Blockade of ubiquitin-conjugating enzyme CDC34 enhances anti-myeloma activity of Bortezomib/Proteasome inhibitor PS-341

Abstract

The ubiquitin-conjugating enzyme CDC34 (UBC3) is linked to cell cycle progression in diverse cell types; however, its role in multiple myeloma (MM) pathogenesis is unclear. Here, we show that CDC34 is highly expressed in patient MM cells and MM cell lines versus normal cells. Blocking CDC34 using a dominant-negative strategy enhances the anti-MM activity of Bortezomib/Proteasome inhibitor PS-341, dexamethasone (Dex) and 2-Methoxyestradiol (2ME2). The expression of wild-type CDC34 reduces Dex-induced cytotoxicity in MM cells. Moreover, inhibition of CDC34 enzymatic activity abrogates interleukin-6-induced protection against Dex-induced apoptosis. Together, these findings provide evidence that (1) CDC34 expression is associated with growth and survival of MM cells and (2) blocking CDC34 activity not only enhances anti-MM activity of Bortezomib and 2ME2 but also overcomes IL-6-triggered Dex-resistance.

Main

Protein ubiquitination and degradation regulate cell cycle progression from the G1 to S phase via the ubiquitin-conjugating enzyme (UBC3)/CDC34 (Verma et al., 1997; Yew and Kirschner, 1997); conversely, mutations in CDC34 gene result in defective transition from the G1 to S phase (Goebl et al., 1988). CDC34 has been implicated in the ubiquitination of p27 (Kip1), I-kappa B-alpha, Wee1, and MyoD (Block et al., 2001), thereby facilitating the degradation of these proteins by 26S proteasomes. Thus, CDC34 modulates cell cycle progression by triggering ubiquitination-dependent protein degradation.

A recent report showed a high expression of CDC34 in pediatric T-cell acute lymphoblastic leukemia (Eliseeva et al., 2001) and hepatocellular carcinoma (Tanaka et al., 2001); however, the mechanism regulating CDC34 in cancer cells has not been characterized. Another study using oligonucleotide arrays demonstrated that CDC34 mRNA is highly expressed in malignant versus normal plasma cells (De Vos et al., 2002). To date, however, the functional significance of CDC34 in multiple myeloma (MM) cells also remains undefined. In the present study, we therefore asked whether (1) CDC34 is differentially expressed in MM versus normal plasma cells; (2) CDC34 expression affects sensitivity to various conventional and novel anti-MM agents, and (3) CDC34 has a role during interleukin-6 (IL-6)-mediated protection against Dex-induced apoptosis.

We first determined whether CDC34 is differentially expressed in MM versus normal cells. Total cellular RNA prepared from purified patient MM and normal cells was subjected to oligonucleotide microarray profiling, followed by data analysis using DNA Chip Analyser (DChip) (Chauhan et al., 2003a). As shown in Figure 1a, CDC34 transcripts were highly expressed in MM cells versus normal cells (three- to four-fold higher; P<0.005, n=3). We next determined whether differences in the CDC34 mRNA levels correlate with alterations in CDC34 protein expression. Total protein extracts from purified patient MM cells and normal cells were subjected to immunoblotting with anti-CDC34 Abs. As seen in Figure 1b (upper panel), MM cells express significantly higher CDC34 protein levels than normal cells. Reprobing the blots with anti-tubulin Ab showed equal protein loading in each lane (Figure 1b, lower panel). Alterations in CDC34 protein levels therefore correlate with changes in mRNA levels. These findings are consistent with a previous study suggesting that CDC34 is upregulated in malignant versus normal plasma cells (De Vos et al., 2002). Moreover, higher levels of CDC34 transcripts have also been reported in acute lymphoblastic leukemia and hepatocellular carcinomas (Tanaka et al., 2001). Together, these findings suggest that CDC34 may regulate the growth of tumor cells.

Figure 1
figure1

(a) Primary cells were freshly isolated from MM patients and normal donors, as in our prior study (Chauhan et al., 2003b). Tumor cells (CD138+ 97±2.0%) were then isolated by CD138 positive selection (Chauhan et al., 2002) using CD138 (Syndecan-(1) Micro Beads and the Auto MACS magnetic cell sorter, according to the manufacturer's instructions (Miltenyi Biotec Inc., Auburn, CA, USA). Total cellular RNA from patient MM and normal plasma cells was subjected to oligonucleotide array (Affymetrix U133A), as previously described (Chauhan et al., 2003a). Data shown are a graphical representation of microarray analysis. The .CEL (cell intensity) files were obtained using Affymetrix Microarray Suite 5.0 software. The DNA-Chip (DChip) is used to normalize all CEL files to a baseline array with overall median intensity, and the model-based expression (PM/MM difference model) is used to compute expression values. The relative fold difference indicates an average difference in the ratio of gene expression in normal versus patient cells (*Confidence interval of 95%). (b) Protein lysates from normal and MM cells were separated by 12.5% SDS–PAGE and analysed by immunoblotting (IB) with anti-CDC34 (upper panel) or antitubulin (lower panels) Abs. Blots are representative of three independent experiments with similar results. (c) MM.1S cells were cultured as previously described (Chauhan et al., 2002; Moalli et al., 1992), and treated with Bortezomib (3 nM) (Millenium Pharmaceuticals, Cambridge, MA, USA), Dex (0.5 μ M), or 2ME2 (3 μ M) (Sigma Chemical Co., St Louis, MO, USA) for 48 h. Protein extracts were subjected to IB with anti-CDC34 (upper panel) and anti-SHP2 (lower panel) Abs (Santa Cruz Biotechnology, CA, USA). Blots are representative of three independent experiments with similar results. Densitometric analysis was performed and CDC34 signal was normalized against an irrelevant protein (SHP2) signal. The mean densitometric value from three independent experiments was converted into fold change relative to control/untreated cells (P<0.05). (d) MM.1S cells were treated with Bortezomib (3 nM), Dex (0.5 μ M), or 2ME2 (3 μ M) for 48 h, and analysed for apoptosis by Annexin V staining, as previously described (Li et al., 1999). Results are mean±s.d. from three independent experiments. The error bars indicate the standard error of the mean. Asterisk (*) indicates significant difference (P<0.005) observed between control (DMSO) and drug-treated MM.1S cells

We next determined whether CDC34 expression is altered during treatment of MM cells with novel anti-MM agents Bortezomib or 2-Methoxyestradiol (2ME2), and with conventional agent dexamethasone (Dex). MM.1S cells were treated with Bortezomib (3 nM), Dex (0.5 μ M), or 2ME2 (3 μ M) for 48 h, and protein extracts were subjected to immunoblotting with anti-CDC34 Abs. As seen in Figure 1c (upper panel), both Bortezomib and 2ME2, but not Dex, significantly (four- to five-fold) decrease CDC34 protein expression, without any alterations in the levels of an irrelevant protein SHP2 (Figure 1c, lower panel). Densitometric analysis was performed on CDC34 normalized against an irrelevant protein (SHP2). The mean of densitometric values from three different experiments was converted into fold change relative to control/untreated cells (P<0.05).

To determine whether Bortezomib, Dex, or 2ME2 triggers apoptosis, MM.1S cells were treated with these agents and analysed for apoptosis by Annexin V staining. As seen in Figure 1d, Bortezomib, Dex, and 2ME2 trigger an increase in the percentage of Annexin V positive cells compared to untreated MM.1S cells, as in our prior studies (Chauhan et al., 2002, 2003c). Importantly, all three agents induce similar degrees of apoptosis (Figure 1d); however, only Bortezomib or 2ME2 triggers decreased CDC34 protein levels, whereas Dex does not. These data exclude the possibility that Bortezomib or 2ME2-induced decreases in CDC34 protein levels are due to increased apoptosis. Together, our findings suggest that downregulation of CDC34 correlates with Bortezomib- and 2ME2-induced apoptosis in MM cells.

We next directly examined whether inhibition of enzymatic activity of CDC34 affects responsiveness to these apoptotic agents. We used the dominant-negative mutant of CDC34 (CL>S), which inhibits the initiation of DNA replication in Xenopus interphase egg extracts (Yew and Kirschner, 1997). MM.1S cells were transiently cotransfected with vector containing GFP alone and either dominant-negative CDC34 (DN-CDC34) or vector alone; GFP-positive cells were sorted by flow cytometry, treated with Bortezomib (3 nM), Dex (0.5 μ M), or 2ME2 (3 μ M) for 48 h, and analysed for cell viability using MTT assays. As seen in Figure 2a, all the three agents significantly decreased cell viability in DN-CDC34-transfected cells compared to vector-transfected cells: the median cell viability after Bortezomib treatment was 45% in vector versus 17% in DN-CDC34-transfected cells; after Dex treatment was 48% in vector versus 19% in DN-CDC34-transfected cells; and after 2ME2 treatment was 51% in vector versus 16% in DN-CDC34-transfected cells (P=0.04, as determined by one-sided Wilcoxon's rank-sum test).

Figure 2
figure2

Blockade of CDC34 by DN-CDC34 enhances anti-MM activity of Bortezomib, Dex, and 2ME2 in MM.1S cells (a) MM.1S cells were transiently cotransfected with GFP and either vector alone or DN-CDC34 using ‘Cell line NucleofectoTM kit V’, according to the manufacturer's instructions (Amaxa Biosystems, Cologne, Germany), as previously described. Following transfections, GFP-positive cells were selected by flow cytometry; treated with Bortezomib (3 nM), Dex (0.5 μ M), or 2ME2 (3 μ M) for 48h; and analysed for cell viability (Hideshima et al., 2000) by MTT assay (P=0.04, as determined by one-sided Wilcoxon's rank-sum test). (b) MM.1S cells were transiently cotransfected as above and analysed for apoptosis by dual staining with PI and HO (percent apoptotic cells: PI− and HO+ cells), as previously described (Chauhan et al., 1999). Results are mean±s.d. from three independent experiments, P<0.005

We next examined whether blocking CDC34 enhances Bortezomib-, Dex-, or 2ME2-induced apoptosis in MM.1S cells. As seen in Figure 2b, all three agents trigger significantly greater apoptosis in DN-CDC34-transfected compared to vector-transfected cells, evidenced by marked increases in PI− and HO+ cells. Transfection of MM.1S cells with DN-CDC34 alone induces only 10–12% apoptosis. Taken together, these findings demonstrate that inhibition of CDC34 enhances the anti-MM activity of both conventional (Dex) and novel (Bortezomib, 2ME2) agents.

As inhibition of CDC34 increases the sensitivity of MM.1S cells to Bortezomib, Dex, or 2ME2, we next asked whether exogenous expression of WT-CDC34 would abrogate apoptosis triggered by these agents. As seen in Figure 3a, Dex-induced proteolytic cleavage of PARP is markedly inhibited in the WT-CDC34-tranfected cells versus vector-transfected cells, whereas Bortezomib or 2ME2-triggered PARP cleavage is only modestly affected by WT-CDC34 expression. PARP cleavage is a hallmark of apoptosis and activation of proteases (Kaufmann et al., 1993). Apoptosis was also quantified using Annexin V staining assays. As seen in Figure 3a (lower panel), ectopic expression of WT-CDC34 significantly abrogates Dex-induced apoptosis compared to vector-transfected cells (median % apoptosis: Dex+vector alone=55% versus Dex+WT-CDC34=13%, P<0.005). Furthermore, Bortezomib or 2ME2-triggered apoptosis is also inhibited by WT-CDC34 expression (median % Annexin V+ cells: Bortezomib+Vector=68% versus Bortezomib+WT-CDC34=45%; 2ME2+Vector=55% versus 2ME2+WT-CDC34=39%, P<0.005).

Figure 3
figure3

Expression of WT-CDC34 abrogates Dex-, but not Bortezomib- or 2ME2-triggered cell death. (a) MM.1S cells were transiently cotransfected with GFP and either vector alone or WT-CDC34. GFP-positive cells were selected by flow cytometry; treated with Bortezomib (3 nM), Dex (0.5 μ M), or 2ME2 (3 μ M) for 48h; and analysed for apoptosis by PARP cleavage (upper panel). Protein extracts were subjected to immunoblot analysis with C-2-10 anti-PARP monoclonal antibody (Desnoyers et al., 1994). Blots are representative of three independent experiments with similar results. FL: full length, CF: cleaved fragment. Cells were also analysed for apoptosis by Annexin V staining (lower panel). Results are mean±s.d. from three independent experiments, P<0.005. (b) Expression of WT-CDC34 in Dex-sensitive MM.1S cells increases resistance to Dex. MM.1S cells were transiently cotransfected with GFP and vector alone or WT-CDC34 or DN-CDC34. GFP-positive cells were selected by flow cytometry; treated with Dex (0.5 μ M) for 24, 48, and 72 h; and analysed for cell viability by MTT assay (*P<0.004)

We next examined the effect of Dex, Bortezomib, or 2ME2 on the viability of WT-CDC34-transfected cells by MTT assays. As seen in Figure 3b, WT-CDC34-transfected cells survive significantly longer after treatment with Dex than control vector-transfected MM.1S cells (P<0.004). In contrast, transient transfection with DN-CDC34 significantly enhances Dex-triggered decreases in cell viability (Figure 3b). WT-CDC34 or vector-transfected cells were also treated with Bortezomib or 2ME2 for 48 h, and analysed for viability by MTT assays. The expression of WT-CDC34 increased survival of Bortezomib or 2ME2-treated cells (median viability: Bortezomib+Vector=32% versus Bortezomib+WT-CDC34=55%; 2ME2+Vector=42% versus 2ME2+WT-CDC34=61% apoptosis, P<0.005) (data not shown). Therefore, the expression of WT-CDC34 conferred protection against Bortezomib or 2ME2, but to a lesser extent than against Dex-induced cytotoxicity (median viability: Dex+vector alone=45% versus Dex+WT-CDC34=11%, P<0.005; Figure 3b). Our results are consistent with a recent study showing that CDC34 can confer resistance to methlymercury in human cells (Hwang et al., 2002). Together, these data demonstrate that (1) CDC34 confers Dex-resistance in MM cells and, conversely, inhibition of CDC34 can enhance sensitivity to Dex; (2) ectopic expression of CDC34 also decreased the anti-MM activity of Bortezomib and 2ME2; and (3) inhibition of CDC34 increases sensitivity of tumor cells to these agents.

We and others have shown that IL-6 is a major growth factor for MM cells and protects against Dex-induced apoptosis (Hardin et al., 1994; Chauhan et al., 1996, 1997, 2000, 2001; Chen et al., 1996; Rowley et al., 2000). We therefore next determined whether IL-6-induced growth in MM cells affects CDC34 expression. MM.1S cells were treated with IL-6 (10 ng/ml) for 24, 48, and 72 h; cells were then harvested and analysed for both cell growth and alterations in the CDC34 expression. As in our prior studies (Chauhan et al., 1996, 2000), IL-6 triggers a 2.3-fold increase (P<0.005) in the growth of MM.1S cells. Total protein extracts from IL-6-treated MM.1S cells were subjected to immunoblot analysis with anti-CDC34 and anti-tubulin Abs. As seen in Figure 4a (upper panel), IL-6 induces CDC34 protein expression in MM.1S cells, without any similar changes in tubulin protein levels (lower panel).

Figure 4
figure4

(a) Interleukin-6 (IL-6) induces CDC34 expression. MM.1S cells were treated with IL-6 (10 ng/ml) and harvested at 24, 48, and 72 h. Proteins were separated by 12.5% SDS–PAGE and analysed by IB with anti-CDC34 (upper panel) or antitubulin (lower panel) Abs. Blots are representative of three independent experiments with similar results. (b) Inhibition of CDC34 abrogates IL-6-mediated protection against Dex-induced apoptosis. MM.1S cells were transiently cotransfected with GFP and either vector alone or DN-CDC34. GFP-positive cells were selected by flow cytometry, treated with Dex in the presence or absence of IL-6 for 48 h, and analysed for apoptosis by DNA fragmentation assay. Results are mean±s.d. from three independent experiments, P<0.005

Since IL-6 also protects against Dex-induced apoptosis in MM cells (Hardin et al., 1994; Chen et al., 1996; Chauhan et al., 1997a, 2001; Rowley et al., 2000), we next asked whether IL-6 similarly prevents Dex-induced apoptosis in DN-CDC34-transfected MM.1S cells. MM.1S cells were cotransfected with GFP and either DN-CDC34 or vector alone; treated with Dex (0.5 μ M) for 48 h in the presence or absence of IL-6 (10 ng/ml); and analysed for apoptosis by DNA fragmentation assay. As seen in Figure 4b, IL-6 prevents Dex-induced apoptosis in control vector-transfected cells, but it fails to block Dex-triggered cell death in DN-CDC34-transfected cells. These data show that DN-CDC34 not only enhances Dex-induced apoptosis but also inhibits the protective effects of IL-6 against Dex-induced apoptosis. Additionally and as expected, the presence of IL-6 did not alter Bortezomib- or 2ME2-triggered lethality in DN-CDC34-transfected cells (data not shown). Together, these findings suggest that (1) DN-CDC34 not only enhances Dex-induced apoptosis but also blocks IL-6-induced protection against Dex-induced apoptosis, and (2) CDC34 mediates, at least in part, IL-6-triggered growth and antiapoptotic signaling in MM cells.

In summary, our results demonstrate the following: (1) CDC34 is highly expressed at both the mRNA and protein level in MM versus normal cells; (2) apoptosis induced by conventional or novel anti-MM agents is associated with decreased CDC34 expression; (3) blocking CDC34 activity using a dominant-negative strategy enhances sensitivity to Bortezomib, Dex, and 2ME2, (4) expression of WT-CDC34 prevents Dex-induced cell death; (5) IL-6-induced growth in MM cells is associated with increased CDC34 expression; and (6) blocking CDC34 activity abrogates IL-6-mediated protection against Dex-induced apoptosis. Our results therefore suggest that an antisense strategy targeting CDC34 may be useful to enhance sensitivity of MM cells to conventional, as well as novel, therapies.

Abbreviations

MM:

multiple myeloma

2ME2:

2-Methoxyestradiol

Dex:

dexamethasone

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Acknowledgements

This investigation was supported by NIH Grants RO-1 CA 50947, PO-1 CA 78373, and IP50CA10070, a Doris Duke Distinguished Clinical Research Scientist Award (KCA), The Myeloma Research Fund, and The Cure Myeloma Fund.

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Correspondence to Kenneth C Anderson.

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Keywords

  • Bortezomib/PS-341
  • multiple myeloma
  • apoptosis
  • 2-Methoxyestradiol
  • dexamethasone

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