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B inhibitor, induces apoptosis in multiple myeloma cells in an IB
-independent manner
1 Department of Hematology, Kumamoto University School of Medicine, Kumamoto, Japan; and 2 Department of Applied Chemistry, Faculty of Science and Technology, Keio University, Tokyo, Japan
Requests for reprints: Hiroyuki Hata, Department of Hematology, Kumamoto University School of Medicine, 1-1-1 Honjo, Kumamoto 860-8556, Japan. Phone: 81-96-373-5156; Fax: 81-96-363-5265. E-mail: hata{at}kumamoto-u.ac.jp
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Abstract |
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B (NF-B) is constitutively activated in multiple myeloma cells. Several proteasome inhibitors have been shown to be effective against multiple myeloma and may act by inhibiting degradation of IB
. Here, we examined the biological effects of a new type of NF-B inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), which is reported to directly inhibit the cytoplasm-to-nucleus translocation of NF-B. A multiple myeloma cell line, 12PE, which is defective for IB protein, was utilized to determine if IB is concerned with the action of DHMEQ. Meanwhile, U266 was used as a multiple myeloma cell line with normal IB. A proteasome inhibitor, gliotoxin, which is an inhibitor of degradation of phosphorylated IB, failed to inhibit translocation of NF-B in 12PE. In contrast, DHMEQ equally inhibited translocation of NF-B to the nucleus and induced apoptosis to both multiple myeloma cell lines, suggesting that apoptosis resulting from DHMEQ is IB independent. DHMEQ also induced apoptosis in freshly isolated multiple myeloma cells. After DHMEQ treatment, cleavage of caspase-3 and down-regulation of cyclin D1 were observed in both cell lines. In addition, administration of DHMEQ resulted in a significant reduction in tumor volume in a plasmacytoma mice model compared with control mice. Our results show that DHMEQ could potentially be a new type of molecular target agent for multiple myeloma. |
Introduction |
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Recent studies have shown that the nuclear factor-B (NF-B) transcription factor family may play a role in the pathogenesis of lymphoid malignancies, including multiple myeloma (2–4). Under normal conditions, NF-B is present in the cytoplasm as an inactive heterodimer consisting of p50, p65, and IB subunits. On activation, IB is degraded in the 26S proteasome after phosphorylation and ubiquitination, allowing NF-B nuclear translocation and binding to specific consensus sequences (B sites), which transactivates its target genes (5). NF-B activity promotes growth, survival, and drug resistance in multiple myeloma cells and these effects can be inhibited by blocking NF-B (6–8). NF-B also induces adhesion molecule and cytokine expression of multiple myeloma cells and bone marrow stromal cells (9, 10). Recently, it was reported that NF-B inhibitors, such as PS-1145 (2), SN50 (11), and curcumin (12), are effective in multiple myeloma cells.
PS-341 (8), a proteasome inhibitor that inhibits the activity of the 26S proteasome, blocks the degradation of IB in the proteasome, thereby inhibiting nuclear translocation of NF-B (13). PS-341 not only inhibits the growth of multiple myeloma cells but also induces apoptosis of multiple myeloma cells. It also inhibits binding of multiple myeloma cells to bone marrow stroma cells, thus disrupting the microenvironment (14). In a phase II clinical trial, the efficacy of PS-341 in patients with relapsed, refractory myeloma was considered promising (15).
Because NF-B is a key molecule for the development of new therapeutic strategies for myeloma, IB is also likely to play an important role in myeloma cells. Recently, mutations and polymorphisms of the IB gene have been reported in lymphoma (16–18) and multiple myeloma (19), respectively. These studies suggest that such mutations prevent the IB protein from interacting with NF-B, leading to transformation to lymphoma or myeloma cells.
Panepoxydone isolated from the basidiomycete Lentinus crinitus was found to inhibit tumor necrosis factor-–induced activation of NF-B (20). Epoxyquinomicin C was originally isolated from Amycolatopsis as a weak antibiotic and anti-inflammatory agent, and has a 4-hydroxy-5,6-epoxycyclohexenone structure like panepoxydone, which prevents type II collagen–induced rheumatoid arthritis in mice (21). 5-Dehydroxymethylepoxyquinomicin (DHMEQ), which is a 5-dehydroxymethyl derivative of epoxyquinomicin C, was recently designed and synthesized. DHMEQ inhibits nuclear translocation of NF-B and has a different mechanism from that of proteasome inhibitors (22). It did not inhibit nuclear translocation of large T antigen or Smad2 (21), suggesting that it inhibits the translocation of NF-B specifically. The target molecule is now being studied by preparing immobilized DHMEQ. DHMEQ showed anti–NF-B activity in Jurkat cells, a human T-cell leukemia line (23), and was also effective against hormone-refractory prostate cancer by inhibiting NF-B (24). These findings suggest that DHMEQ may be a new therapeutic agent for multiple myeloma by inhibiting NF-B activity.
Therefore, we analyzed the apoptotic effects of DHMEQ utilizing multiple myeloma cell lines and freshly isolated multiple myeloma cells. We utilized an IB-deficient multiple myeloma cell line to confirm the role of IB in DHMEQ-induced cytotoxicity. Apoptosis and cell cycle–related molecules, such as caspase-3, Bcl-2, Bcl-xL, A1/Bfl-1 (25), and cyclin D1, were simultaneously examined to explain the apoptotic pathway induced by DHMEQ. Finally, we evaluated the efficacy of DHMEQ in vivo.
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Materials and Methods |
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B protein, is a subclone of KMS-12PE. Nucleotide sequence analysis showed that there was a genomic mutation in the splicing acceptor site of intron 4 (AG to TG) of IB gene in 12PE. Sequence analysis revealed that the splicing donor site of intron 4 is connected to a new cryptic splicing acceptor site located at the 128th nucleotide of exon 5, resulting in 129 bp deletion of exon 5. Because this mutation generates a truncated mRNA, it is suggested that the absence of IB in 12PE cells is possibly due to instability of the truncated mRNA or IB protein.
Patient Samples
After obtaining informed consent, myeloma cells were obtained from the bone marrow of myeloma patients admitted to Kumamoto University Hospital. Myeloma cells were purified using immunomagnetic beads as previously described (28).
Agents
DHMEQ (a 5-dehydroxymethyl derivative of epoxyquinomycin C) was synthesized in the Faculty of Science and Technology, Keio University. For in vivo experiments, a chiral form of DHMEQ, (–)-DHMEQ, was used by dissolving in DMSO to prepare a 10 mg/mL solution. This solution was then diluted in culture medium for further experiments. A proteasome inhibitor, gliotoxin, was purchased from Calbiochem (La Jolla, CA) and dissolved in chloroform at a concentration of 10 mg/mL.
Electrophoretic Mobility Shift Assay
Cells were treated with 10 µg/mL DHMEQ. Cells were harvested after various intervals, then washed with PBS, suspended in 400 µL of lysis buffer [10 mmol/L HEPES-KOH (pH 7.9), 5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride], and incubated on ice for 1 hour. Nuclei were pelleted by centrifugation for 1 minute at 14,000 rpm, then resuspended in 40 µL of washing buffer [20 mmol/L HEPES-KOH (pH 7.9), 1.5 mmol/L MgCl2, 420 mmol/L NaCl, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.2 mmol/L phenylmethylsulfonyl fluoride]. After 20 minutes incubation on ice, the sample was centrifuged for 2 minutes at 14,000 rpm at 4°C. The supernatant was used as a nuclear extract. The binding reaction mixture containing 5 mg of nuclear extract, 2 µg of poly-deoxyinosinic-dCMP, and 32P-labeled probe, was incubated for 20 minutes at room temperature. The DNA probe used for NF-B binding was a double-strand oligonucleotide containing the interleukin-6 B binding sequence (5'-TCGACATGTGGGATTTTCCCATGAC-3'). The complexes were separated from free DNA on 4% polyacrylamide gel and visualized by autoradiography.
Western Blot Analysis
Cells were lysed in radioimmunoprecipitation assay buffer (0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, protease inhibitor cocktail; Roche Diagnostics GmbH, Mannheim, Germany). Cell lysates were subjected to electrophoresis on a SDS polyacrylamide gel and transferred onto nitrocellulose membranes. The membranes were blocked with TBS containing 0.05% Tween and 5% nonfat milk at 4°C overnight. Subsequently, they were incubated for 1 hour with anti–caspase-3, anti-Bcl-2, anti–Bcl-Xs/L (Santa Cruz Biotechnology, Santa Cruz, CA), anti–A1/Bfl-1 (Santa Cruz), antiactin (Sigma), anti-IB (Cell Signal, Beverly, MA), or anti–phosphorylated IB (Cell Signal) antibodies. The membranes were then incubated for 1 hour with peroxidase-labeled anti-rabbit or anti-mouse secondary antibody, and developed using an enhanced chemiluminescence system (Amersham Life Science, Inc., Little Chalfont, United Kingdom).
Detection of Apoptosis
Apoptosis was quantified by using the Annexin V/propidium iodide staining kit (Medical and Biological Laboratories, Nagoya, Japan; ref. 29). Briefly, after treatment with various concentrations of DHMEQ or gliotoxin, cells were harvested, washed, and then incubated with Annexin V-FITC and propidium iodide for 15 minutes in the dark. Fluorescence was analyzed by flow cytometry (EPICS V, Coulter, Miami, FL).
Mouse Model
All procedures involving animals and their care described in this study were approved by the Animal Care Committee of Kumamoto University in accordance with institutional and Japanese government guidelines for animal experiments. CB17/Icr female mice (6 weeks old) were obtained from Clea Japan (Tokyo, Japan). 12PE cells were implanted s.c. in the flank of each mouse. When animals developed palpable tumors, they were randomly assigned into two groups. DHMEQ was administered i.p. at a dosage of 12 mg/kg once daily for 14 consecutive days. Control animals received only a vehicle medium injection. Each experimental group consisted of five mice. Tumor size was measured twice a week over 7 weeks. Tumor volume was calculated according to the following formula: (smallest diameter)2 x (largest diameter) x 0.52.
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Results |
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B ActivityB inhibitory effect of DHMEQ by electrophoretic mobility shift assay. DNA-binding activity of NF-B was found in both U266 and 12PE cell lines before DHMEQ treatment (Fig. 2A). DHMEQ treatment equally reduced DNA binding activity of NF-B in both U266 and 12PE at 24 and 36 hours after treatment (Fig. 2A, left). In contrast, a proteasome inhibitor, gliotoxin, failed to inhibit DNA-binding activity of NF-B in 12PE at 8 hours after treatment, whereas inhibition was seen in U266 (Fig. 2A, right). The sensitivity of 12PE to gliotoxin (IC50 > 8 µmol/L) was significantly less than that of U266 (IC50 0.5-1.0 µmol/L).
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B inhibitory effect of DHMEQ in freshly isolated myeloma cells. Translocation of NF-B to nucleus was inhibited by DHMEQ treatment also in fresh myeloma cells (Fig. 2B).
DHMEQ Caused Apoptosis Independent of IB
Because IB is thought to be critical in the NF-B pathway, we next investigated IB and phosphorylated-IB protein levels before and after treatment with DHMEQ or gliotoxin (Fig. 3). Gliotoxin is a well-characterized proteasome inhibitor that blocks degradation of phosphorylated IkB, leading to accumulation of phosphorylated and unphosphorylated IkB protein. In U266, DHMEQ did not change the protein level of IB or phosphorylated IB, whereas phosphorylated IB accumulated after treatment with gliotoxin. Meanwhile, there was no expression of IB in 12PE cells. Because DHMEQ can induce apoptosis in 12PE, these results indicate that DHMEQ-induced apoptosis is IB independent.
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B (30, 31), we examined the effects of DHMEQ on the expression of these proteins (Fig. 4). DHMEQ decreased Bcl-2 and Bcl-xL expression level in U266 but not in 12PE, suggesting that regulation of translation of Bcl-2 and Bcl-xL is different in U266 compared with 12PE although both cell lines similarly undergo apoptosis after treatment with DHMEQ. Bfl-1, a novel Bcl-2 family protein, was expressed in both cell lines and its expression was not changed by treatment with DHMEQ.
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B induces mdr-1 gene expression (33). However, neither expression nor induction of mdr-1 was detected by reverse transcription-PCR in either cell line (data not shown).
Another hallmark of apoptosis is activation of caspases. Therefore, we next examined whether blocking the NF-B pathway with DHMEQ activated the caspase pathway. Western blot analysis showed that DHMEQ activated caspase-3 in both cell lines (Fig. 4), indicating that DHMEQ-induced apoptosis is at least partly mediated by the caspase-3 pathway. Significant activation of caspase-3 was found at 24 hours in 12PE but not in U266; however, activation was found at 36 hours in U266. This is compatible with the fact that 12PE undergoes apoptosis sooner than U266 after treatment with DHMEQ (Fig. 1D).
DHMEQ Inhibits Tumor Growth In vivo
The efficacy of DHMEQ treatment in the plasmacytoma in vivo model was examined. We s.c. transplanted 12PE cells into severe combined immunodeficient mice. After the mice developed palpable tumors, they received DHMEQ at a dosage of 12 mg/kg daily for 14 days. Tumor growth in treated mice was significantly inhibited (2.91 ± 1.39 mL) compared with those of control mice (7.91 ± 1.54 mL) 21 days after the start of the treatment (P < 0.01; Fig. 5). No apparent toxic effect was noted in the mice during treatment. These data suggested that DHMEQ may be an applicable agent for multiple myeloma treatment in vivo.
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Discussion |
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B inhibitor, DHMEQ, on multiple myeloma cell lines and patient myeloma cells. The major findings of the present study were as follows: (a) DHMEQ induced apoptosis of multiple myeloma cell lines and multiple myeloma patient cells, (b) DHMEQ suppressed constitutive NF-B activation in an IB-independent manner, (c) down-regulation of cyclin D1 and activation of caspase-3 were involved in apoptosis by DHMEQ, and (d) DHMEQ effectively reduced plasma cell tumor growth in vivo.
A proteasome inhibitor, gliotoxin, did not induce apoptosis in the 12PE cell line, whereas apoptosis was significantly induced in the U266 cell line 8 hours after application. Resistance of 12PE to gliotoxin is recognized as an absence of NF-B blocking in 12PE cells by electrophoretic mobility shift assay. Moreover, gliotoxin induced an accumulation of phosphorylated IB in U266, indicating gliotoxin actually inhibited degradation of IB at the proteasome as previously reported (13). Because IB was not detected in 12PE, these results suggest that IB should be critical for gliotoxin-induced apoptosis.
Although 12PE was defective in IB, the sensitivity of 12PE to DHMEQ was equal to that of U266, suggesting that DHMEQ induces apoptosis independent of IB. Electrophoretic mobility shift assay analysis actually showed that DHMEQ inhibited translocation of NF-B in both cell lines. This finding suggests that DHMEQ inhibit NF-B by direct interaction but not by an IB-mediated mechanism as previously reported in other proteasome inhibitors (22).
It is clear that the absence of IB is not critical for the growth of 12PE cells. However, regulation of NF-B in 12PE is unknown. It has been suggested that a defect in IB leads to constitutive translocation of NF-B. However, whether this abnormal regulation of NF-B causes unexpected physiologic phenomena is unknown. The 12PE cell line seems to have very efficient proliferation with a doubling time of 
24 hours. The possibility that mechanisms regulating NF-B other than IB may function in 12PE cannot be ruled out. Further detailed analysis is needed to clarify the mechanisms regulating NF-B without IB. Emmerich et al. (17) also reported a mutation of IkB in a Reed Sternberg cell-derived cell line, L428. However, detailed mechanisms for regulating NF-B in the cell line were not examined.
To elucidate the downstream pathway of DHMEQ-induced apoptosis, we next evaluated caspase-3, Bcl-2, Bcl-xL, Bfl-1, and cyclin D1 protein levels after treatment with DHMEQ. Caspase-3 was cleaved after DHMEQ exposure as reported with other NF-B inhibitors (11, 12), suggesting that DHMEQ causes apoptosis of multiple myeloma cells through the caspase-3 pathway. In contrast, down-regulation of Bcl-2 was found only in U266 and not in 12PE despite more significant cleavage of capase-3 in 12PE, suggesting that Bcl-2 is not involved with caspase-3 cleavage by DHMEQ in 12PE although the Bcl-2 and Bcl-xL genes are reported to be regulated by NF-B (30, 31). Because inhibition of NF-B was observed in 12PE, absence of reduction of Bcl-2 and Bcl-xL in 12PE cannot be simply explained. It may be that expression of Bcl-2 and Bcl-xL is regulated by different pathways in U266 and 12PE.
It was reported that Bfl-1, a member of the Bcl-2 family of proteins, regulates apoptosis (34). Expression of Bfl-1 is regulated by NF-B (35, 36), indicating that expression of Bfl-1 in myeloma cells after activation of NF-B in myeloma cells is frequently found. However, Tarte et al. (25) reported that Bfl-1 is repressed in myeloma, suggesting that regulation of Bfl-1 could be independent of NF-B in myeloma cells. Our finding that Bfl-1 was expressed but not influenced by the treatment with DHMEQ supports this hypothesis.
We also found that U266 and 12PE overexpressed cyclin D1, which was down-regulated by DHMEQ. Because NF-B regulates transcription of cyclin D1, it is reasonable to find down-regulation of cyclin D1 by treatment with DHMEQ. Because cyclin D1 is needed for the G1-S transition during the cell cycle, down-regulation of cyclin D1 by DHMEQ may lead to growth arrest in myeloma cells. Indeed, Yata et. al. (37) reported that treatment of myeloma cells with an antisense oligonucleotide of cyclin D1 inhibited the growth rate of cyclin D1–overexpressing myeloma cells. We found a variant form of cyclin D1 with a different molecular weight in 12PE and its expression was also inhibited by DHMEQ. As assessed by its molecular weight, this variant form of cyclin D1 is thought to be cyclin D1b (32), which is derived from an alternate transcript of cyclin D1. A recent study reported that cyclin D1b protein is constitutively localized in the nucleus (32) and a potential inducer of cellular transformation. DHMEQ efficiently inhibited both the normal and variant forms of cyclin D1. Moreover, given the fact that 12PE has t(11;14) and U266 has an insertion in the IgH enhancer region on chromosome 11q13 (38), overexpression of cyclin D1 should result. It is also noteworthy that DHMEQ efficiently shut off the expression of cyclin D1, which is powerfully driven by chromosome translocation or insertion.
Taken together, DHMEQ may induce apoptosis of multiple myeloma cells at least through activation of the caspase-3 pathway and down-regulation of cyclin D1. Further analysis is needed to elucidate mechanisms of apoptosis of multiple myeloma cells, including the relationship to activation of the caspase-3 pathway and down-regulation of cyclin D1.
Finally, we evaluated the efficacy of DHMEQ in an in vivo plasma cell tumor model. DHMEQ treatment using doses at a concentration far lower than the toxic dosage induced marked reduction in tumor volume compared with vehicle treatment. Treatment of severe combined immunodeficient mice with DHMEQ did not result in any apparent side effects or death throughout the evaluation period. Because the toxic dose of DHMEQ is 30 times that utilized here,3 DHMEQ is believed to be a very safe compound. These findings suggest that treatment at higher doses and for longer durations should be examined in future studies. Further analysis of the metabolism of DHMEQ is needed to select the most appropriate therapeutic regimen.
Analysis of fresh myeloma cells from 11 cases revealed that DHMEQ induced apoptosis even in cases refractory to conventional chemotherapy, including MP, VAD, high-dose melphalan, or thalidomide. Indeed, melphalan was not effective in this 12PE mouse model (data not shown), suggesting that DHMEQ should be a very useful agent for chemoresistant myeloma cells.
In conclusion, we have shown in the present study that DHMEQ could be used as a new molecular-targeted chemotherapeutic agent for multiple myeloma. Analysis of fresh myeloma cells indicates that DHMEQ has a potential to overcome refractoriness to various chemotherapeutic drugs. Our in vivo model showed that DHMEQ was safe and effective. Further analysis of the exact mechanisms of the inhibitory effects of DHMEQ on nuclear translocation of NF-B is currently under way.
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Acknowledgments |
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Footnotes |
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3 K. Umezawa, unpublished result from in vivo experiments. 
Received 8/ 5/04; revised 3/21/05; accepted 4/29/05.
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References |
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B as a therapeutic target in multiple myeloma. J Biol Chem 2002;277:16639–47.B (NF-B) in multiple myeloma: down-regulation of NF-B induces apoptosis. Br J Haematol 2001;115:279–86.[CrossRef][Medline]
Bharti AC, Shishodia S, Reuben JM, et al. Nuclear factor-{}B and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and their suppression leads to apoptosis. Blood 2003;103:3175–84.
Gilmore TD. The Rel/NF-B signal transduction pathway: introduction. Oncogene 1999;18:6842–4.[CrossRef][Medline]
Berenson JR, Ma HM, Vescio R. The role of nuclear factor-B in the biology and treatment of multiple myeloma. Semin Oncol 2001;28:626–33.[CrossRef][Medline]
Landowski TH, Olashaw NE, Agrawal D, Dalton WS. Cell adhesion-mediated drug resistance (CAM-DR) is associated with activation of NF-B (RelB/p50) in myeloma cells. Oncogene 2003;22:2417–21.[CrossRef][Medline]
Mitsiades N, Mitsiades CS, Richardson PG, et al. The proteasome inhibitor PS-341 potentiates sensitivity of multiple myeloma cells to conventional chemotherapeutic agents: therapeutic applications. Blood 2003;101:2377–80.B. Blood 1996;87:1104–12. in the pathophysiology of human multiple myeloma: therapeutic applications. Oncogene 2001;20:4519–27.[CrossRef][Medline]
Mitsiades N, Mitsiades CS, Poulaki V, et al. Biologic sequelae of nuclear factor-B blockade in multiple myeloma: therapeutic applications. Blood 2002;99:4079–86.B and IB kinase in human multiple myeloma cells, leading to suppression of proliferation and induction of apoptosis. Blood 2003;101:1053–62.B without inhibition of NF-B activity and mutations in the IB gene in Reed-Sternberg cells. Blood 1999;94:3129–34.B
mutations in Hodgkin/Reed-Sternberg cells. J Pathol 2003;201:413–20.[CrossRef][Medline]
Cabannes E, Khan G, Aillet F, Jarrett RF, Hay RT. Mutations in the IkBa gene in Hodgkin's disease suggest a tumour suppressor role for IB. Oncogene 1999;18:3063–70.[CrossRef][Medline]
Parker KM, Ma MH, Manyak S, et al. Identification of polymorphisms of the IB gene associated with an increased risk of multiple myeloma. Cancer Genet Cytogenet 2002;137:43–8.[CrossRef][Medline]
NF-B and STAT3 are constitutively active in CD138+ cells derived from multiple myeloma patients, and suppression of these transcription factors leads to apoptosis. Blood 2004;103(8):3175–84. Epub 2003 Dec 18.
Matsumoto N, Iinuma H, Sawa T, et al. Epoxyquinomicins A, B, C and D, new antibiotics from Amycolatopsis. II. Effect on type II collagen-induced arthritis in mice. J Antibiot (Tokyo) 1997;50:906–11.[Medline]
Ariga A, Namekawa J, Matsumoto N, Inoue J, Umezawa K. Inhibition of tumor necrosis factor--induced nuclear translocation and activation of NF-B by dehydroxymethylepoxyquinomicin. J Biol Chem 2002;277:24625–30.B activation inhibitors derived from epoxyquinomicin C. Bioorg Med Chem Lett 2000;10:865–9.[CrossRef][Medline]
Kikuchi E, Horiguchi Y, Nakashima J, et al. Suppression of hormone-refractory prostate cancer by a novel nuclear factor B inhibitor in nude mice. Cancer Res 2003;63:107–10. is produced by human multiple myeloma (MM) cells and its expression correlates with bone lesions in patients with MM. Br J Haematol 2003;120:53–5.[CrossRef][Medline]
Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J Immunol Methods 1995;184:39–51.[CrossRef][Medline]
Pahl HL. Activators and target genes of Rel/NF-B transcription factors. Oncogene 1999;18:6853–66.[CrossRef][Medline]
Sevilla L, Zaldumbide A, Pognonec P, Boulukos KE. Transcriptional regulation of the bcl-x gene encoding the anti-apoptotic Bcl-xL protein by Ets, Rel/NFB, STAT and AP1 transcription factor families. Histol Histopathol 2001;16:595–601.[Medline]
Solomon DA, Wang Y, Fox SR, et al. Cyclin D1 splice variants. Differential effects on localization, RB phosphorylation, and cellular transformation. J Biol Chem 2003;278:30339–47.B confers chemo-sensitization and apoptosis in prostate cancer cells. Int J Oncol 2003;23:317–23.[Medline]
D'Sa-Eipper C, Subramanian T, Chinnadurai G. bfl-1, a bcl-2 homologue, suppresses p53-induced apoptosis and exhibits potent cooperative transforming activity. Cancer Res 1996;56:3879–82.B-mediated up-regulation of Bcl-x and Bfl-1/A1 is required for CD40 survival signaling in B lymphocytes. Proc Natl Acad Sci U S A 1999;96:9136–41.B that blocks TNF-induced apoptosis. Genes Dev 1999;13:382–7.
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, U266; 
, 12PE. Points, mean of at least three individual experiments; bars, SD. Sensitivity of cells to DHMEQ is not significantly different. D, time course analysis of DHMEQ-induced apoptosis. Cells were treated with 10 µg/mL of DHMEQ and incubated for various periods. Columns, mean of at least three individual experiments; bars, SD. 
