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Published as doi: 10.1096/fj.06-8050com.
 (The FASEB Journal. 2008;22:1369-1379.)
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Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RAR{alpha}

Oliver H. Krämer1, Sylvia Müller, Marc Buchwald, Sigrid Reichardt and Thorsten Heinzel

Institute of Biochemistry and Biophysics, University of Jena, Jena, Germany

1Correspondence: Institute of Biochemistry and Biophysics, University of Jena, Philosophenweg 12, D-07743 Jena, Germany. E-mail: o.kraemer{at}uni-jena.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The chromosomal translocation products AML1-ETO and PML-RAR{alpha} contribute to the pathogenesis of leukemias. Here, we demonstrate that both AML1-ETO and PML-RAR{alpha} are degraded by the ubiquitin-proteasome system and that their turnover critically depends on the E2-conjugase UbcH8 and the E3-ligase SIAH-1. Contrary to its role in HDAC2 degradation, the E3-ligase RLIM does not target AML1-ETO and PML-RAR{alpha} for ubiquitin-dependent elimination. RLIM rather is a substrate of SIAH-1, which indicates that these E3-ligases operate in a hierarchical order. Remarkably, proteasomal degradation of leukemia fusion proteins, in addition to the block of histone deacetylase (HDAC) enzymatic activity is a consequence of HDAC-inhibitor treatment. The former requires the induction of UbcH8 expression and each of these processes might be beneficial for leukemia treatment. Our observations shed light on the mechanism determining the interplay between E2-conjugases, E3-ligases, and their substrates and suggest a strategy for utilizing the ubiquitylation machinery in a therapeutic setting.—Krämer, O. H., Müller, S., Buchwald, M., Reichardt, S., Heinzel, T. Mechanism for ubiquitylation of the leukemia fusion proteins AML1-ETO and PML-RAR{alpha}.


Key Words: proteasomal degradation • RLIM • Siah-1 • UbcH8 • HDAC-inhibitor VPA


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEVERAL DISEASES ARE LINKED to changes in gene expression caused by the conversion of transcriptional activators into repressors due to chromosomal translocations. Their occurrence correlates with certain leukemia subtypes, and fusion proteins likely are the initiating event in leukemia development. In ~10–15% of all de novo acute myeloid leukemia (AML) patients, and especially in the M2 subtype (FAB, French-American-British classification), the AML1 (acute myeloid leukemia, RUNX1) gene located on chromosome 21 is fused to the ETO (eight-twenty-one, MTG8, CBF2T1) gene on chromosome 8 (1) . This translocation t(8;21) replaces the transactivation domain of AML1, a crucial factor for definitive hematopoiesis, with almost the complete ETO protein. The chimeric AML1-ETO protein interferes with normal AML1-dependent transcription by constitutive repression of genes for hematopoietic differentiation via the ETO part, which recruits repressor complexes containing N-CoR, mSin3, and histone deacetylases (HDACs). Association of AML1-ETO with these proteins thereby blocks hematopoietic gene expression, differentiation, and apoptosis, and provides a reservoir of preleukemic cells (1 2 3 4 5) .

Similar events occur in the M3 subtype (FAB) of AML, acute promyelocytic leukemia (APL). APL is predominantly associated with the chromosomal translocation t(15;17), resulting in the fusion protein PML-RAR{alpha} (promyelocytic leukemia-retinoic acid receptor {alpha}). This oncoprotein binds to RAR target genes and recruits HDACs via N-CoR and mSin3. The recruitment of these factors is insensitive to physiological levels of retinoic acid and blocks differentiation and PML-dependent apoptosis of promyelocytes (6) .

The roles of AML1-ETO and PML-RAR{alpha} during leukemogenesis, the signaling pathways they affect, and factors determining their turnover are under intense investigation (1 , 6) . PML-RAR{alpha} has been shown to undergo proteasomal degradation, which involves polyubiquitylation exerted by the hierarchical action of an E1-enzyme, an E2-conjugase and an E3-ligase (7) . The E3-ligase SIAH-1 was identified as an enzyme mediating PML-RAR{alpha} degradation (8) . Moreover, turnover of PML-RAR{alpha} can be increased by chemotherapy, which is appreciated as a mechanism accounting for its effectiveness in APL treatment (6) . A small-molecule approach that would eliminate the initiating factor predisposing myeloid precursors for transformation could also be beneficial for patients with t(8;21) leukemia.

Inhibitors of HDACs (HDACi) might be a possible treatment option for cancer and have recently received appreciable attention. Considering the important role of HDACs in M2 AML and M3 APL, HDACi are promising therapeutic agents for leukemias. Recent findings show that these compounds not only affect HDAC activity, but also protein stability (reviewed in 9). Both effects may explain the molecular actions of HDACi in vitro as well as in experimental therapy (9 10 11 12 13 14 15) . We previously showed that valproic acid (VPA) not only inhibits HDACs but also induces proteasomal degradation of the histone deacetylase HDAC2, which is regulated by limiting amounts of the E2-conjugase UbcH8 and the RING-finger E3-ligase RLIM (12) . Selective VPA-induced attenuation of HDAC2 has been confirmed in murine tissues and in patient material (10 , 12 , 16) . Analysis of the HDACi-induced protein turnover may hence be useful for molecular monitoring and for the development of therapeutic strategies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture, Western blot, immunoprecipitations, pull-down experiments, in vivo ubiquitylation analysis and real-time quantitative PCR analysis
Cell culture, lysate preparation, and transfections are described in refs. 12 , 17 . Cells were grown in Dulbecco modified Eagle medium (DMEM) (293T) or Roswell Park Memorial Institute (RPMI) medium supplemented with 10% fetal calf serum (FCS), 2% L-glutamine, antibiotics and 10 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF; SKNO-1). N-ethyl-maleimide (10 mM; Sigma, St. Louis, MO, USA) was added to lysates to preserve protein modifications and UbcH8 complex formation. MG-132 (1 µM) was added for 4–16 h to prevent the degradation of polyubiquitylated leukemia fusion proteins. For pull-down experiments, glutathione S-transferase (GST) -UbcH8 was expressed in Escherichia coli BL21-Gold (DE3) pLysS (Stratagene, La Jolla, CA, USA). Bacteria were lysed in PBS containing 0.5 mg/ml lysozyme, 1 mM dithiothreitol (DTT), 5 mM EGTA, 5 mM EDTA, and protease inhibitors. GST fusion proteins were immobilized and purified on glutathione-Sepharose 4B beads (Amersham, Piscataway, NJ, USA). Two hundred micrograms of cell lysate and 50 µg of the GST fusion protein were incubated in hypotonic lysis buffer (17) . To detect the ubiquitylation of leukemia fusion proteins, lysates were prepared under stringent conditions in radioimmunoprecipitation assay (RIPA) buffer. All immunoprecipitations were done with 1 µg of antibody and 40 µl protein A/G beads (Amersham). After incubation for 16 h at 4°C, beads were washed, and bound proteins were eluted with Laemmli buffer and analyzed by immunoblotting. Antibodies for Western blot analysis were from Santa Cruz Biotechnology (Santa Cruz, CA, USA; AML1-ETO, sc-9797/-9737; caspase-3, sc-7272; Gal4, sc-510; GFP, sc-9996; GST, sc-138; HDAC2, sc-7899, sc-9959; HA, sc-7392; HSP90, sc-13119; Myc, sc-40; PML, sc-966; SIAH-1, sc-5506; antiacK, sc-8663), Sigma (actin, A2066/A5060; Flag, F3165; {alpha}-tubulin, T5168; ubiquitin, U5379), Abgent (San Diego, CA, USA; UbcH8, AP2118b), Chemicon (Temecula, CA, USA; ubiquitin, MAB1510), Upstate (Lake Placid, NY, USA; antiacK), New England Biolabs (Beverly, MA, USA; antiacK, 9441), Invitrogen (Carlsbad, CA, USA; V5-tag, 46–0705). Immune sera against RLIM and PML-RAR{alpha} were described elsewhere (18 , 19) . Western blots were probed for actin to ensure equal sample loading. Real-time quantitative RT-PCR analyses determining delta-delta-Ct were carried out with SYBRGreen as described (20) . 18S rRNA primer sequences were 5'-CGGCTACCACATCCAAGGA and 5'-CCAATTACAGGGCCTCGAAA.

Plasmids, transfections, and apoptosis assays
Plasmids for UbcH8, RLIM, SIAH-1, AML1-ETO, and PML-RAR{alpha} are described elsewhere (4 , 8 , 12 , 21 , 22) . 293T cells were transfected with lipofectamine or polyethylenimine (PEI) (10 mM; 2.7 µl/µg DNA). Empty vector pcDNA3.1 was used to obtain equal amounts of transfected DNA. Transfection efficiencies were around 80–100% for 293T cells, as measured by green fluorescent protein (GFP) expression. UbcH8 and SIAH-1 levels were lowered by lipofectamine-mediated transfection of siRNAs directed against the UbcH8 mRNA or the SIAH-1/2 mRNAs (sc-44102) (12) as specified by Invitrogen. Irrelevant siRNAs (sc-37007, (12) served as controls. Kasumi-1 cells were transfected with lipofectamine on seven consecutive days. 293T cells were transfected once, and lysates were prepared 48–72 h later. NB-4 cells were electroporated as recommended by Amaxa (Cologne, Germany). Dominant-negative SIAH-1 was generated by site-directed mutagenesis (Stratagene) and verified by sequencing. The primers (Thermo, San Jose, CA, USA) 5'-GCCCAAAGCTCACATGTTCTCCAACTTGCCGG and 5'-CCGGCAAGTTGGAGAACTGTGGC-TTGGGC exchange C72 to S72. Apoptosis assays were performed as described (17) . 105 cells were fixed in 1 ml 70% EtOH with 0.05% Tween-20 overnight at 4°C. The following day, cells were washed with 3 ml 38 mM sodium citrate, pH 7.4, and incubated for 20 min at 37°C in 0.5 ml 38 mM sodium citrate, pH 7.4, supplemented with 50 µg/ml propidium iodide and 10 µg/ml RNase-A. DNA contents of the cells were measured by fluorescence-activated cell sorter (FACS). This method allows detection of the apoptotic subG1-fraction, which has a DNA content below 2n due to apoptotic cleavage of DNA. Equally, the cell cycle profile can be assessed with this method. Nucleosomal laddering was performed according to the method described by Yeung (23) .


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Proteasomal degradation of AML1-ETO is induced by VPA
There is increasing evidence that apart from inhibiting the catalytic activity of HDACs, HDACi can affect protein stability (9 , 12) . Because knowledge about leukemia fusion protein stability in the presence of HDACi is limited, we investigated whether the well-tolerated HDACi valproic acid affects the stability of endogenous AML1-ETO in the human t(8;21)-positive AML cell line Kasumi-1. Figure 1 A shows a significant decrease of AML1-ETO at the protein level in Kasumi-1 cells treated with the therapeutically achievable concentration of 1.5 mM VPA (24 , 25) for 24 h. Compared to the leukemia fusion protein, the intact proteins AML1 and ETO were clearly less susceptible to VPA-induced degradation (Supplemental Fig. S1).


Figure 1
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  		Figure 1. VPA reduces AML1-ETO protein levels via the ubiquitin-proteasome pathway. A) Kasumi-1 AML cells were left untreated (–) or treated for 24 h with the HDACi VPA (+; 1.5 mM). Untreated 293T cells served as a control for the specificity of AML1-ETO detection. Amounts of AML1-ETO and the loading control actin were determined by Western blot analysis. Molecular weight markers are indicated on the right. B) Kasumi-1 cells were treated for 24 h with 1.5 mM VPA (+) or left untreated (–). MG-132 (MG; 10 µM) was added during the last 4 h of incubation. AML1-ETO and actin protein levels were determined by Western blot analysis. C) Kasumi-1 cells were incubated for 24 h with 1.5 mM VPA or 1 µM MG-132 (MG), or left untreated. AML1-ETO and 18 S RNA levels were determined by real-time RT-PCR. Relative AML-ETO mRNA expression levels are shown. D) Precipitation of endogenous polyubiquitylated AML1-ETO with anti-AML1-ETO antiserum and Western blot analysis with an antibody against ubiquitin. Kasumi-1 cells were left untreated (–) or treated with 1.5 mM VPA (+) for 24 h. The proteasome inhibitor MG-132 (MG; 10 µM) was added 4 h before cell harvest. Nonimmune serum (pre) served as control. E) Same as in D, except that an antiubiquitin antibody was used for immunoprecipitation and high-molecular-weight AML1-ETO-reactive proteins were detected with an AML1-ETO antibody.

Because VPA destabilizes HDAC2 via a proteasomal mechanism (9 , 12) , we investigated whether AML1-ETO degradation in Kasumi-1 cells equally depends on ubiquitin-dependent elimination by the 26 S proteasome. We found that the proteasomal inhibitors MG-132 or ALLN significantly increased the AML1-ETO level and abolished its VPA-induced degradation (Fig. 1B and data not shown). Real-time quantitative RT-PCR revealed that AML1-ETO mRNA transcripts also decreased in response to VPA (Fig. 1C ). However, MG-132 treatment also reduced the mRNA levels of AML-ETO, although its protein stability was increased. Thus, a mechanism involving enhanced proteasomal turnover is the most likely explanation for AML1-ETO degradation after VPA treatment.

We obtained direct evidence for AML1-ETO ubiquitylation when AML1-ETO immunoprecipitates were probed with an antibody against ubiquitin. This experiment showed that Kasumi-1 cells contain ubiquitylated AML1-ETO, which can be detected on proteasomal inhibition (Fig. 1D ). Thus, a basal level of AML1-ETO ubiquitylation occurs independently of VPA-treatment. Cotreatment with VPA and MG-132 led to the occurrence of dramatically increased amounts of polyubiquitylated AML1-ETO.

To confirm these observations, we probed antiubiquitin immunoprecipitates with an antibody against AML1-ETO. In this direction, AML1-ETO-reactive high-molecular-weight bands migrating more slowly than unmodified AML1-ETO were detected in Kasumi-1 cells treated with both VPA and MG-132 (Fig. 1E ). We concluded that ubiquitylation of endogenous AML1-ETO occurs in vivo and can be increased by HDACi.

UbcH8 critically regulates the degradation of AML1-ETO
Polyubiquitylation of proteins requires the concerted action of an E2-conjugase and an E3-ligase (7) . HDACi up-regulate the E2 UbcH8 at the mRNA and protein level, which results in the proteasomal degradation of HDAC2 (12) . We hypothesized that UbcH8 also regulates the turnover of AML1-ETO. Because Kasumi-1 cells undergo apoptosis in response to HDACi (see below) and have a very low DNA transfection efficiency, we transiently expressed AML1-ETO and UbcH8 in 293T cells. This approach is commonly used to investigate protein turnover (8) . Indeed, AML1-ETO expression was strongly reduced on cotransfection of UbcH8 (Fig. 2 A), whereas the ectopically expressed Gal4 protein under the control of the same promoter was unaffected (Supplemental Fig. S2). This showed that the abundance of UbcH8 determines the stability of AML1-ETO.


Figure 2
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  		Figure 2. The E2-conjugase UbcH8 induces AML1-ETO degradation. A) 293T cells were transfected with 0.4 µg of Gal-AML1-ETO and 0.2 µg UbcH8 or 0.2 µg of a myc-tagged dominant-negative RING-finger mutant form of RLIM (dnRLIM). Empty vector pcDNA3.1 was added if appropriate to achieve 1 µg DNA per 12 wells. Expression of AML1-ETO, UbcH8, actin, and RLIM were detected by Western blot analysis 48 h after transfection; the lower band represents endogenous RLIM (arrowhead: overexpressed dnRLIM). Actin protein levels were determined to verify equal loading. B) 293T cells were transfected with Gal-AML1-ETO (1 µg) and two siRNAs directed against human UbcH8 (U8) or murine Ubce8 (Ctl) as a control (40 pmol). AML1-ETO, UbcH8, and actin levels were determined 48 h later by Western blot analysis. C) Endogenous UbcH8 levels were lowered in Kasumi-1 cells by transfection of two siRNAs directed against the UbcH8 mRNA (U8) or a nonrelated mRNA (Ctl). Cells were left untreated or were incubated with 1.5 mM VPA for 24 h. AML1-ETO, UbcH8, and actin levels were determined by Western blot analysis. Apoptosis rates were measured by PI-FACS-analysis (right; subG1, apoptotic cells with a DNA content <2n). D) Kasumi-1 cells were treated for 24 h with 1.5 mM VPA or left untreated (Ctl). The occurrence of apoptosis was determined by PI-FACS-analysis (left) and by assessment of nucleosomal laddering on a 2% agarose TAE-gel (right; kb, kilobase pairs). E) Kasumi-1 cells were treated with 1.5 mM VPA, 100 nM TSA (T), 1.5 mM butyrate (B), 5 µM MS-27–275 (MS), or MG-132 (MG; 1 µM) alone or with 1.5 mM VPA for 24 h. AML1-ETO, UbcH8, RLIM, and actin levels were determined by Western blot analysis.

To obtain further proof for the role of UbcH8 in the turnover of AML1-ETO, AML1-ETO was transfected together with siRNAs specific for UbcH8 or murine Ubce8 as a nontargeting control in human 293T cells. Data obtained with this experiment confirmed that AML1-ETO is a UbcH8 target (Fig. 2B ). Next, we transfected Kasumi-1 cells with these siRNA oligonucleotides. We found that UbcH8 siRNAs specifically decreased UbcH8 and augmented AML1-ETO levels in Kasumi-1 cells. Moreover, in VPA-treated Kasumi-1 cells, the siRNAs directed against human UbcH8 significantly reduced the degradation of the leukemic fusion protein (Fig. 2C , left). These data together with those depicted in Fig. 1D, E suggest a basal as well as a VPA-induced proteasomal degradation of AML1-ETO via UbcH8.

Similar to other HDACi, VPA can induce apoptosis and cell-cycle arrest in transformed hematopoietic progenitor cells and leukemic blasts from AML patients in vitro and in vivo (24 , 26 , 27) . Kasumi-1 AML cells underwent apoptosis in response to VPA (Fig. 2D and Supplemental Fig. S3), which correlated with the induction of UbcH8 and the reduction of AML1-ETO (Fig. 2C , left, and Supplemental Fig. S3). Furthermore, depletion of UbcH8 by siRNA protected these cells from VPA-induced apoptosis (Fig. 2C , right), indicating a role of UbcH8 in the HDACi-mediated induction of apoptosis in this cell line.

UbcH8 serves as an E2 for the RLIM E3 and both act together on HDAC2 as a substrate. The HDACi trichostatin A (TSA) and MS-27–275, unlike VPA and butyrate, induce proteasomal degradation of RLIM and thus fail to induce HDAC2 degradation in 293T and F9 cells (12) . In contrast, AML1-ETO levels decreased after application of each of these HDACi to Kasumi-1 cells (Fig. 2E ). Therefore, we analyzed RLIM expression in these cells. We observed that UbcH8 levels increased and RLIM levels decreased after incubation with HDACi. The proteasomal inhibitor MG-132 prevented the reduction of RLIM, which indicates its HDACi-enhanced proteasomal degradation. Because all HDACi that we tested reduce RLIM (Fig. 2E ), it appears unlikely that this E3 is crucial for the proteasomal degradation of AML1-ETO. To test this hypothesis, we transfected dominant-negative RLIM (19) together with AML1-ETO and UbcH8. This RLIM mutant was abundantly expressed but failed to stabilize AML1-ETO (Fig. 2A ). Hence, RLIM is not critical for the degradation of AML1-ETO. Moreover, Kasumi-1 cells had stable HDAC2 levels in the presence of VPA (Figs. 2E and 3 A), which confirms that VPA blocked RLIM functions.


Figure 3
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  		Figure 3. The E3-ligase SIAH-1 induces degradation of AML1-ETO. A) Kasumi-1 cells were left untreated or incubated with 1.5 mM VPA for 2–24 h. AML1-ETO, UbcH8, SIAH-1, HDAC2, caspase-3, and actin levels were determined by Western blot analysis (fl, full length; cf, cleaved form). B) 293T cells were transfected with expression vectors for Gal-AML1-ETO (1 µg), UbcH8 (0.5 µg), SIAH-1 (0.5 µg), both UbcH8 and SIAH-1 (0.25 µg each), and empty vector pcDNA3.1 if appropriate. Whole-cell extracts for Western blot analysis were prepared 48 h later and analyzed for AML1-ETO, UbcH8, SIAH-1, caspase-3, and actin. Endogenous SIAH-1 could not be detected with the protein amount loaded. C) 293T cells were transfected with expression vectors for Gal-AML1-ETO (0.4 µg), UbcH8 (0.2 µg), dominant-negative SIAH-1C72S (dnSIAH-1; 0.05 µg), and pcDNA3.1 if appropriate. Lysates for Western blot analysis were prepared 48 h later and analyzed for the expression of AML1-ETO, UbcH8, SIAH-1, and actin. D) 293T cells were transfected with AML1-ETO (0.5 µg) and with either control siRNA (si Ctl, 40 pmol) or increasing amounts of siRNA against SIAH-1/2 (10–40 pmol). GFP expression from a cotransfected vector served as internal control. Forty-eight hours after transfection, AML1-ETO, SIAH-1, GFP and actin levels in whole-cell lysates were determined by Western blot analysis. E) 293T cells were transfected with control siRNA (Ctl) or siRNA against SIAH-1/2 (40 pmol) together with expression vectors for Gal-AML1-ETO and UbcH8. Forty-eight hours after transfection, AML1-ETO, UbcH8, SIAH-1, and actin levels were determined by Western blot analysis. F) Endogenous SIAH-1 interacts with endogenous AML1-ETO and induces polyubiquitylation in vivo. Interaction of SIAH-1 with AML1-ETO was tested by coimmunoprecipitation from Kasumi-1 and SKNO-1 cell lysates. AML1-ETO coprecipitated with the SIAH-1 antibody was detected by Western blot analysis. Control immunoprecipitations were performed with normal goat serum (pre; IgG, immunoglobulin heavy chain; arrowheads: ubiquitylated AML1-ETO).

Next, we investigated the time course of AML1-ETO degradation in Kasumi-1 cells. After a 7-h exposure to 1.5 mM VPA, AML1-ETO started to decrease (Fig. 3A ). VPA concentrations required for AML1-ETO reduction are similar to those for the inhibition of HDAC enzymatic activity, and the delayed degradation of AML1-ETO before the onset of apoptosis argues against a direct destabilization by VPA. Moreover, unlike the apoptosis-inducing compound MG-132, the pan-caspase inhibitor Z-VAD-FMK could not rescue AML1-ETO from HDACi-induced destabilization (Fig. 1B and data not shown). These observations indicate that the intermediary induction of proteins such as UbcH8 leads to the degradation of AML1-ETO (Figs. 2E and 3A) .

SIAH-1 is a crucial factor for the degradation of AML1-ETO
Previous work showed that the E3-ligase SIAH-1 is involved in proteasomal degradation of nuclear proteins (8 , 21 , 28) , and UbcH8 was identified as the E2-conjugase preferentially interacting with SIAH-1 (29) . Therefore, we analyzed whether this E3 regulates AML1-ETO stability. First, we found that in Kasumi-1 cells SIAH-1, in contrast to RLIM, remained stable in the presence of HDACi (Figs. 2E and 3A) . More important, SIAH-1 strongly reduced AML1-ETO in transient transfection experiments (Fig. 3B ), and a dominant-negative SIAH-1 molecule (SIAH-1C72S) prevented AML1-ETO degradation by UbcH8 (Fig. 3C ). To challenge the role of SIAH-1 in the turnover of AML1-ETO, we transfected siRNA oligonucleotides against SIAH-1/2 together with AML1-ETO into 293T cells. We observed that a highly efficient knockdown of SIAH-1/2 blocked the degradation of this oncoprotein by UbcH8 and SIAH-1 (Fig. 3D, E ; compare lane 2 and lane 4 in E). The expression of a GFP construct, which is like AML1-ETO under control of a CMV promoter, served as a control for specificity. These experiments clearly indicate that equally to a reduction of UbcH8 (Fig. 2B, C ), the attenuation of SIAH-1 stabilizes AML1-ETO.

Since the transfer of ubiquitin to a target protein requires interaction with its E3-ligase, we next tested whether SIAH-1 interacts with AML1-ETO in coimmunoprecipitation experiments. Endogenous SIAH-1 was precipitated from lysates of t(8;21)-positive Kasumi-1 and SKNO-1 cells, and Western blots were probed against AML1-ETO. This approach provides clear evidence of a physical interaction between AML1-ETO and SIAH-1 (Fig. 3F ). Remarkably, AML1-ETO coprecipitated with SIAH-1 shows slower migrating bands indicative of mono- and polyubiquitylation. In vitro experiments confirmed that SIAH-1 in conjunction with UbcH8 induces polyubiquitylation of AML1-ETO (Supplemental Fig. S4). We also precipitated SIAH-1 from lysates of 293T cells transfected with SIAH-1 and AML1-ETO or PML-RAR{alpha} and confirmed the association of both proteins with SIAH-1 (Supplemental Fig. S5).

HDACi, UbcH8, and SIAH-1 determine PML-RAR{alpha} turnover
Similar to AML1-ETO in Kasumi-1 cells, PML-RAR{alpha} was degraded in t(15;17)-positive NB-4 APL cells treated with various HDACi (Fig. 4 A). HDACi attenuated this oncoprotein, with clear effects detectable after ~16 h, and this correlated with UbcH8 induction (Fig. 4B ). PML-RAR{alpha} degradation induced by HDACi hence correlates with HDAC-inhibition and the intermediary induction of protein expression. Real-time RT-PCR revealed that as AML1-ETO, PML-RAR{alpha} mRNA levels decreased in response to VPA (data not shown). In sharp contrast to PML-RAR{alpha}, levels of RAR{alpha} were unaffected, and PML was even induced by VPA (Fig. 4A ).


Figure 4
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  		Figure 4. UbcH8 and SIAH-1 induce degradation of PML-RAR{alpha}. A) NB-4 cells were left untreated (Ctl) or treated with 1.5–5 mM VPA, 100 nM TSA (T) or 1.5 mM butyrate (B) for 24 h. PML-RAR{alpha}, RAR{alpha}, PML, and actin levels were determined by Western blot analysis. B) NB-4 cells were left untreated or incubated with 1.5 mM VPA for 24 h. PML-RAR{alpha}, UbcH8, RLIM, SIAH-1, HDAC2, caspase-3, and actin levels were assessed by Western blot analysis (casp3, caspase-3; fl, full length; cf, cleaved form). C) NB-4 cells were transfected with 2 µg of UbcH8 or GFP as a control. PML-RAR{alpha}, UbcH8, RLIM, SIAH-1, and tubulin levels were analyzed by Western blot analysis. D) 293T cells were transfected with Flag-PML-RAR{alpha} (1 µg) and two siRNAs (40 pmol) directed against human UbcH8 (U8) or murine Ubce8 (Ctl). PML-RAR{alpha}, UbcH8 and actin levels were determined 48 h later by Western blot analysis. E) 293T cells were transfected with Flag-PML-RAR{alpha} (1 µg) and either UbcH8 (0.5 µg), SIAH-1 (0.5 µg), or both (0.25 µg each). Empty vector was added when appropriate. Some dishes were also transfected with a myc-tagged dominant-negative RLIM mutant (dnRLIM; 0.5 µg). Expression of PML-RAR{alpha}, dnRLIM, UbcH8, and actin were detected by Western blot analysis 48 h post-transfection. F) 293T cells were transfected with expression vectors for Flag-PML-RAR{alpha} (0.4 µg), UbcH8 (0.2 µg), dominant-negative SIAH-1C72S (0.05 µg), and pcDNA3.1 if appropriate. Extracts for Western blot analysis were prepared 48 h later and analyzed for PML-RAR{alpha}, UbcH8, SIAH-1, and actin.

To analyze whether UbcH8 affects PML-RAR{alpha} stability, we transfected NB-4 cells with UbcH8. A strong decrease of endogenous PML-RAR{alpha} occurred under these conditions (Fig. 4C ). In an independent approach, we cotransfected 293T cells with PML-RAR{alpha} and siRNAs against UbcH8. This approach showed that a knockdown of UbcH8 increased PML-RAR{alpha} levels and confirmed that equally to AML1-ETO, PML-RAR{alpha} is a target of UbcH8 (Fig. 4D ). In agreement with this, PML-RAR{alpha} was degraded by UbcH8 and SIAH-1 (Fig. 4E ). This degradation mechanism does not appear to target another leukemia fusion protein, STAT5-RAR{alpha}, since this protein remained stable in the presence of overexpressed UbcH8 and SIAH-1 (Supplemental Fig. S6). These observations indicate that SIAH-1 and UbcH8 selectively determine protein stability.

Equally to Kasumi-1 cells, VPA-treated and UbcH8-transfected NB-4 cells had stable levels of SIAH-1, though not of RLIM (Fig. 4B, C ). Experiments with the dominant-negative RLIM construct showed that analogous to AML1-ETO, PML-RAR{alpha} levels were decreased by UbcH8 and SIAH-1, but not by RLIM (Fig. 4E ). Therefore, RLIM is unlikely to be an E3-ligase for both, AML1-ETO and PML-RAR{alpha}. If RLIM is functionally inactivated in NB-4 cells treated with VPA, its target HDAC2 should remain stable in these cells (12) . Indeed, HDAC2 was unaffected by VPA in NB-4 cells (Fig. 4B ).

We then tested whether the dominant-negative SIAH-1C72S protein affects PML-RAR{alpha} and its degradation by UbcH8. This SIAH-1 mutant completely abolished the degradation of PML-RAR{alpha} by UbcH8. Similar results were obtained with siRNA against SIAH-1/2 (Fig. 4F and data not shown). We concluded that UbcH8 and SIAH-1 abundance modulate PML-RAR{alpha} turnover.

Functional and physical interaction between AML1-ETO, PML-RAR{alpha}, UbcH8, SIAH-1 and RLIM
We speculated that SIAH-1 not only targets leukemia fusion proteins but also RLIM for proteasomal degradation. To test this, 293T cells were transfected with SIAH-1 and assessed for endogenous RLIM levels. It became clear that the E3 RLIM, which is required for proper embryonic development (19) , is subject to degradation induced by SIAH-1 (Fig. 5 A). Transfection of HA-RLIM and SIAH-1 confirmed this result (Fig. 5B ). On the other hand, ectopic expression of RLIM or a dominant-negative RLIM did not alter SIAH-1 stability (data not shown). These results suggest that SIAH-1 is the E3 for RLIM, though not vice versa. Since E3-ligases have to interact with substrates to induce their ubiquitylation, immunoprecipitates of endogenous SIAH-1 were probed for the presence of RLIM. RLIM was readily detectable in these precipitates (Fig. 5C ), consistently indicating that SIAH-1 acts as an E3 for RLIM.


Figure 5
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  		Figure 5. Interplay between leukemia fusion proteins, UbcH8, RLIM, and SIAH-1. A) 293T cells were transfected with 1 µg of SIAH-1 (+) or pcDNA3.1 (–). Forty-eight hours after transfection, lysates were prepared, and RLIM, SIAH-1, and actin levels were determined by Western blot analysis. B) 293T cells were transfected with HA-RLIM (0.5 µg) and 0.5 µg of SIAH-1 or pcDNA3.1. Forty-eight hours after transfection, whole-cell lysates were prepared, and HA-RLIM, SIAH-1, and actin levels were determined by Western blot analysis. C) Endogenous SIAH-1 was precipitated from 293T whole-cell lysates, and coprecipitated endogenous RLIM was detected by Western blot analysis (pre, nonimmune serum; IP, immunoprecipitation; IgGL, immunoglobulin light chain). Molecular weight standards are indicated on the right. D) Comparison of the expression of RLIM in untreated 293T, Kasumi-1, SKNO-1 and NB-4 cells. Actin serves as a loading control. E) 293T cells were transfected with SIAH-1 (1 µg) and treated with 1 µM MG-132 (16 h). NEM-treated cell lysates were analyzed with a SIAH-1 antibody (arrowheads: ubiquitylated SIAH-1). F) 293T cells were transfected with siRNAs against mRNAs for human UbcH8 (U8) or murine Ubce8 (Ctl) as a control (40 pmol). Forty-eight hours after transfection, SIAH-1, UbcH8, and actin levels were determined by Western blot analysis.

In contrast to Kasumi-1 and NB-4 cells, 293T cells have stable RLIM levels in the presence of VPA and when UbcH8 is overexpressed (Figs. 2E and 4B and ref. 12 ). We analyzed whether this is due to a differential expression of RLIM in these cells and observed far higher RLIM amounts in 293T cells compared to hematopoietic cells (Fig. 5D ). Subsequently, we analyzed whether our previous observation that TSA, though not VPA, induces RLIM degradation correlates with a differential induction of SIAH-1 by these compounds. Indeed, a 24-h incubation with TSA but not VPA induced SIAH-1 and decreased RLIM in 293T cells (Supplemental Fig. S7 and ref. 12 ). Apparently, RLIM levels in 293T cells are too high to be significantly lowered by VPA.

SIAH-1 is highly unstable in vivo because of autoubiquitylation followed by proteasomal degradation (30 , 31) . In agreement with these findings, a SIAH-1 antibody detected unmodified SIAH-1 and incremental increases of molecular weight by 8 kDa, indicative of ubiquitylation (Fig. 5E ). As SIAH-1 preferentially interacts with UbcH8 (29) , UbcH8 may well be the E2-conjugase responsible for this process. To test this, we lowered UbcH8 levels with siRNAs and analyzed SIAH-1 expression in 293T cells. This experiment showed that the specific reduction of UbcH8 increased the amount of endogenous SIAH-1 (Fig. 5F ).

Finally, we analyzed whether leukemia fusions and proteins of the ubiquitylation machinery occur in a complex. Since UbcH8 and SIAH-1 critically determine leukemia fusion protein stability in a concerted action, these proteins should be in close proximity. We performed subcellular fractionation and immunocytochemical analysis of Kasumi-1, NB-4, and AML1-ETO- or PML-RAR{alpha}-transfected 293T cells. Our data show that in cells harboring these fusion proteins, SIAH-1 predominantly resides in the nucleus and UbcH8 is distributed between the cytoplasmic and nuclear compartment. Hence, these proteins of the ubiquitylation machinery can potentially interact with the strictly nuclear proteins AML1-ETO and PML-RAR{alpha} (Fig. 6 A and data not shown). To further prove this, recombinant GST-UbcH8 was incubated with cellular lysates and Western blots were probed for SIAH-1 and the leukemia fusion proteins. This assay clearly showed the physical interaction of these proteins with UbcH8 (Fig. 6B ). Next, UbcH8 immunoprecipitates were probed for SIAH-1, AML1-ETO, and PML-RAR{alpha}. Results obtained with this experiment indicate the presence of a complex between UbcH8, SIAH-1, and the leukemia fusion proteins in vivo (Fig. 6C ). As expected (12) , HDAC2 and RLIM were also found in these immunoprecipitates.


Figure 6
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  		Figure 6. Interaction of AML1-ETO, PML-RAR{alpha}, HDAC2, UbcH8, RLIM, and SIAH-1. A) Cytoplasmic and nuclear extracts of Kasumi-1 and NB-4 cells were analyzed for the presence of AML1-ETO, PML-RAR{alpha}, SIAH-1, and UbcH8. Tubulin served as a marker for the fractionation. 293T cells transfected with AML1-ETO or PML-RAR{alpha} were analyzed in the same way. B) Interaction of GST-UbcH8 with SIAH-1, AML1-ETO, and PML-RAR{alpha}. 293T cells were transfected with the appropriate expression constructs. Lysates of these cells were incubated with GST or GST-UbcH8. Interaction of AML1-ETO, PML-RAR{alpha}, SIAH-1, and UbcH8 was assessed by Western blot analysis (IN, input representing 10% (AML1-ETO) or 20% (SIAH-1, PML-RAR{alpha}) of lysate used for the pull-down). C) 293T cells were transfected with AML1-ETO, PML-RAR{alpha}, SIAH-1, V5-UbcH8 (5 µg each), or pcDNA3.1. UbcH8 was precipitated with a V5 antibody. Presence of AML1-ETO, PML-RAR{alpha}, SIAH-1, RLIM, HDAC2, and UbcH8 in these immunoprecipitates was analyzed by Western blot analysis (pre, nonimmune serum; IP, immunoprecipitation; IN, 10% of IP input, IgG, immunoglobulin heavy chain). D) Schematic representation of the mechanism of AML1-ETO, PML-RAR{alpha}, SIAH-1, and RLIM degradation mediated by UbcH8. Proteasomal degradation of these proteins involves the E3-ligase SIAH-1, and limiting amounts of the E2-conjugase UbcH8. UbcH8 is induced in HDACi-treated leukemia cells and can mediate oncoprotein depletion. HDAC2 degradation occurs depending on the basal abundance and stability of the ubiquitin ligase RLIM.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The chimeric fusion proteins AML1-ETO and PML-RAR{alpha} are crucial for the transformation of myeloid precursors (1 , 6) . Our study shows that the polyubiquitylation and subsequent proteasomal degradation of AML1-ETO and PML-RAR{alpha} can be increased by HDACi. Several independent lines of evidence obtained from ectopic expression, dominant-negative molecules, siRNA experiments, and coimmunoprecipitation approaches coherently indicate that this process is mediated by the E2-conjugase UbcH8 and the E3-ligase SIAH-1. Hence, the leukemia fusion proteins AML1-ETO and PML-RAR{alpha} represent key targets of the E2 UbcH8, the E3 SIAH-1 and HDACi. Moreover, we also identified UbcH8 as an E2 mediating autoubiquitylation of SIAH-1, and SIAH-1 as the E3 for RLIM in a previously undescribed hierarchical order (Fig. 6D ).

HDACi appear to act as inhibitors of leukemia fusion proteins indirectly via the induction of their degradation and directly by inhibiting the catalytic activity of their associated HDACs. The HDACi-induced proteasomal degradation of AML1-ETO and PML-RAR{alpha} may be particularly relevant, since they compete as oligomers with the remaining intact transcription factors for proper hematopoiesis (1 , 6 , 32) , which prevents the expression of their target genes in response to appropriate stimuli. Furthermore, AML1-ETO directly binds, sequesters, and competes with further transcriptional regulators of hematopoietic differentiation, e.g., c-Jun, PLZF, C/EBP, SMAD3, and VDR (1) ; PML-RAR{alpha} interferes with C/EBP transcription factors and cell cycle restricting proteins (6) . Since the expression of leukemia fusion proteins is highly restricted to leukemic cells, the degradation of such proteins may become a molecularly defined, selective intervention strategy.

Our results show that the well-tolerated drug VPA (24) induces leukemia fusion protein degradation. This HDACi might therefore be particularly suited for leukemia therapy. However, the screening of additional parameters such as the degradation of HDAC2 and RLIM or the induction of Stat1 (10, 12–14, 17) is required to identify patients with cancer traits susceptible to HDACi. Greater benefits may equally be achieved when VPA is used in conjunction with drugs targeting DNA-replication (33 , 34) . Moreover, several newly developed HDACi currently undergo clinical trials. Remarkably, Depsipeptide has been shown to be beneficial for patients with t(8;21) leukemia, and this HDACi induces AML1-ETO degradation (35) . These reports confirm and support our results and conclusions. Our data additionally extend these observations to PML-RAR{alpha} and provide detailed information on the enzymology of these processes.

Since HDACi also induce nonhistone protein acetylation, the stability of leukemia fusion proteins may also be influenced by their acetylation status or via Hsp90 acetylation (36) . However, VPA is a class 1-selective HDACi, which does not inhibit HDAC6 (24 , 37) , the class 2 deacetylase targeting Hsp90 (38 , 39) . It is therefore not surprising that several attempts failed to detect Hsp90 acetylation with three antiacetyl-lysine antibodies in VPA-treated cells at various time points and concentrations. Similarly, AML1-ETO was not detectable with antiacetyl-lysine antibodies (ref. 35 and data not shown). Furthermore, the siRNA-mediated knockdown of Ubc8 prevented the degradation of endogenous AML1-ETO, even in the presence of HDACi (Fig. 2C ). On the other hand, leukemia fusion proteins were degraded under conditions that do not inhibit HDAC activity, such as UbcH8 or SIAH-1 overexpression (Figs. 2A ; 3B, E ; 4C, E and ref. 8 ). Still, Hsp90 acetylation may be below the detection limit and could be masked by its HDACi-induced cleavage (Supplemental Fig. S8). Nevertheless, acetylation of Hsp90 is unlikely to be a prerequisite for the degradation of AML1-ETO and PML-RAR{alpha} in the context of our experiments. Consistent with this assumption, we observed that Hsp90-bound AML1-ETO is not protected but also degraded on HDAC-inhibition (Supplemental Fig. S8). Moreover, a knockdown of HDAC6 induces Hsp90 acetylation and impairs its function but hardly affects the stability of the leukemia fusion protein Bcr-Abl (38) . This study also implies that limiting amounts of enzymes of the ubiquitylation machinery determine oncoprotein degradation.

As VPA induces apoptosis in Kasumi-1 and NB-4 cells (Figs. 2C, D ; 3A ; 4B and Supplemental Figs. S3 and S10), caspases could equally cleave AML1-ETO and PML-RAR{alpha}. However, HDACi trigger the degradation of these proteins before the onset of caspase cleavage, a pan-caspase inhibitor did not block the HDACi-induced decrease of AML1-ETO levels, and no cleavage products typical for caspase-induced degradation were observed. In contrast, MG-132 is a strong proapoptotic stimulus, which stabilized the AML1-ETO protein (Figs. 1B and 2E and data not shown). Furthermore, UbcH8 and SIAH-1 did not induce apoptosis in 293T cells, although they destabilized AML1-ETO and PML-RAR{alpha} (Figs. 3A, B and 4E) , and MG-132 blocked this degradation (data not shown). Nevertheless, it is possible that caspases cleave leukemia fusion proteins at late stages of HDACi treatment.

HDACi counteract transcriptional repression and also selectively affect protein stability, but only limited knowledge exists about the E2 and E3 enzymes involved in HDACi-induced ubiquitylation (9) . UbcH8 and RLIM appear to be crucial for at least a subset of these processes, and we could confirm our initial observation that HDACi induce UbcH8 expression in several different cell lines (Figs. 2E and 4B , data not shown, and ref. 12 ). The E3 RLIM together with UbcH8 induces proteasomal degradation of HDAC2 (12) and high HDAC2 levels correlate with certain neoplastic malignancies. Nevertheless, RLIM does not contribute to AML1-ETO and PML-RAR{alpha} degradation (Figs. 2A, E ; 3A ; and 4B, E ), and RLIM is expressed at much lower levels in Kasumi-1, NB-4, and SKNO-1 cells compared to 293T cells (Fig. 5D ). Apparently, an increased expression of UbcH8 on VPA treatment suffices to trigger the proteasomal degradation of such low RLIM levels in hematopoietic cells via the E3 SIAH-1. As expected from these results, VPA does not induce proteasomal degradation of the RLIM target HDAC2 in Kasumi-1 and NB-4 cells (Figs. 3A and 4B) (12) . Hence, the molecular mechanism through which UbcH8 controls protein degradation depends on the abundance of its E3-ligases and not on HDACi treatment per se. On the basis of these data, we propose that a hierarchical ubiquitylation system modulates E3-ligases via cross-regulation. This, in turn, leads to the degradation of different substrates, including E3 enzymes themselves.

E3 enzymes typically target multiple substrates, and therefore proteins in addition to RLIM, HDAC2, PML-RAR{alpha}, and AML-ETO could be subject to VPA-induced proteasomal degradation in a cell-type-specific manner. Furthermore, RLIM and SIAH-1 may not be the only E3-ligases downstream of the E2-conjugase Ubc8, and such pleiotropic effects of an E2 may explain why a knockdown of UbcH8 protects Kasumi-1 cells from VPA-induced apoptosis (Fig. 2C ). Such complex interplays between ubiquitin E2-conjugases and different E3-ligases may control multiple regulatory networks in health, disease, and development.

In contrast to RLIM, SIAH-1 appears to bind and directly control the proteasomal turnover of AML1-ETO and PML-RAR{alpha} (Figs. 3 and 4E, F ). This is in agreement with the presence of consensus motifs for SIAH-1 binding in these proteins (8 , 40) . Four of these motifs are clustered in the AML1-ETO fusion protein. This fact could explain why the intact AML1 and ETO proteins, each containing only two SIAH-1 binding sites, are far less susceptible to VPA-induced degradation (Supplemental Fig. S1). A similar situation occurs in PML-RAR{alpha} (Fig. 4A ), where the SIAH-1 binding sites of RAR{alpha} are brought into close proximity of the PML coiled coil, which also recruits this E3-ligase (12) . Neither the clustering of SIAH-1 binding sites nor a PML-type coiled coil exist in STAT5-RAR{alpha}. Accordingly, this fusion protein is not degraded via UbcH8 and SIAH-1 (Supplemental Fig. S6).

All of the cell lines that we used endogenously express UbcH8 and SIAH-1, which are detectable on loading of high amounts of protein, immunoprecipitation, or subcellular fractionation (Figs. 3D, E ; 5 ; and 6A and Supplemental Fig. S9 for comparison). Previously published data indicating that certain cell lines are SIAH-1 negative may be due to lower detection sensitivities of earlier antibody generations. Difficulties in detecting SIAH-1 also reflect its high turnover (30 , 31) . Our experiments with UbcH8 siRNAs indicate that SIAH-1 stability depends on its E2 UbcH8 (Fig. 5E, F ). Nevertheless, UbcH8 induction was not sufficient to cause SIAH-1 degradation (Figs. 2E , 3A , and 4B, C and Supplemental Fig. S7). This discrepancy could be explained by a model in which the stability of SIAH-1 depends on the abundance of substrates to which ubiquitin can be transferred. Such a model of an E3 autoinactivation has been proposed before and is consistent with much higher expression rates of enzymatically inactive SIAH-1 RING-finger mutants (28 , 30 , 41) .

Considering our results on the role of the UbcH8-SIAH-1 axis in leukemia fusion protein degradation, this pathway could be shared by the basal and the HDACi-induced proteasomal degradation of oncoproteins (30 , 36 , 42) . Future studies are required to determine whether UbcH8 is a tumor suppressor similar to SIAH-1 (43) . The dependence of transformed cells on oncoproteins and their HDACi-induced degradation could be one reason why these compounds are more toxic to leukemic cells than to normal cells (24) . Further analyses are also required to clarify whether HDACi impose post-translational modifications other than ubiquitylation on leukemia fusion proteins and whether they affect protein stability. Remarkably, it was reported that a truncated variant of AML1-ETO that does not interact with corepressors promotes leukemia development (44) . Similarly, transformation by PML-RAR{alpha} depends on its cleavage by neutrophil elastase (45) . One could thus speculate that the benefits of HDACi in leukemia treatment rely on the elimination of leukemia fusion proteins as well as on HDAC inhibition. Regulation of such processes by HDACi is likely to be relevant for the activity of these drugs in cultured cells, animal models, and patients (10 , 12 , 14 , 16 , 42) .


   ACKNOWLEDGMENTS
 
We thank A. Schimpf and G. Greiner for excellent technical assistance and M. Grez, R. Marschalek, C. Wichmann, and E. Jandt for helpful discussion. S. Drube and O. Rudeschko kindly helped with FACS, T. Kamradt and B. Groner provided access to FACS facilities. S. Knauer and R. Stauber generously helped with immunocytochemical analyses and provided access to microscopical facilities. K. Volling and H.-P. Saluz kindly provided advice for nucleosomal laddering assays. Expression constructs and antibodies were generously provided by Manuel Grez, Georg-Speyer-Haus, Frankfurt, Germany (AML1-ETO; STAT5-RAR); Rolf Marschalek, University of Frankfurt, Frankfurt, Germany (SIAH-1); Saverio Minucci, European Institute of Oncology, Milan, Italy (PML-RAR); Robert M. Krug, University of Texas, Austin, TX, USA (pGEX4T1-UbcH8); Frank Böhmer, University of Jena, Jena, Germany (pGEX4T1); Hinrich Gronemeyer, IGBMC, Illkirch, France (PML-RAR{alpha} antibody); and Ingolf Bach, University of Massachusetts, Worcester, MA, USA (RLIM constructs and antibody). This work was supported by the Deutsche Forschungsgemeinschaft (SFB 604) and a grant of the German National Genome Research Network to T.H. (N1KR-S31T30).

Received for publication January 12, 2007. Accepted for publication November 8, 2007.


   REFERENCES
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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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