Cancer Research SABCS Protein Translation and Cancer
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boitano, A.
Right arrow Articles by Opipari, A. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boitano, A.
Right arrow Articles by Opipari, A. W., Jr.
[Cancer Research 63, 6870-6876, October 15, 2003]
© 2003 American Association for Cancer Research

Regular Articles

The Proapoptotic Benzodiazepine Bz-423 Affects the Growth and Survival of Malignant B Cells1

Anthony Boitano, Jonathan A. Ellman, Gary D. Glick and Anthony W. Opipari, Jr.2

Departments of Chemistry [A. B., G. D. G.] and Obstetrics and Gynecology [A. W. O.] and Graduate Program in Immunology [G. D. G.], University of Michigan, Ann Arbor, Michigan 48109, and Department of Chemistry, University of California-Berkeley, 724 Latimer Hall, Berkeley, California 94720-1460 [J. A. E.]


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bz-423 is a novel proapoptotic 1,4-benzodiazepine that induces cell death via a superoxide signal. Previous work has shown that Bz-423 ameliorates disease in animal models of systemic lupus erythematosus that also have features of lymphoproliferative disease. Here we describe the effects of Bz-423 against a group of malignant B-cell lines derived from Burkitt’s lymphoma. These experiments demonstrate that Bz-423 has cytotoxic activity against all B-cell lines tested, regardless of EBV status or Bcl-2 and Bcl-xL expression levels. In addition to its cytotoxic properties, we found that Bz-423 is also a potent antiproliferative agent that induces a G1-phase arrest independent of p53. Mechanistically, both the cytotoxicity and growth arrest are mediated by increased reactive oxygen species levels and appear independent of binding to the peripheral benzodiazepine receptor. This work further defines the biological activities of Bz-423 that are consistent with those of other compounds in clinical development for antineoplastic therapies.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Benzodiazepines are widely used therapeutic agents best known for their action as anxiolytics, anticonvulsants, antispasmodics, and hypnotics. Their physiological effects are mediated by binding to the central benzodiazepine receptor, which is associated with the {gamma}-aminobutyric acid receptor in the central nervous system (1) . Benzodiazepines also bind to an 18-kDa protein known as the PBR3 (2 , 3) . The PBR is present in nearly all cell types and is primarily located in mitochondria, but it can also be found on the plasma membrane and within the nucleus (4 , 5) . In mitochondria, the PBR forms part of the PTP, a multiprotein complex that forms junctions between the inner and outer membranes (6) . The PBR has been implicated in numerous biological processes (e.g., proliferation, calcium channel activity, immune responses, porphyrin transport, heme biosynthesis, anion transport, and regulation of both steroid biosynthesis and phosphorylation) and has been extensively characterized pharmacologically and biochemically (7) . Nevertheless, the precise function of the PBR remains an enigma (8) .

We previously identified a novel 1,4-benzodiazepine (designated Bz-423; Fig. 1Citation ) that induces cellular responses independent of the central benzodiazepine receptor or PBR. Bz-423 possesses significant and selective cytotoxicity toward lymphocytes in vivo (9 , 10) . The mechanism of cytotoxicity involves a rapid increase in intracellular superoxide, and this ROS response is essential for inducing the subsequent apoptotic process characterized by cytochrome c release, mitochondrial depolarization, and caspase activation. Cell fractionation and inhibitor experiments reveal that the superoxide response results from the interaction of Bz-423 with a target in mitochondria that is not the PBR (9) . To examine the therapeutic potential of this compound, Bz-423 was administered to mice with autoimmune disease dominated by lymphoproliferative changes. In the (NZB x NZW)F1 (NZB/W) mouse model, Bz-423 eliminated the pathological expansion of GC B cells (9) . In MRL/MpJ-Faslpr (MRL-lpr) mice, where peripheral autoreactive T cells drive a lupus-like process, Bz-423 caused a shift in the Th1/Th2 cell balance and decreased specific T-cell subsets (10) . In both models, treatment decreased autoimmune damage to the kidneys and other organs and increased apoptosis in secondary lymphoid tissues. The reductions in pathogenic lymphocytes in Bz-423-treated mice were consistent with the ability of this compound to kill lymphoid cells in vitro. However, because lymphoid cell homeostasis is a balance between proliferation and cell death, an antiproliferative response to Bz-423 might also contribute to disease improvement in these models.



View larger version (14K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 1. Structure of Bz-423.

 
Other benzodiazepines have been shown to have antiproliferative activity, and this effect has been correlated with PBR binding characteristics only in some cases (11, 12, 13) . However, because the antiproliferative effects of PBR ligands occur only at concentrations dramatically above those necessary to saturate PBR binding (e.g., 1000-fold), it is likely that binding to the PBR does not mediate this response (14 , 15) . For example, PK11195 [1-(2-chlorophenyl)-N-(1-methylpropyl)-3-isoquinolinecarboxamide] and 4-ClDz bind to the PBR with nanomolar dissociation constants, but the antiproliferative effects of these agents are usually observed at micromolar concentrations. In addition, genetically silencing PBR expression does not alter normal cellular proliferation (16) . Therefore, the mechanistic basis of the antiproliferative effects of benzodiazepines remains uncertain.

Because Bz-423 selectively targeted pathologically expanded GCs in NZB/W mice and kills Ramos cells derived from BL, a disease that originates from the malignant conversion GC B cells, we hypothesized that Bz-423 would have activity against other BL-derived cells. BL occurs as a EBV-associated malignancy among Africans, as a common neoplasm in HIV-infected patients, and in sporadic cases not associated with EBV. All BL cells carry reciprocal chromosomal translocations that activate the c-myc oncogene through juxtaposition to one of the immunoglobulin loci and many carry mutations in the p53tumor suppressor gene (17 , 18) . In vitro, the expression of EBV-latent gene products in BL cells is associated with a lymphoblastoid phenotype, the expression of cellular survival genes, and increased resistance to apoptosis (19, 20, 21) . Although intensive chemotherapy results in long-term survival for nearly 70% of children with BL, survival for adults remains less than 25%, and innovative therapeutic approaches are an urgent priority for this disease (22) .

In this report, we extend our characterization of Bz-423, demonstrating and comparing its cytotoxic function among a group of malignant B-cell lines, including cells transfected to express high-levels of the antiapoptotic proteins Bcl-2 and Bcl-xL. In addition to cytotoxic activity, we demonstrate potent antiproliferative effects of Bz-423 that are independent of cell death. In fact, Bz-423 is significantly more effective at inhibiting cell growth than other antiproliferative benzodiazepines and PK11195. Mechanistic experiments demonstrate that Bz-423-induced ROS are essential for growth inhibition and that the magnitude of this response may arbitrate between death and G1 growth arrest.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents.
Bz-423 was synthesized as described previously and dissolved in aqueous DMSO as the vehicle (23) . DMSO was present at a final concentration of 0.5% (v/v) in all experiments.

Cells and Culture.
Ramos, ST486, Raji, Daudi, Namalwa, and CA46 were purchased from American Type Culture Collection (Manassas, VA). Cells (106 cells/ml) were maintained in RPMI 1640 (Mediatech, Herndon, VA) supplemented with penicillin (100 units/ml), streptomycin (100 µg/ml), L-glutamine (290 µg/ml), and heat-inactivated FBS (HyClone, Logan, UT). Viability experiments were conducted in this same media at 37°C in a CO2 (5%) atmosphere.

Transfection and Selection of Cells Expressing Bcl-2 and Bcl-xL.
Expression plasmids for Bcl-2 and Bcl-xL were provided by G. Nunez (24) . Ramos cells (107) were transfected with pSFFVneo (vector only control), pSFFVneo-Bcl-2, and pSFFVneo-Bcl-xL by electroporation. Stably transfected cells were selected and maintained in complete media containing Geneticin (1.5 mg/ml; Life Technologies, Inc., Rockville, MD).

Assessment of Viability and Cell Number.
Cells were incubated with PI (1 µg/ml, 15 min), and viability was assessed by flow cytometry based on PI exclusion (25) . Data were collected and analyzed using a FACSCalibur (BD Bioscience, San Diego, CA). For each sample, 10,000 events were recorded, and the data were analyzed excluding aggregates. In experiments to determine growth, the number of viable and dead cells was determined after indicated treatments using trypan blue (0.4% w/v) staining, and cells were quantified by microscopy using a hemocytometer. In experiments where Bz-423 was washed out, cells were treated with this compound or vehicle for 3 days, washed by centrifugation and three media exchanges, and then replated in fresh media at 100,000 cells/ml.

Titration of BSA to Culture Media.
Certified FBS used in this study contains BSA at a concentration of 27 mg/ml, meaning an approximate BSA concentration of 2.7 mg/ml in culture media containing 10% FBS (v/v). To achieve a total BSA concentration equivalent to that found in media containing 5%, 7.5%, or 10% FBS, media with 2% FBS were supplemented with purified BSA (Sigma, St. Louis, MO) added at 0.81, 1.49, and 2.16 mg/ml, respectively. Before use, media were sterilized by filtration.

Monitoring Cell Divisions using PKH67.
PKH67 was used according to the manufacturer’s protocol (Sigma). Briefly, cells (2 x 107) were washed in serum-free media and suspended in PKH67 (1 ml of a 0.5 µM solution) for 5 min at room temperature. Cells were then washed suspended in complete media at 0.5 x 106 cells/ml. Immediately after staining, and after indicated treatment for 3 days, fluorescence intensity was determined by flow cytometry on a FACSCalibur. Analysis excluded any dead cells based on forward/side scatter characteristics.

Detection of Intracellular ROS.
DCHF-DA (Molecular Probes, Eugene, OR) was prepared as a 10 mM stock solution in DMSO before each use. Cells (106 cells/ml) were loaded (30 min, 37°C) with DCHF-DA (100 µM), washed by centrifugation, and resuspended in fresh media before treatment. After the indicated treatments, the fluorescence of the oxidized product DCF was monitored on a FACSCalibur.

Statistical Analysis.
Statistical significance was assessed using Student’s t test unless otherwise noted. Ps are two-tailed, and data are given as mean ± SD of the mean.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Bz-423 Kills BL-Derived B-Cell Lines.
The apoptotic properties of Bz-423 were first evaluated using Ramos cells, a human malignant B-cell clone derived from BL that shares many characteristics with activated GC B cells. Other benzodiazepines are significantly less cytotoxic than Bz-423 despite binding more tightly to the PBR (Table 1)Citation . Other BL cell lines were also sensitive to Bz-423 (Fig. 2, A and B)Citation . The sensitivity of the different cell lines to Bz-423-induced death was fairly similar, with the LC50 ranging from 6 µM (ST486) to 10 µM (Namwala cells). To determine the potential impact of survival genes encoded or induced by latent EBV infection on the effectiveness of Bz-423, these experiments included EBV-negative cells and EBV-positive cells known to express latent viral gene products. The BL cells that do not contain EBV (Fig. 2ACitation , Ramos, CA46, and ST486) are only slightly more sensitive than the group III EBV-positive cells (Fig. 2BCitation , Daudi, Namwala, and Raji). Experiments assessing colony formation by Ramos and Raji cells in methylcellulose-based semisolid media for 1 week after treatment with Bz-423 for 24 h confirmed that treatment dose-dependently reduces clonogenic survival (data not shown).


View this table:
[in this window]
[in a new window]

  		Table 1 Comparison of Bz-423 with other benzodiazepines and high-affinity ligands of the PBR

Data for PK11195, 4-ClDz, and clonazepam come from Ref. 61 . IC50 is the concentration of each compound required to inhibit 50% binding of radiolabeled PK11195 to the PBR. LC50 is the concentration required to kill 50% of the cells in media with 2% FBS, determined by PI exclusion. GI50 is the concentration that reduces growth after 3 days in media with 10% FBS to 50% of control as determined by cell counting.

 


View larger version (26K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 2. Bz-423 has cytotoxic activity in BL cell lines. Dose-dependent killing in media with 2% FBS was determined by PI exclusion. A, ST486 ({blacksquare}), Ramos ({bullet}), and CA46 ({blacktriangleup}) cells; B, Daudi ({bullet}), Raji ({blacksquare}), and Namalwa ({blacktriangleup}) cell lines. C, Ramos cells stably transfected as indicated express Bcl-2 or Bcl-xL at levels substantially greater than wild-type or vector control-transfected cells. D, cell death determined after 24 h of exposure to CDDP in media with 2% FBS for nontransfected Ramos cells ({bullet}), vector control-transfected cells ({blacksquare}), Bcl-2-expressing cells ({circ}), and Bcl-xL-expressing cells ({square}). E, cell death after 24 h caused by Bz-423 using the same cells and conditions as in C.

 
Previous work has established that Bz-423-induced cell death is executed through mitochondria-dependent events beginning with superoxide production within 1 h of treatment. Superoxide production is the first detectable signal that triggers subsequent collapse of the mitochondrial electrochemical gradient and cell death events consistent with opening of the PTP (9) . To determine whether the antiapoptotic proteins Bcl-2 and Bcl-xL, which contribute to the regulation of mitochondrial pore function and cellular redox balance, limited the effectiveness of Bz-423, Ramos cells were generated that overexpress these gene products (Fig. 2C)Citation . The gene product-overexpressing cells acquired significant resistance to the toxic effects of CDDP (Fig. 2D)Citation . In contrast, the gene product-overexpressing cells remained sensitive to Bz-423, with only a modest increase in the LC50 (Fig. 2ECitation ; Table 2Citation ). Thus, the death mechanism engaged by Bz-423 is sufficiently robust to overcome resistance from high-level expression of these antiapoptotic genes that produce extreme drug resistance to conventional agents.


View this table:
[in this window]
[in a new window]

  		Table 2 MnTBAP modulates Bz-423-induced death and antiproliferative responses

LC50 was determined in media with 2% FBS, by PI exclusion. GI50 is based on growth inhibition after 3 days in media with 10% FBS and was determined by cell counting.

 
Binding to Serum Albumin Modulates the Cytotoxic Effects of Bz-423.
It is well established that nonspecific binding to serum albumin regulates the bioavailability of benzodiazepines by altering the free concentration of drug (26) . In tissue culture media containing 10% (v/v) FBS with 10 µM Bz-423, nearly 99% of the compound is bound to BSA (data not shown). Therefore, initial studies with Bz-423 were performed with culture media containing 2% (v/v) FBS. However, media with less serum have lower levels of growth factors and other components that could also affect cell survival. To investigate the role of culture conditions on the activity of Bz-423, purified BSA was added to media containing 2% FBS in amounts to achieve a total BSA concentration equivalent to that contained in media with 5%, 7.5%, or 10% FBS. Titrating in BSA shifted the Bz-423 killing curve. When BSA was added to the culture medium, to yield the total amount contained in media supplemented with 10% FBS, the dose-response curve overlapped the dose response in media actually containing 10% FBS (Fig. 3)Citation . From these results, we conclude that binding to BSA accounts for the lower cytotoxicity (higher LC50) of Bz-423 observed in culture media with 10% versus 2% FBS.



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 3. Serum albumin limits the bioavailability of Bz-423. Dose-dependent cytotoxicity of Bz-423 against Ramos cells was determined in culture media with 2% FBS containing final BSA concentrations of 0.54 mg/ml ({blacksquare}), 1.35 mg/ml ({triangleup}), 2.03 mg/ml ({circ}), 2.7 mg/ml ({square}), or 10% FBS that also contains 2.7 mg/ml BSA ({bullet}). Viability after 24 h was determined by PI exclusion.

 
Higher Serum Conditions Reveal the Antiproliferative Effects of Bz-423.
To examine the effects of Bz-423 on proliferation, Ramos cells cultured in media with 10% FBS were continuously exposed to Bz-423 (20 µM) or vehicle control and analyzed over 3 days to determine the total number of live and dead cells by vital dye microscopy. Bz-423 dramatically reduced the increase in cell number (growth) over 3 days without causing a significant increase in dead cells over the same time (Fig. 4)Citation . To be certain that this effect on cell number truly resulted from reduced proliferation versus increased cell death balanced by ongoing proliferation, cell divisions were monitored using the membrane-specific fluorescent compound PKH67. An important feature of PKH67 is that after labeling a cell, the probe is partitioned equally between daughter cells upon cell division, making it possible to quantify divisions based on fluorescence intensity (27) . PKH67 itself does not affect the viability or proliferation of Ramos cells (data not shown). To determine how Bz-423 affects cell divisions, cells were labeled with PKH67, treated with Bz-423 for 3 days, and analyzed by flow cytometry. Over this time frame, the maximal concentration of Bz-423 used in these experiments (20 µM) killed <10% of cells. Mean fluorescence intensity (gating on live cells) was compared for cells immediately after labeling (e.g., time 0) with cells treated as indicated for 3 days after dye labeling. As seen in Fig. 5Citation , the decrease in mean fluorescence intensity after 3 days from the time 0 value is greater in control cells than in cells treated with Bz-423, and in additional dose-response experiments, the magnitude of the difference from control is directly proportional to the concentration of Bz-423 used (data not shown). Thus, Bz-423 dose-dependently prevented the decrease in PKH67 fluorescence intensity, demonstrating that it blocks proliferation. In sharp contrast, the PKH67 fluorescence of cells after treatment with an equal concentration (20 µM) of clonazepam or PK11195 is indistinguishable from that of vehicle control-treated cells (Fig. 5)Citation . Although 4-ClDz also blocks proliferation, it is significantly less potent than Bz-423. These data are consistent with the results of cell counting experiments summarized in Table 1Citation , where the drug concentration reducing growth by 50% (GI50) for Bz-423 is significantly less than that for other benzodiazepines and PK11195.



View larger version (11K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 4. Bz-423 inhibits growth of Ramos cells in culture. Viable ({square}) and nonviable ({blacksquare}) Ramos cell number after 24, 48, and 72 h of treatment with vehicle control or Bz-423 (20 µM) as indicated. Experiment was performed in media containing 10% FBS. Cells were quantified by trypan blue staining and cell counting. The results show mean values of three separate determinations. P < 0.05 for the differences between Bz-423 and control for each end point.

 


View larger version (23K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 5. Bz-423 reduces Ramos cell proliferation. Cell proliferation after 72 h in media containing 10% FBS and either vehicle control (blue histograms) or experimental agent (red histograms) including 20 µM Bz-423, 20 µM 4-ClDz, 20 µM PK11195, and 20 µM clonazepam. Cell proliferation is reflected by the change in the mean fluorescence of PKH67. White histograms shows fluorescence intensity immediately after staining. The results here are representative of five separate experiments.

 
We next used a clonogenic assay to determine whether growth inhibition is permanent or whether it is reversed when the compound is removed from cells. Ramos cells were treated with vehicle control or Bz-423 (10 and 20 µM) for 3 days, during which time cell number was monitored daily by staining with trypan blue and counting using a hemocytometer (Fig. 6A)Citation . After 72 h, cells were removed from culture, washed extensively, and replated at equal concentrations (1 x 105 cells/ml) in media without any compound or vehicle. During this washout phase, cell number was again determined daily, and, as indicated in Fig. 6ACitation , no differences in growth were observed between control cells and those treated previously with Bz-423. These results demonstrate that the antiproliferative effects of Bz-423 depend on the continuous presence of the drug, thus arguing that an irreversible genotoxic mechanism does not underlie its antiproliferative effects.



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 6. Growth inhibition is reversible and associated with G1 arrest. A, Ramos viable cell number during treatment with vehicle control ({bullet}), 10 µM Bz-423 ({blacksquare}), and 20 µM Bz-423 ({blacktriangleup}) for 1, 2, and 3 days determined by cell counting. After 3 days, cells were washed and replated without any treatment, and cell number was further monitored for 3 additional days. The experiment was performed in media containing 10% FBS, and data shown are the mean ± SD. Cell cycle distribution from cultures treated for 24 h with vehicle control (B) or 20 µM Bz-423 (C) in media containing 10% FBS is shown. Cell cycle assignment was based on DNA content detected with PI by flow cytometry.

 
To determine whether the antiproliferative effects involved arrest of the cell cycle at a specific checkpoint, cells treated with Bz-423 for 24, 48, and 72 h were analyzed for DNA content to reveal the distribution of cells within the cell cycle. By 24 h, treatment with Bz-423 caused a significant increase in cells with G1-G0 DNA content and corresponding reductions in S and G2 cells (Fig. 6, B and C)Citation . No increase in hypodiploid DNA, a marker of apoptosis, was observed under these treatment conditions. In the continuous presence of Bz-423, the G1-G0 arrest persisted for up to 72 h, the longest point at which this response was measured (data not shown). Together, these results demonstrate that Bz-423 blocks the proliferation of Ramos B cells independent of its cytotoxic effects through a mechanism that arrests the cell cycle at the G1 checkpoint.

ROS Are Involved in the Antiproliferative Response to Bz-423.
Because intracellular ROS are increased by Bz-423 as an early, necessary step in the cytotoxic response, we hypothesized that growth arrest may also be mediated by ROS. To test this possibility, ROS was measured using DCHF-DA in cells treated with Bz-423 in media with 10% FBS (28) . Similar to the data obtained in media with 2% FBS, treatment with Bz-423 leads to the dose-dependent production of ROS within 1 h (Fig. 7A)Citation . The fluorescent signal reflects the presence of superoxide because addition of the superoxide dismutase mimetic, MnTBAP (29) , abrogates the fluorescence change (Fig. 7B)Citation .



View larger version (16K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 7. Bz-423 induces a ROS response inhibited by MnTBAP. Ramos cells were treated for 1 h in media with 10% FBS. In A, ROS response to vehicle control (thin line) is compared with 20 µM Bz-423 (thick line). In B, the response to vehicle control (thin line) and 20 µM Bz-423 (thick line) in the presence of 100 µM MnTBAP is shown. ROS response is detected by measuring DCF fluorescence using flow cytometry.

 
To establish a functional role between Bz-423-induced ROS and growth arrest, cells were treated with MnTBAP before the addition of Bz-423. As seen in Table 2Citation , MnTBAP blocked the antiproliferative effects of Bz-423. In separate experiments, nonlymphoid cells (MCF-7 breast cancer line) transfected to express high levels of manganese superoxide dismutase were less sensitive to Bz-423-induced growth inhibition than control cells (data not shown). From these experiments, we conclude that ROS are essential for Bz-423-mediated growth arrest. We also tested whether Ramos cells expressing high levels of Bcl-2 or Bcl-xL were less sensitive to growth inhibition by Bz-423. These experiments were predicated on the observation that, in some cases, these two gene products have antioxidant properties (30 , 31) . However, in the model tested here, neither of these antiapoptotic proteins changed the antiproliferative response to Bz-423 (Table 2)Citation .

Because the antiproliferative actions of Bz-423 require its ongoing presence, we hypothesized that critical second messengers mediating growth arrest were likely persistently affected as well. When measured as a function of time during treatment under antiproliferative conditions (10% FBS), the ROS response demonstrated precisely this characteristic. An early burst of ROS was detected by a maximal increase of mean fluorescence intensity at 1 h, followed by sustained continuous elevation for periods exceeding 8 h (Fig. 8)Citation ; immediately after drug wash-out, the signal fell to control levels (data not shown).



View larger version (12K):
[in this window]
[in a new window]
[Download PPT slide]
  		Fig. 8. Bz-423 induces a sustained increase in ROS levels. ROS in Ramos cells treated with 10 µM Bz-423 ({blacksquare}) or 20 µM ({bullet}) in media containing 10% FBS as a function of time were monitored by DCF fluorescence. The results presented are the mean of five separate determinations. P < 0.05 for the differences between Bz-423 and control for each end point.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Bz-423 was identified previously on the basis of its ability to induce apoptosis in Ramos B lymphocytes, an EBV-negative cell line established from BL. BL is thought to originate from a GC B cell, and here we show that, like primary GC cells in NZB/W mice and Ramos cells, Bz-423 has proapoptotic effects against several BL cell lines. Some of these cell lines do not contain EBV (Ramos, ST486, and CA46) and maintain close phenotypic approximations of GC cells, including a tendency to easily undergo apoptosis to a variety of stimuli (32, 33, 34, 35, 36) . In contrast, those BL lines that contain EBV and express the viral latency-associated proteins (Daudi, Raji, and Namalwa) are resistant to apoptotic triggers, including calcium ionophores, serum deprivation, and IgM surface receptor cross-linking (19) . Bz-423 triggers apoptosis in both groups of cells with only a modest increase in LC50 observed for the group III EBV-positive compared with the EBV-negative lines. Therefore, the resistance mechanisms resulting from the expression of EBV latent proteins do not significantly affect the mechanism of Bz-423.

Mutation of the p53tumor suppressor gene is an important factor in the malignant transformation, progression, and therapeutic response to chemotherapy of BL (37 , 38) . In each of the cell lines tested, p53 function is abnormal. For example, in Ramos cells, one copy of the p53gene is deleted, and the remaining allele encodes a mutant protein that is incapable of activating transcription or mediating apoptosis (39) . Because Bz-423 is active in cells that express only mutant p53, its ability to induce apoptosis and inhibit cell proliferation is independent of p53 status.

The oncoproteins Bcl-2 and Bcl-xL can diminish therapeutic effectiveness of many cytotoxic drugs by preventing apoptosis (40 , 41) . Several mechanisms may be involved in the antiapoptotic effects of Bcl-2 and Bcl-xL. Paramount among these is their ability to keep the mitochondria PTP closed (42) . Although their exact role in BL is not clear, Bcl-2 may be necessary to block the apoptotic effects of deregulated c-myc expression, which is a hallmark of BL (43 , 44) . We tested whether high-level expression of Bcl-2 or Bcl-xL blocked Bz-423 killing and observed only a slight increase in the LC50 and no change in the ability of Bz-423 to induce ROS (data not shown). By way of comparison, cells overexpressing these gene products are completely resistant to CDDP. Collectively, these experiments indicate that Bz-423 circumvents an important group of drug resistance mechanisms.

The mechanisms of action mediating apoptosis in response to 4-ClDz and PBR ligands such as PK11195 are not well understood. Some studies have, however, associated the cytotoxicity of these agents with increases in ROS or inhibition of coupled mitochondrial respiration. Using HL60 and KG1A leukemia cells, Fennell et al. (45) have shown that PK11195 induces a ROS response required for the subsequent collapse of mitochondrial transmembrane gradient, which is itself necessary for the apoptotic response. 4-ClDz and PK11195 reduce cellular oxygen consumption and coupled mitochondrial respiration, both effects that are often linked to increased ROS production (12 , 46 , 47) . At concentrations substantially greater than their Kd values for binding to the PBR, both compounds also reduce oxygen consumption in isolated mitochondria under conditions similar to those in which we found that Bz-423 induces superoxide [i.e., respiratory state 3 (12) ]. These similarities suggest that cytotoxic activity of Bz-423, PK11195, and 4-ClDz could arise from a common mechanism and possibly a common molecular target.

At subapoptotic concentrations, Bz-423 has profound antiproliferative effects. Because the proportion of cells in the G1-G0 phase significantly increased with treatment, Bz-423 appears to affect the G1 checkpoint. Experimental agents that cause cell cycle arrest are presently attracting considerable interest as an approach to control cancer. For example, flavopiridol and 7-hydroxystaurosporine block Cdk functions causing G1 and G2 arrest; both are currently being tested as novel anticancer drugs in human trials (48) .

Under conditions where Bz-423 induces cell cycle arrest (and not apoptosis), we observed a sustained ROS. This signal is functionally significant in the antiproliferative mechanism because antioxidants (MnTBAP) attenuate the efficacy of Bz-423-mediated growth inhibition. Thus, just as ROS are necessary for Bz-423-mediated apoptosis, they also appear to regulate Bz-423-induced cell cycle arrest. ROS can cause withdrawal from the cell cycle at the G1 checkpoint. For example, peroxides suppress S-phase entry in a dose-dependent manner by inhibiting cyclin E/Cdk2 activity, which controls the G1 checkpoint (49) . In addition, the ataxia telangiectasia mutated protein (pATM) is thought to function, in part, as a sensor of oxidative stress. pATM is required for both the G1 and G2 checkpoint responses and causes induction of p53 in cells treated with tert-butyl hydroperoxide (50) . p53 transcriptionally activates genes including p21 (Cip1/Waf1), which binds to and inhibits the activity of cyclin E/Cdk2 (51) . Because the mutated p53 present in Ramos cells is transcriptionally inactive, other mechanisms must be involved in Bz-423-mediated growth arrest (39) . In this regard, it is interesting to note that p21 can be induced independently of p53 in response to certain chemicals (52) .

Other benzodiazepines and ligands of the PBR have been reported to affect cell cycle progression, resulting in growth arrest via undefined mechanisms (53, 54, 55, 56) . However, several lines of evidence suggest that direct PBR binding is not involved in this response. First, Leydig cells in which PBR expression is silenced by antisense transfection survive and continue to proliferate normally (16) . Second, to affect proliferation or survival, PBR ligands must be present at concentrations greatly exceeding those necessary to saturate the PBR [e.g., 1000 times the Kd (14 , 15) ]. Third, among the PBR ligands, binding affinity for the PBR has not been consistently shown to correlate with effectiveness at inhibiting cell growth (13) . Thus, these reports and our data with Bz-423 suggest that benzodiazepines have antiproliferative effects via an alternative target.

Second-wave signaling responses that intervene between benzodiazepines binding and processes leading to growth arrest are also not well understood. One possibility is that polyamine metabolism is involved in this signaling. The antiproliferative effects of 4-ClDz are associated with a rapid reduction in ODC activity in Nb 2 lymphoma cells (57) . This response is expected to reduce intracellular polyamines, including putrescine, spermine, and spermidine, which play key roles in the regulation of cell proliferation. Consequently, antiproliferative signals induced by benzodiazepines may stem from reduced ODC expression or increased expression of antizyme 1, a negative regulator that targets ODC for proteosomal degradation. Of particular interest, under conditions that favor increased ROS, ODC activity is reduced, in part through oxidation of active site thiol groups (58 , 59) . Thus, these observations suggest that polyamine pathways may be the critical mediators between Bz-423-induced ROS and growth arrest.

In conclusion, Bz-423 is a novel 1,4-benzodiazepine that has both cytotoxic and antiproliferative properties against transformed B cells. Increased ROS are essential for mediating both cell death and growth arrest, and the magnitude of the ROS response may determine the fate of a Bz-423-treated cell. Low concentrations of Bz-423 produce levels of ROS that ultimately result in growth arrest. Higher amounts of Bz-423 may cause proportionally greater levels of ROS that trigger apoptosis. Compounds that act on mitochondria to induce apoptosis are currently being explored as pharmacological candidates to use against cancer and related disease processes (60) . Bz-423 is a promising therapeutic candidate because cells respond to it regardless of Bcl-2 or Bcl-xL levels or EBV and p53 status, factors that limit the effectiveness of current chemotherapeutics. Further work is now needed to identify the molecular target that responds to Bz-423 and orchestrates the cellular response.


   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.

1 Supported by NIH Grants AI-47450 and GM-42168 (to G. D. G.) and GM-50353 (to J. A. E.). Back

2 To whom requests for reprints should be addressed, at Department of Obstetrics and Gynecology, University of Michigan, 1500 East Medical Center Drive, L4602 WH, Ann Arbor, MI 48109. Phone: (734) 764-9106; Fax: (734) 615-8902; E-mail: aopipari{at}umich.edu Back

3 The abbreviations used are: PBR, peripheral benzodiazepine receptor; BL, Burkitt’s lymphoma; 4-ClDz, 4-chlorodiazepam; CDDP, cis-diamineplatinum(II)dichloride; LC50, drug concentration killing 50% of cells; DCF, 2',7'-dichlorofluorescin; DCHF-DA, 2',7'-dichlorodihydrofluorescin diacetate; FBS, fetal bovine serum; GC, germinal center; ODC, ornithine decarboxylase; PTP, permeability transition pore; PI, propidium iodide; ROS, reactive oxygen species; Cdk, cyclin-dependent kinase; MnTBAP, Mn(III)tetrakis (4-Benzoic acid)porphyrin Chloride. Back

Received 5/ 6/03. Revised 7/ 1/03. Accepted 7/25/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Haefely W. The GABA-benzodiazepine interaction fifteen years later. Neurochem. Res., 15: 169-174, 1990.[Medline]
  2. Braestrup C., Squires R. F. Specific benzodiazepine receptors in rat brain characterized by high-affinity (3H)diazepam binding. Proc. Natl. Acad. Sci. USA, 74: 3805-3809, 1977.[Abstract/Free Full Text]
  3. Joseph-Liauzun E., Farges R., Delmas P., Ferrara P., Loison G. The Mr 18, 000 subunit of the peripheral-type benzodiazepine receptor exhibits both benzodiazepine and isoquinoline carboxamide binding sites in the absence of the voltage-dependent anion channel or of the adenine nucleotide carrier. J. Biol. Chem., 272: 28102-28106, 1997.[Abstract/Free Full Text]
  4. Oke B. O., Suarez-Quian C. A., Riond J., Ferrara P., Papadopoulos V. Cell surface localization of the peripheral-type benzodiazepine receptor (PBR) in adrenal cortex. Mol. Cell. Endocrinol., 87: R1-R6, 1992.[Medline]
  5. Hardwick M., Fertikh D., Culty M., Li H., Vidic B., Papadopoulos V. Peripheral-type benzodiazepine receptor (PBR) in human breast cancer: correlation of breast cancer cell aggressive phenotype with PBR expression, nuclear localization, and PBR-mediated cell proliferation and nuclear transport of cholesterol. Cancer Res., 59: 831-842, 1999.[Abstract/Free Full Text]
  6. Papadopoulos V., Boujrad N., Ikonomovic M. D., Ferrara P., Vidic B. Topography of the Leydig cell mitochondrial peripheral-type benzodiazepine receptor. Mol. Cell. Endocrinol., 104: R5-R9, 1994.[Medline]
  7. Casellas P., Galiegue S., Basile A. S. Peripheral benzodiazepine receptors and mitochondrial function. Neurochem. Int., 40: 475-486, 2002.[Medline]
  8. Gavish M., Bachman I., Shoukrun R., Katz Y., Veenman L., Weisinger G., Weizman A. Enigma of the peripheral benzodiazepine receptor. Pharmacol. Rev., 51: 629-650, 1999.[Abstract/Free Full Text]
  9. Blatt N. B., Bednarski J. J., Warner R. E., Leonetti F., Johnson K. M., Boitano A., Yung R., Richardson B. C., Johnson K. J., Ellman J. A., Opipari A. W., Jr., Glick G. D. Benzodiazepine-induced superoxide signals B cell apoptosis: mechanistic insight and potential therapeutic utility. J. Clin. Investig., 110: 1123-1132, 2002.[Medline]
  10. Bednarski J. J., Warner R. E., Rao T., Leonetti F., Yung R., Richardson B. C., Johnson K. J., Ellman J. A., Opipari A. W., Jr., Glick G. D. Attenuation of autoimmune disease in Fas-deficient mice by treatment with a cytotoxic benzodiazepine. Arthritis Rheum., 48: 757-766, 2003.[Medline]
  11. Wang J. K., Morgan J. I., Spector S. Benzodiazepines that bind at peripheral sites inhibit cell proliferation. Proc. Natl. Acad. Sci. USA, 81: 753-756, 1984.[Abstract/Free Full Text]
  12. Zisterer D. M., Gorman A. M., Williams D. C., Murphy M. P. The effects of the peripheral-type benzodiazepine acceptor ligands, Ro 5-4864 and PK 11195, on mitochondrial respiration. Methods Find. Exp. Clin. Pharmacol., 14: 85-90, 1992.[Medline]
  13. Zisterer D. M., Hance N., Campiani G., Garofalo A., Nacci V., Williams D. C. Antiproliferative action of pyrrolobenzoxazepine derivatives in cultured cells: absence of correlation with binding to the peripheral-type benzodiazepine binding site. Biochem. Pharmacol., 55: 397-403, 1998.[Medline]
  14. Gorman A. M., O’Beirne G. B., Regan C. M., Williams D. C. Antiproliferative action of benzodiazepines in cultured brain cells is not mediated through the peripheral-type benzodiazepine acceptor. J. Neurochem., 53: 849-855, 1989.[Medline]
  15. Camins A., Diez-Fernandez C., Pujadas E., Camarasa J., Escubedo E. A new aspect of the antiproliferative action of peripheral-type benzodiazepine receptor ligands. Eur. Pharmacol., 272: 289-292, 1995.
  16. Kelly-Hershkovitz E., Weizman R., Spanier I., Leschiner S., Lahav M., Weisinger G., Gavish M. Effects of peripheral-type benzodiazepine receptor antisense knockout on MA-10 Leydig cell proliferation and steroidogenesis. J. Biol. Chem., 273: 5478-5483, 1998.[Abstract/Free Full Text]
  17. Hecht J. L., Aster J. C. Molecular biology of Burkitt’s lymphoma. J. Clin. Oncol., 18: 3707-3721, 2000.[Abstract/Free Full Text]
  18. Gaidano G., Ballerini P., Gong J. Z., Inghirami G., Neri A., Newcomb E. W., Magrath I. T., Knowles D. M., Dalla-Favera R. p53 mutations in human lymphoid malignancies: association with Burkitt lymphoma and chronic lymphocytic leukemia. Proc. Natl. Acad. Sci. USA, 88: 5413-5417, 1991.[Abstract/Free Full Text]
  19. Gregory C. D., Dive C., Henderson S., Smith C. A., Williams G. T., Gordon J., Rickinson A. B. Activation of Epstein-Barr virus latent genes protects human B cells from death by apoptosis. Nature (Lond.), 349: 612-614, 1991.[Medline]
  20. Finke J., Fritzen R., Ternes P., Trivedi P., Bross K. J., Lange W., Mertelsmann R., Dolken G. Expression of bcl-2 in Burkitt’s lymphoma cell lines: induction by latent Epstein-Barr virus genes. Blood, 80: 459-469, 1992.[Abstract/Free Full Text]
  21. Fries K. L., Miller W. E., Raab-Traub N. Epstein-Barr virus latent membrane protein 1 blocks p53-mediated apoptosis through the induction of the A20 gene. J. Virol., 70: 8653-8659, 1996.[Abstract]
  22. Evens A. M., Gordon L. I. Burkitt’s and Burkitt-like lymphoma. Curr. Treat. Options Oncol., 3: 291-305, 2002.[Medline]
  23. Bunin B. A., Plunkett M. J., Ellman J. A. The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library. Proc. Natl. Acad. Sci. USA, 91: 4708-4712, 1994.[Abstract/Free Full Text]
  24. Oltvai Z. N., Milliman C. L., Korsmeyer S. J. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell, 74: 609-619, 1993.[Medline]
  25. Darzynkiewicz Z., Juan G., Li X., Gorczyca W., Murakami T., Traganos F. Cytometry in cell necrobiology: analysis of apoptosis and accidental cell death (necrosis). Cytometry, 27: 1-20, 1997.[Medline]
  26. Fehske K. J., Muller W. E., Wollert U. The location of drug binding sites in human serum albumin. Biochem. Pharmacol., 30: 687-692, 1981.[Medline]
  27. Rousselle C., Barbier M., Comte V. V., Alcouffe C., Clement-Lacroix J., Chancel G., Ronot X. Innocuousness and intracellular distribution of PKH67: a fluorescent probe for cell proliferation assessment. In Vitro Cell. Dev. Biol.-Animal, 37: 646-655, 2001.[Medline]
  28. LeBel C. P., Ischiropoulos H., Bondy S. C. Evaluation of the probe 2',7'-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress. Chem. Res. Toxicol., 5: 227-231, 1992.[Medline]
  29. Day B. J., Shawen S., Liochev S. I., Crapo J. D. A metalloporphyrin superoxide dismutase mimetic protects against paraquat-induced endothelial cell injury, in vitro. J. Pharmacol. Exp. Ther., 275: 1227-1232, 1995.[Abstract/Free Full Text]
  30. Korsmeyer S. J., Yin X. M., Oltvai Z. N., Veis-Novack D. J., Linette G. P. Reactive oxygen species and the regulation of cell death by the Bcl-2 gene family. Biochim. Biophys. Acta, 1271: 63-66, 1995.[Medline]
  31. Fang W., Rivard J. J., Ganser J. A., LeBien T. W., Nath K. A., Mueller D. L., Behrens T. W. Bcl-xL rescues WEHI 231 B lymphocytes from oxidant-mediated death following diverse apoptotic stimuli. J. Immunol., 155: 66-75, 1995.[Abstract]
  32. Rowe M., Rooney C. M., Edwards C. F., Lenoir G. M., Rickinson A. B. Epstein-Barr virus status and tumour cell phenotype in sporadic Burkitt’s lymphoma. Int. J. Cancer, 37: 367-373, 1986.[Medline]
  33. Milner A. E., Johnson G. D., Gregory C. D. Prevention of programmed cell death in Burkitt lymphoma cell lines by bcl-2-dependent and -independent mechanisms. Int. J. Cancer, 52: 636-644, 1992.[Medline]
  34. Park E., Kalunta C. I., Nguyen T. T., Wang C. L., Chen F. S., Lin C. K., Kaptein J. S., Lad P. M. TNF-{alpha} inhibits anti-IgM-mediated apoptosis in Ramos cells. Exp. Cell Res., 226: 1-10, 1996.[Medline]
  35. Klein G., Giovanella B., Westman A., Stehlin J. S., Mumford D. An EBV-genome-negative cell line established from an American Burkitt lymphoma; receptor characteristics, EBV infectibility and permanent conversion into EBV-positive sublines by in vitro infection. Intervirology, 5: 319-334, 1975.[Medline]
  36. Magrath I. T., Pizzo P. A., Whang-Peng J., Douglass E. C., Alabaster O., Gerber P., Freeman C. B., Novikovs L. Characterization of lymphoma-derived cell lines: comparison of cell lines positive and negative for Epstein-Barr virus nuclear antigen. I. Physical, cytogenetic, and growth characteristics. J. Natl. Cancer Inst. (Bethesda), 64: 465-476, 1980.
  37. Levine A. J., Perry M. E., Chang A., Silver A., Dittmer D., Wu M., Welsh D. The 1993 Walter Hubert Lecture: the role of the p53 tumour-suppressor gene in tumorigenesis. Br. J. Cancer, 69: 409-416, 1994.[Medline]
  38. Fan S., el-Deiry W. S., Bae I., Freeman J., Jondle D., Bhatia K., Fornace A. J., Jr., Magrath I., Kohn K. W., O’Connor P. M. p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA-damaging agents. Cancer Res., 54: 5824-5830, 1994.[Abstract/Free Full Text]
  39. Allday M. J., Inman G. J., Crawford D. H., Farrell P. J. DNA damage in human B cells can induce apoptosis, proceeding from G1/S when p53 is transactivation competent and G2/M when it is transactivation defective. EMBO J., 14: 4994-5005, 1995.[Medline]
  40. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat. Med., 3: 614-620, 1997.[Medline]
  41. Paradiso A., Simone G., Lena M. D., Leone B., Vallejo C., Lacava J., Dellapasqua S., Daidone M. G., Costa A. Expression of apoptosis-related markers and clinical outcome in patients with advanced colorectal cancer. Br. J. Cancer, 84: 651-658, 2001.[Medline]
  42. Kroemer G. Mitochondrial control of apoptosis: an overview. Biochem. Soc. Symp., 66: 1-15, 1999.[Medline]
  43. Komano J., Takada K. Role of bcl-2 in Epstein-Barr virus-induced malignant conversion of Burkitt’s lymphoma cell line Akata. J. Virol., 75: 1561-1564, 2001.[Abstract/Free Full Text]
  44. Ruf I. K., Rhyne P. W., Yang H., Borza C. M., Hutt-Fletcher L. M., Cleveland J. L., Sample J. T. EBV regulates c-MYC, apoptosis, and tumorigenicity in Burkitt’s lymphoma. Curr. Top. Microbiol. Immunol., 258: 153-160, 2001.[Medline]
  45. Fennell D. A., Corbo M., Pallaska A., Cotter F. E. Bcl-2 resistant mitochondrial toxicity mediated by the isoquinoline carboxamide PK11195 involves de novo generation of reactive oxygen species. Br. J. Cancer, 84: 1397-1404, 2001.[Medline]
  46. Larcher J. C., Vayssiere J. L., Le Marquer F. J., Cordeau L. R., Keane P. E., Bachy A., Gros F., Croizat B. P. Effects of peripheral benzodiazepines upon the O2 consumption of neuroblastoma cells. Eur. Pharmacol., 161: 197-202, 1989.
  47. Hirsch J. D., Beyer C. F., Malkowitz L., Beer B., Blume A. J. Mitochondrial benzodiazepine receptors mediate inhibition of mitochondrial respiratory control. Mol. Pharmacol., 35: 157-163, 1989.[Abstract]
  48. Senderowicz A. M. The cell cycle as a target for cancer therapy: basic and clinical findings with the small molecule inhibitors flavopiridol and UCN-01. Oncologist, 7 (Suppl. 3): 12-19, 2002.[Abstract/Free Full Text]
  49. Deshpande N. N., Sorescu D., Seshiah P., Ushio-Fukai M., Akers M., Yin Q., Griendling K. K. Mechanism of hydrogen peroxide-induced cell cycle arrest in vascular smooth muscle. Antioxid. Redox Signal., 4: 845-854, 2002.[Medline]
  50. Shackelford R. E., Innes C. L., Sieber S. O., Heinloth A. N., Leadon S. A., Paules R. S. The ataxia telangiectasia gene product is required for oxidative stress-induced G1 and G2 checkpoint function in human fibroblasts. J. Biol. Chem., 276: 21951-21959, 2001.[Abstract/Free Full Text]
  51. Bartek J., Lukas J. Pathways governing G1/S transition and their response to DNA damage. FEBS Lett., 490: 117-122, 2001.[Medline]
  52. Serfas M. S., Goufman E., Feuerman M. H., Gartel A. L., Tyner A. L. p53-independent induction of p21WAF1/CIP1 expression in pericentral hepatocytes following carbon tetrachloride intoxication. Cell Growth Differ., 8: 951-961, 1997.[Abstract]
  53. Landau M., Weizman A., Zoref-Shani E., Beery E., Wasseman L., Landau O., Gavish M., Brenner S., Nordenberg J. Antiproliferative and differentiating effects of benzodiazepine receptor ligands on B16 melanoma cells. Biochem. Pharmacol., 56: 1029-1034, 1998.[Medline]
  54. Carmel I., Fares F. A., Leschiner S., Scherubl H., Weisinger G., Gavish M. Peripheral-type benzodiazepine receptors in the regulation of proliferation of MCF-7 human breast carcinoma cell line. Biochem. Pharmacol., 58: 273-278, 1999.[Medline]
  55. Maaser K., Hopfner M., Jansen A., Weisinger G., Gavish M., Kozikowski A. P., Weizman A., Carayon P., Riecken E. O., Zeitz M., Scherubl H. Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human colorectal cancer cells. Br. J. Cancer, 85: 1771-1780, 2001.[Medline]
  56. Sutter A. P., Maaser K., Hopfner M., Barthel B., Grabowski P., Faiss S., Carayon P., Zeitz M., Scherubl H. Specific ligands of the peripheral benzodiazepine receptor induce apoptosis and cell cycle arrest in human esophageal cancer cells. Int. J. Cancer, 102: 318-327, 2002.[Medline]
  57. Laird H. E., II, Gerrish K. E., Duerson K. C., Putnam C. W., Russell D. H. Peripheral benzodiazepine binding sites in Nb 2 node lymphoma cells: effects on prolactin-stimulated proliferation and ornithine decarboxylase activity. Eur. Pharmacol., 171: 25-35, 1989.
  58. Babal P., Ruchko M., Ault-Ziel K., Cronenberg L., Olson J. W., Gillespie M. N. Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells. Am. J. Physiol. Lung Cell. Mol. Physiol., 282: L840-L-846 2002.
  59. Guarnieri C., Flamigni F., Rizzuto S., Vaona I., Caldarera C. M. Altered thiol group status in the heart ornithine decarboxylase inactivated following perfusion with t-butylhydroperoxide. Int. J. Biochem., 19: 931-938, 1987.[Medline]
  60. Costantini P., Jacotot E., Decaudin D., Kroemer G. Mitochondrion as a novel target of anticancer chemotherapy. J. Natl. Cancer Inst. (Bethesda), 92: 1042-1053, 2000.[Abstract/Free Full Text]
  61. Doble A., Ferris O., Burgevin M. C., Menager J., Uzan A., Dubroeucq M. C., Renault C., Gueremy C., Le Fur G. Photoaffinity labeling of peripheral-type benzodiazepine-binding sites. Mol. Pharmacol., 31: 42-49, 1987.[Abstract]



This article has been cited by other articles:


Home page
 J. Pharmacol. Exp. Ther.Home page
N. Bhagavathula, K. C. Nerusu, A. Hanosh, M. N. Aslam, T. B. Sundberg, A. W. Opipari Jr., K. Johnson, S. Kang, G. D. Glick, and J. Varani
7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(naphthalen-2-ylmethyl)-4,5-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423), a Benzodiazepine, Suppresses Keratinocyte Proliferation and Has Antipsoriatic Activity in the Human Skin-Severe, Combined Immunodeficient Mouse Transplant Model
J. Pharmacol. Exp. Ther., March 1, 2008; 324(3): 938 - 947.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
T. B. Sundberg, G. M. Ney, C. Subramanian, A. W. Opipari Jr., and G. D. Glick
The Immunomodulatory Benzodiazepine Bz-423 Inhibits B-Cell Proliferation by Targeting c-Myc Protein for Rapid and Specific Degradation
Cancer Res., February 1, 2006; 66(3): 1775 - 1782.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
Y. Fernandez, T. P. Miller, C. Denoyelle, J. A. Esteban, W.-H. Tang, A. L. Bengston, and M. S. Soengas
Chemical Blockage of the Proteasome Inhibitory Function of Bortezomib: IMPACT ON TUMOR CELL DEATH
J. Biol. Chem., January 13, 2006; 281(2): 1107 - 1118.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
J. Varani, N. Bhagavathula, K. C. Nerusu, H. Sherzer, K. Fay, A. E. Boitano, G. D. Glick, K. Johnson, S. Kang, and A. W. Opipari Jr.
A Novel Benzodiazepine Selectively Inhibits Keratinocyte Proliferation and Reduces Retinoid-Induced Epidermal Hyperplasia in Organ-Cultured Human Skin
J. Pharmacol. Exp. Ther., April 1, 2005; 313(1): 56 - 63.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boitano, A.
Right arrow Articles by Opipari, A. W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boitano, A.
Right arrow Articles by Opipari, A. W., Jr.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online