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Induction of Cell Cycle Arrest and Apoptosis by the Proteasome Inhibitor PS-341 in Hodgkin Disease Cell Lines Is Independent of Inhibitor of Nuclear Factor-B Mutations or Activation of the CD30, CD40, and RANK Receptors

Departments of Lymphoma/Myeloma, Bioimmunotherapy, and Cancer Biology, M. D. Anderson Cancer Center, Houston, Texas

	ABSTRACT

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Purpose: The malignant Hodgkin and Reed-Sternberg cells of Hodgkin disease (HD) are known to constitutively express high levels of activated nuclear factor B (NF-B), which plays an important role in their survival. The proteasome inhibitor PS-341 has been recently shown to modulate tumor cell proliferation and survival by inhibiting NF-B and modulating critical cellular regulatory proteins, but its activity in cells carrying IB gene mutations has not been reported previously.

Experimental Design: The activity of PS-341 in four well-characterized, HD-derived cell lines. Cell proliferation and apoptosis were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) and Annexin-V binding methods, respectively. Cell cycle analysis was determined by flow cytometry. Intracellular protein levels were determined by Western blot.

Results: PS-341 demonstrated a strong antiproliferative activity, which was irrespective of the status of mutations in IB and even the presence of CD30, CD40, or RANK receptor activation. This effect was attributable to the induction of apoptosis and cell cycle arrest at the G₂-M phase. PS-341 not only inhibited nuclear localization of NF-B but also activated the caspase cascade, increased p21 and Bax levels, and decreased Bcl-2 levels. Furthermore, PS-341 enhanced the effect of gemcitabine chemotherapy and potentiated the effect of tumor necrosis factor-related apoptosis-inducing ligand/APO2L and two agonistic antibodies to tumor necrosis factor-related apoptosis-inducing ligand death receptors R1 and R2.

Conclusions: The in vitro activity of PS-341 against HD-derived cell lines suggests that PS-341 may have a therapeutic value for the treatment of HD.

	INTRODUCTION

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Nuclear factor B (NF-B) plays a central role in regulating the expression of various genes involved in cell survival, apoptosis, carcinogenesis, and inflammation, making it a potential therapeutic target (1 , 2) . The NF-B family of proteins (including p50/p105, p52/p100, p65, RelB, and c-Rel) exists in homodimers and heterodimers. In mammalian cells, the dominant responsive form is the p50/p65 heterodimer. In unstimulated cells, NF-B is present in the cytoplasm in an inactive form, bound to inhibitors of NF-B (IB, IBß, and IB). On activation, IB is rapidly phosphorylated and ubiquinated and subsequently degraded by the proteasome pathway. Consequently, the active p50/p65 heterodimer is translocated to the nucleus and binds to a specific DNA sequence to induce gene transcription (3) .

The malignant Hodgkin/Reed-Sternberg (H/RS) cells of Hodgkin disease (HD) aberrantly express the activated p50/p65 (RelA) heterodimer form of NF-B (4, 5, 6, 7) . This expression is observed in HD cell lines and the vast majority of primary H/RS cells in HD lymph node sections (7) . Several mechanisms have been implicated in the aberrant activation of NF-B in H/RS cells, including genetic defects in the IB gene, Epstein-Barr viral infection, and stimulation by cytokines (8, 9, 10, 11, 12, 13) . Inhibition of NF-B activation by a dominant-negative IB in HD-derived cell lines has been shown to interfere with cell cycle progression and inhibit proliferation in vitro and in severe combined immunodeficient mice (7) . Thus, the pharmacological inhibition of this pathway may prove to be of clinical value for the treatment of patients with HD.

PS-341 (Velcade, bortezomib) is a small molecule inhibitor of the ubiquitin-proteasome pathway. Proteasome plays an essential role in the degradation of most intracellular proteins, including those that regulate the cell cycle, cell survival and apoptosis, cell adhesion and trafficking, and transcription factor activation (14) . By inhibiting the degradation of cytoplasmic inhibitor of nuclear factor-B (IB), PS-341 inhibits the activation of NF-B. Furthermore, PS-341 has been reported to alter the levels of p21, p27, Bcl-2, Bax, XIAP, survivin, and p53, leading to cell cycle arrest and apoptosis in several tumor types (15) . PS-341 has also been shown to enhance tumor cell sensitivity to chemotherapy (16, 17, 18) . Because of these favorable antitumor properties, PS-341 was rapidly developed to treat human cancer (15 , 19, 20, 21) .

In this study, we investigated the activity of PS-341 in HD-derived cell lines and determined the molecular mechanisms for its biological activity

	MATERIALS AND METHODS

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Cell Lines and Cell Culture.
The human HD-derived cell lines HD-MYZ, HD-LM2, L-428, and KM-H2 were obtained from the German Collection of Microorganisms and Cell Cultures, Department of Human and Animal Cell Cultures (Braunschweig, Germany). Two cell lines are known to have mutations in the IB gene (L-428 and KMH-2). The phenotypes and genotypes of these cell lines have been published previously (22) . All cell lines were cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD), L-glutamine, and penicillin/streptomycin in a humid environment of 5% CO₂ at 37°C.

Reagents, Antibodies, and Recombinant Proteins.
PS-341 was kindly provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). Recombinant human RANK ligand (RANKL, TRANCE), CD40 ligand (CD40L, CD154), and CD30 ligand (CD30L, CD153) and the pan-caspase inhibitor Z-VAD-FMK were obtained from Alexis Corp. (San Diego, CA). Recombinant human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/APO2L) trimer was kindly provided by Dr. Avi Ashkenazi (Genentech Corp., South San Francisco, CA). Agonistic monoclonal antibodies to the TRAIL death receptors R1 (ETR1) and R2 (ETR2) were provided by Human Genome Sciences, Inc. (Rockville, MD). Antibodies to extracellular signal-regulated kinase (ERK)1/2 and phosphorylated ERK1/2 (Thr202, Tyr204) and antibodies to procaspases 7, 8, 9, and 10 to cleaved caspase 3 and polyadenosine-5'-diphosphate-ribose polymerase were all from Cell Signaling Technology (Beverly, MA). A phospho p53 antibody sampler kit containing antibodies to total p53 and phospho-specific antibodies to Ser6, Ser9, Ser15, Ser20, Ser37, Ser46, and Ser392 was also obtained from Cell Signaling Technology. Antibodies to Mcl-1, Bcl-x, Bcl-2, Bax, Bak, cIAP2, p14^ARF, p21^WAF, and p27^Kip1 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); antibodies to cFLIP, cIAP1, survivin, NAIP, and BID were from R&D Systems (Minneapolis, MN); antibodies to XIAP were obtained from Transduction Laboratories (San Diego, CA); antibodies to ß-actin were obtained from Sigma Chemical Co. (St. Louis, MO); antihuman p65 antibody was obtained from Santa Cruz Biotechnology. Three NF-B inhibitors were prepared: (a) resveratrol (Sigma Chemical) was prepared as a 100-mM stock solution in DMSO; (b) Pyrrolidine dithiocarbamate (PDTC) (Alexis) was prepared as a 25 mM stock solution in PBS; and (c) N-acetyl-L-leucinyl-L-leucinyl-norleucinal (LLnL) (Sigma Chemical) was prepared as a 25 mM stock solution in DMSO

In Vitro Proliferation Assay.
Cells were cultured in 6-, 12-, and 24-well plates at a concentration of 0.5 x 10⁶ cells/ml for all cell lines and assays. Cell viability was assessed with a nonradioactive cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay using CellTiter 96 Aqueous One Solution Reagent (Promega, Madison, WI) as described previously (23) . Briefly, 80 µl of cell suspension were added to 20 µl of the reagent and incubated in 96-well plates for 1 h at 37°C in 5% CO₂, and light absorbance of formazan was measured at 495 nm on a µQuant plate reader equipped with KC4 software (Biotek Instruments, Winooski, VT). Each measurement was made in triplicate, and the mean value was determined.

Flow Cytometry.
Cell cycle fractions and apoptosis were determined by staining with propidium iodide (Sigma-Aldrich) and Annexin-V-Fluos (Roche Molecular Biochemicals, Indianapolis, IN), as described previously (24, 25, 26) . Data were collected on a Becton Dickinson FACScan flow cytometer using CellQuest software (BD Biosciences, San Jose, CA) and analyzed using WinMDI 2.8 software (Joseph Trotter, Scripps Research Institute, La Jolla, CA).

Western Blot Analysis.
Whole-cell protein extract was prepared by incubating the cells in lysis buffer (Cell Signaling Technology) for 30 min at 4°C and then centrifuging to remove cellular debris. The protein in the resulting supernatant was quantified by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s instructions, diluted 1:1 in protein SDS loading buffer (Cell Signaling Technology), and boiled for 30 min. A total of 30 µg of protein was loaded onto 10 or 15% Tris-HCl SDS-PAGE Ready Gels (Bio-Rad, Hercules, CA), transferred to a nitrocellulose transfer membrane (Pierce Chemical), and detected using SuperSignal West Dura Extended Duration Substrate (Pierce Chemical).

Immunocytochemistry for NF-B p65 Localization.
Cellular localization of the p65 subunit was performed as described previously (27) . Briefly, HD cells treated with PS-341 were plated on a glass slide by centrifugation at 750 rpm using a Cytospin 4 (Thermoshandon, Pittsburgh, PA), air dried for 1 h at room temperature, and fixed with cold acetone. After brief washing in PBS, slides were blocked with 5% normal goat serum for 1 h and then incubated with rabbit polyclonal antihuman p65 antibody (dilution 1:100). After overnight incubation, the slides were washed and then incubated with goat antirabbit IgG (Alexa Fluor 594;1:100) for 1 h and counterstained for nuclei with Hoechst (50 ng/ml) for 5 min. Stained slides were mounted with mounting medium (Sigma Chemical) and analyzed under an epifluorescence microscope (Labophot-2; Nikon, Tokyo, Japan). Pictures were captured using Photometrics Coolsnap CF color camera (Nikon, Lewisville, TX) and MetaMorph version 4.6.5 software (Universal Imaging Corp., Downingtown, PA).

Electrophoretic Mobility Shift Assay.
Electrophoretic mobility shift assay was performed to determine the activation and nuclear translocation of NF-B as described previously but using minor modifications (28) . Briefly, 4 µg of nuclear protein extract were incubated with 16 fmol of a ³²P-labeled, 45-mer double-stranded DNA oligonucleotide derived from the human immunodeficiency virus long terminal repeat (5'-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3'; italicized areas indicate NF-B-binding sites) for 30 min at 37°C. The resulting complex was resolved from free oligonucleotide using electrophoresis on 6.6% native polyacrylamide gels.

	RESULTS

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PS-341 Inhibits HD Cell Proliferation in Vitro Irrespective of IB Gene Mutation.
It is well established that H/RS cells constitutively express activated NF-B (4 , 6) . On the basis of the antiproliferative activity of PS-341 in other NF-B-expressing lymphoid malignancies, including multiple myeloma (16, 17, 18) , we first investigated whether PS-341 had a similar antiproliferative effect on HD-derived cell lines. Results were compared between cell lines that are known to have mutations in the IB gene (L-428 and KMH-2) and cell lines that do not have such mutations (HD-MYZ and HD-LM2; Ref. 10 ). For these experiments, HD cells were incubated with increasing concentrations of PS-341 (0–50 nM) for 48 h, and the viable cell number was determined using an MTS assay. As shown in Fig. 1A , PS-341 had antiproliferative activity in all four HD cell lines in a dose-dependent manner. The most sensitive cell line was the HD-LM2 line, with an IC₅₀ between 2.5 and 5 nM. The other three cell lines had an IC₅₀ ranging between 10 and 20 nM. Thus, a PS-341 concentration of 20 nM was used, unless otherwise specified. Using this concentration, PS-341 inhibited HD cell proliferation in a time-dependent manner, with significant antiproliferative activity being observed as early as 24 h after treatment and lasting for 72 h (Fig. 1B) . Thus, PS-341 had antiproliferative activity in HD-derived cell lines irrespective of IB mutations.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Activity of PS-341 in Hodgkin disease (HD) cell lines is independent of inhibitor of nuclear factor- mutation. In A, four HD cell lines (HD-MYZ and HD-LM2 have I-B mutation) were incubated with increasing doses of PS-341 for 48 h, and the viable cell number was determined using an MTS assay. The dose-response curve shows the IC50 to be between 2.5 and 5 nM for the HDL-M2 cell line and between 10 and 20 nM for the other three cell lines. In B, HD cell lines were incubated with 20 nM PS-341 for 24, 48, and 72 h. PS-341 had an antiproliferative effect in all four cell lines. Each value represents a mean of three independent experiments performed in triplicate. There was no significant difference in the activity of PS-341 in cell lines that had inhibitor of nuclear factor-B mutation (HD-MYZ and HD-LM2) and those that did not. In C, PS-341 maintained its antiproliferative activity in HD cells in the presence of 1 µg/ml CD30L, CD40L, or RANKL. L-428 cells were incubated with one of these ligands with or without PS-341 for 48 h, and viable cell numbers were determined using an MTS assay. This is a representative of two independent experiments performed in triplicate. Similar results were observed in the other three cell lines.

PS-341 Induces Cell Cycle Arrest and Apoptosis in HD-Derived Cell Lines Irrespective of Ik-B Gene Mutation.
To determine whether the antiproliferative activity induced by PS-341 was caused by cell cycle arrest or induction of apoptosis, we examined the effect of PS-341 on cell cycle fractions. For these experiments, HD-derived cells were incubated with increasing concentrations of PS-341 (2.5–50 nM) for 48 h. The percentages of cells in the G₀-G₁, S, and G₂-M phases were determined by flow cytometric analysis of propidium iodide-stained cells as described previously (31) . In addition, the percentage of dead cells in the sub-G₀ fraction was also calculated. PS-341 induced cell cycle arrest at the G₂-M phase in three cell lines, reaching a maximum at 10 nM (Fig. 2A) . When higher PS-341 concentrations were used, cell death rather than cell cycle arrest predominated. In contrast, PS-341 induced cell death in the HD-LM2 cells without significant G₂-M cell cycle arrest (Fig. 2A) . The ability of PS-341 to induce cell cycle arrest at the G₂-M phase was observed as early as 24 h after treatment in two cell lines (L-428 and KM-H2) and became more prominent after 48 h, after which the cells died (Fig. 2B) .

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Effect of PS-341 on cell cycle. In A, HD cells were incubated with increasing concentrations of PS-341 (2.5–50 nM) for 48 h before cell cycle fractions were determined using the propidium iodide staining method. In the HD-LM2 cells, PS-341 induced cell death (sub-G0 fraction) without G2-M arrest. In the other three cell lines, cell cycle arrest at the G2-M phase was followed by cell death. Percentages in the left top quadrant, dead cells; percentage in the right top quadrant, cells in the G2-M phase. In B, HD cells were incubated for 24 and 48 h with medium or 20 nM PS-341. Cell cycle fraction was determined using the propidium iodide staining method. PS-341 induced accumulation of the cells in the G2-M phase in the L-428 and KM-H2 cells as early as 24 h after treatment.

View larger version (42K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Induction of apoptosis by PS-341. Hodgkin disease (HD) cells were incubated with medium or PS-341 (20 nM) for 48 h before the percentage of dead cells was determined using dual staining with Annexin-V and propidium iodide. PS-341 induced apoptosis in all four HD cell lines to a variable degree. Left bottom quadrant, viable cells. Percentages of dead cells in each quadrant are shown.

PS-341 Inhibits NF-B Activation in HD-Derived Cell Lines.

View larger version (51K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Effect of PS-341 on nuclear localization of nuclear factor -B (NF-B) subunit p65. In A, L-428 cells, which are known to carry inhibitor of nuclear factor-B mutations, were incubated with 20 nM PS-341 for 2–8 h. P65 was visualized under a fluorescence microscope using Alexa-Fluor 594 secondary antibody (top panel). The figure shows that PS-341 inhibited nuclear localization of p65 in L-428 cells within 2 h of incubation. Hoechst staining (bottom panel) shows that the nuclei were intact. In B, PS-341 shows a similar effect on the HD-MYZ cells, which carry wild-type IB. In C, inhibition of NF-B activation in the HD-LM2 cells was achieved by the antioxident PDTC but not resveratrol or LLnL (left panel). NF-B activity was determined after 48 h of incubation using electrophoretic mobility shift analysis as described in "Materials and Methods." However, PDTC remained relatively ineffective compared with PS-341 (right panel).

PS-341 Activates the Caspase Cascade and Alters the Levels of Cell Cycle Regulatory Proteins.
On the basis of the ability of PS-341 to induce apoptosis of HD cells, we investigated whether this process was caspase dependent. For these experiments, HD-derived cell lines were incubated with medium or PS-341 (20 nM) for 6, 24, or 48 h. Whole-cell lysates were then examined using Western blotting. PS-341 cleaved caspases 8, 9, and 3 within 48 h, with subsequent cleavage of polyadenosine-5'-diphosphate-ribose polymerase (Fig. 5A) . In contrast, PS-341 had no effect on caspase 7, 10, or 6 (data not shown). In one cell line (HD-LM2), PS-341 decreased cFLIP levels. In two cell lines (HD-MYZ and HD-LM2), BID protein was detected in unstimulated cells. In these two cell lines, PS-341 reduced BID levels, indicative of BID cleavage (Fig. 5A) .

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. PS-341-induced apoptosis in Hodgkin disease (HD) cells is caspase mediated. In A, HD cells were incubated with medium or 20 nM PS-341 for 6–48 h. Whole-cell lysates were examined by Western blot for changes in intracellular proteins. PS-341 cleaved caspases 8, 9, and 3 and poly(ADP-ribose) polymerase (PARP). Furthermore, PS-341 down-regulated c-FLIP in the HDL-M2 cells. In two cell lines that expressed BID (HD-MyZ and HD-LM2), PS-341 also decreased BID levels, suggesting its cleavage. In B, PS-341-induced cell death of HD cells was either partially or completely blocked by the pan-caspase inhibitor Z-VAD-FMK. HD cells were incubated with medium, Z-VAD-FMK (20 µM), PS-341 (20 nM), or a combination of PS-341 and Z-VAD-FMK. After 48 h in culture, the viable cells were counted using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay. Each value represents a mean of three independent experiments done in triplicate.

PS-341 has been reported previously to regulate the level of several proteins that control the cell cycle. Here, we found that PS-341 increased p21^WAF levels in all four HD cells lines without a significant effect on p27^Kip1 (Fig. 6A) . In addition, PS-341 increased cyclin D2 levels in the L-428 cell line and cyclin D1 levels in the HD-MYZ and L-428 cells (Fig. 6A) . Furthermore, PS-341 did not change the total cellular content of p53 but increased its phosphorylation status at the serine 15 site in one cell line (KM-H2) and decreased the MDM2 protein level in two cell lines (HD-LM2 and KM-H2; Fig. 6A ).

View larger version (55K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Effect of PS-341 on Bcl-2 family, IAPs, and cell cycle proteins. In A, PS-341 up-regulated p21 and increased p53 phosphorylation (serine 15) without inducing changes in total p53. Cyclins D2 and D1 also were each up-regulated in two cell lines. In B, PS-341 up-regulated Bax in the HD-LM2 cells and down-regulated Bcl-2 in the L-428 cells. PS-341 had little or no effect on Bcl-x or Mcl-1. In C, PS-341 had little or no effect on the IAP family.

Finally, we examined the effect of PS-341 on several members of the IAP family (Fig. 6C) . All cell lines expressed detectable levels of XIAP, c-IAP1, and c-IAP2. PS-341 slightly decreased c-IAP1 in one cell line (L-428) but had no significant effect on the level of other IAP family members in any of the cell lines.

PS-341 Enhances the Activity of Chemotherapy, TRAIL/APO2L, and Agonistic Antibodies to TRAIL Death Receptors R1 and R2.
PS-341 has been reported to have synergy with chemotherapy in several tumor models. To explore whether a similar synergistic effect could be achieved in HD cells, we incubated HD cells with submaximal concentrations of PS-341 (5 nM), with or without submaximal concentrations of gemcitabine (0.05 µM), for 72 h. The viable cells were counted using the MTS assay. A synergistic effect was observed only in the HD-LM2 cells (Fig. 7A) , whereas an additive effect was observed in the remaining cell lines (data not shown).

View larger version (19K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. PS-341 enhances the effect of chemotherapy and TRAIL in Hodgkin disease cell lines. In A, Hodgkin disease-LM2 cells were incubated with medium, PS-341 (5 nM), gemcitabine (0.01 nM), or a combination of PS-341 and gemcitabine for 24–72 h. The viable cells were counted using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymeth-oxy-phenyl)-2-(4-sulfonyl)-2H-tetrazolium (MTS) assay. Each value represents the mean of three independent experiments performed in triplicate (±SE). In B, synergy between PS-341 (5 nM) and tumor necrosis factor-related apoptosis-inducing ligand/APO2L (0.1 µg/ml) and agonistic anti-TRAIL receptor R1 (ETR1) and R2 (ETR2) antibodies (0.1 µg/ml) is demonstrated in the Hodgkin disease-LM2 cells. The viable cells were counted after 48 h in culture using an MTS assay. Each value represents the mean of three independent experiments performed in triplicate ±SE.

	DISCUSSION

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This is the first study demonstrating the activity of PS-341 in cell lines known to have IB gene mutation. In this study, we found that PS-341 was effective as an antiproliferative agent in all four HD-derived cell lines that were tested. Two of these cell lines are known to have mutations in the IB gene, resulting in a truncated, nonfunctional IB protein (10) . Thus, it would seem that proteasome inhibition might not be effective in these cell lines. However, we found that IB mutation had no effect on PS-341 activity, which suggests that IBß or other unknown inhibitors can substitute for the IB protein. Furthermore, some PS-341 activities may be mediated by NF-B-independent mechanisms. In any case, the presence of IB mutations was therapeutically irrelevant.

An additional concern was whether PS-341 could maintain its activity in the presence of activating cytokines (13) . It is well established that malignant H/RS cells express several receptors that belong to the tumor necrosis factor family, including CD30, CD40, and RANK (28 , 32 , 33) . Activating these receptors by their ligands activates several shared signaling pathways, including ERK and NF-B (4 , 31) . We found that although PS-341 did not alter ERK levels or phosphorylation status, it maintained its activity in the presence of these ligands, suggesting that PS-341 would be active in vivo. This also suggests that PS-341 alters protein levels downstream from p-ERK that may be crucial for its function.

In this study, PS-341 induced apoptosis and cell cycle arrest at the G₂-M phase (16 , 17 , 34, 35, 36) . Apoptosis was mediated by cleavage and activation of apical caspases, including caspases 8 and 9 and the excutionary caspase 3, with subsequent cleavage of polyadenosine-5'-diphosphate-ribose polymerase. It is not clear how PS-341 can activate caspase 8, which is normally activated through death receptor activation. It is also not clear whether caspase 9 activation in HD cell lines is independent of caspase 8 activation or linked through BID cleavage or an increase in Bax levels. This possibility may be supported by the fact that PS-341 did not activate caspase 9 or increase Bax levels in either of the two HD-derived cell lines that did not express detectable levels of BID (L-428 and KM-H2). Interestingly, when these cells were incubated with the pan-caspase inhibitor Z-VAD-FMK, PS-341-induced apoptosis was either completely or partially inhibited. This finding suggests that caspase cleavage is the dominant mechanism for the induction of apoptosis in these HD-derived cell lines.

The ability of PS-341 to accumulate HD cells in the G₂-M phase was associated with accumulation of proteins that are known to regulate cell cycle progression, e.g., p21 levels increased in response to PS-341 in all four HD cell lines, whereas p27 levels were not significantly affected. Cyclin D1 levels increased in two cell lines (HD-MYZ and L-428), and the cyclin D2 level increased in one (L-428). PS-341 had no significant effect on total cellular content of wild-type p53, but it increased the phosphorylation level of p53 at serine 15 in one cell line (KM-H2). A similar observation was reported recently in multiple myeloma cells.

In this study, we found that all HD lines produced members of the IAP family of proteins (XIAP, c-IAP1, and c-IAP2). PS-341 treatment either had no effect or only slightly decreased the level of one or more proteins in these cells, e.g., PS-341 decreased c-IAP1 in the L-428 cells. Interestingly, PS-341 reduced the XIAP level in the HD-LM2 cells within 6 h after treatment but lost this effect with extended incubation time.

Using a submaximal concentration of gemcitabine or doxorubicin in combination with a submaximal concentration of PS-341 (5 nM), we found that PS-341 enhanced the activity of gemcitabine only in the HD-LM2 cells. However, this enhancement was modest. Similarly, PS-341 enhanced the activity of TRAIL/APO2L only in the HD-LM2 cells but had no significant effect in the other three cell lines. The ability of PS-341 to enhance TRAIL/APO2L and agonistic TRAIL death receptor antibodies in the HD-LM2 cells may have been related to its ability to down-regulate cFLIP levels (37) .

Collectively, these data show that PS-341 has significant antiproliferative activity against HD-derived cell lines in vitro, irrespective of IB gene mutation and in the presence of activating cytokines. Evaluating PS-341 activity in vivo in patients with relapsed HD is warranted.

	FOOTNOTES

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

Requests for reprints: Anas Younes, the Department of Lymphoma/Myeloma, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: (713) 792-2860; Fax: (713) 794-5656; E-mail: ayounes{at}mdanderson.org

Received 10/29/03; revised 1/20/04; accepted 1/28/04.

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