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B Activation in Mantle Cell Lymphoma B Cells Leads to Induction of Cell Cycle Arrest and Apoptosis1
Department of Hematopathology, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030
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Abstract |
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B has been documented to be involved in the pathogenesis of many human malignancies, including hemopoietic neoplasms. In this study, we examined the status of NF-B in two non-Hodgkin’s lymphoma cell lines derived from mantle cell lymphoma (MCL) samples and in patient MCL biopsy specimens by EMSA and confocal microscopic analysis. We observed that NF-B is constitutively activated in both the MCL cell lines and in the MCL patient biopsy cells. Since NF-B has been shown to play an important role in a variety of cellular processes, including cell cycle regulation and apoptosis, targeting the NF-B pathways for therapy may represent a rational approach in this malignancy. In the MCL cell lines, inhibition of constitutive NF-B by the proteasome inhibitor PS-341 or a specific pIB
inhibitor, BAY 11-7082, led to cell cycle arrest in G1 and rapid induction of apoptosis. Apoptosis was associated with the down-regulation of bcl-2 family members bcl-xL and bfl/A1, and the activation of caspase 3, that mediates bcl-2 cleavage, resulting in the release of cytochrome c from the mitochondria. PS-341or BAY 11-induced G1 cell cycle arrest was associated with the inhibition of cyclin D1 expression, a molecular genetic marker of MCL. These studies suggest that constitutive NF-B expression plays a key role in the growth and survival of MCL cells, and that PS-341 and BAY 11 may be useful therapeutic agents for MCL, a lymphoma that is refractory to most current chemotherapy regimens. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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NF-B, a centrally important transcription factor involved in immune and inflammatory cellular responses affecting both cell growth and survival, appears to be pivotally involved in the pathogenesis of aggressive lymphoid malignancies (7). In mammalian cells, the NF-B family consists of five members, c-rel, p65 (Rel A), Rel B, p50/p105 (NF-B1), and p52/p100 (NF-B2), that can form various hetero- or homodimers (8). Normally, NF-B is transcriptionally inactive in the cytoplasm of most cells because it is bound to its cytoplasmic inhibitor IB. Upon stimulation with proinflammatory cytokines, such as TNF- or IL-1, IB protein is phosphorylated, ubiquitinated, and subsequently degraded by the proteasome. This process exposes the previously masked nuclear localization signal of NF-B that upon proteolysis of IB allows NF-B to translocate into the nucleus and subsequently activate the expression of important target genes involved in cell growth, survival, adhesion, etc. (9, 10).
Proteasome inhibitors have recently emerged as an interesting and potentially new group of chemotherapeutic agents for a variety of human cancers, including breast, prostate, and lung carcinomas, that function, in part, by stabilizing the IB protein and, finally, inhibiting NF-B activation (11, 12, 13). More recently, a series of dipeptide boronic acid analog proteasome inhibitors that specifically inhibit the chymotryptic enzyme activity of the proteasome have been synthesized (14, 15). One of these, PS-341, is a low molecular weight, water-soluble dipeptide that targets the chymotryptic site in the 20S proteasome. Recent reports have shown that PS-341 inhibits tumor cell growth by inhibiting NF-B activation, particularly in tumors constitutively expressing this pivotal transcription factor (16, 17). PS-341 is currently undergoing clinical trials as a therapeutic agent for several human cancers in which preliminary studies have been promising (18, 19). In this study, we have used PS-341 to target the possible modulation of NF-B in MCL cells. Although the proteasome inhibitor PS-341 is capable of inhibiting NF-B activation, it also has a variety of other activities, since the proteasome is an enzymatic structure that degrades many important proteins, particularly protooncogenes and cell cycle regulators in the cell (20, 21). To examine specific effects of NF-B down-regulation on MCL cell growth and survival, we have used a specific inhibitor of IB phosphorylation, BAY 11-7082 (BAY 11), an organic compound that selectively inhibits the TNF--inducible phosphorylation of IB (22).
In this study, we demonstrate that NF-B is constitutively active in MCL cell lines as well as in primary MCL cells. Inactivating NF-B in the MCL cell lines with the proteasome inhibitor PS-341 or the pIB inhibitor BAY 11 blocks MCL cell growth, leading to tumor cell death. Our studies show that PS-341 or BAY 11 inhibits MCL tumor cell growth through two important control mechanisms: cell cycle arrest and induction of cell death, both of which involve inhibition of constitutive NF-B activation. We also used a dominant-negative (DN) form of IB to transfect into MCL cells to determine whether all of the observed effects of PS-341 or BAY 11 treatments of MCL can be attributed to NF-B inhibition. Our findings suggest that constitutive NF-B activation plays a key role in the survival of MCL cells and that PS-341 and BAY 11 may be useful future therapeutic agents for clinical trials in MCL.
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Materials and Methods |
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Human MCL cell lines (Mino and DB (sp53)) were established from patient biopsy samples as previously described (23, 24). The Molt-4 T cell leukemia cell line (American Type Culture Collection, Manassas, VA) and the Hodgkin’s lymphoma cell line (L-428; courtesy of Dr. V. Diehl, University of Cologne, Cologne, Germany) were cultured in RPMI 1640 (Life Technologies, Rockville, MD) containing 10% FCS (HyClone Laboratories, Logan, UT). Fresh biopsy-derived MCL cells were obtained from biopsy specimens from our Tissue Procurement and Banking core facility. Lymphoma cells were enriched by SRBC rosetting, followed by "Rosette Sep" and contained 98% CD20+ and <1% CD3+ T cells by flow cytometry.
Ab reagents and materials
The following Abs were used: bcl-xL, Bfl-1/A1, bcl-2, bax, cyclin (D1, D2, and D3), p27, p21, NF-B p50, p52, p65, Rel-B, and c-rel, IB (Santa Cruz Biotechnology, Santa Cruz, CA); bcl-2 and p50 (Upstate Biotechnology, Lake Placid, NY); and phospho-IB (New England Biolabs, Beverly, MA). The proteasome inhibitor PS-341 was kindly supplied by Dr. J. Adams (Millennium, Cambridge, MA). The IB inhibitor BAY 11-7082 was purchased from Calbiochem (San Diego, CA). The following kits were used: caspase 3 (Calbiochem), 20S proteasome (Boston Biochem, Cambridge, MA), and cell fractionation (Clontech Laboratories, Palo Alto, CA).
Cell proliferation assay
In vitro thymidine incorporation proliferation assays were performed as described previously (25). Briefly, cells were plated (in triplicate) at 4.0 x 104 cells/well in 200 µl of RPMI 1640 with 10% FCS and the indicated reagents in a 96-well plate and incubated in 5% CO2 at 37°C. After 24 h, each well was pulsed with 0.5 µCi/10 µl of [3H]thymidine (Amersham, Arlington Heights, IL) for 16 h. Cells were harvested and the radioactivity was measured.
EMSAs
Nuclear protein extraction and gel shift assay procedures were performed as previously described (26). Briefly, a 45-mer double-stranded NF-B oligonucleotide 5'TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3' was labeled with [
-32P]ATP. Underlined sequence indicates consensus B binding sites. Nuclear extract (10 µg) was incubated 1x binding buffer, 2 µg poly(dI:dC), 0.5% Nonidet P-40, and the labeled probe (4 fmol/µl) at 37°C for 15 min. The samples were then subjected to 6% nondenaturing acrylamide gel, electrophoresis and visualized with a Typhoon phosphor imager (Amersham Biosciences, Piscataway, NJ). For supershift analysis, 1 µl of Ab was added to the nuclear extract 15 min before the labeled probe was added. For specificity, 800 fmol of unlabeled oligonucleotide were used to compete out the labeled probe.
Immunoblot analysis
Whole cell extracts were solubilized with 1.0% SDS sample buffer and electrophoresed on a 4–15% SDS-PAGE gel (Bio-Rad, Richmond, CA). Proteins were transferred onto a polyvinylidene difluoride membrane and were probed with various specific primary Abs and then the appropriate HRP-conjugated secondary Abs. Proteins were visualized by ECL (Amersham, Piscataway, NJ).
Apoptosis detection assay and cell cycle analysis
MCL cells were washed and stained with Annexin VFITC and propidium iodide in accordance with the manufacturer’s procedure (BD PharMingen, San Diego, CA). Apoptosis was quantified with a FACS and CellQuest software (BD Biosciences, Mountain View, CA). For cell cycle analysis, the MCL cells were fixed with cold methanol overnight and then treated with propidium iodide (2 µg/ml) and RNase before FACS analysis.
Immunofluorescence
MCL cells were fixed with 2% paraformaldehyde in PBS and deposited onto poly-L-lysine-coated glass slides, preblocked with 5% FCS in PBS for 15 min, and stained with a 1/200 dilution of the appropriate primary Abs for 1 h. After three washes with PBS, the cells were stained with the appropriate donkey secondary Abs conjugated to Cy2, Cy3, or Cy5 (1/200 dilution) for 45 min and washed with PBS. Coverslips were applied with Slow Fade reagent (Molecular Probes, Eugene, OR). The cells were visualized with an Olympus FV500 laser scanning confocal microscope (Olympus, Melville, NY). Images were captured with a x64 objective and with the appropriate filter sets.
Transfection
Transient transfections of MCL cells with pCMV-IB or pCMV-IBM (Clontech Laboratories) were conducted using the "Nucleofector" protocol from AMAXA Biosystems (Cologne, Germany). MCL cells (5 x 106) were suspended in 100 µl of Nucleofector T solution with 15 µg of plasmid DNA and then electroporated using program O-17. Cells were transferred to 2.5 ml of medium containing 15% FCS and harvested 24 h after transfection. For transfection efficiency, MCL cells were cotransfected with green fluorescent protein and analyzed by flow cytometry. The transfection efficiency ranges between 30 and 40% for each experiment.
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Results |
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B activation in MCL
Because NF-B plays an important role in regulating cell growth and cell viability in many tumors, including other aggressive NHL-Bs, such as diffuse large B cell lymphomas, we determined its biochemical status in MCL. Fig. 1A shows that in Mino and DB cell lines, and in primary MCL cells, NF-B is constitutively activated by EMSA analysis. When analyzed by Western blotting, the nuclear proteins of MCL cells contain all of the NF-B components (p50, p52, p65, c-rel, and relB) (Fig. 1B). Confocal microscopic analysis indicates that p50 and p65 are expressed in the nucleus of Mino and DB MCL cells and that in some areas they are colocalized (Fig. 1C). A supershift EMSA assay using specific Rel family Abs indicates that the p50, p65, and c-rel subunits of NF-B are expressed and bound to DNA in MCL cells, while p52 and relB do not show binding (Fig. 2A). Because the NF-B complex pattern observed in patient MCL cells is slightly different from that of the MCL cell lines, we performed a supershift assay on a representative MCL patient biopsy sample. As indicated in Fig. 2B, MCL patient cells, like the MCL cell lines, also expressed p50, p65, and c-rel but not p52 and relB. However, there was an unidentified additional band present in the patient cells that did not appear in the cell lines.
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B activation in MCL cells
To ascertain whether PS-341 and BAY 11 have an effect on NF-B components expressed in MCL cells, we performed EMSA with nuclear extracts from PS-341-treated or BAY 11-treated Mino cells. NF-B DNA-binding activity gradually declined between 6 and 12 h after the addition of PS-341 and was barely detectable at 24 h (Fig. 3A). PS-341 blocked IB degradation, resulting in the accumulation of pIB after 3 and 6 h of treatment (Fig. 3B). However, there was little pIB present after 12 h of PS-341 treatment. PS-341 treatment also inhibited IB protein expression in a time-dependent manner (Fig. 3B). BAY 11 inhibits NF-B DNA-binding activity after 6 and 20 h of treatment (Fig. 3A) and also blocks IB phosphorylation (Fig. 3B). Confocal microscopic analysis indicates that after PS-341 or BAY 11 treatment, the p50 and p65 components of NF-B are prevented from translocating to the nucleus; i.e., consistent with the NF-B inhibition observed by EMSA (Fig. 3C).
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To evaluate the effects of PS-341 and BAY 11 on MCL cell growth, proliferation assays were performed in vitro on two MCL cell lines (Mino and DB). Both PS-341 (Fig. 4A) and BAY 11 (Fig. 4B) inhibited cell proliferation in Mino and DB MCL cell lines in a dose-dependent response. The sensitivity to PS-341 and BAY 11 was very similar in the two MCL cell lines. More importantly, PS-341, at doses above 25 nM, and BAY 11, at doses above 5 µM, completely inhibited MCL growth, as evidenced by low or virtually no tritiated thymidine uptake (Fig. 4, A and B). PS-341 and BAY 11 had significantly more growth inhibitory activity on the MCL cells than the other well-known biotherapeutic agents, with known negative growth effects on lymphoid tumor cells, including TGF-
(Fig. 4C) and IFN- (Fig. 4D). These results suggest that even though Mino and DB MCL cells are relatively refractory to other common biotherapeutic agents, they are sensitive to the inhibitory effect of PS-341 or BAY 11. To demonstrate differing roles of these inhibitors on the proteasome, we subjected cell lysates from PS-341-treated and BAY 11-treated Mino cells to a 20S proteasome proteolysis assay. As shown in Fig. 4E, the proteasome activity was substantially inhibited (>50%) after 3 h of PS-341 treatment and was completely inactive after 6 h of treatment, whereas BAY 11 treatment did not affect the proteasome activity.
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One likely target of cellular growth inhibitory activity of PS-341 or BAY 11 involves the cell cycle, where NF-B has been shown to play a key role in cell cycle regulation. As shown in Fig. 5A, both PS-341- and BAY 11-treated MCL cells in vitro accumulated in the G0-G1 phase of the cell cycle. Because cyclin D1 is ectopically overexpressed in MCL and involved in the early stage of the G1 phase of the cell cycle, we evaluated the effects of PS-341 or BAY 11 in modulating the ectopic expression of cyclin D1 in MCL cells. Fig. 5B shows that cyclin D1 gradually decreased in a time-dependent manner with PS-341 or BAY 11 treatments, whereas cyclin D2 was not affected by BAY 11 treatment but progressively increased for 12 h after PS-341 treatment and then leveled off for the next 12 h. We further explored the cell cycle effects of PS-341 and BAY 11 on later G1 cell cycle components. PS-341 treatment up-regulated the cyclin-dependent kinase inhibitor p21waf1 during the first 6 h of treatment, but was barely detectable at 12 and 24 h after treatment (Fig. 5B). Another cyclin-dependent kinase inhibitor p27kip1, usually expressed later in G1, also gradually accumulated after PS-341 treatment. BAY 11 treatment did not affect p21 or p27 in MCL cells (Fig. 5B).
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To investigate whether PS-341 or BAY 11 could be inhibiting MCL cell growth by inducing cell death, we treated Mino MCL cells with 50 nM PS-341 for 6, 12, and 24 h or with 5 µM BAY 11 for 6 and 20 h and assayed for apoptosis. These experiments show that >75% of the MCL cells underwent apoptosis after 24 h of PS-341 treatment or 20 h of BAY 11 treatment (Fig. 6A). To verify that these cells actually underwent apoptosis, we measured the generation of caspase 3 activity. Mino MCL cells treated with PS-341 or BAY 11 activate caspase 3 activity after 12 h of treatment, and the caspase 3 activity was blocked with pretreatment of a caspase 3 inhibitor (DEVD) but not with a caspase 1 inhibitor (VAD) (Fig. 6B). In addition, a known caspase substrate, poly(ADP-ribose) polymerase, was cleaved after PS-341 or BAY 11 treatment (Fig. 6C).
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We next determined whether PS-341 or BAY 11 treatment in MCL cells involves bcl-2 family proteins in the induction of apoptosis. As shown in Fig. 7A, PS-341 or BAY 11 treatment of Mino cells down-regulated the protein expression of bcl-xL and bfl/A1. We also evaluated two other members of the bcl-2 family proteins: bcl-2, an apoptosis antagonist, and bax, an apoptosis agonist. Interestingly, PS-341 or BAY 11 treatment in Mino cells cleaves the bcl-2 protein from a 29-kDa product to a 22-kDa product, suggesting that these agents may induce apoptosis by inactivating the cell death antagonist or by producing a proapoptotic form of bcl-2 (Fig. 7A). Bax protein expression increased as treatment of PS-341 duration increased, whereas BAY 11 has little or no effect on bax.
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Inhibition of constitutive NF-B by the dominant-negative IB
Next, we wished to determine whether the specific inhibition of NF-B is sufficient to block cell growth in MCL cells. The super-repressor form of IB (pCMV-IB or pCMV-IBM), when overexpressed, binds to NF-B and prevents it from translocating to the nucleus. Unlike pCMV-IB, pCMV-IBM binds to NF-B but cannot be phosphorylated on the basis of alanine substitution for serines 32 and 36 as dominant negative, thereby blocking the NF-B signaling pathway (29). As shown in Fig. 8A, transient transfection of the DN-IB in Mino MCL cells suppresses constitutive NF-B expression. Suppression of NF-B by the DN-IBM also leads to inhibition of MCL cell viability (Fig. 8B). These results suggest that NF-B inhibition is linked to the antiproliferative effects in MCL cells.
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To this point, we have established that the transcription factor NF-B is an important factor in cell survival in MCL cell lines. Next, we examined the effect of PS-341 or BAY 11 on NF-B expression and apoptosis on primary MCL cells. As shown in Fig. 9A, PS-341 or BAY 11 treatment suppresses constitutive NF-B expression in T cell-depleted primary MCL cells derived from two patients (patients 1 and 2). Control and treated cells were also examined for apoptosis by annexin/propidium iodide staining and flow cytometric analysis. As shown in Fig. 9B, the apoptotic cell population increased from 30% (control) to 87 and 57% after PS-341 and BAY 11 treatments, respectively, in patient 1 and from 42% (control) to 86 and 78% after PS-341 and BAY 11 treatments, respectively, in patient 2. These results demonstrate that both of these NF-B inhibitors, PS-341 and BAY 11, are effective not only in MCL cells from cell lines but also effective in primary MCL cells.
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Discussion |
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B has been demonstrated in a variety of other human malignancies, including pancreatic cancer, colon cancer, breast cancer, T cell leukemia, Hodgkin’s lymphoma, and large B cell, NHL-B (30, 31, 32, 33, 34). In this study, we demonstrate that MCL cells from two established cell lines and from freshly obtained MCL patient specimens constitutively express activated NF-B (Fig. 1A). The molecular basis of constitutive NF-B activation in many malignant cells is still unclear, but the constitutive expression of NF-B is virtually always associated with the parallel constitutive expression of another important transcription factor, AP-1, that together are thought to mediate neoplastic transformation in a number of experimental oncogenesis systems (35). These findings suggest that constitutive activation of NF-B and AP-1 in aggressive B cell neoplasms could be important participants in whatever the oncogenic event(s) are that result in malignant transformation in these tumors. Our studies also suggest that constitutive activation of NF-B may also serve as a surrogate marker for malignancy in MCL (as well as in the other B cell malignancies that display this activity), which may be valuable in assessing the effectiveness of potential therapeutic agents. Recently, we have shown that in another aggressive NHL-B, diffuse large B cell lymphoma, that "ectopic" expression of CD154 by the lymphoma cells initiated NF-B activation through an anchored CD40 signalosome in cell membrane lipid rafts (34). Our preliminary data have shown that MCL cells may also use similar mechanism(s) for NF-B activation (L. V. Pham, A. T. Tamayo, L. C. Yoshimura, R. J. Ford, et al., manuscript in preparation). Constitutive NF-B activation in MCL cells is of particular importance because it implies that the cells use NF-B for constant cycling through the cell cycle and to maintain MCL cell survival through antiapoptotic members of the bcl-2 family. Our finding that agents with known inhibitory effects targeting NF-B inhibit the growth of MCL cells in vitro suggests that inhibition of NF-B can alter continuous neoplastic cell cycling as well as MCL tumor cell survival.
Our data show that NF-B inactivation with the proteasome inhibitor PS-341 or the pIB inhibitor BAY 11 leads to G1 cell cycle arrest in MCL cells. The impaired cell cycle progression following NF-B inactivation was associated with the reduction of cyclin D1, but not cyclin D2 or D3. In MCL cells, cyclin D1 (bcl-1, PRAD 1) is a well-described protooncogene product, occurring as a result of gene dysregulation, and enforced overexpression due to t(11;14) chromosomal translocation. This nonrandom chromosomal anomaly is considered by most clinicians and investigators as the molecular signature of this B cell lymphoma (36). The t(11;14) translocation places the cyclin D1 gene under the control of the strong heterologous Ig H chain (IgH) enhancer, presumably resulting in unrestrained production of cyclin D1 protein (37). Cyclin D1 is also, however, a target gene of the transcription factor NF-B(38) that under the constitutive activation of NF-B is maintained in the expressed state. In MCL cells, ectopic cyclin D1 is overexpressed, as normal lymphocytes usually express cyclin D2 and/or D3 (but not D1), that are required for G1 traversal, in concert with cyclin-dependent kinases 4 and 6, resulting in the phosphorylation of Rb before subsequent entry into S phase (39). It has been unclear however whether in MCL overexpressed cyclin D1 plays a direct or facilitative role in driving (or even streamlining) G1 traversal by possibly replacing cyclin D2 and/or D3, as has been previously hypothesized (40). Alternatively, it has been argued that cyclin D1 is accompanied by a down-regulation in cyclin D3 and is not related to the proliferative activity of the MCL cells (41). Our studies however indicate that the expression of ectopic cyclin D1 is actually involved cell cycle progression and that its expression in MCL as a result of t(11;14), is down-regulated by both PS-341 and Bay 11, resulting in G1 arrest. The cyclin-dependent kinase inhibitors p21waf1 and p27kip1 are substrates of the proteasome that accumulates after proteasomal inhibition (42, 43). Large B cell lymphomas and MCL cells generally show low levels of p21 and p27 due to rapid proteasomal degradation (44). Consistent with these findings, we have shown that both p21 and p27 proteins are up-regulated after PS-341 treatments, whereas, both p21 and p27 proteins are not affected by BAY 11 treatments. These results suggest that NF-B inhibition-mediated G1 cell cycle arrest may not be involved with the cyclin-dependent kinase inhibitors p21 or p27.
A number of reports have demonstrated that NF-B activation can maintain tumor cell viability and that inhibiting NF-B activity alone can be sufficient to induce cell death (45, 46, 47). PS-341 and BAY 11 were recently reported to induce apoptosis via NF-B inhibition in a variety of tumor cells (22, 48). In MCL cells, PS-341 and BAY 11 appear to affect cell viability indirectly not only by inhibiting bcl-2 family members, including bfl1/A1 and bcl-xL, that are regulated at the transcriptional level by NF-B (49, 50, 51), but also through its activity in the mitochondria involving bcl-2 cleavage and cytochrome c release. This result suggests that initiation of caspase 3 activation appears as a result of bcl-xL and bfl/A1 down-regulation, because either can suppress cytochrome c release (52).
A recent study suggested that PS-341 treatment of cells from another human B cell neoplasm, multiple myeloma, appears to produce apoptosis without affecting bcl-2 or bax (53). In MCL, however, we show that PS-341 or BAY 11 cleaves the 29-kDa bcl-2 protein to a 22-kDa product by activating caspase 3. Our studies agree with previous studies that bcl-2 can be cleaved by caspase 3 activation, transforming it from an antiapoptotic protein into a proapoptotic protein (27, 28). How the bcl-2 protein functions as a proapoptotic protein is still unknown. One possibility is that the BH4 domain of bcl-2 stabilizes a voltage-dependent anion channel in the mitochondria, preventing the release of cytochrome c, a critical molecule that binds to Apaf1 and caspase 9 to form an apoptosome that leads to the activation of caspase 3 and, subsequently, to apoptosis (54). Another possibility is that when the BH4 domain is cleaved, the 22-kDa product of bcl-2 may heterodimerize with bax or allow bax to homodimerize to release cytochrome c, as both bcl-2 and bax are capable of creating pores in an artificial membrane (55). The cleaving of bcl-2 seems to be an amplification mechanism for facilitating the apoptotic pathway in MCL cells, as bcl-2 cleavage occurs only late in apoptosis.
The expression of NF-B as well as the effect of the proteasome inhibitor PS-341 or pIB inhibitor BAY 11 have not yet been established in MCL. This is the first demonstration that NF-B is constitutively active in MCL cells and that either PS-341 or BAY 11, two NF-B inhibitory agents, can suppress NF-B expression and induce apoptosis, both in MCL cell lines and in primary MCL cells. In fact, Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies has been conducted and has shown promising results, especially against refractory multiple myeloma and NHL (56).
In summary, this study describes an important aberrant signaling pathway involved with constitutive activation of NF-B that maintains the growth and survival of MCL cells in vitro. This study also explores the antitumor mechanism(s) of PS-341 and BAY 11, that theoretically should target and affect the NF-B pathway, and provides supporting evidence indicating that these agents (or other agents with similar mechanisms of action) may also be useful as potential therapeutic agents for MCL.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Richard J. Ford, Department of Hematopathology, Box 54, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston TX 77030. E-mail address: rford{at}mdanderson.org
3 Abbreviations used in this paper: MCL, mantle cell lymphoma; NHL, non-Hodgkin’s lymphoma; DN, dominant negative; DEVD, acetyl-Asp-Glu-Val-Asp; VAD, Val-Ala-Asp.
Received for publication January 27, 2003. Accepted for publication April 25, 2003.
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E. Ortega-Paino, J. Fransson, S. Ek, and C. A. K. Borrebaeck Functionally associated targets in mantle cell lymphoma as defined by DNA microarrays and RNA interference Blood, February 1, 2008; 111(3): 1617 - 1624. [Abstract] [Full Text] [PDF]
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H.-J. Zhou, L. V. Pham, A. T. Tamayo, Y.-C. Lin-Lee, L. Fu, L. C. Yoshimura, and R. J. Ford Nuclear CD40 interacts with c-Rel and enhances proliferation in aggressive B-cell lymphoma Blood, September 15, 2007; 110(6): 2121 - 2127. [Abstract] [Full Text] [PDF]
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M. Wang, L. Zhang, X. Han, J. Yang, J. Qian, S. Hong, F. Samaniego, J. Romaguera, and Q. Yi Atiprimod inhibits the growth of mantle cell lymphoma in vitro and in vivo and induces apoptosis via activating the mitochondrial pathways Blood, June 15, 2007; 109(12): 5455 - 5462. [Abstract] [Full Text] [PDF]
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R. J. Ford, L. Shen, Y. C. Lin-Lee, L. V. Pham, A. Multani, H.-J. Zhou, A. T. Tamayo, C. Zhang, L. Hawthorn, J. K. Cowell, et al. Development of a murine model for blastoid variant mantle-cell lymphoma Blood, June 1, 2007; 109(11): 4899 - 4906. [Abstract] [Full Text] [PDF]
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S. J. Strauss, K. Higginbottom, S. Juliger, L. Maharaj, P. Allen, D. Schenkein, T. A. Lister, and S. P. Joel The Proteasome Inhibitor Bortezomib Acts Independently of p53 and Induces Cell Death via Apoptosis and Mitotic Catastrophe in B-Cell Lymphoma Cell Lines Cancer Res., March 15, 2007; 67(6): 2783 - 2790. [Abstract] [Full Text] [PDF]
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G. N.C. Chiu, L. A. Edwards, A. I. Kapanen, M. M. Malinen, W. H. Dragowska, C. Warburton, G. G. Chikh, K. Y.Y. Fang, S. Tan, J. Sy, et al. Modulation of cancer cell survival pathways using multivalent liposomal therapeutic antibody constructs Mol. Cancer Ther., March 1, 2007; 6(3): 844 - 855. [Abstract] [Full Text] [PDF]
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J. An and M. B. Rettig Epidermal growth factor receptor inhibition sensitizes renal cell carcinoma cells to the cytotoxic effects of bortezomib Mol. Cancer Ther., January 1, 2007; 6(1): 61 - 69. [Abstract] [Full Text] [PDF]
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A Belch, C. Kouroukis, M Crump, L Sehn, R. Gascoyne, R Klasa, J Powers, J Wright, and E. Eisenhauer A phase II study of bortezomib in mantle cell lymphoma: the National Cancer Institute of Canada Clinical Trials Group trial IND.150 Ann. Onc., January 1, 2007; 18(1): 116 - 121. [Abstract] [Full Text] [PDF]
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M. Rudelius, S. Pittaluga, S. Nishizuka, T. H.-T. Pham, F. Fend, E. S. Jaffe, L. Quintanilla-Martinez, and M. Raffeld Constitutive activation of Akt contributes to the pathogenesis and survival of mantle cell lymphoma Blood, September 1, 2006; 108(5): 1668 - 1676. [Abstract] [Full Text] [PDF]
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Y.-C. Lin-Lee, L. V. Pham, A. T. Tamayo, L. Fu, H.-J. Zhou, L. C. Yoshimura, G. L. Decker, and R. J. Ford Nuclear Localization in the Biology of the CD40 Receptor in Normal and Neoplastic Human B Lymphocytes J. Biol. Chem., July 7, 2006; 281(27): 18878 - 18887. [Abstract] [Full Text] [PDF]
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M. C. Ralstin, E. A. Gage, M. T. Yip-Schneider, P. J. Klein, E. A. Wiebke, and C. M. Schmidt Parthenolide Cooperates with NS398 to Inhibit Growth of Human Hepatocellular Carcinoma Cells through Effects on Apoptosis and G0-G1 Cell Cycle Arrest Mol. Cancer Res., June 1, 2006; 4(6): 387 - 399. [Abstract] [Full Text] [PDF]
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L. Fu, Y.-C. Lin-Lee, L. V. Pham, A. Tamayo, L. Yoshimura, and R. J. Ford Constitutive NF-{kappa}B and NFAT activation leads to stimulation of the BLyS survival pathway in aggressive B-cell lymphomas Blood, June 1, 2006; 107(11): 4540 - 4548. [Abstract] [Full Text] [PDF]
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S. J. Strauss, L. Maharaj, S. Hoare, P. W. Johnson, J. A. Radford, S. Vinnecombe, L. Millard, A. Rohatiner, A. Boral, E. Trehu, et al. Bortezomib Therapy in Patients With Relapsed or Refractory Lymphoma: Potential Correlation of In Vitro Sensitivity and Tumor Necrosis Factor Alpha Response With Clinical Activity J. Clin. Oncol., May 1, 2006; 24(13): 2105 - 2112. [Abstract] [Full Text] [PDF]
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A. Sors, F. Jean-Louis, C. Pellet, L. Laroche, L. Dubertret, G. Courtois, H. Bachelez, and L. Michel Down-regulating constitutive activation of the NF-{kappa}B canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis Blood, March 15, 2006; 107(6): 2354 - 2363. [Abstract] [Full Text] [PDF]
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A. Tanaka, S. Muto, M. Konno, A. Itai, and H. Matsuda A New I{kappa}B Kinase {beta} Inhibitor Prevents Human Breast Cancer Progression through Negative Regulation of Cell Cycle Transition Cancer Res., January 1, 2006; 66(1): 419 - 426. [Abstract] [Full Text] [PDF]
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P. Perez-Galan, G. Roue, N. Villamor, E. Montserrat, E. Campo, and D. Colomer The proteasome inhibitor bortezomib induces apoptosis in mantle-cell lymphoma through generation of ROS and Noxa activation independent of p53 status Blood, January 1, 2006; 107(1): 257 - 264. [Abstract] [Full Text] [PDF]
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L. V. Pham, A. T. Tamayo, L. C. Yoshimura, Y.-C. Lin-Lee, and R. J. Ford Constitutive NF-{kappa}B and NFAT activation in aggressive B-cell lymphomas synergistically activates the CD154 gene and maintains lymphoma cell survival Blood, December 1, 2005; 106(12): 3940 - 3947. [Abstract] [Full Text] [PDF]
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A. Hachem and R. B. Gartenhaus Oncogenes as molecular targets in lymphoma Blood, September 15, 2005; 106(6): 1911 - 1923. [Full Text] [PDF]
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Y. Fernandez, M. Verhaegen, T. P. Miller, J. L. Rush, P. Steiner, A. W. Opipari Jr., S. W. Lowe, and M. S. Soengas Differential Regulation of Noxa in Normal Melanocytes and Melanoma Cells by Proteasome Inhibition: Therapeutic Implications Cancer Res., July 15, 2005; 65(14): 6294 - 6304. [Abstract] [Full Text] [PDF]
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A. Tanaka, M. Konno, S. Muto, N. Kambe, E. Morii, T. Nakahata, A. Itai, and H. Matsuda A novel NF-{kappa}B inhibitor, IMD-0354, suppresses neoplastic proliferation of human mast cells with constitutively activated c-kit receptors Blood, March 15, 2005; 105(6): 2324 - 2331. [Abstract] [Full Text] [PDF]
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A. Goy, A. Younes, P. McLaughlin, B. Pro, J. E. Romaguera, F. Hagemeister, L. Fayad, N. H. Dang, F. Samaniego, M. Wang, et al. Phase II Study of Proteasome Inhibitor Bortezomib in Relapsed or Refractory B-Cell Non-Hodgkin's Lymphoma J. Clin. Oncol., February 1, 2005; 23(4): 667 - 675. [Abstract] [Full Text] [PDF]
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O. A. O'Connor, J. Wright, C. Moskowitz, J. Muzzy, B. MacGregor-Cortelli, M. Stubblefield, D. Straus, C. Portlock, P. Hamlin, E. Choi, et al. Phase II Clinical Experience With the Novel Proteasome Inhibitor Bortezomib in Patients With Indolent Non-Hodgkin's Lymphoma and Mantle Cell Lymphoma J. Clin. Oncol., February 1, 2005; 23(4): 676 - 684. [Abstract] [Full Text] [PDF]
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 M. Lun, P. L. Zhang, N. Siegelmann-Danieli, T. M. Blasick, and R. E. Brown Intracellular Inhibitory Effects of Velcade Correlate with Morphoproteomic Expression of Phosphorylated-Nuclear Factor-{kappa}B and p53 in Breast Cancer Cell Lines Ann. Clin. Lab. Sci., January 1, 2005; 35(1): 15 - 24. [Abstract] [Full Text] [PDF]
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M. Lun, P. L. Zhang, P. K. Pellitteri, A. Law, T. L. Kennedy, and R. E. Brown Nuclear Factor-kappaB Pathway as a Therapeutic Target in Head and Neck Squamous Cell Carcinoma: Pharmaceutical and Molecular Validation in Human Cell Lines Using Velcade and siRNA/NF-{kappa}B. Ann. Clin. Lab. Sci., January 1, 2005; 35(3): 251 - 258. [Abstract] [Full Text] [PDF]
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R. Lahav, M.-L. Suva, D. Rimoldi, P. H. Patterson, and I. Stamenkovic Endothelin Receptor B Inhibition Triggers Apoptosis and Enhances Angiogenesis in Melanomas Cancer Res., December 15, 2004; 64(24): 8945 - 8953. [Abstract] [Full Text] [PDF]
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B. Zheng, G. V. Georgakis, Y. Li, A. Bharti, D. McConkey, B. B. Aggarwal, and A. Younes 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-{kappa}B Mutations or Activation of the CD30, CD40, and RANK Receptors Clin. Cancer Res., May 1, 2004; 10(9): 3207 - 3215. [Abstract] [Full Text] [PDF]
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Y. Dai, X.-Y. Pei, M. Rahmani, D. H. Conrad, P. Dent, and S. Grant Interruption of the NF-{kappa}B pathway by Bay 11-7082 promotes UCN-01-mediated mitochondrial dysfunction and apoptosis in human multiple myeloma cells Blood, April 1, 2004; 103(7): 2761 - 2770. [Abstract] [Full Text] [PDF]
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) and DB (
) cells were cultured in the presence of increasing doses of PS-341 (A, 0–50 nM), BAY 11 (B, 0–5 µM), TGF-
