ToxSci Advance Access originally published online on May 28, 2003
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Toxicological Sciences 74, 1-3 (2003)
Copyright © 2003 by the Society of Toxicology
TOXICOLOGICAL HIGHLIGHT |
The Mitochondrial Benzodiazepine Receptor as a Potential Target Protein for Drug Development: Demonstration of Functional Significance with Cell Lines Exhibiting Differential Expression of Bcl-2
Department of Pharmacology, Wayne State University School of Medicine, Detroit, Michigan 48201
Received May 2, 2003; accepted May 5, 2003
ABSTRACT
The article highlighted in this issue is "Reversal of Bcl-2 Mediated Resistance of the EW36 Human B-Cell Lymphoma Cell Line to Arsenite and Pesticide-Induced Apoptosis by PK11195, a Ligand of the Mitochondrial Benzodiazepine Receptor" by Donna E. Muscarella, Kerry A. O’Brien, Ann T. Lemley, and Stephen E. Bloom from Cornell University in Ithaca, NY (pp. 66–73). The following brief review summarizes their findings, highlights the novel biological model and experimental approach used, and explores potential mechanistic and therapeutic implications of these findings.
Muscarella and colleagues (2003)
report their most recent findings on signaling pathways involved in the response of human Burkitt’s lymphoma cells to induction of apoptosis by mitochondrial toxicants and other cytotoxic chemicals. This is an elegant study using two human B lymphocyte-derived cell lines, one that expresses relatively low levels of the anti-apoptotic protein Bcl-2, ST486 cells, and a cell line that overexpresses Bcl-2, EW36 cells. ST486 cells are highly sensitive to apoptosis induced by a variety of mitochondrial toxicants and other chemicals whereas EW36 cells are relatively resistant to induction of apoptosis. Moreover, the two cell lines also differ in their requirement for activation of the c-Jun-N-terminal kinase (JNK) pathway in the induction of apoptosis. Hence, the novelty of using these two cell lines rests not only in the quantitative difference in the expression of Bcl-2, but also in the qualitative or mechanistic difference in how the cell lines respond to exposure to toxicants.
The key components of the mitochondrial permeability transition (PT) pore are now known to include the adenine nucleotide translocase, the voltage dependent ion channel, cyclophilin D, mitochondrial hexokinase, and the mitochondrial or so-called peripheral benzodiazepine receptor (mBzR). The study focuses on the mBzR and its function in mediating the PT and apoptosis. Other proteins are associated with the PT pore, and include members of the Bcl-2 family of proteins. It is thought that the antiapoptotic effect of Bcl-2 is mediated, at least in part, by modulation of the PT pore (Kantrow and Piantadosi, 1997; Susin et al., 1996; Zanzami et al., 1996). A functional relationship between the mBzR and Bcl-2 is suggested by several studies showing that treatment of various cell types, including those of B- and T-lymphoid lineages, with antagonists of the mBzR reverses the anti-apoptotic effects of Bcl-2 overexpression (Constantini et al., 2000; Hirsch et al., 1998; Larochette et al., 1999). One such mBzR antagonist is 1-(2-chlorophenyl)-N-(1-methylpropyl)-3-isoquinoline-carboxamide (PK11195), which is relatively nontoxic alone but synergizes with several agents to induce apoptosis in otherwise resistant cells that overexpress Bcl-2 (Hirsch et al., 1998; Larochette et al., 1999). Although these studies indicated that PK11195 directly affects mitochondrial function by facilitating the loss of mitochondrial membrane potential (
m) and release of apoptogenic factors, it is not known whether the PK11195-mediated reversal of Bcl-2 protection also occurs with environmental chemicals. This is critical because several pesticides, such as rotenone and antimycin A, interact directly with mitochondria and their cytotoxicity appears to be regulated by proteins associated with the PT pore. Accordingly, the logical hypothesis from putting these observations together is that modulation of the mBzR with antagonistic ligands such as PK11195 can modulate pesticide-induced cytotoxicity.
Previous work by these investigators and their colleagues (Muscarella and Bloom, 2002, 2003; O’Brien et al., 2001) established the differential sensitivity to apoptotic induction of the two cell lines used in the present study (Lee and Shacter, 1997; O’Brien et al., 2001). They further showed that ST486 cells treated with low concentrations of carbonyl cyanide m-chlorophenylhydrazone (mClCCP) or arsenite undergo rapid mitochondrial depolarization and extensive apoptosis in the absence of JNK pathway activation. In contrast, the EW36 cells are relatively resistant to chemically induced mitochondrial depolarization and apoptosis, but do exhibit these toxic responses when exposed to relatively high concentrations of arsenite. Moreover, these toxic responses in EW36 cells are always associated with JNK-pathway activation.
To address their hypothesis that antagonism of the mBzR alters susceptibility to chemically-induced apoptosis, Muscarella and colleagues used several toxicants, including two pesticides or insecticides that inhibit complex I of the respiratory chain (pyridaben, rotenone), an inhibitor of complex III (antimycin A), an uncoupler of mitochondrial oxidative phosphorylation (mClCCCP), the genotoxic herbicide alachlor, and the metal sodium arsenite (NaAsO2), which inhibits mitochondrial respiration, reacts with protein sulfhydryl groups, and is genotoxic. State-of-the-art techniques were used to quantify the cytotoxic responses of the two cell lines. A double-fluorescence staining technique they previously validated (Muscarella and Bloom, 1997; Muscarella et al., 1998) was used to determine the extent of apoptosis and necrosis. The method uses staining with propidium iodide (red fluorescence emission) and Hoechst 33342 (blue fluorescence emission). Mitochondrial depolarization was monitored at the single-cell level by JC-1 fluorescence (O’Brien et al., 2001; Reers et al., 1991).
The studies are presented in a clear, insightful manner. The authors first show that PK11195 sensitizes EW36 cells to apoptosis induced by the wide range of toxicants studied. The potential importance of this observation is enhanced by the use of multiple agents with diverse molecular targets. Besides morphological data, increased cleavage of poly (ADP ribose) polymerase and pro-caspase-9 was also shown. This finding of enhanced apoptosis is followed up by demonstration that PK11195 also sensitizes EW36 cells to loss of m following exposure to mitochondrial toxicants. As noted earlier, the differences between the responses of ST486 and EW36 cells do not appear to be simply quantitative, but also encompass qualitative differences that implicate a fundamentally different mechanism of action in each cell type. Although the apoptosis that results from exposure of EW36 cells to the various toxicants appears to be obligatorily associated with activation of the JNK pathway and protein synthesis, prior treatment with PK11195 enables apoptosis to occur in the absence of JNK-pathway activation. Whereas cycloheximide normally causes significant decreases in the extent of chemically-induced apoptosis due to its inhibition of protein synthesis, PK11195-treated EW36 cells were able to undergo apoptosis in the presence of cycloheximide, suggesting a disconnect between apoptosis and the normal requirement for new protein synthesis. Although there are obvious, potential pitfalls with the use of a pharmacological approach involving a single agent, the authors provide convincing discussion that the effects of PK11195 are relatively specific for the mBzR and not due to some of the other known effects of this compound.
The mechanistic implications of these studies are wide-ranging and significantly move the field forward. The mBzR is an approximately 18-kD protein that spans the mitochondrial outer membrane. Several diverse, cellular functions have been associated with the mBzR, including that as a mitochondrial sensor for reactive oxygen species (Casellas et al., 2002; Cotter, 2000; Verma and Snyder, 1989). This suggested function raises some very intriguing questions about mitochondrial redox status, mitochondrial function, and apoptosis. Although the relationship between alterations in mitochondrial redox status, particularly that involving glutathione, and the induction of mitochondrial toxicity or apoptosis have been known for some years, the precise mechanism by which changes in redox status are transmitted and result in pathological changes has remained unclear. These and previous findings on potential functions of the mBzR in regulating the PT pore and its interactions with Bcl-2, provide some additional clues as to how redox changes are transmitted in the mitochondria.
In summary, this is a timely and highly relevant report that identifies a role for the mBzR in modulating the activity of the mitochondrial PT pore in response to exposure to environmental chemicals. The findings have important mechanistic and pharmacological implications. Ultimately, a new class of therapeutic agents, with the mBzR as its target, may result.
NOTES
1 To whom correspondence should be addressed at the Department of Pharmacology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, MI 48201. Tel: 313-577-0475. Fax: 313-577-6739. E-mail: l.h.lash{at}wayne.edu. 
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