The relative importance of the activator-binding and effector-binding functions of antiapoptotic proteins is not completely established. It may well be that the importance varies with type of cell or insult. It is worth making an important technical note in the quantitation of complexes between antiapoptotic proteins and BAX and BAK. The conformational change in BAX and BAK can be elicited simply by exposure to nonionic detergents commonly used in the preparation of cell lysates such as Triton X-100 or NP-40. Other detergents, such as 3-[3-cholamidopropyl- dimethylammonio]-1-propane-sulfonate (CHAPS), lack this property. This induction of conformational change is furthermore associated with an increased binding of BAX and BAK to antiapoptotic proteins like BCL-2. Therefore, incautious use of detergents can result in a very significant artifactual overestimation of complexes between antiapoptotic proteins and BAX and BAK. When detergents like CHAPS are used to make wholecell lysates, only a small minority of BAX and BAK appear to be sequestered by antiapoptotic proteins, suggesting that factors distinct from antiapoptotic protein binding are critical in controlling BAX and BAK activation.

Other proteins have been suggested to inhibit BAX or BAK activation. Humanin, a small but interesting polypeptide for which an open reading frame is present in both the nuclear and mitochondrial genomes, has been found to inhibit activation of BAX by BID by binding BID. VDAC2 has been shown to bind to BAK and negatively regulate its activation.

4. INHIBITING THE INHIBITORS

An additional layer of modulation exists for the BCL- 2 family control of MOMP, composed of the sensitizer BH3-only proteins. These proteins all possess a BH3 domain, but they lack the ability to activate BAX or BAK and thus are classified as sensitizers. Unlike activators, sensitizers demonstrate a more selective pattern of interaction with antiapoptotic proteins (Table 5-1). For instance, the sensitizer BH3-only protein BAD binds to BCL-2, BCL-XL, and BCL-w, but not to BFL-1 or MCL- 1. In contrast, NOXA binds to MCL-1 but not to BCL-2, BCL-XL, or BCL-w. The sensitizers are all pro-death proteins, but they exert their effect by being inhibitors of the antiapoptotic proteins. If the antiapoptotic protein is previously unbound by activators, then the interaction with a sensitizer serves to simply neutralize its function and decrease the remaining antiapoptotic reserve. If, alternatively, the antiapoptotic protein is already bound by an activator, then the binding of a sensitizer will displace the activator, allowing it to activate BAX or BAK.

It can be seen, therefore, that the commitment to apoptosis depends on a balance of proand antiapoptotic proteins, but one significantly more complex than originally described in a simple rheostat model, when only BAX and BCL-2 were participants. The interactions described previously are summarized in Figure 5-2.

5. ACTIVATING THE ACTIVATORS – CONNECTING

THE INSULT TO THE BCL-2 FAMILY

There are many treatments that are known to commit cells to the fate of apoptosis. Yet very often, the details of how the initiating insults communicate a death signal to the BCL-2 family are poorly understood. Examples follow in which some of the important steps have been identified. These examples demonstrate the wide variety of mechanisms that are employed in provoking cell death by apoptosis.

In response to many types of DNA damage, p53 drives a response that results in either senescence or apoptosis. The apoptosis response ultimately uses the intrinsic, mitochondrial apoptotic pathway, resulting in MOMP. Much of the p53-generated signaling is due to the transcriptional upregulation of the proapoptotic BH3-only protein, PUMA. Whether PUMA acts primarily as an activator or a sensitizer is a matter of debate. However, in its sensitizer role, PUMA is particularly potent because it can bind to and neutralize all of the identified antiapoptotic proteins. In certain models of DNA damage, loss of PUMA alone can nearly phenocopy loss of p53. Transcriptional induction of other proapoptotic proteins, including NOXA and BAX, also contribute to the apoptotic response.

Intriguingly, there is some evidence that p53 can induce apoptosis independent of any modulation of transcription. Some have found that in response to DNA damage, p53 can migrate to the mitochondrion and act as both a sensitizer and an activator to directly induce MOMP. It is notable that p53 lacks a discernible BH3 domain, so that this interaction with BCL-2 family proteins is likely different from that involving BH3 domains.

In many cells, particularly those designated as type II cells, incorporation of the mitochondrial apoptotic pathway is necessary for induction of apoptosis downstream of ligation of the tumor necrosis factor (TNF) family of receptors. The key connector of the extrinsic and intrinsic pathways in this situation is the activator BH3-only protein BID. In response to ligation of their extracellular domains, receptors that include CD95/FAS, TNF, and TNF-related apoptosis-inducing ligand (TRAIL) assemble a death-inducing signaling complex (DISC). A component, the protein FADD/MORT, recruits

procaspase-8 to the complex. Perhaps via an “induced proximity” mechanism, procaspase-8 is then autoproteolytically cleaved into its active caspase-8 form. Caspase-8 can then cleave and activate effector caspases like caspase-3 and -7. In type I cells, such as thymocytes, no further involvement of the mitochondrial pathway is necessary to commit the cell to apoptosis, and BCL-2 cannot inhibit apoptosis. However, in type II cells, BCL-2 can inhibit death, and the mitochondrial amplification loop is required for apoptosis. Cleavage of BID into the 15-kDa truncated BID (tBID) can efficiently induce activation of BAX and BAK and cause MOMP. The activator function of tBID can be further enhanced by enzymatic myristoylation of the glycine residue on the new amino-terminus of the tBID molecule, as it facilitates targeting to the mitochondrion.

Although BH3-only proteins are usually the most dynamic players in the BCL-2 family in response to noxious stimuli, MCL-1 levels can also dramatically change in response to select death signaling events. A prominent example is growth factor withdrawal. When interleukin (IL)-3 is removed from the culture of IL-3–dependent cell lines, including FL5.12, 32D, and Ba/F3, there is a reduction in PI3K activity. This results in decreased activated AKT, resulting in a release of increased glycogen synthase kinase 3 (GSK-3) activity. GSK-3 phosphorylates MCL-1, targeting it for ubiquitinylation and proteasomal degradation. The shortening of the already short half-life (on the order of minutes) of MCL-1 results in a dramatic lowering of MCL-1 levels, freeing BIM that had been sequestered by MCL-1 to activate BAX and BAK and commit the cell to apoptosis. In contrast, in an IL-6– dependent model, withdrawal of IL-6 yielded a decrease in MCL-1 levels as a result of decreased transcription of MCL-1.

Subcellular localization can also play an important role in modulating function of BCL-2 family proteins. For

instance, in its phosphorylated state, the proapoptotic sensitizer BH3-only protein BAD is sequestered by 14–3- 3 in the cytoplasm. In IL-3–dependent cells, presence of IL-3 induced BAD phosphorylation. When dephosphorylated, BAD migrates to the mitochondrion to bind to antiapoptotic proteins such as BCL-2 and BCL-XL to promote death. It has been suggested that the BH3-only proteins BIM and BMF promote death when displaced from their cytoskeletal locations on the microtubule dynein motor complex and actin-based myosin V motor complex, respectively.

Kinase inhibitors are playing an increasing role in the oncologist’s pharmacopeia. They induce a death that can almost uniformly be inhibited by BCL-2 or related antiapoptotic proteins, designating the intrinsic apoptotic pathway as the key arbiter of cell death downstream of kinase inhibition. BIM protein levels increase in cancer cells that are sensitive to inhibitors of the epidermal growth factor receptor. This increase is due to increased transcription, but the details regarding this transcriptional control remain to be elucidated.

6. THE BCL-2 FAMILY AND CANCER

Even before anything was known about its role in controlling cell death, it was evident that BCL-2 played a role in cancer. BCL-2 first caught the attention of molecular biologists solely due to its location at the breakpoint of the t(14;18) translocation present in nearly all cases of follicular lymphoma, an indolent malignancy of germinal center B-lymphocytes. This translocation placed the BCL-2 gene on chromosome 18 under the control of the heavy chain promoter on chromosome 14, resulting in high levels of BCL-2 expression in cells in the B-lymphocyte lineage. Subsequent to its cloning, numerous experiments indicated its role in inhibiting cell death and its ability to act as an oncogene.

High levels of BCL-2 expression in cancer may be found most consistently in the lymphoid cancers follicular lymphoma and chronic lymphocytic leukemia (CLL). The presence of a t(14;18) is very rare in CLL, however. The loss of miR15 and miR16, micro-RNA loci that can suppress BCL-2 mRNA levels, may be responsible for increased BCL-2 expression in some cases of CLL, but it is not clear what proportion. Of the antiapoptotic proteins, levels of BCL-2, MCL-1, or BCL-XL have been best examined in cancer. One or more of these proteins may be found in a very wide range of cancers of all kinds. Much depends on the level of detection, and in most cases, it is not possible to know the level of expression once a certain threshold detectable by immunohistochemistry is reached. When expression of antiapoptotic protein is compared with clinical outcomes, the record is quite mixed across cancers, with certain studies showing inferior prognosis with higher antiapoptotic protein expression and others showing superior prognosis.

At first, it might be expected that expression of antiapoptotic proteins would universally confer clearly inferior prognosis. After all, antiapoptotic proteins like BCL- 2, when overexpressed in cancer cell lines in vitro, induce resistance to a very wide variety of types of chemotherapy and radiation. However, it is very important to consider the basis of expression in cell culture models in vitro versus cancer cells in vivo. In most cell culture studies of BCL-2, a cell line that is growing well is supplemented by extra BCL-2 via forced over-expression. In this case, the BCL-2 is very likely to provide extra antiapoptotic reserve and promote resistance to apoptotic signaling from applied toxins. In the case of a cancer cell in vivo, however, BCL-2 can be selected for, but not over-expressed in the expectation of a subsequent chemotherapy treatment. The selection pressure for increased antiapoptotic protein expression can be driven by nearly ubiquitous cancer phenotypes such as genomic instability and oncogene activation. The subsequent death signaling, ultimately conducted by proapoptotic proteins, can be blocked by expression of antiapoptotic proteins like BCL-2, which can bind and sequester the proapoptotic proteins. But BCL-2 in this instance is now unable to sequester subsequent additional proapoptotic signaling. Indeed, the BCL-2 is now primed with pro-death proteins that can be released to kill the cell should BCL-2 function be abrogated in any way. To simplify, in the case of the over-expression cell culture model, the excess BCL-2 is largely “empty,” whereas in the case of the cancer cell, it is largely “full” (Figure 5-3).

In consequence, overexpression of BCL-2 that is concurrently laden with pro-death proteins may in fact predispose to chemosensitivity. For example, follicular

- BIM or BID

- sensitizer BH3-only proteins - cytochrome c

- BCL-2 protein

- BAX/BAK protein

Figure 5-3. Illustration representing an unprimed mitochondrion versus a primed mitochondrion. Although it may express more BCL-2 than the normal mitochondrion, the primed mitochondrion has less antiapoptotic reserve as a result of significant priming by activator BH3-only proteins. With permission from Springer Science+Business Media: Del Gaizo Moore V and Letai A. Rational design of therapeutics targeting the BCL-2 family: are some cancer cells primed for death but waiting for a final push? Adv Exp Med Biol. 2008;615:159–75 (Figure 3), Copyright C 2008. See Color Plate 7.

lymphoma and CLL express very high levels of BCL-2 but are also extremely chemosensitive. Although they are difficult to cure permanently, initial treatment of either disease with modern chemotherapy is usually rewarded by a complete response with no evidence of residual disease. Therefore, the expression of antiapoptotic proteins alone is usually insufficient to predict the response of the mitochondrial apoptotic pathway to death signaling from chemotherapy. Instead, strategies that can simultaneously weigh the input of all antiand proapoptotic BCL-2 family proteins are needed. One such strategy is BH3 profiling, which uses synthetic BH3 domain peptides to apply standardized death signals to mitochondria so that the readiness of a mitochondrion to undergo apoptosis can be objectively measured in a controlled fashion.

Of course, once subjected to chemotherapy, cancer cells may select for higher BCL-2 expression, and increased BCL-2 may indeed be an important source of secondary chemoresistance in clinical cancer therapy. However, the longitudinal studies comparing protein expression in de novo chemosensitive tumors with that in relapsed and chemorefractory samples from the same patients have not been performed. Therefore, the highly plausible hypothesis that overexpression of BCL- 2 family proteins can contribute to acquired chemoresistance in cancer in vivo must still be considered formally unproven.

However, the abundant evidence from preclinical studies of the potential for BCL-2 and related antiapoptotic proteins to confer chemoresistance has fostered

considerable enthusiasm for the clinical targeting of BCL-2. At this writing, at least four individual molecules that target BCL-2 have entered clinical trials. As these trials mature and progress into combinations with conventional chemotherapy agents, the importance of BCL- 2 in promoting chemoresistance and supporting cancer cell survival will be tested.

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