Finally, we demonstrate that the capability of RARalpha fusion proteins to induce apoptosis is linked to As2O3-induced hypermodification by PIC-1/SUMO-1 or immunologically cross-reactive proteins, arguing for a role of this modification in the control of cell death.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Preparation of anti-RARalpha antibodies. The cDNA encoding the RARalpha F domain was cloned into the bacterial expression plasmid pGEX-2T (Pharmacia, Uppsala, Sweden) after PCR-based creation of an in-frame BamHI site. Bacterial cultures expressing pGEX vectors were grown in LB containing 50 mg of ampicillin per ml, induced with 1 mM isopropyl-beta -D-thiogalactopyranoside (IPTG), for 3 to 6 h, and the induced bacteria were lysed by sonication in 1% Triton X-100 in phosphate-buffered saline (PBS). The GST-RARalpha -F fusion protein was purified using glutathione-agarose (Pharmacia, Uppsala, Sweden) and eluted by using 15 mM glutathione. Anti-RARalpha antibodies were prepared by immunizing New Zealand White rabbits with the purified GST-RARalpha fusion protein.

Cell lines, cell culture, Western blotting, and induction of differentiation and apoptosis. NB4 and U937 cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (GIBCO). The U937 MTB45, PLZF-RARalpha -positive B412, and PML-RARalpha -positive P/R9 cells were obtained as described previously (19, 20, 44). The PML/Delta RARalpha clones P/Delta R B321 and B327 were obtained by limiting dilution from the P/Delta R 12 and 14 cells described previously (gift from P. G. Pelicci) (20). Expression of the exogenous protein was induced by treatment for 6 to 12 h with 100 µM ZnSO4 (Zn) as described previously (19, 20, 44). For induction of apoptosis, the cells were extensively washed with PBS after Zn treatment, diluted to a concentration of 105 cells/ml, and exposed to a final concentration of 1 µM As2O3 or all trans-RA (both from Sigma, St. Louis, Mo.) with a 1:1,000 dilution of a 1 mM stock solution in PBS or absolute ethanol, respectively. Expression of the exogenous protein was evaluated by Western blotting after 6 to 12 h of Zn treatment by using the anti-RARalpha -antibody described above according to established procedures. Blocking and antibody incubations were performed in 5% low-fat dry milk, and washing was carried out in PBS containing 0.1% Tween 20. Anti-PLZF, anti-PML, or anti-Sp100 antibodies were used as described elsewhere (22, 44, 49, 50). PIC-1/SUMO-1 and ubiquitin-specific monoclonal antibodies (MAbs) (anti-GMP-1, 21C7, and Ubi-1, respectively) were purchased from Zymed Laboratories, Inc. (WAK-Chemie, Bad Homburg, Germany). Anti-poly(ADP ribose) polymerase (PARP) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif.). Quantitation of immunoblots was performed by using the TINA 2.09g bioimaging software (RAYTEST, Straubenhardt, Germany) on TIFF images of low- or medium-density-exposure X-ray films. No electronic modifications of the images, such as contrast or brightness adjustment, were performed prior to quantitation.

Immunofluorescence staining. Cells were cytocentrifuged and fixed in methanol at -20°C for 5 min, followed by acetone at -20°C for 20 s. PML, PLZF, RARalpha , and SUMO-1/PIC1 stainings were performed with the antibodies mentioned above as described elsewhere (22, 44, 49, 50). After extensive washes in PBS, cells were stained with fluorescein isothiocyanate (FITC)-, DTAF- or LRSC-conjugated donkey anti-mouse immunoglobulin (Ig), anti-rabbit Ig, or anti-rat Ig (DIANOVA, Hamburg, Germany). Microscopic analysis was performed with an Olympus BX-60 fluorescence microscope equipped with a chilled 3CCD color camera (C5810; Hamamatsu Photonics, Hamamatsu City, Japan). Images were captured with a 24-bit board (Image Grabber 24; Neotech, London, United Kingdom) on a 8100/80 Power Macintosh computer (Apple, Cupertino, Calif.). Distinct cubes for FITC (excitation filter, 470 to 490 nm; dichroic mirror, 505 nm; barrier filter, 515 to 550 nm) and Texas red or LRSC (excitation filter, 510 to 550 nm; dichroic mirror, 570 nm; barrier filter LP, 590 nm) were used and the images were either directly superimposed by the C5810 3CCD control unit or were merged electronically by using Adobe Photoshop 4.01 software (Adobe Systems, San Jose, Calif.).

Apoptosis assay. For staining of apoptotic and dead cells, the 7-amino-actinomycin D (7-AAD) method was used (45). After 36 to 72 h of As2O3 exposure, the cells were harvested by centrifugation and incubated with 20 µg of 7-AAD per ml in PBS, without Ca2+ and Mg2+, containing 2% calf serum and 0.1% sodium azide (Sigma) (PBSAz) for 20 min at 4°C protected from light; the cells were then analyzed on a FACScan flow cytometer (Becton Dickinson, San Jose, Calif.) in the manufacturer's staining solution. All data were collected, stored and analyzed by Lysis II software (Becton Dickinson).


    RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

As2O3-induced apoptosis in APL blasts is genetically determined by the presence of t(15;17). To answer the question of whether the As2O3-induced apoptosis is specifically mediated by PML-RARalpha and whether PLZF-RARalpha -positive APLs could also be potentially treated with this agent, we analyzed the effect of As2O3 on PML-RARalpha - and PLZF-RARalpha -expressing U937 cells. This model system was used because, to date, no APL patient-derived cell line harboring the t(11;17) translocation exists.

U937 cells are myeloid precursors blocked at the promonocytic stage that do not undergo As2O3-induced apoptosis (6). In our experiments, we used U937 cells transfected with an expression vector containing PML-RARalpha (P/R9 cells) or PLZF-RARalpha (B412 cells) under the control of the ZnSO4 (Zn)-inducible metallothionine (MT-1) promoter and compared them with U937 control cells, MTB45 (B45), transfected with the empty expression vector as described elsewhere (19, 20, 44). As a positive control for As2O3-induced apoptosis, we used NB4 cells (30). A schematic drawing of the PML-RARalpha and PLZF-RARalpha proteins expressed in these cells is given in Fig. 1A. Expression of the transgenes was confirmed by immunoblotting (Fig. 1B). For As2O3 treatment, the cells were exposed to 1 µM As2O3. The apoptosis rate was measured after 36 to 72 h of As2O3 exposure by FACScan analysis of the cells stained with 7-AAD (45). Unfixed cells were stained with 7-AAD for discrimination of live from early apoptotic cells and from cells which have lost membrane integrity (late apoptotic or necrotic, dead cells). After 36 to 72 h of exposure to As2O3, the majority of NB4 cells (47%) showed signs of early apoptosis (R2 gate in Fig. 2A). The inclusion of late apoptosis (gate R3 in Fig. 2A) revealed that 72% of cells were aptotic. Here we show the results from one experiment out of three that gave nearly identical results. Similarly, the 7-AAD FACScan analysis of U937 cells clearly distinguished two cell populations, apoptotic and viable cells, respectively. For simplification, the U937 cell data are represented as columns (Fig. 2B). In the absence of Zn, without protein expression from the transgenes as determined by control experiments (data not shown), B45, B412, and P/R9 cells, similar to U937 wild-type cells, showed no significant apoptosis upon As2O3 exposure. When the cells were treated for 12 h with Zn for induction of protein expression prior to As2O3 exposure, a high incidence of apoptosis induction was seen only in the P/R9 clone, even to a larger extent than in NB4 cells (about 96% of cells were apoptotic). The 23% apoptosis in the non-Zn-induced P/R9 cells was likely due to low-level expression of PML-RARalpha in these cells (Fig. 2B). Zn treatment alone did not induce significant apoptosis with respect to untreated control cells (Fig. 2B). To confirm these data, growth curves assessed by cell number were performed. In the presence of Zn, As2O3 inhibited growth temporarily in the B45 and B412 clones, due probably to some combined toxicity of As2O3 plus Zn, whereas growth was absolutely blocked in the P/R9 clone. In the absence of Zn, As2O3 exposure had no significant effect on growth of either of B45, B412, or P/R9 clones (data not shown).


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  		FIG. 1.   (A) Structure of the t(11;17) and t(15;17) fusion proteins. RARalpha is subdivided into its conserved functional domains. C and E indicate the DNA binding and the ligand binding domains, respectively. In the fusion, PML retains three novel zinc fingers, the RING domain (R) and the B boxes 1 and 2 (B1 and B2). In the alpha  helix, PML presents a coiled-coil region, which is its homodimerization interface. The PLZF POZ domain and the retained two zinc fingers are also shown. The breakpoints (bp) where PML and PLZF fuse to RARalpha are indicated by black arrows. (B) Zn-induced PML-RARalpha and PLZF-RARalpha expression in U937 cells. Western blot analysis from U937 cells stably transfected with a Zn-inducible PLZF-RARalpha or PML-RARalpha expression vector in the presence (+) or absence (-) of Zn induction. Blots were stained with an anti-RARalpha polyclonal antibody directed against the RARalpha F domain. Molecular weight markers are given to the left (in thousands). Each lane was loaded with lysates from 2 × 105 cells. The positions of PLZF-RARalpha and PML-RARalpha polypeptides are indicated.


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  		FIG. 2.   (A) Apoptotic effect of As2O3 on NB4 cells (cell line derived from an APL patient [30]) and NB4 cells treated with As2O3 in the presence of ZVAD, as shown by 7-AAD analysis (one of three experiments that gave nearly identical results). (B) Apoptotic effect of As2O3 on PML-RARalpha - and PLZF-RARalpha -expressing U937 cells, as shown by 7-AAD analysis (one of three experiments that gave nearly identical results): MTB45-control cells transfected with the empty MT expression vector; B412, PLZF-RARalpha -expressing cells; P/R9, PML-RARalpha -expressing cells. The U937 cells are treated with Zn alone (Zn+) and with As2O3 (As+) in the absence or presence of Zn-induced protein expression. Also represented are Zn-induced P/R9 cells exposed to As2O3 in the presence of ZVAD-FMK (ZVAD). (C) bcl-2 and PARP expression of the P/R9 clone in the absence of As treatment (As-) and after 12, 24, and 48 h of As2O3 (As+) or 12 and 24 h of RA treatment (t-RA + or -) as a control for PARP cleavage. The PARP and bcl-2 proteins are indicated.

In PML-RARalpha -expressing U937 cells, As2O3-induced apoptosis is independent of PARP-cleaving caspase activity and bcl-2 expression. When cell extracts of PML-RARalpha -expressing cells were probed with very sensitive anti-RARalpha antibodies, a characteristic ladder of at least four high-molecular-mass species of PML-RARalpha with relative electrophoretic mobilities of approximately 120, 135, 160, and 180 kDa was detected (Fig. 1B, 3A, and B, and 4).


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  		FIG. 3.   Western blot analysis of PML-RARalpha - and PLZF-RARalpha -expressing U937 cells. (A) P/R9 and B412 clones in the presence of Zn-induced PML-RARalpha or PLZF-RARalpha expression, respectively, in the presence or absence of 12 and 24 h of RA treatment (t-RA - or +). (B) P/R9 and B412 clones in the absence or presence of Zn induced PML-RARalpha or PLZF-RARalpha expression (Zn - or +), respectively, and in the absence or presence of 12 h of As2O3 exposure (As - or +). Blots were stained with an anti-RARalpha polyclonal antibody (alpha -RARalpha ). The positions of PLZF-RARalpha and PML-RARalpha polypeptides are indicated. (C) PML-RARalpha lanes of panel B stained with anti-Sp100 antibody (alpha -Sp100). The positions of Sp100 and PIC-1/SUMO-1-modified Sp100 are indicated.


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  		FIG. 4.   PIC-1/SUMO-1 modification of PML/RARalpha upon As2O3 exposure. (A) P/R9 clone in the absence or presence of Zn-induced PML-RARalpha expression (Zn - or +) and in the absence or presence of 12 h of As2O3 exposure (As - or +). Blots were stained with an anti-RARalpha polyclonal antibody (alpha -RARalpha ) and anti-SUMO-1 monoclonal antibody (alpha -Sumo1). (B) Electronic juxtaposition of the lanes from panel A stained with anti-RARalpha antibody with that stained with anti-SUMO-1 antibody in the absence or presence of Zn-induced PML-RARalpha expression in the P/R9 clone (Zn - or +). The SUMO-1-conjugated bands are indicated by bars. (C) Electronic quantification of wild-type and As2O3-modified PML-RARalpha protein from the lanes stained with anti-RARalpha antibody shown in panel A. (D) Electronic comparison between single wild-type PML-RARalpha bands and the smear of PIC-1/SUMO-1-modified PML-RARalpha resulting from As2O3 treatment.

In agreement with previously reported results from RA-mediated degradation of PML-RARalpha and PLZF-RARalpha (29, 56, 57), an immunoblot probed with an anti-RARalpha antibody directed against the RARalpha F domain (see Materials and Methods) showed a progressive degradation of both PML-RARalpha or PLZF-RARalpha in Zn-induced U937 cells upon RA treatment. The degradation was nearly complete after 12 h of incubation with RA (Fig. 3A). The RA-induced down-regulation of both APL fusion proteins has recently been shown to be due to posttranscriptional modification by caspase 3-like activity induced by RA (38). The caspases are a family of cysteine proteases with aspartic acid substrate specificity, thought to be key effectors of cellular apoptosis in multicellular organisms (reviewed in reference 55). The different responses of PML-RARalpha and PLZF-RARalpha to As2O3 prompted us to address the question of whether in As2O3-induced apoptosis, caspase 3 activity could play a role and explain the activation of apoptosis cascades. Evidence that caspase 3 activity is not involved during As2O3 apoptosis is given by the fact that neither PML-RARalpha nor PLZF-RARalpha is degraded by As2O3 (Fig. 3B). To further exclude the involvement of caspase 3 activity that might be induced by the presence of PML-RARalpha , we probed the immunoblot filters with an antibody specific for PARP, which is a known substrate for several caspases including caspase 3. Cleavage of the PARP protein by caspase 3-like activity is seen when the cells are treated with RA (38). We investigated PARP cleavage after 12, 24, and 48 h of As2O3 in the Zn-treated P/R9 clone, and no cleavage of endogenous PARP was seen, which was different from the results with Zn-treated P/R9 cells upon RA exposition used as a control in these experiments (Fig. 2C).

The fact that PARP is not cleaved during As2O3-induced apoptosis in PML-RARalpha -expressing cells prompted us to investigate whether caspases are involved at all in arsenic-induced apoptosis. This was analyzed by incubating the NB4 cells and the P/R9 clone with 100 mM ZVAD-FMK (Bachem, Basel, Switzerland), a potent global caspase inhibitor, 1 h prior to As2O3 exposure, and after 3 days, 7-AAD staining of both PML-RARalpha -expressing U937 and NB4 cells was quantitated by FACScan analysis. The number of apoptotic cells in As2O3-exposed cells was significantly reduced in the presence of ZVAD in both NB4 cells (43 and 72%) (Fig. 2A) and Zn-induced P/R9 cells (76% and 96%) (Fig. 2B). Nevertheless, the ZVAD-treated cells did not recover with prolonged culture. Thus, the effect of ZVAD has to be seen as a delay of cell death.

Previously, it has been reported that in NB4 cells, As2O3-induced apoptosis is correlated with the down-regulation of bcl-2 after 48 h of As2O3 exposure (6, 16). Both NB4 and PML-RARalpha -expressing U937 cells showed early apoptosis-related modifications after 36 to 48 h. The As2O3-related modifications of PML-RARalpha were nearly complete after 3 h of treatment (37). To investigate whether there are differences in bcl-2 expression between PML-RARalpha - or PLZF-RARalpha -positive and U937 control cells upon As2O3 treatment, we compared the levels of bcl-2 expression in the B45, B412, and P/R9 clones in the presence and absence of Zn-induced protein expression. We performed immunoblots of cellular lysates after 12, 24, and 48 h of As2O3 treatment probed with a MAb directed against bcl-2 (Santa Cruz). At this time point, no modification of bcl-2 expression was observed either in NB4 cells (not shown) or in PML-RARalpha -positive U937 cells. Cells of all three clones (B45, P/R9, and B412) expressed very similar levels of bcl-2, independent of prior exposure to Zn and/or As2O3 (shown representatively for P/R9 cells) (Fig. 2C).

Taken together, these data shown that As2O3-induced apoptosis critically depends on the presence of the PML-RARalpha fusion protein and is independent of bcl-2 and caspase 3-like activity. As2O3 induces the apoptosis signalling pathway independently from caspase activities.

Upon As2O3 treatment, PML-RARalpha is modified to high-molecular-weight species. Recently it has been reported that PML-RARalpha is significantly degraded also when NB4 cells are treated with As2O3 (37, 46, 57). For this reason, we compared the effects of As2O3 on the expression level of either PML-RARalpha or PLZF-RARalpha in Zn-treated P/R9 and B412 cells, respectively. After 12 h of As2O3 exposure, there was a significant decrease of all high-molecular-weight PML-RARalpha species, with the exception of a band with a molecular mass of about 180 kDa (Fig. 3B and 4A). The 120-kDa band, probably the nonmodified PML-RARalpha , showed only a minor decrease in intensity. Furthermore, a smear of anti-RARalpha staining higher than the 180-kDa band was detected, which represented other PML-RARalpha high-molecular-weight species that could not be separated on the denaturating acrylamide gel (Fig. 3B and 4A). The quantitative evaluation of the intensity of all high-molecular-mass ladder bands, including that of the smear and the 120- and the 180-kDa bands, by a bioimager revealed that the overall signal intensity for PML/RARalpha was not reduced significantly by As2O3 treatment, but the signals had shifted to a higher molecular mass (Fig. 4C and D). The PLZF-RARalpha protein, on the contrary, was neither degraded nor modified as a consequence of As2O3 treatment (Fig. 3B).

PML-RARalpha is progressively modified by PIC-1/SUMO-1. It is known that PML is modified by PIC-1/SUMO-1 (26, 37, 50). To determine whether the size shift of PML-RARalpha following As2O3 is due to a PIC-1/SUMO-1 modification similar to that of PML, the blots were probed with an anti-SUMO-1 antibody, thereby revealing that three of five PML-RARalpha bands detected with the anti-RARalpha antibody in non-As2O3-treated cells are also detected by the anti-SUMO-1 antibody (Fig. 4A). To elucidate whether the PML-RARalpha "ladder" is due to some PIC-1/SUMO-1 modification in the absence of As2O3, the blot from Fig. 4A is presented in an electronically modified form to juxtapose the PML-RARalpha bands stained with anti-RARalpha and anti-SUMO-1 antibodies, respectively. As is obvious from the staining pattern, PIC-1/SUMO-1-modified proteins different from PML-RARalpha are also detected by the antibody and in part overlap with the PML-RARalpha modification ladder. In the As2O3-treated PML-RARalpha cells, the 180-kDa band described above is detected as a strong signal also by the anti-SUMO-1 antibody (arrow in Fig. 4A). Furthermore, the smear over the 180-kDa band was also strongly stained by the anti-SUMO-1 antibody. The correlation between the intensity of the anti-RARalpha and anti-SUMO-1 staining in this case was striking. Thus, we conclude that the 180-kDa band that intensified upon As2O3 exposure contains exclusively or predominantly PML-RARalpha with covalently bound PIC-1/SUMO-1 or closely related proteins.

Taken together, these data indicate that PML-RARalpha is not posttranslationally degraded but is modified by multiple covalent attachment of multiple PIC-1/SUMO-1 proteins or immunologically cross-reactive polypeptides upon As2O3 treatment (hyperSUMOylation).

PML and PML-RARalpha are As2O3-specific targets of PIC-1/SUMO-1 modification. In addition to PML, the Sp100 protein, another component of the PML-NBs (51), and RanGAP1, a factor involved in nuclear import (34, 36), are known to be covalently modified by PIC-1/SUMO-1 or immunologically cross-reactive proteins. To check the influence of As2O3 on PIC-1/SUMO-1 modification of other proteins in PML-RARalpha -positive cells, we stained the blots with the PML-RARalpha -positive cell lysates with an anti-Sp100 antibody. In addition, the relative amount of PIC-1/SUMO-1-modified RanGAP1 protein (35, 36) was determined by measuring the intensity of the dominant 90-kDa band visible on the immunoblots by using the PIC-1/SUMO-1-specific antibody. The modification pattern of both Sp100 and RanGAP1 was not altered upon As2O3 treatment (Fig. 3C and 4A, respectively). Thus, it seems that hyperSUMOylation induced by As2O3 is highly specific for PML and PML-RARalpha . Taken together, these data suggest that the PIC-1/SUMO-1-modified PML-RARalpha alone is able to mediate As2O3-induced apoptosis. This is supported by the fact that the PML modification in PML-RARalpha -negative U937 cells does not lead to apoptosis.

Upon As2O3 exposure, PIC-1/SUMO-1 is recruited to the PML-NBs and changes the immunostaining pattern from prevalent nuclear diffused to speckled. Treatment of PML-RARalpha -positive NB4 cells with As2O3 leads to a reconstitution of the PML-NBs disrupted by the expression of PML-RARalpha (37, 57). The fact that PML-RARalpha is modified covalently by PIC-1/SUMO-1 prompted us to investigate whether PIC-1/SUMO-1 is completely dislocated into the PML-NBs or whether some PML-RARalpha -PIC-1/SUMO-1 complexes carrying microspeckles are detectable. Furthermore, we analyzed whether PIC-1/SUMO-1 and PML-RARalpha or PLZF-RARalpha colocalize in Zn-treated P/R9 or B412 clones, respectively, in the presence or absence of As2O3 treatment. Double immunostaining with an anti-PIC-1/SUMO-1 antibody (Fig. 5, red fluorochrome) and rat anti-PML (22) or rabbit anti-PLZF (44) antibodies (Fig. 5, green fluorochrome) was performed. As a control, we used NB4 cells. In the absence of As2O3, no difference in PIC-1/SUMO-1 (red fluorochrome) localization between Zn-treated and untreated cells in both clones was seen. The Zn-treated cells exhibited a PIC-1/SUMO-1 nuclear diffused immunostaining pattern (red fluorochrome) identical to that of NB4 cells (Fig. 5). The patterns obtained with anti-PML antibodies were identical to the reported microspeckled anti-PML pattern (green fluorochrome) but were slightly more intense in U937 cells than those in NB4 cells (Fig. 5). The anti-PLZF immunostaining pattern (green fluorochrome) in the B412 clone was microspeckled, as described previously (29, 43). Superimposition of anti-PML/anti-PLZF staining with the anti-SUMO-1 stainings revealed no significant colocalization (yellow) (Fig. 5). Upon As2O3 exposure, the anti-PML staining of Zn-induced P/R9 identical to that of As2O3 treated NB4 cells revealed 5 to 10 nuclear dots per cell, slightly different from typical PML-NBs, as described previously (green fluorochrome) (9, 15, 28) (Fig. 5). Anti-SUMO-1 staining drastically changed in all As2O3-treated cells from a nuclear diffuse pattern to a prevalently speckled pattern similar to that of PML or PLZF (red fluorochrome). The anti-PLZF staining in the Zn-induced B412 clone, however, revealed no difference between As2O3-treated and untreated B412 cells. Superimposition of anti-PML and anti-SUMO-1 staining in the P/R9 clone revealed perfect colocalization between PML, PML-RARalpha , and PIC-1/SUMO-1 (Fig. 5, yellow). Superimposition of anti-PLZF and anti-SUMO-1 staining in B412 cells on the contrary revealed no colocalization between PLZF-RARalpha microspeckles and PIC-1/SUMO-1 speckles.


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  		FIG. 5.   Immunofluorescence analysis of PML, PML-RAR, PLZF-RARalpha , and PIC-1/SUMO-1 protein localization in the U937 P/R9 and B412 cell clones and the NB4 cells. Results for the P/R9 and B412 clones are reported in the absence (-Zn) and presence (+Zn) of Zn-induced PML-RARalpha or PLZF-RARalpha expression with or without As2O3 (+ or -As) NB4 cells are reported in the presence or absence of As2O3. Cells were stained with the indicated antibodies: the anti-PML (alpha -PML) and anti-PLZF (alpha -PLZF) polyclonal antibodies (green fluorochrome) and the anti-SUMO-1 MAb (alpha -SUMO) (anti-GMP-1 21C7) (red fluorochrome). Colocalization images of PIC-1/SUMO-1 and PML or PLZF were obtained by electronic overlapping of the images recorded (for merge, colocalization of fluorochromes yields a yellow color). Phaco, phase-contrast images.

Taken together, these data indicate that only PML and PML-RARalpha are modified by PIC-1/SUMO-1. No interaction of PIC-1/SUMO-1 with PLZF-RARalpha is seen. As2O3 leads to a reorganization of slightly modified PML-NBs in PML-RARalpha -expressing cells. A microspeckled subnuclear structure was seen in neither PML-RARalpha -positive U937 cells nor NB4 cells, implying that PML-RARalpha is completely recruited into the PML-NBs upon As2O3 treatment.

PML-RARalpha -mediated As2O3-induced apoptosis is independent of the RARalpha DNA binding activity. PML-RARalpha is dislocated into the PML-NBs upon As2O3 exposure. Both PML and PML-RARalpha are hyperSUMOylated by As2O3. The fact that the modification of endogenous PML upon As2O3 exposure is not associated with apoptosis prompted us to investigate the role of the RARalpha portion of the APL fusion protein. For that reason, we analyzed the As2O3 response of U937 expressing a PML-RARalpha mutant (P/Delta R) lacking the two RARalpha zinc fingers representing the RARalpha DNA binding domain. It has been previously shown that the deletion of the RARalpha DNA binding domain abolishes the biological activities of PML-RARalpha , such as differentiation blocking and mediation of RA sensitivity in U937 cells (20). When the effects of this construct on the As2O3 response in U937 cells were examined, no significant differences with respect to the PML-RARalpha -expressing P/R9 cells were seen. Here are reported the results from one of three experiments performed that gave similar results, with two U937 P/Delta R clones, B321 and B327, derived from limiting dilution of two different clones described previously (20) (a gift from P. G. Pelicci). In the absence of Zn, without protein expression, both P/Delta R cell clones B321 and B327 behave upon As2O3 exposure identically to U937 control cells (B45), showing no significant apoptosis (Fig. 6). When the cells were treated for 12 h with Zn for induction of protein expression, subsequent exposure of both B321 and B327 clones to As2O3 resulted in apoptosis to an extent similar to that of the P/R9 cells (about 60% of apoptotic cells) in these experiments (Fig. 6). Sensitivity of promyelocytic blasts to the action of As2O3, therefore, seems to strictly depend on the presence of the PML portion of the t(15;17) fusion protein and is independent of the DNA binding and transactivating properties of the RARalpha portion.


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  		FIG. 6.   7-AAD analysis of the U937 clones B321 and B327 expressing the PML-Delta RARalpha mutant (one out of three experiments that gave nearly identical results). MTB45, control cells transfected with the empty MT expression vector; P/R9, PML-RARalpha -expressing cells; B321 and B327, PML-Delta RARalpha -expressing clones. The U937 cells are treated with Zn alone (+Zn) and with As2O3 (- or + As) in the absence or presence of Zn-induced protein expression.


    DISCUSSION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

PML-RARalpha and PLZF-RARalpha are the abnormal fusion products of APLs with either t(15;17) or t(11;17). They differ in their responses to RA. PML-RARalpha -positive APLs achieve complete remission in 90 to 95% of cases (11, 18, 52). Despite the small number of t(11;17) APL patients, this variant APL has attracted the attention of many investigators, because patients harboring the t(11;17) translocation do not respond to an RA-based regimen (31). Recently it has been shown that As2O3 induces complete remission in relapsed and/or RA-resistant APLs (5, 6, 48). To determine a potential pathophysiological basis for treatment of t(11;17) APLs with As2O3, we compared the effect of As2O3 on PML-RARalpha - and PLZF-RARalpha -expressing U937 cells with that of PML-RARalpha -positive NB4 cells.

In our report, we demonstrate that As2O3-induced apoptosis is not a general feature of bone marrow cells at the promyelocytic stage of differentiation but is genetically determined by the presence of the t(15;17)-specific chimeric gene product, PML-RARalpha . Initial support for the hypothesis of a genetic determination of the As2O3 response was given previously by the fact that only NB4 cells, and not HL60 cells, undergo As2O3-induced apoptosis (6). Both are promyelocyte-like cell lines that differ in their origin. One, NB4 derived from a patient with APL (FAB-M3) (30), is PML-RARalpha positive, and the other, HL-60, is PML-RARalpha negative (8). With the induction of PML-RARalpha -dependent As2O3-induced apoptosis in cells (U937 cells) that do not respond to As2O3 in the absence of PML-RARalpha , we unequivocally show that the response to As2O3 does not depend on the promyelocytic stage of differentiation, but on expression of the PML-RARalpha fusion protein.

We extended the analysis of the biological behavior of PML-RARalpha and PLZF-RARalpha with regard to their capability to mediate apoptosis by As2O3. In contrast to PML-RARalpha , PLZF-RARalpha is not able to mediate a response to As2O3. Sensitivity of promyelocytic blasts to the action of As2O3, therefore, is strictly dependent on the type of fusion protein present and thus is genetically restricted to the t(15;17)-positive APLs.

To investigate whether known mechanisms of apoptosis induction are involved in As2O3 apoptosis, we studied the effect of As2O3 in PML-RARalpha -expressing cells on two major regulation points of apoptosis in mammalian cells, bcl-2 and caspase 3. Our data confirm evidence from RA-resistant NB4 cells that underwent apoptosis without down-regulation of bcl-2. (16). In our studies, neither NB4 cells nor PML-RARalpha -expressing U937 cells have shown clear evidence of apoptosis after 24 to 48 h of As2O3 treatment, and after 12 h, the PML-RARalpha modification is completed and the fusion protein is dislocated into the PML-NBs, whereas no effect on bcl-2 expression was observed until 72 h of As2O3 treatment.

When treated with RA, both PML-RARalpha and PLZF-RARalpha are degraded by a PARP-cleaving activity (38). The fact that in U937 neither PML-RARalpha nor PLZF-RARalpha is degraded by As2O3 excludes an activation of caspase 3-like activity. However, the fact that the global inhibition of caspase activity by the ZVAD tetrapeptide temporarily prevents As2O3-mediated apoptosis in PML-RARalpha -positive U937 cells suggests that other members of the caspase family may be involved in the process of As2O3-induced apoptosis.

Unlike reported previously (16, 37, 57), we have demonstrated that PML-RARalpha is not degraded during As2O3 exposure but is hypermodified by covalent binding to PIC-1/SUMO-1 molecules. Our results suggest that the hyperSUMOylated PML-RARalpha might be involved directly in the induction of apoptosis, because only in the presence of PML-RARalpha protein are hemopoietic cells able to undergo As2O3-induced apoptosis. The differences between our results and that reported previously may be due to the different sensitivity and specificity of the anti-RARalpha antibodies used. The anti-RARalpha antibody used in this study with appropriate blocking solution was able to identify the 180-kDa band and the high-molecular-mass smear of PML-RARalpha probably not detected by other anti-RARalpha antibodies. Other possible explanations may be differences in electrophoretic separation of the PML-RARalpha -SUMO-1 conjugates in the various gel systems used or differences in the production of the protein extracts.

Recently, it has been shown that PML is covalently modified by PIC-1/SUMO-1 (26, 37, 50). This modification is strongly increased when the cells are exposed to As2O3, resulting in formation of high-molecular-weight species of PML (37, 57). It has been reported that in nonhemopoietic cell lines, these modifications seem, first, to shift the nucleoplasmic fraction of PML onto the nuclear matrix, as evident by the appearance of brighter speckles of PML-NBs, and then to degrade PML (57).

We have shown that PIC-1/SUMO-1 is recruited to the PML-NBs in U937 and NB4 cells upon As2O3 treatment. This recruitment leads to brighter speckles in U937, but does not interfere in absence of PML-RARalpha with mechanisms of apoptosis. In PML-RARalpha -expressing NB4 and U937 cells, PIC-1/SUMO-1 is recruited to the speckled subnuclear structures both by PML and by PML-RARalpha . Proof of PML-RARalpha recruitment of PIC-1/SUMO-1 to the PML-NBs is derived by the fact that the anti-RARalpha immunofluorescence staining of PML-RARalpha -expressing NB4 or U937 cells evidenced a staining pattern identical to that of the anti-PML speckles (data not shown). In RA-treated cells, the reconstitution of the PML-NBs is probably due to the release of sequestered PML from the heterodimerization with PML-RARalpha because of the degradation of PML-RARalpha (43, 56). In contrast, the reconstitution of the PML-NBs upon As2O3 treatment is caused by the physical transfer of PML-RARalpha onto PML-NBs. In none of our experiments have we seen a decrease of the anti-PML staining as a sign of PML down-regulation by As2O3, as described previously (6, 57). These data indicate that the PIC-1/SUMO-1 modification of PML-RARalpha leads to its delocalization into the PML-NBs.

To exclude the possibility that another known target of PIC-1/SUMO-1 modification is involved in As2O3-induced apoptosis, we investigated whether modification of other proteins is modulated by As2O3 treatment. We found that PML and PML-RARalpha are the major targets to be PIC-1/SUMO-1 hypermodified after As2O3 treatment. Furthermore, our immunofluorescence analysis argues that PML-NBs are the major cellular structure for PIC-1/SUMO-1 targeting after As2O3 treatment. Together with the fact that only PML-RARalpha -positive cells undergo As2O3-induced apoptosis, these data led us to the conclusion that the PIC-1/SUMO-1-modified PML-RARalpha species might mediate As2O3-induced apoptosis by delocalizing PML-RARalpha from the not-well-defined microspeckles into the PML-NBs, where it can exert its effect.

One could speculate that As2O3 induced a direct effect of one of the components of the fusion protein on apoptosis mechanisms. A convincing hypothesis for the role of PML-RARalpha in As2O3-induced apoptosis would be a direct influence of PML-RARalpha on one of the apoptosis-inducing pathways mediated directly by the PML moiety of the fusion protein. This hypothesis is supported by the fact that PLZF-RARalpha lacking the functional domains of PML is not able to mediate apoptosis despite the presence of the identical portion of RARalpha in the fusion protein. A PML-mediated effect on apoptosis could be triggered by its interaction with hypophosphorylated Rb (1). Rb phosphorylation regulates cell cycle progression and activation of E2F-induced transcription. The PML-Rb interaction is interrupted when PML-RARalpha is expressed and PML is dislocated in the microspeckles (1). The dislocation of PML-RARalpha from microspeckles to reconstituted PML-NBs by PIC-1/SUMO-1 modification could reestablish the interaction between Rb and PML and PIC-1/SUMO-1-modified PML-RARalpha and lead to abnormal cell cycle regulation followed by apoptosis. The lack of interaction between PLZF and PIC-1/SUMO-1 might be responsible for As2O3 nonresponsiveness of t(11;17) APLs.

It remains to be shown whether the apoptosis-promoting activity of the fusion protein is due to a new function introduced into the PML-NBs by PML-RARalpha or is due to a simple increase in the quantity of PML in the PML-NBs.

In conclusion, our data demonstrate that As2O3-induced apoptosis in APL blasts is genetically determined by PML-RARalpha and therefore depends on the presence of t(15;17) translocation. A prerequisite for As2O3-induced apoptosis appears to be the dislocation of PML-RARalpha into the PML-NBs by conjugation to PIC-1/SUMO-1. It will be interesting to investigate whether hypermodification of PML by SUMO-1 or related proteins also occurs in situations different from arsenic treatment and thus might represent a more common mechanism involved in apoptosis induction by other stimuli as well.


    ACKNOWLEDGMENTS

We are grateful to Clara Nervi for helpful suggestions and critical reading of the manuscript and Pier Giuseppe Pelicci for critical reviewing of the manuscript. We thank J. Löhler and O. Utermöhlen for help with cytocentrifuge cell preparations.

This work was supported by a grant from the Deutsche Krebshilfe. The Heinrich-Pette-Institut is supported by the Freie und Hansestadt Hamburg and the Bundesministerium für Forschung und Gesundheit. E.P. is supported by a fellowship of "Deutsche José Carreras Leukämie Stiftung e.V." (DJCLS-99/NAT-1).

T.S. and E.P. contributed equally to this work.


    FOOTNOTES

* Corresponding author. Mailing address: Med. Klinik III/Abtl. Hämatologie, Klinikum der J. W. Goethe-Universität Frankfurt, Theodor Stern Kai 7, 60590 Frankfurt, Germany. Phone: 49-69-6301-6129. Fax: 49-69-6301-6131. E-mail: ruthardt{at}em.uni-frankfurt.de.


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Abstract
Introduction
Materials and Methods
Results
Discussion
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Molecular and Cellular Biology, July 1999, p. 5170-5178, Vol. 19, No. 7
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Copyright © 1999, American Society for Microbiology. All rights reserved.


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