MODULATION OF APOPTOSIS

Abstract: Methods and compositions for modulating apoptosis in cells and patients are provided. One method comprises selecting a compound which affects the ability of a channel comprised of a member of the BCL-2 family to allow passage of cytochrome c, then administering the compound to the cell or patient. Another method comprises selecting a compound which changes ion conductance properties of the channel, then administering the compound to the cell or patient. Compounds which affect these channel characteristics are also provided. Additionally, methods for identifying apoptosis-modulating compounds using lipid bilayers are provided. One method involves contacting a compound of interest with a lipid bilayer which contains an ion-channel formed by an anti-apoptotic or pro-apoptotic polypeptide of the BCL-2 family and assaying for changes in the ability of the pore to allow passage of cytochrome c. Changes in ion conductance properties of the channel, including ion selectivity, single channel conductance and rectification are also useful characteristics for identifying apoptosis-modulating compounds. A second method identifies compounds which can form ion channels in planar lipid bilayers and determines the ability to allow passage of cytochrome c, the ion selectivity and the pH dependence of such channels, where apoptosis modulating activity is predicted based on comparing these channel forming characteristics with those of BCL-2 family members.

Description: Modulation of Apoptosis Reference to Government Grant This invention was made with U.S. government support under Grant Number ADRC #AG05681-14. The government has certain rights in this invention. Cross-Reference to Related Applications This application is a continuation-in-part of U.S. application 09/127,048, filed on 31 July 1999, which claims priority to U.S. Provisional Application Serial No. 60/061,823, filed on 14 October 1997. Both applications are herein incorporated by reference. Background of the Invention (1) Field of the Invention This invention relates generally to the regulation of apoptosis and to compounds which modulate apoptosis, both antagonists and agonists, and more particularly, to methods for modulating apoptosis in cells and in animals, and to methods for identifying compounds with pro-apoptotic or anti-apoptotic activity. (2) Description of the Related Art Programmed cell death, also referred to herein as apoptosis, plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms (Raff, Nature 35(5:397-400, 1992). Genetic and molecular analysis from nematodes to humans indicates that the apoptotic pathway of cellular suicide is highly conserved (Hengartner and Horvitz, Cell 76: 1107-1114, 1994). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer. The BCL-2 family of proteins constitutes an intracellular checkpoint of apoptosis. The founding member of this family is the apoptosis-inhibiting protein encoded by the bcl-2 protooncogene which was initially isolated from a follicular lymphoma (Bakhshi et al., Cell 47:889-906, 1985; Tsujimoto et al, Science 229:1390-1393, 1985; Cleary and Sklar, Proc NatlAcad Sci USA ,52:7439-7443, 1985). The Bcl-2 protein is a 25 kD, integral membrane protein localized to intracellular membranes including mitochondria. This factor extends survival in many different cell types by inhibiting apoptosis elicited by a variety of death- inducing stimuli (Korsmeyer, Blood 80: 879-886, 1992). The family of BCL-2-related proteins is comprised of both anti-apoptotic and pro-apoptotic members that function in a distal apoptotic pathway common to all multi- cellular organisms. It has been suggested that the ratio of anti-apoptotic (e.g., Bcl-2, Bcl-x L , Mcl-1 and Al) to pro-apoptotic (e.g., Bax, Bak, Bcl-x s , Bad, Bik and Bid) molecules may be involved in determining whether a cell will respond to a proximal apoptotic stimulus (Oltvai et al., Cell 74:609-619, 1992; Farrow, et al, Curr. Opin. Gen. Dev. 6: 45-49, 1996). Because members of this family can form both homodimers and heterodimers, the latter often between anti- and pro-apoptotic polypeptides, the balance of these homodimers and heterodimers could play a role in regulating apoptosis (Oltvai and Korsmeyer, Cell 79: 189- 192, 1994). Members of the BCL-2 family are defined by sequence homology that is largely based upon conserved motifs termed BCL-Homology domains. (Yin et al, Nature 369:321- 323, 1994). BCL-Homology domains 1 and 2 (BH1 and BH2) are important in dimerization and in modulating apoptosis (Yin et al, supra). A third homology region, BH3, is found in some family members and is important in dimerization as well as promoting apoptosis (Boyd et al, Oncogene 77:1921-1928; Chittenden et al., Embo J 74:5589-5596, 1995). BH4, the most recently identified homology domain, is present near the amino terminal end of some pro-apoptotic family members (Farrow et al, supra). All known members of the BCL-2 family other than Bad and Bid have a C- terminal membrane-anchoring tail (TM). BCL-2 family members with a TM are intracellular integral membrane proteins most convincingly localized to mitochondria, the endoplasmic reticulum and the nuclear membrane. The intracellular membrane localization of BCL-2 family members together with the identification of structural similarity between the Bcl-x L monomer and the ion channel forming toxins of colicin and diphtheria toxin B fragment (Muchmore et al. Nature 357:335-341, 1996) has prompted electrophysiological studies by several groups on the ability of BCL-2 family members to form ion channels in artificial lipid membranes. These studies have reported that the anti-apoptotic family members Bcl-x L and Bcl-2 lacking the TM (Bcl-x L ΔTM and Bcl-2ΔTM) insert into synthetic lipid vesicles at pH values below 5.5, but have little or no detectable ability to form ion channels when added to lipid vesicles at pH values above 5.5 (Minn et al. Nature 355:353-356, 1997; Schendel et al, PNAS USA 94:5113, 1997; and Antonsson et al. Science 277:370-372, 1997). When added to planar lipid bilayers at physiological pH, Bcl-x ΔTM forms channels which exhibit multiple conductance states and which have an ion selectivity sequence of K + =Na + >Ca 2+ >Cl " (Minn et al, supra). Bcl-2ΔTM also forms a channel in lipid bilayers at physiological pH having multiple channel conductance states, with a primary conductance of 18 ± 2 pS being consistent with channel formation by Bcl-2 homodimers (Schendel et al, supra). Schendel et al reported that the Bcl-2ΔTM channel is cation selective at pH 5.4 but did not test ion selectivity at neutral pH. It has also been reported that BaxΔTM has an intrinsic ability to firm ion channels in liposomes and that Bcl-2 antagonizes this effect at physiological pH (Antonsson et al, supra). This reference also reported that BaxΔTM forms voltage-dependent channels in planar lipid bilayers at pH 7.0 that are slightly cation selective, with a permeability ratio of Na + to Cl " of about 2.1. None of these electrophysiological studies have shown any functional connection between the ion channel forming ability of Bcl-2 family members and apoptosis. Antonsson et al. speculated that the differences between the intrinsic channel forming properties of Bcl-x and Bcl-2 may be related to the pro-apoptotic and anti-apoptotic functions of these substances, however, what the connection might be between ion channel activity and apoptosis was not disclosed or suggested in this reference. Schendel et al. suggested that Bcl- 2 may allow transport of an ion or protein across membranes in a direction that is cytoprotective whereas Bax does the opposite, however, this reference indicated that what Bcl-2 is intended to transport across membranes remains unanswered. Minn et al. speculated that although Bcl-x L forms an ion-selective channel , it is also possible that Bcl-x L regulates the passage of proteins and cytochrome c was mentioned as a protein that resides in the mitochondrial inter membrane space and its redistribution was noted by this reference to have been suggested to promote apoptosis. Nevertheless, this group provided further speculation that the ability of Bcl-x L to promote cell survival may not result solely from the ability to form a channel so that no clear mechanism connecting the channel forming ability of Bcl-x L and apoptosis was suggested by this reference. Furthermore Minn et al. provided no experimental results suggesting what the connection between ion channel formation and apoptosis might be. Other groups have provided evidence suggesting that cytochrome c may be involved in apoptosis. The release of cytochrome c from mitochondria has been shown to be an important early event in Bax-induced apoptosis (Kluck et al. Science 275: 1132-1136, 1997; Shimizu et al. Nature 3PP:483-487, 1999). Nevertheless, none of these studies provided any suggestion that Bcl-2 family members, acting alone, are responsible for cytochrome c release. Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals. Furthermore, some DNA viruses such as Epstein- Barr virus, African swine fever virus, and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, Crohn's disease, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation. In such disease conditions it would be desirable to promote apoptotic mechanisms. Conversely, in other disease conditions it would be desirable to inhibit apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like. In the treatment of such diseases it would be desirable to inhibit apoptotic mechanisms. Thus, it would be desirable to elucidate the biochemical mechanisms involved in the regulation of apoptosis by BCL-2 family members and to utilize these mechanisms as a basis for identifying compounds which promote or inhibit death. Such compounds would be useful in developing treatment regimens for advantageously modulating the apoptotic process in disease conditions involving either inappropriate repression or inappropriate enhancement of cell death. Summary of the Invention Accordingly, the inventors herein have succeeded m discoveπng that anti- apoptotic and pro-apoptotic BCL-2 family members form channels m artificial membranes that have distinct characteristics, including the ability to allow passage of cytochrome c, ion selectivity, conductance, voltage dependence and rectification and that compounds which modulate these characteristics also modulate apoptosis. In particular, lipid membranes (including proteol posomes) with incorporated anti-apoptotic or pro-apoptotic BCL-2 family members, at neutral pH result m channels which have the inability or ability, respectively, to allow passage of cytochrome c through the membrane. Additionally, these channels are K + - selective or Cl ' -selective channels, respectively, with anti-apoptotic BCL-2 family members often displaying a complex multichannel conductance pattern having no rectification and pro- apoptotic family members generally displaying a simpler single channel conductance pattern having mild rectification. Upon exposure of anti-apoptotic channels (e.g., anti-apoptotic channel comprised of Bcl-2) to the 11-mer polypeptide ECLKRIGDELD (SEQ ID NO:l), which is a fragment of the Bax BH3 domain, and the channels acquire characteristics more similar to those of pro-apoptotic channels, including the ability to allow passage of cytochrome c, Cl " selectivity, a simpler single channel conductance pattern, and rectification. Analogously, when pro-apoptotic channels (e.g., pro-apoptotic channel comprised of Bax) are exposed to the 11-mer polypeptide, LTLRQAGDDFS (SEQ ID NO:2), which is a fragment of the Bcl-2 BH3 domain, the channels acquire characteristics more similar to those of anti- apoptotic channels, including an inability to allow passage of cytochrome c and K + selectivity. The relationship between apoptotic activity and ion selectivity is also established by channel forming experiments performed with mutant Bcl-2 and Bax polypeptides having altered apoptotic activity. In particular, a mosaic Bcl-2 polypeptide m which the BH3 domain is substituted with a Bax BH3 domain both promotes cell death and forms a Cf-selective channel in a planar lipid bilayer. Conversely, a naturally occurring mutant Bax polypeptide which has lost its apoptotic activity forms a K + -selectιve channel. Accordingly, one aspect of the present invention provides a method of inducing apoptosis m a cell. The method compπses selecting a compound which induces the formation of a pore capable of transporting cytochrome c out of mitochondria, then administering the compound to the cell. The selecting step in this method compπses testing the compound for the induction of the pore formation. In one embodiment, the compound can be a polypeptide or derivative, including SEQ ID NO:l. In other embodiments, the cell is a cell of a patient with a condition mediated by excessive down-regulation of apoptosis. Another aspect of the present invention provides a method of inhibiting apoptosis m a cell. The method comprises selecting a compound which inhibits the formation of a pore capable of transporting cytochrome c out of mitochondria, then administering the compound to the cell. The selecting step in this method comprises testing the compound for the inhibition of said pore formation. In one embodiment, the compound can be a polypeptide or derivative, including SEQ ID NO:2. In other embodiments, the cell is a cell of a patient with a condition mediated by excessive apoptosis. An additional aspect of the present invention provides methods for treating a patient having a condition mediated either by excessive down-regulation of apoptosis or excessive apoptosis The methods comprise selecting a compound which either induces the formation of a pore capable of transporting cytochrome c out of mitochondria (for conditions mediated by excessive down-regulation of apoptosis) or inhibits the formation of a pore capable of transporting cytochrome c out of mitochondria (for conditions mediated by excessive apoptosis), then administering the compound to the patient. Additionally, the present invention is directed to methods for inducing or inhibiting apoptosis in a cell. The method comprises selecting a compound which changes the ion selectivity of an anti-apoptotic BCL-2 family member from K + selective to Cl " selective (when induction of apoptosis is desired) or selecting a compound which changes the ion selectivity of a pro-apoptotic BCL-2 family member in the cell from Cl " selective to K + selective (when inhibition of apoptosis is desired), then administering the compound to the cell. The selecting step in these embodiments compπses testing the compound for activity m changing the ion selectivity as desired. These methods can also be used for treating a patient having a condition mediated by excessive down-regulation of apoptosis (where induction of apoptosis is desired) or excessive apoptosis (where inhibition of apoptosis is desired). Further aspects of the present invention are directed to selecting and identifying apoptosis-modulating compounds, which comprises providing a lipid bilayer comprising a channel compπsed of a polypeptide from the BCL-2 family, contacting the bilayer with a compound of interest, and determining ion selectivity of the channel. A cell death agonist can be identified by its effect in changing the selectivity of a channel comprised of an anti- apoptotic polypeptide from cation-selective to amon-selective. Alternatively, if the channel comprises a pro-apoptotic polypeptide, a cell death antagonist will change the channel from amon-selective to cation-selective. Anti-apoptotic polypeptides of the BCL-2 family include the mammalian proteins Bcl-2, Bcl-x L , Mcll, Al, and NR-13, while pro-apoptotic polypeptides of this family include Bax, Bak, Bik, Bid, and Bad (Figure 10). In one embodiment, the lipid bilayer comprises a planar bilayer in the presence of an ion concentration gradient and the determining ion selectivity step comprises measuring the reversal potential of the channel. In another embodiment, the lipid bilayer comprises a proteoliposome loaded with a mixture of cations and anions in known amounts and the determining ion selectivity step comprises measuring the relative rates of cation and anion efflux from the proteoliposome using ion specific electrodes. The channel forming activities of Bcl-2 and Bax are also distinguished by their different responses to pH in mediating release of Cl " from KC1 loaded lipid vesicles. While the same quantities of each protein initiate rapid release of Cl " (75%) when added at pH 4.0, Bcl-2 is twice as active as Bax at pH 3.5, e.g., Bcl-2 and Bax release 81% and 41% of the Cl " , respectively, but Bax is more.active than Bcl-2 at pH 5.0, e.g., releasing 41% of the Cl " as opposed to 7% released by Bcl-2. The structural basis for this distinction is due to the composition of helices α-5 and α-6 in BCL-2 family members. The acidic content of the α-5 and α-6 helices of Bax and other pro-apoptotic family members define their low pH activity and will always give them an insertion ratio of greater than 1 when comparing insertion in lipid bilayers at low pH and neutral pH values. Conversely, these helices in Bcl-2 and other anti-apoptotic family members are loaded with more of the basic lysine, arginine and histidine residues, which will decrease in polarity with increasing pH. Thus, Cl " release from liposomes in the presence of an anti-apoptotic BCL-2 family member will not decrease as much with neutral pH. Thus, another aspect of the invention provides a method for identifying apoptosis-modulating compounds which comprises both evaluating ion selectivity of a channel formed by a test compound and assessing pH dependence of channel formation by the test compound. Evaluating ion selectivity comprises providing a lipid bilayer, contacting the bilayer with a compound of interest, and assaying for ion-selective channel formation. Assessing pH-dependence of channel formation comprises (1) providing lipid vesicles in a solution having a pH of 3.5, pH 4.0, pH 5.0, or pH 7.0, the vesicles containing a channel indicator, (2) contacting the test compound with each of the vesicles, and (3) assaying for release of the channel indicator from the vesicles. If, like Bax, the compound forms an anion- selective channel, shows a ratio of channel indicator release at pH 3.5 and pH 4.0, (R 3 . 5 0 ) of less than 1, and shows a ratio of indicator release at pH 4.0 and 5.0 (Rt. 0 /5.0) of between 1 and 2, the compound will have apoptotic activity. Conversely, the compound is anti-apoptotic if it behaves like Bcl-2 in these tests, i.e., it forms a cation-selective channel and produces a R 3 . 5/ .o of about 1.0 and a t.o/s.o of greater than 7.0. Additional aspects of the invention provides compositions for inducing apoptosis or inhibiting apoptosis, comprising polypeptides of no more than 50 contiguous ammo acids which contain SEQ ID NO: 1 or SEQ ID NO:2, respectively, or derivatives thereof. In one embodiment, the composition is in a pharmaceutically acceptable preparation. Methods of inhibiting or inducing apoptosis cells comprising administering these compositions are also provided. Among the several advantages found to be achieved by the present invention, therefore, may be noted the provision of methods for modulating apoptosis in cells, methods for modulating apoptosis m patients having a condition which is mediated by excessive apoptosis or excessive down-regulation of apoptosis; the provision of methods for screening compounds for apoptosis-modulating activity that allow compounds to be evaluated without regard to their ability to penetrate into cells or stability m a cell-based assay; the provision of methods that can be used to rapidly screen large numbers of compounds for apoptosis- modulating activity; and the provision of death agonists and antagonists identified by these pid-bilayer screening methods. Brief Description of the Drawings Figure 1 illustrates the effect of pH on Bax and Bcl-2 induced release of Cl " ions from synthetic lipid vesicles, showing (FIG. 1A) vesicles incubated with BaxΔTM at pH 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0, (FIG. IB) vesicles incubated with Bcl-2ΔTM at pH 3.5, 4.0, 4.5 and 5.0, (FIG. 1C) vesicles incubated with BaxΔTM at pH 6.0 shifted to pH 4.0 at the time point indicated with the vertical arrow, and (FIG. ID) vesicles incubated with Bcl-2ΔTM at pH 5.0 shifted to pH 4.0 at the time point indicated with the vertical arrow; Figure 2 illustrates the low pH formation of Bax channels m planar lipid bilayers as characterized by the currents appearing after addition of BaxΔTM to the cis chamber of an established planar lipid bilayer in a 450/150 mM cis to trans KC1 gradient at pH 4.0, showing (FIG. 2A) a tracing of an initial, inward Cl- current that appeared spontaneously at 0 volts (O s ), (FIG. 2B) tracings of the currents that occurred at +40 mV (upper tracing) or -40 mV (lower tracing) after removal of soluble BaxΔTM from the cis chamber showing the transition from the O s to a large open pore with * denoting a direct C-O s to 0 2 transition, and (FIG. 2C and 2D) current-voltage (I/V) plots for the mature pore at pH 4.0 (FIG. 2C) and following a shift to pH 7.0 in the presence of the 450/150 mM KC1 gradient (open circle) or m symmetric 150 mM KC1 (filled square) (FIG. 2D); Figure 3 illustrates the incorporation of Bax into planar lipid bilayer membranes following addition of BaxΔTM-containmg unilamellar proteoliposomes to the cis chamber of an established lipid bilayer, showing (FIG. 3 A) a tracing of an initial inward Cl " -selectιve current in the presence of a 450/150 mM KC1 gradient, (FIG. 3B) tracings of the current of a single Bax channel in symmetrical 150 mM KC1 at different voltages, (FIG. 3C) an I/V plot of the Bax channel, and (FIG. 3D) longer tracings of the current which demonstrate rectification at 70 mV (upper tracing) and closures at -70 mv (lower tracing); Figure 4 illustrates the low pH formation of Bcl-2 channels m planar lipid bilayers as characteπzed by the currents appearing after addition of Bcl-2ΔTM to the cis chamber of an established planar lipid bilayer m a 450/150 mM cis to trans KC1 gradient at pH 4.0, showing (FIG. 4A) a tracing of an initial, outward K + current that appeared spontaneously at 0 volts (O s ), (FIG 4B) channel transitions between the open (O s ) and closed (c) states at 0 volts for three duplicate experiments, (FIG. 4C) a histogram of the distribution of current amplitudes for the channel over 120 seconds, (FIG. 4D) an I/V plot of the Bcl-2 channel from (B), and (FIG. 4E) an I/V plot of the activity of the large pore that formed over time at pH 4.0 (closed square) and following a shift to pH 7.0 (open square) or in symmetric 150 mM KC1 (filled square); Figure 5 illustrates the incorporation of Bcl-2 into planar lipid bilayer membranes following addition of Bcl-2ΔTM-contammg unilamellar proteoliposomes to the cis chamber of an established lipid bilayer, showing (FIG. 5 A) a tracing of the initial K + current of two Bcl-2 channels that simultaneously appeared m the presence of a 450/150 mM KC1 gradient, (FIG. 5B) tracings of the channel current of an established single Bcl-2 channel (upper tracing) resulting from application of a series of voltage steps (lower tracing) to the channel m a 450/150 mM KC1 gradient, (FIG. 5C) tracings of a single Bcl-2 channel in symmetrical 150 mM KC1 at different voltages, and (FIG. 5D) an I/V plot of the Bcl-2 channel; Figure 6 illustrates models of the three-dimensional structure of the positively charged surface of the α-5 and α-6 helices of Bax (left side) and the negatively charged surface of the same region of Bcl-2 (right side) showing darker surfaces for the most highly charged regions, with linear interpolation for values in-between; Figure 7 illustrates the aligned sequences of α-5 and α-6 helices in two anti- apoptotic (Bcl-x L and Bcl-2) (SEQ ID NOS.2-3) and m two pro-apoptotic molecules (Bax and Bak) (SEQ ID NOS:4-5), Figure 8 illustrates the effect of a death-promoting 11-mer Bax peptide on the conductance characteristics of a Bcl-2 channel m a planar lipid bilayer showing the conductance patterns of Bcl-2 (FIGS. 8A-D) and Bax (FIG 8E) channels at 80 mV under voltage clamp conditions with the frequency that each channel is present at one of several conductance levels plotted against the conductance m picoamps in the absence (FIGS. 8 A and 8E) or presence of the 11-mer peptide at 20 μM (FIG. 8B), 50 μM (FIG. 8C), or 100 μM (FIG. 8D); Figure 9 illustrates the effect of the Bax death agonist peptide on the conductance and ion selectivity of a Bcl-2 channel showing an IV plot of channel activity at 150 mM symmetrical KC1 and the pK/pCl calculated from the reversal potential in a 450: 150 mM cιs:trans KC1 gradient for (FIG. 9A) a Bcl-2 channel, (FIG. 9B) a Bcl-2 channel after the addition of the Bax death agonist peptide to the cis side of the bilayer, and (FIG. 9C) a Bax channel, with the slope of the conductance at negative potentials indicated; Figure 10 illustrates the members of the BCL-2 family showing the presence and location of homology domains BH1, BH2, BH3 and BH4, as well as the transmembrane domain (TM); Figure 11 illustrates the aligned sequences of mouse and human Bax (SEQ ID NOS:6-7) and human and mouse Bcl-2 (SEQ ID NOS:8-9), with identical residues and conservative substitutions shaded dark and light, respectively, exon boundaries indicated by vertical dashed lines, and the BH1 and BH2 domains boxed; Figure 12 illustrates the kinetics of Bax pore formation, comparing the release of carboxyfluorescein (short dashes) and FITC-cytochrome C (long dashes) from hposomes; and Figure 13 illustrates the measurement of time constants for dequenchmg of carboxyfluorescein via Bax pores by various sizes of dextran, at three concentrations of Bax in hposomes. Descnption of the Preferred Embodiments The present invention is based on the surprising discovery that BCL-2 family members form pores that can regulate cytochrome c passage out of mitochondria that the ability of cytochrome c to pass through channels formed by BCL-2 family members m lipid membranes is dependent upon whether the polypeptide has anti-apoptotic activity or pro- apoptotic activity. Specifically, channels formed by pro-apoptotic BCL-2 family members can allow cytochrome c to pass through, whereas channels formed by anti-apoptotic BCL-2 family members cannot allow passage of cytochrome c. Furthermore, it has been discovered that the ion-selectivity of channels formed by BCL-2 family members in lipid membranes is dependent upon whether the polypeptide has anti-apoptotic activity or pro-apoptotic activity, specifically anti-apoptotic polypeptides are K + -selectιve while pro-apoptotic polypeptides are Cl ' -selective, and (3) that a compound which modulates the above selectivities can modulate apoptosis. These discoveries were unexpected because there was no previous indication that the channels made by pro-apoptotic family members are large enough to allow passage of cytochrome c, and because channels formed by Bax, which is pro-apoptotic, had previously been reported to be cation selective at pH 7.0 with a permeability ratio of Na + to Cl " of about 2.1 (Antonsson et al, supra), whereas it is now herein reported that Bax channels are anion selective. Also, no connection had previously been made between the ability of a channel to allow cytochrome c to pass, or the ion selectivity of a channel formed by a BCL-2 family member, and apoptotic activity. The term "ion selective" is used herein to mean a characteristic of a membrane channel or pore where a particular ion is favored for passage through the channel over another ion. For example, "K + selective" refers to a channel which favors passage of K + over another ion, for example Cl " . The degree of ion selectivity between two ions can be expressed as a ratio, also known as a permeability ratio. For example, a channel which has a permeability ratio of Na + to Cl " of 2.1 allows 2.1 times as much Na + as Cl " to pass though over a given time period and can be designated Na + selective or cation selective. Therefore, the present invention provides novel methods for modulating apoptosis which involves administering to cells compounds which change the cytochrome c passage or the ion selectivity of a pores made by members of the BCL-2 family. If a decrease in apoptotic activity is desired, the cells are administered a compound which inhibits the formation of a cytochrome c transporting pore of a pro-apoptotic BCL-2 family member (for example, Bax, Bad, or Bid) or changes the ion selectivity of a pore of a pro-apoptotic BCL-2 family member from Cl " to K + ; if an increase in apoptotic activity is desired, the cells are administered a compound which induces the formation of a cytochrome c transporting pore of an anti-apoptotic BCL-2 family member (for example, Bcl-2 or Bcl-x L ) or changes the ion selectivity of an anti-apoptotic BCL-2 family member from K + to Cl " . As used herein, the term "compound" is intended to include inorganic or organic chemical compounds as well as biochemical molecules such as nucleic acids, proteins, lipids, lipoproteins, and carbohydrates. Without being held to a particular mechanism, it is believed that the pro-apoptotic and anti- apoptotic family members normally form separate ion channels having distinct pro-apoptotic and anti-apoptotic activities and that altering the balance of these activities, such as by enhancing the first activity or reducing the second activity, will modulate apoptosis. In preferred embodiments, when apoptosis is to be induced in a cell, the cell is administered an 11-mer polypeptide with the sequence ECLKRIGDELD, presented herein as SEQ ID NO:l. This compound is a fragment of the BH3 domain of Bax corresponding to amino acids 61-71 of full length Bax (SEQ ID NO:3). As demonstrated in Examples 8 and 9, this compound, when added to a membrane comprising a anti-apoptotic BCL-2 family member, changes the ion selectivity of the pore formed by the anti-apoptotic BCL-2 family member from Cl " selective to K + selective Analogous polypeptides from other pro-apoptotic BCL-2 family members are also encompassed within these embodiments. As used herein, the term "polypeptide" encompasses a linear sequence of two or more ammo acids, linked by peptide bonds. In other preferred embodiments, when apoptosis is to be inhibited in a cell, the cell is administered an 11-mer polypeptide with the sequence LTLRQAGDDFS, presented herein as SEQ ID NO:2. This compound is a fragment of the BH3 domain of Bcl-2 corresponding to ammo acids 95-105 of full length Bcl-2 (SEQ ID NO:4) As demonstrated in Example 9, this compound, when added to a membrane compπsing a pro-apoptotic BCL-2 family member, changes the ion selectivity of the pore formed by the proapoptotic BCL-2 family member from K + selective to Cl selective. Analogous polypeptides from other anti- apoptotic BCL-2 family members are also encompassed by these embodiments. In other preferred embodiments, a polypeptide or polypeptide derivative is administered to a cell, wherein the polypeptide comprises SEQ ID NO: 1 or SEQ ID NO:2. In these embodiments, the polypeptide can be any length, including a full length BCL-2 family member. As used herein, the term "administer" includes indirect administration of the compound, for example, treatment with a nucleotide sequence which encodes the polypeptide compound operably linked to a promoter which allows the expression of the polypeptide. As used herein, a polypeptide derivative includes any functional variant or conservative substitution of the polypeptide. A functional variant of a naturally-occurπng polypeptide contains one or more ammo acid substitutions in that sequence which do not destroy the ability of the resulting polypeptide to function to modulate apoptosis. Preferably, ammo acid substitutions in functional variants are conservative ammo acid substitutions, which refer to the mterchangeabihty of residues having similar side chains. Conservatively substituted ammo acids can be grouped according to the chemical properties of their side chains. For example, one grouping of amino acids includes those ammo acids have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those ammo acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those ammo acids having basic side chains (K, R, and H); another grouping is those ammo acids having acidic side chains (D and E), another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those ammo acids having ahphatic- hydroxyl side chains (S and T); another grouping is those amino acids having amine- contammg side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those ammo acids having sulfur- containing side chains (C and M). Preferred conservative ammo acid substitutions groups are: R-K; E-D, Y-F, L-M; V-I, and Q-H. A functional variant as used herein can also include modified sequences in which one or more ammo acids have been inserted, deleted, or replaced with a different amino acid or a modified ammo acid or unusual amino acid, as well as modifications such as glycosylation or phosphorylation so long as the polypeptide containing the modified sequence retains the ability to modulate apoptosis. By retaining the biological activity, it is meant that the modified polypeptide can function to modulate apoptosis.. Because of the universality of the apoptotic mechanisms which are the subject of the present invention, it is believed that apoptotic mechanisms of any cell of any multicellular organism would be affected by the methods presented herein. Included are cells from any invertebrate or vertebrate, including humans. Preferred are cells from a patient with a deleterious condition which is mediated by impaired regulation of apoptosis - either excessive apoptosis or excessive down-regulation of apoptosis. The term "patient" is used herein to include any vertebrate. Most preferred are cells from a human patient which has a condition mediated by impaired regulation of apoptosis. Examples of conditions which may be mediated by excessive down-regulation of apoptosis are neoplasias, diseases caused by Epstem-Barr virus, African swine fever virus, and adenovirus, lymphoproliferative conditions, cancer, arthritis, Crohn's disease, inflammation, and autoimmune disease. Examples of conditions which may be mediated by excessive apoptosis are immunodeficiency diseases, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and conditions characterized by slow wound healing. Compositions comprising the compound which modulates apoptosis can be administered to a cell ex vivo. For example, if a patient has a metastatic cancer which is mediated by down-regulation of apoptosis, the patient can be treated by removing bone marrow cells, treating the patient with a sublethal dose of radiation to kill the cancer in the patient, treating the bone marrow cells with a pro-apoptotic compound of the present invention to kill the cancer cells in the bone marrow, then transplanting the bone marrow back into the patient. Preferably, however, the compounds of the present invention are administered to cells which are withm a living patient The invention compounds can be administered by any suitable route known in the art including, for example, intravenous, subcutaneous, intramuscular, transdermal, lntrathecal, or intracerebral. The compositions can also be administered to target cells directly in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of a slow release formulation. For treating cells m the central nervous system, administration can be by injection into the cerebrospinal fluid. In embodiments in which it is desirable to administer polypeptides to cells or to a patient, these can be administered by administering a polynucleotide encoding the polypeptide to the cell or patient. It is contemplated that the compounds of the present invention are usually administered as compositions in the form of a pharmaceutical preparations. Such preparations are made m a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological salme solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used. It may also be desirable that a suitable buffer be present the composition. Such solutions can, if desired, be lyophihzed and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection The primary solvent can be aqueous or alternatively non- aqueous. The carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolaπty, viscosity, clarity, color, steπhty, stability, rate of dissolution, or odor of the formulation. Similarly, the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-bram barrier. Such excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion. It is also contemplated that certain formulations comprising the agent are to be administered orally. Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms. Some examples of suitable earners, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, manmtol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalhne cellulose, polyvinylpyrrohdone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like. The formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents The compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art. The formulations can also contain substances that dimmish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents. Alternatively, the carrier can be a gelatin capsule, or coated tablet, or the like which is suitable for administering a unit dosage to a patient. The agent is administered to the organism in an amount effective to modulate apoptosis of target cells within the organism. The specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied. The dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropπate dosage for treatment is routinely made by those of ordinary skill m the art. Such calculations can be made without undue experimentation by one skilled m the art in light of the activity disclosed herein m apoptosis assays. Exact dosages are determined in conjunction with standard dose-response studies. It will be understood that the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used. In some embodiments of the present invention, the compound which is used to mediate apoptosis in a cell is first selected wherein the selecting comprises testing the compound for the ability to either (a) affect the ability of a channel formed by a BCL-2 family member to transport cytochrome c out of the mitochondria, or (b) affect the ion selectivity of such a channel. This selecting step is intended to apply to all compounds which are known or not known to mediate apoptosis, and comprises, in whole or in part, testing for the ability or inability of a particular compound to mediate apoptosis. Such testing can involve either determining or confirming previously suspected channel forming activity. The process of testing and selecting a compound which mediates apoptosis is also intended as an additional embodiment of the invention comprising methods for identifying apoptosis-modulating compounds. Based on the structural features of the critical ammo acid sequence of the peptides of the present invention that permit either formation of a pore for cytochrome c passage or binding to a BCL-2 family member to form the desired pore, one can develop non- peptide derivatives that are capable of such action. The techniques for development of such peptide mimetics are well known in the art (see, for example, Navia et al. Trends Phartn Sci 74: 189-195, 1993; Olson et al, J Med Chem. 3(5:3039-3049, which are herein incorporated by reference). Typically this involves identification and characterization of the protein target as well as the protein ligand using X- ray crystallography and nuclear magnetic resonance technology such as has been done with BCL-2 family members (see, e.g., Figure 6). Using information learned from the structure of the target protein and ligand, a pharmacophore hypothesis is developed and compounds are made and tested in an assay system. Thus, in some embodiments of the invention, apoptosis-modulating compounds are conveniently selected or identified using methods which involve assaying the effect of candidate compounds on the ion selectivity of a channel in a lipid bilayer, the channel being comprised of a polypeptide from the BCL-2 family. The lipid bilayer assay described herein has several advantages over a whole cell assay to identify candidate death agonists and antagonists. First, it provides specific information about the biochemical effect of compounds on what is believed to be an underlying biochemical mechanism of the regulation of apoptosis by BCL-2 family members. In addition, a lipid bilayer assay will identify more candidate death agonists and antagonists than the whole cell assay. For example, some compounds that have apoptotic activity are not detected by the whole cell assay due to cell uptake problems or degradation of the compound by the cell. However, such problems might be overcome in further development or modification of a lead compound identified by a lipid bilayer assay. The lipid bilayer used in the method comprises a double layer of lipid in a hydrophilic environment. In some embodiments, the lipid bilayer comprises a planar bilayer, while in other embodiments, the lipid bilayer comprises a proteoliposome. Preparation of such lipid bilayers can be accomplished using biochemicals and techniques typically used in bilayer reconstitution experiments. See, e.g., Hanke, W. and Schlue, W.-R, Planar Lipid Bilayers, D.B. Sattelle, ed. Academic Press, 1993. The lipids in the bilayer can comprise natural lipids purified from biological membranes and/or synthetic lipids. Examples of natural lipids include but are not limited to Azolectin, a commercially available lipid extract from soybeans (Sigma, St. Louis, MO), and phosphatidylcholine and phosphatidylethanolamine purified from egg yolk. Synthetic lipids can be lipids which are pure in their head-group and hydrocarbon composition or lipids that are not normally present in biological membranes. Preferably, the lipid bilayer comprises at least 10% negatively charged lipids. The lipid composition of planar bilayers preferably comprises about 10-20% choline and 80-90% negatively charged lipids. Liposomes preferably comprise about 60% 1,2 dioleoyl phophatidyl choline and about 40% 1,2 dioleoyl phosphatidyl glycerol. A planar lipid bilayer is established using standard techniques (Hanke and Schlue, supra). In brief, a double compartment chamber with an aperture having a diameter of about 0.1 to about 1 mm and a thickness of about 1 mm or less between the compartments is filled with an aqueous solution to completely cover the aperture. The aqueous solution is typically a KC1 solution although other salt solutions can be used, including NaCl and CaCl 2 . Preferably, the salt solution is comprised of monovalent cations and monovalent anions. A drop of lipid dissolved in an organic nonpolar solvent is then spread across the aperture and allowed to thin to the thickness of a lipid bilayer. The concentration of lipids in the lipid solution is between about 1 and 100 mg/ml, preferably between 20 and 80 mg/ml, and more preferably between 30 and 70 mg/ml. Most preferably, the lipid concentration is 50 mg/ml. Decane is generally used as the solvent, but the solvent can also be other short chain alkanes such as hexane as well as long-chain alkanes and alkenes such as squalene. When the solvent is decane, the lipid bilayer will have a specific capacity of about 0.4 μF/cm 2 . Preferably, the aperture is pretreated with lipid by covering it with the lipid solution and then evaporating the solvent. After pretreatment, the compartments are filled with the aqueous solution and a second drop of lipid solution is spread on the aperture for forming the lipid bilayer. The planar lipid bilayer can also be prepared by other techniques known in the art such as by forming a folded bilayer across the aperture from two monolayers, one in each compartment, and apposition of two monolayers at the tip of a patch pipet (Hanke and Schlue, supra). The lipid bilayer comprises at least one channel comprised of an anti-apoptotic or pro-apoptotic polypeptide from the BCL-2 family (Fig. 10). The sequences of these polypeptides are readily available on GenBank and in the scientific literature. The Bcl-2 related polypeptide can be a conservatively substituted variant of the naturally-occurring sequence, or a fragment thereof. Fragments of BCL-2 related polypeptides useful in the invention should have sufficient length to form a channel in a lipid bilayer. Construction of such fragments and testing them for channel forming activity or for alteration of channels provided by a BCL-2 family member, either endogenously present or administered with the fragment, can be done using well-known deletion-mutagenesis procedures and the procedures described herein. Such fragments of BCL-2 related polypeptides can, in some embodiments, include polypeptides having a length up to and including the full length BCL-2 family member. The channel can also be formed by any subsequently discovered BCL-2 family member that has channel forming activity in vitro and has anti-apoptotic or pro-apoptotic activity. For the formation of pores of sufficient size to allow passage of cytochrome c, renaturation of purified bacteπally expressed Bax should be m 8M urea with gradual refolding by dialysis. See, e.g. Anfinsen et al. Adv. Protein Chem. 29:205-300, 1975. Preferably, the polypeptide used to form the channel lacks a C-termmal membrane-anchoring tail (TM) to eliminate interference by this hydrophobic anchor with the channel forming activity of other portions of the polypeptide dunng in vitro studies with lipid bilayers. All known mammalian BCL-2 family members other than BID and BAD contain a TM (Fig. 10). A polypeptide lacking the TM (ΔTM) can be prepared by expression from a cDNA in which the DNA sequences encoding the TM have been removed from a cDNA encoding the full-length BCL-2 family member using standard deletion mutagenesis techniques. cDNA clones encoding full-length BCL-2 family member proteins are either publicly available or readily obtainable using published sequences. A preferred anti-apoptotic polypeptide for forming the channel is Bcl-2ΔC21 which compnses ammo acids 1-218 or 1-221 of human or mouse Bcl-2 shown in FIG. 11 or a conservatively substituted variant thereof. For pro-apoptotic channels, the preferred pro- apoptotic polypeptide is BaxΔC19, which comprises ammo acids 1-173 of human or mouse BAX as shown in FIG. 11, or a conservatively substituted variant thereof. The channel can be formed by adding the polypeptide to the aqueous solution on either or both sides of the bilayer and then mixing the solution. Preferably, the polypeptide is added to only one side of the bilayer which is designated as the cis side. Typically, the concentration of the polypeptide in the aqueous solution is between about 5 and 500 nM, preferably between about 10 and 250 nM, more preferably between about 20 and 125 nM, and even more preferably between about 40 and 60 nM. The aqueous solution is buffered to a pH of between about 4.0 and 7.2. Because low pH promotes insertion into planar bilayers of polypeptides from the BCL-2 family, the aqueous solution is preferably between pH 4.0 and 6.0. The pH of the solution is preferably adjusted to between 6.8 and 7.2 before determining the ion selectivity of the channel. The polypeptide is preferably incorporated into the planar lipid bilayer at neutral pH by fusing the bilayer with a reconstituted unilamellar proteoliposome comprising the polypeptide. Proteoliposomes can be prepared from purified polypeptides and lipids by standard techniques known m the art, including dialysis, gel filtration and dilution techniques. A membrane fragment vesicle preparation of an intracellular membrane where the BCL-2 family member polypeptide is found in vivo can also be used as a source of proteoliposomes. Preferably, the dialysis approach is used and involves adding about 7.5 to 15 μmole of a polypeptide from the BCL-2 family to about 25 to 75 mg lipid m a buffered solution at a pH of about 6.8 to 7.2. This protein-hpid solution is placed in a dialysis bag with a molecular weight cutoff size selected to make the dialysis bag impermeable to lipid and polypeptide but is permeable to any detergent in the polypeptide preparation. Dialysis is performed m a large volume of aqueous solution buffered at about pH 6.8 to 7.2 until unilamellar proteoliposomes form, usually about 10 to 20 hours. Once prepared, the proteoliposomes are added to one or both sides, preferably the cis side, of a pre-established planar bilayer with mixing until fusion occurs. As an alternative or in addition to mixing, fusion can be initiated by electrofusion, in which a high electπc field pulse is applied to the bilayer chamber. Preferably, about 5-20 μl of proteoliposomes, compnsmg about 2 0 x l0 4 to l0 x l0 "4 nmoles polypeptide incorporated in Azolectin vesicles, are added to the cis side of a planar lipid bilayer. Formation of a channel by the polypeptide is verified by applying a voltage and assaying for a current above noise and leak values. Once an initial channel current is identified, soluble polypeptide or proteoliposomes unincorporated m the bilayer can then be removed from the chamber by exchanging the chamber contents with fresh buffer Prior to or following addition of the channel-forming polypeptide, the concentration of ions in one or both compartments of the bilayer chamber is adjusted to establish an ion concentration gradient between the compartments so that ion selectivity of the channel can be determined. A concentration gradient means that the concentration of cations and the concentration of ions on one side of the bilayer are both higher than on the other side of the bilayer. The concentrations of cations and anions on a particular side of the bilayer need not be equivalent to each other but it is preferable that they are approximately equal. If the concentration gradient is set up before addition of the polypeptide, the ion selectivity of the initial currents can be identified. The ion concentration gradient is generally between 2- and 100-fold cιs:trans and preferably is between about 3-fold and 10-fold cιs:trans. Alternatively, similar gradients can be set up trans is. Examples of ion concentration gradients typically used are 150:15 μM, 450:150 μM, and 1.0:0.5 M To determine if a compound of interest has cell death promoting or cell death antagonizing activity, the compound is added to either side of a planar bilayer comprising a channel comprised of an anti-apoptotic or pro-apoptotic polypeptide of the BCL-2 family. The ion selectivity of the channel is determined in the presence of an ion concentration gradient which can be established before or after addition of the compound. If the ion selectivity of an anti-apoptotic channel changes from cation-selective to amon-selective in the presence of the compound, the compound is a cell death agonist. Conversely, if the compound changes the ion selectivity of a pro-apoptotic channel from amon-selective to cation-selective, the compound is a cell death antagonist. Preferably, the compound is tested at several different concentrations to determine the lowest amount that is effective in changing ion selectivity. The compound's activity can be further characterized by determining whether it affects the conductance pattern and rectification of the channel. These channel properties are assessed by measuring the current at different negative and positive voltages when equivalent ion concentrations are present on both sides of the planar bilayer. Based on the distinct characteristics of Bcl-2 and Bax channels and the behavior of the Bcl-2 channel in the absence and presence of an 11 -mer peptide with death agonist activity, it is believed that a compound with death agonist activity will impart a pro-apoptotic conductance pattern and rectification to an anti-apoptotic channel. In a preferred embodiment, the compound is also tested for its ability to insert into membranes to form a channel and for the effect of pH on any such channel forming activity. This can be accomplished by the incubating the compound with lipid vesicles in solutions at different pH values between 3 5 and 7.0 as described below. If the compound has both cation selectivity and its channel forming activity is affected by pH in a manner similar to that of Bax, the compound is more likely to be pro-apoptotic under conditions that activate native apoptosis agonists. Conversely, if the compound has both anion selectivity and its channel forming activity at acidic and neutral pH is similar to that of Bcl-2, the compound is more likely to be anti-apoptotic under conditions that activate native apoptosis antagonists. In one embodiment of the invention, the lipid bilayer comprises a unilamellar proteoliposome loaded with a mixture of cations and anions in known amounts and the effect of a compound on the relative rates of cation and anion efflux from the proteoliposome is determined. The unilamellar proteoliposome can be prepared as described above or by any other technique known in the art. Preferably the mixture of cations and ions comprises equal amounts of K + and Cl " . Cation or anion efflux can be measured with cation- or amon-specific electrodes or alternatively by a dye which reacts with the cation or anion m a concentration dependent manner. The relative rates of cation and anion efflux rates from the vesicles is compared in the absence and presence of a compound of interest. If the compound induces a faster rate of anion efflux from a proteoliposome comprising an anti-apoptotic channel, it has death agonist activity. Conversely, if the compound induces a faster rate of cation efflux from a proteoliposome comprising a pro-apoptotic channel, it has death antagonist activity. The invention also provides a method for selecting or identifying apoptosis modulating compounds based on their ability to both form a cation- or amon-selective channel in a lipid bilayer and by the effect of pH on their channel forming activity in lipid vesicles. The lipid bilayer lacks preformed channels and can be a planar bilayer or a hposome loaded with cations and anions. A compound of interest is added to the bilayer and ion selectivity is determined as described m the planar bilayer and proteoliposome expenments described above. Preferably, the method comprises assaying for formation of K + and/or Cl " - selective channels. The effect of pH on channel forming activity is quantified by incubating the test compound with lipid vesicles in separate solutions at pH values between 3.5 and 7.0 (e.g., pH 4.0, 5.0, 6.0, 7.0 and others as required). The lipid vesicles comprise a lipid bilayer surrounding an aqueous interior which contains a channel indicator. The channel indicator is any substance that is detectable upon release from the vesicle. For example, the indicator can be a detectable ion or a substrate that forms a detectable reaction product upon contact with an enzyme located outside the vesicle, or a fluorescent compound such as carboxyfluorescein or fluorescem-dextran that increases intensity when its release from the vesicle removes concentration dependent quenching. The test compound is incubated with vesicles at the desired pH values for the same length of time. At the end of the incubation period, the amount of channel indicator released at each pH is then determined. The channel indicator is detected by an electrode or spectrophotometer appropriate for the type of indicator used. If no channel indicator is released, then the compound does not form channels. If the ratio of release at pH 3.5 to that at pH 4.0 (R 3 5 4 o) is less than 1 and the ratio of release at pH 4.0 to that at pH 5.0 (R 40 5 0 ) is between 1 and 2, the compound is inserting under pH conditions that favor channel formation by anti-apoptotic compounds. An R 3 5 40 of approximately 1 accompanied by an (R t 05 0 ) of greater than 7 indicates the compound is inserting under pH conditions that favor channel formation by anti-apoptotic compounds. This information allows the prediction of channel forming characteristics of compounds having ion selectivities appropπate to pro- or anti- apoptotic action. With compounds that vary in structure it may be useful to assess the pH dependence of channel formation using a broader pH range, for example between pH 3.5 and 8.0. In other embodiments of the present invention, apoptosis-modulating compounds are selected (and identified) by testing for their ability to either (a) induce the formation of a pore capable of transporting cytochrome c out of mitochondria (when the compound is utilized to induce apoptosis), or (b) inhibit such pore formation (when the compound is utilized to inhibit apoptosis). Testing for this ability is preferably performed by applying the compound to a BCL-2 family pore formed in either a planar bilayer or a proteoliposome as discussed above, then determining whether the pore has the cytochrome c-transporting characteristics desired. For example, if a compound is being selected or identified for its ability to induce apoptosis, a pore is first formed using a pro-apoptotic BCL-2 family member such as Bax. The test compound is then added to the pore and the pore is tested for its ability to allow cytochrome c to pass through. That ability is evaluated, for example, by preparing the vesicles or planar lipid bilayers with labeled cytochrome c or a labeled compound of the approximate size of cytochrome c, such as dextran, then determining whether the label can pass through the pore. A preferred label for this method is a fluorescein compound such as fluorescein isothiocyanate. Other embodiments of this method would be apparent to those skilled in the art in light of the disclosure herein, including Example 11. Industrial Application: The methods and compositions of the present invention provide remedies for disorders which are mediated by excessive apoptosis or excessive inhibition of apoptosis, including Epstein-Barr virus infection, African swine fever virus infection, adenovirus infection, lymphoproliferative conditions, cancer, arthritis, Crohn's disease, inflammation, autoimmune diseases, immunodeficiency diseases, senescence, neurodegenerative diseases, ischemic and reperfusion cell death, infertility, and poor wound healing. These remedies represent potentially improved treatments for these disorders. Also provided are improved methods for evaluating compounds for their effect on apoptosis. Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which follow the examples. Example 1 This example illustrates that Bax and Bcl-2 induce the release of ions from synthetic lipid vesicles. The Bax and Bcl-2 polypeptides used in the ion channel studies reported herein were recombinant murine Bcl-2 and Bax polypeptides lacking the C-terminal signal-anchor membrane targeting sequence (Bcl-2ΔC21, amino acids 1-218 of SEQ ID NO:9 and BaxΔC19, amino acids 1-173 of SEQ ID NO:6). These deletion derivatives, also referred to herein as BaxΔTM and Bcl-2ΔTM, were used to avoid any interference of the C-terminal signal-anchor membrane targeting sequence with the capacity of internal α helices to mediate insertion into artificial lipid bilayers. To produce Bcl-2ΔC21 and BaxΔC19A, cDNAs encoding amino acids 1-218 of murine Bcl-2 (SEQ ID NO:9) and amino acids 1-173 of murine Bax (SEQ ID NO:6) were cloned into pGEX-KG. The resulting recombinant vectors were transformed into XL-1 cells and expression of GST-Bcl-2ΔC21 and GST-BaxΔC19A fusion proteins was induced by adding 0.1 mM IPTG. The bacterial pellets were resuspended in lysis buffer (0.5mM EDTA, ImM DTT, 1% Triton X-100, O.lmg/ml PMSF, 2 ug/ml aprotinin, 2 ug/ml leupeptine and 1 ug/ml pepstatin A in PBS) and sonicated. After centrifugation at 20,000 x g for 20 minutes, the supernatant was applied to glutathione-agarose beads (Sigma). The beads were washed with buffer and treated with 10 units of thrombin/original liter. Cleaved BaxΔC19 and Bcl- 2ΔC21 were eluted from beads and the cleavage reaction was terminated by adding 80 ug of Na-p-tosyl-L-lysine choloromethyl ketone (TLCK). The cleaved eluent was dialyzed against buffer (20 mM Tris pH 8.5, 5mM EDTA, ImM DTT, 0.1% Triton X-100). To remove the GST protein and incompletely cleaved fusion proteins, the dialyzed preparation was further purified on a monoQ column and the proteins were eluted with a NaCl gradient. The examples described below utilized 3 independent protein preparations of Bax and Bcl-2. Example 2 This example illustrates that Bax and Bcl-2 form channels in planar lipid bilayers at low pH. Unilamellar vesicles composed of 40% 1,2 dioleoyl phosphatidyl glycerol and 60% 1,2 dioleoyl phosphatidyl choline (Avanti Polar Lipids) were prepared in 100 mM KC1, 2 mM CaN0 3 and 10 mM dimethylglutarate, pH 5.0 as previously described (Peterson et al, J. Membr. Biol. 99:197-204, 1987). The resulting Hposomes were diluted 200-fold to a concentration of 0.05 mg/ml in 100 mM KN0 3 , 2 mM CaN0 3 and 10 mM dimethylglutarate which was titrated to pH 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 with NaOH or with acetic acid. BaxΔC19 and Bcl-2ΔC21 were added at a concentration of 500 ng/ml and Cl " efflux was measured with a Cl " combination ion selective electrode (Accumet). Triton X-100 (0.1%) was added to release the total encapsulated Cl " at the times indicated by the arrows in Figure 1. The total amount of Cl " released was quantitated by a calibration curve produced by successive additions of 25 μM KC1. As shown in FIG. 1A-D, Bax and Bcl-2 mediated release of Cl " from KC1 loaded lipid vesicles is dependent on pH (Fig. IB-E). The maximum release of Cl " by Bax occurs at pH 4.0-4.5, decreasing to 50% at pH 3.5 or 5.0 and to less than 10% at a pH of 5.5 (Fig. 1A). When Bax added to vesicles at pH 6.0 was subsequently shifted to pH 4.0 a rapid release of Cl resulted indicating the reversibility of the pH influence (Fig. 1C). Bcl-2 displays a more narrow pH dependence of Cl " release with complete mactivation occurring by pH 5.0 (Fig. IB). Shifting from pH 5.0 to 4.0 activated the release of Cl " by Bcl-2 (Fig. ID). Thus, both purified Bax and Bcl-2 proteins are capable of pH dependent macroscopic ion release requiring activity of the bulk population of Bcl-2 and Bax proteins. Example 3 This example illustrates the formation of Bax channels in planar lipid bilayers at low pH. Planar lipid bilayers were prepared from soybean lipids by chloroform extraction of Azolectin Type II (10-20% choline, 80-90% negatively charged lipids) (Sigma). The chloroform was removed with a stream of nitrogen and the lipids stored under N 2 until dissolved in decane at 30 mg/ml. This preparation was then stored under nitrogen. The 0.25 mm orifice of a polystyrene cuvette (Warner Instruments) was pretreated with 2μl of the decane lipid solution and the solvent allowed to evaporate. The cuvette was then placed into a bilayer chamber and connected to Bilayer Clamp BC525-a (Warner Instruments) by Ag/AgCl electrodes via agar salt bridges. Data were collected using an Axoscope (Axon Instruments Software), archived on video tape using a Neurocorder DR-484 (Neuro Data Instruments), and analyzed using Origin (Microcal) and pClampό (Axon Instruments Software). Slope conductance was calculated by the method of least squares and the variance is given. Ion selectivities were calculated using the reversal potential and the Goldman equation. The reversal potential means the zero-current potential of a bilayer membrane. The reversal potential is zero with no ion gradient and the magnitude of the potential m the presence of an ion gradient reflects the cation versus anion selectivity of the bilayer. Bilayers were formed by spreading with a polished glass rod and allowed to thm to a capacitance of 0.4 μF/cm at which point the noise was typically 0.2 pA and the leak conductance was 20 pS. The salt concentrations were initially 450 mM KCl m the cis chamber (1.0 ml) and 150 mM KCl in the trans chamber (0.5 ml) to permit identification of spontaneous initial currents Outward (positive) currents were defined as K + moving cis-to trans. All solutions were buffered to pH 4.0 with 10 mM K-acetate. BaxΔTM or Bcl-2ΔTM (approx. 1 μg) was added to the cis chamber with mixing. After initial currents were identified the soluble protein was usually removed by exchanging the cis chamber contents with ahquots of buffer. Four minutes after BaxΔTM was added to the cis chamber of an established planar lipid bilayer in a 450/150 mM (cis to trans) KCl gradient at pH 4.0, an initial, inward current appeared spontaneously (O s ), reflecting Cl " moving down the KCl gradient (Fig. 2A). The initial current of the Bax channel had consistent characteristics in multiple experiments: it was always inward, usually appeared within 10 minutes following BAXΔTM addition, and had a conductance of 22 (s.d. 5, n = 5) pS. As shown in FIG. 2B, a characteristic pattern of Bax currents occurred during a transition from Os to a large open pore. At +40 mV four current levels were noted during a typical Bax channel transition which took ~5 mins.: 0 pA (C and Os), 2.36 pA (Oi), 6.4 pA (0 2 ) and 8.81 pA (Oι +2 ) (Fig. 2B, upper panel). The largest current appears to be the sum of the two smaller levels (Oi and 0 2 ). The closed (C) and O s levels are indistinguishable at +40 mV as it is close to the chloride reversal potential (E R =29mV). Direct transitions between these levels, which occur in both directions, suggest a random mechanism for movement between the open states (FIG. 2B). At -40 mV there were also four levels: 0 pA (C), -2.14 pA (O s ), -10.9 pA (O,), -30.5 pA (0, +2 ), but no observable O s to 0 2 transition (FIG. 2B, lower panel). Hyperpolarization to -40 mV increased the channel activity, demonstrating voltage dependent behavior of the channel. Computing the E R for each of these current levels gives a substantial Cl " selectivity that averages Pκ/Pcι ≡ 0.10. The final and apparently stable BAX channel at pH 4.0 was Cl " selective, with a slope conductance of 0.731 ± 0.01 nS and was characteristically open. This progression of Bax channels proceeded after the removal of protein from the cis chamber. Increasing the pH to 7.0 altered the conductance of the Bax channel to 0.329 ± 0.002 nS in a 450/150 mM KCl gradient or to 0.302 ± 0.008 nS when both chambers were 150 mM KCl (FIG. 2C, 2D). The Bax channel was observed in this activity state at pH 7.0 for prolonged periods. At pH 7.0, the current of the Bax channel was linear with voltage and retained a mild selectivity for Cl " (P /Pc O.5). Example 4 This example illustrates the formation of Bax channels in planar lipid bilayers at pH 7.0 following fusion of the planar bilayer with unilamellar proteoliposomes reconstituted with BaxΔTM. Proteoliposomes for fusion with a planar lipid bilayer were prepared as follows. Purified protein (0.25 μg) was added to 50 mg lipid (azolectin) in 20 μl of KCl buffer (150 mM KCl, 10 mM HEPES, pH 7.0). This mixture was placed into a dialysis bag with a molecular weight cutoff of 12 KDa. After 16 hours dialysis in 3000 volumes of 150 mM KCl (10 mM HEPES, pH 7.0) the proteoliposome suspension was removed and placed on ice for immediate use. The bilayer was set up with a 450: 150 cis to trans KCl gradient to identify the ion selectivity of the initial currents. Fusion was initiated by adding 5 - 20 μl of lipid vesicles (only 5 - 20 ng of protein) to the cis bilayer chamber with mixing. Non-fused proteoliposomes were removed from the compartments and solutions exchanged as described above. BaxΔTM was incorporated into lipid vesicles using standard techniques. The resulting Bax proteoliposomes were added to the cis chamber of an established lipid bilayer with a 450/150 mM KCl gradient at pH 7.0 to permit identification of initial currents. The inward (Pκ<Pcι) current observed was characteristically open and large. Transitions between one and two channels open (0 → 0 2 , Fig. 3A) that were identical m amplitude suggested that 0 represented the single channel conductance (Fig. 3A). A mild Cl " selectivity (P R /P CI = 0.32) was calculated from the reversal potential The concentration of KCl was adjusted to 150 mM KCl in both cis and trans compartments and voltage dependence of the current was determined when one channel was present (Fig. 3B and 3C). Mild outward rectification in the symmetrical KCl solution was observed. At negative voltages the channel conductance averaged 1.5 (sd 0.3, n=3) nS (FIG. 3C), while at positive voltages (+70 mV) the rapid flickering was consistent with channel block as a mechanism for the rectification seen in FIG. 3B. At -70 mV there was no flickering but periodic closing of 20-30 msec duration was observed (Fig. 3C), a pattern similar to that reported for poπn type channels (Benz, Biochim. Biophys. Ada 7797:167-196, 1994). Example 5 This example illustrates the formation of Bcl-2 channels m planar lipid bilayers at low pH. Bcl-2ΔTM was added to the cis chamber of an established bilayer in a 450/150 mM KCl gradient at pH 4 0 An initial outward (P κ /Pcι = 3.9) current (O s ) consistently appeared within 5 minutes in multiple experiments (Fig. 4A). The magnitude of the current was 0.85+0.06 pA which flickered open and shut m the first few seconds and subsequently remained open. Under these conditions, the Bcl-2 channel had a conductance of 80.3 ± 0.06 Subsequently, the Bcl-2 channel remained open for long periods (5-10 seconds) with brief closures (Fig. 4B, 4C). This initial state was present for only 2 - 5 minutes under these conditions and then the Bcl-2 channel progressed to a stable open pore at pH 4.0 with a 1.90 ± 0.06 nS conductance (Fig. 4E). After the pH was shifted to 7.0, the Bcl-2 channel remained a large pore with a 2.14 ± 0.04 nS conductance and K + selectivity of P κ /Pcι = 6.5 (Fig. 4E). Example 6 This example illustrates the formation of Bcl-2 channels in planar lipid bilayers at pH 7.0 following fusion of the planar bilayer with unilamellar proteoliposomes reconstituted with Bcl-2ΔTM. Bcl-2ΔTM proteoliposomes were prepared as described in Example 4 and added to the cis chamber of an established bilayer with a 450/150 mM KCl gradient. In multiple experiments fusion of Bcl-2 proteoliposomes to the bilayer always resulted in an outward (K + ) current (Fig. 5A). The closures in Fig. 5A indicate a single channel current of 5 pA (O), although multiple channels could open simultaneously (O 2 ). When a series of voltage steps were applied to a single channel established in the bilayer, channel closures were observed at voltages greater than ± 50 mV (Fig. 5B). These closures became more complete as the voltage increased. The voltage dependence of the open channel current in symmetrical 150 mM KCl was linear with a slope conductance of 1.08 (s.d. 0.10, n = 5) nS (FIGS. 5C and 5D). However, with hyperpolarization to more than -70 mV, a channel closure occurs (Fig. 5C). The reversal potential in KCl gradients indicated a mild K + selectivity (P κ /P C ι=2.4). The small channels and time dependent changes noted when Bcl-2ΔTM was inserted into planar lipid bilayers at low pH were not observed when reconstituted proteoliposomes containing Bcl-2ΔTM were fused to planar lipid bilayers at pH 7.0. Example 7 This example illustrates models of the three-dimensional structure of the α-5 and α-6 helices of Bax and Bcl-2. The models were generated using LNSIGHTII (Biosym, San Diego) from the crystallographic model of Bcl-x L (PDB entry 1MAZ). Views of the positively and negatively charged surfaces of the α-5 and α-6 helices of Bax and Bcl-2, respectively, were calculated and displayed as shown in FIG. 6 using GRASP (Nicholls et al. Protein Struct. Fund. Genet. 77:281-296, 1991). The darker surfaces represent the most highly charged regions. The experiments described in Examples 1-6 demonstrate that Bax and Bcl-2 have distinct channel forming properties. The inventors herein found that both Bax and Bcl-2 initiate rapid release of ions from Hposomes when added at low pH. However, Bax demonstrated a broader pH optimum, retaining activity as high as pH 5.5. It is believed this could be due to the higher α5-helιx pi of 10.64 for Bax versus 4.55 for Bcl-2 (FIG. 6). If the insertion of the putative transmembrane α5, α6-hehces of these apoptotic regulators benefits from charge reduction, the lower pH requirement for Bcl-2 may reflect glutamic acid residues that would be prone to lomzation with increasing pH in contrast to the presence of lysine and argimne residues m Bax (FIG. 6). The ion channels formed by Bax and Bcl-2 m planar lipid bilayers have charactenstics that depend in part on the method of incorporation. When BaxΔTM or Bcl-2ΔTM is inserted into bilayers at low pH the initial currents were small with conductances of 22 pS and 80 pS respectively. Like Bcl-x L (Minn et al, supra), and consistent with other observations on Bcl- 2 (Schendel et al, supra), the experiments described herein showed a mild cation selectivity for anti-apoptotic Bcl-2. However, the pro-apoptotic molecule Bax surpπsmgly has a consistent anion selectivity. If the α5, α6-helιces contribute to the channel these selectivities may reflect the positively charged residues of Bax and negatively charged residues of Bcl-2. While modest differences m ion selectivity are unlikely to be the sole explanation for opposite influences on apoptosis, these charge reversals appear to be consistent in the α5, α6-helιces of anti- versus pro-apoptotic members (Fig. 7) Bax channels respond to shifting the pH to 7.0 after insertion at pH 4.0 consistent with previous observations on toxms and porms (M dell et al, Biophys J 62:41-44, 1992 and Todt et al, Biochem. 31, 10471-10478, 1992). The changes described herein could also relate to pH dependent lomzation of charged residues in these channels. A striking progression of the Bax channel in planar bilayers occurred withm 2-4 minutes of its initial appearance. This included I) an early Cl " selective small channel, n) a transition phase with multiple subconductance levels and moderate Cl " selectivity, and in) an apparently stable ohmic pore of large conductance that is mildly Cl " selective and open continuously (Fig. 2). The Bcl-2 channel activity also progressed from an early K + selective small channel that opened and closed spontaneously to a large ohmic pore (Fig. 4). Removal of protein from the chamber and alteration of salt concentrations did not prevent Bax and Bcl- 2 channel transition which may represent mtra-membranous organization of Bcl-2 or Bax into its mature form. Of note, shifting from pH 4.0 to 7.0 altered the conductance and selectivity of Bax, but not Bcl-2. In contrast, the overnight reconstitution of Bax or Bcl-2 into lipid vesicles which were subsequently fused to planar bilayers resulted immediately m large, open pores (Fig. 3B and 5C). In addition to differences in ion selectivity, Bcl-2 and Bax channels display other unique characteristics, including conductance, voltage dependence and rectification. Example 8 This example illustrates that the conductance pattern of a Bcl-2 channel is altered by a Bax peptide death agonist (SEQ ID NO:l) to appear similar to that of a Bax channel. Control bilayers containing Bcl-2 or Bax channels were prepared by fusion of unilamellar proteoliposomes with incorporated Bcl-2ΔTM or BaxΔTM with an established planar bilayer as described in Example 4. Test bilayers containing Bcl-2 channels were prepared in the same manner to which an 11 mer Bax peptide (SEQ ID NO: 1) with known death agonist activity was added at 20, 50 or 100 μM. A voltage of 80 mV was applied to each control and test channel and its conductance measured for a period of 12 to 16 seconds and the frequency that each channel was measured at a particular conductance level (pA) was calculated. The data, representing about 500,000 measurements, are shown as histograms in FIGS. 8A-8E. As seen in FIG. 8A, the Bcl-2 control channel moves between several conductance levels, clustering around preferred open states at about 80 pA, 102 pA, and 130 pA. Bax displays a simpler conductance pattern, with two preferred conductance levels between about 45 and 55 pA (FIG. 8E). The addition of the 11 mer Bax peptide to a Bcl-2 channel changes its characteristic conductance pattern in a progressive manner as the concentration is increased from 20 to 100 μM (FIG. 8B-D). At 100 μM the single remaining peak is not coincident with any of the levels seen in Bcl-2 but coincides with one of the less frequent Bax peaks at about 65 pA. When murine T cell hybridoma 2B4 cells are treated with 100 μM of this peptide fused with the HIV tat peptide, which increases cell uptake of the Bax peptide, a significant number of the cells are dead in less than 4 hr as compared to treatment with the tat peptide alone (data not shown). Thus, the complex conductance pattern of a Bcl-2 channel is altered to a simpler Bax-like conductance pattern by the same concentration of peptide that promotes cell death in vitro. The activity of Bax channels in the lipid bilayer was not affected by addition of the Bax 11-mer peptide at concentrations between 20 and 100 μM. In addition, the Bax 11-mer peptide exhibited no channel forming activity upon addition to planar lipid bilayers which lack BCL-2 related proteins. Example 9 This example illustrates the effect of the Bax death agonist peptide (SEQ ID NO:l) on ion selectivity and conductance of a Bcl-2 channel, as well as the effect of the Bcl-2 death antagonist (SEQ ID NO:2) and full length Bcl-2 on ion selectivity of a Bax channel. Bilayers containing Bcl-2 and Bax channels were prepared as descπbed m Example 4 with 150 mM KCl on both sides of the bilayer. The Bax death agonist peptide (SEQ ID NO: l) was added to the cis side of a Bcl-2 channel at 150 μM. The voltage dependence of the currents and the ion selectivity for the Bcl-2 channel in the absence and presence of the death agonist peptide, as well as the Bax channel were determined and the data are shown in FIGS 9A-C. The IV plots show that the death agonist peptide imparted rectification to the voltage dependence of the Bcl-2 channel current, similar to the rectification displayed by the Bax channel, as indicated by the curving lines at positive potentials m the plots in FIGS. 9B and 9C The slope of Bcl-2 channels in the presence of the death agonist peptide and at negative potentials is linear and intermediate between that of Bcl-2 and Bax alone. Of note, addition of the death agonist peptide reversed the ion selectivity of the Bcl-2 channel from K + selective with a pK/pCl=2.4) to Cl- selective with a pK/pCl value, 0.26+0.05, within the range of that measured for the Bax channel (0.32 pK/pCl). The insertion of Bax into a planar lipid bilayer produced a Cl " selective channel with 1.5 nS conductance and P κ /Pcι of 0.3. When full length Bcl-2 (SEQ ID NO:4) was added to the bilayer, the conductance increased to 2 5 ± 0.5 nS and the P κ /Pcι shifted to 1.4 ± 0.5. When 50-200 μM Bcl-2 death antagonist 11-mer (SEQ ID NO:2) was added to the Bax-contaimng planar lipid bilayer, currents at positive potentials decreased consistent with a shift towards cation selectivity. Example 10 This example illustrates that the ion conductance patterns of planar lipid bilayers containing Bad or Bid pro-apoptotic BCL-2 family members are similar to Bax-contaimng bilayers, and that a loss-of- function Bax variant which does not induce apoptosis has ion conductance characteπstics similar to anti-apoptotic BCL-2 family members. Bad channels in azolectin, pH 7.0, were formed and conductance charactenstics of these channels were charactenzed as disclosed in Example 4. The C1 " :K + selectivity of this composition was 4:1, which is very similar to the selectivity established for Bax channels. The conductance of the channels were about 1 nS with gating similar to Bax channels. Bid channels were also formed as with the Bad channels discussed above. A C1 " :K + selectivity of 3.7:1, with a maximum conductance of 4 nS was established. The channel closed in steps of 1 nS. These characteπstics are also similar to those of Bax channels. A loss-of- function form of Bax (ScBax G67R ) has been isolated from human lymphoma cells (Meijeπnk et al. Leukemia 9:1828-1832, 1995; Meijeπnk et al. Blood 97:2991-2997, 63783120 doc 1998). This Bax is unable to promote apoptosis due to an ammo acid change from gly to arg at position 67 (withm the BH3 region). When incorporated into planar lipid bilayers as in Example 4, ScBax G67R formed channels having the gating and size of wild-type Bax, but with K + :C1 " selectivity of 3.2.1, which is distinct from channels formed with wild-type (pro- apoptotic) Bax, and similar to channels formed with anti-apoptotic BCL-2 family members. Example 11 This example illustrates the characterization of the kinetics and size of the apoptotic pore formed by Bax, and, in particular, establishes that the Bax pore can act as a channel capable of conducting cytochrome c Liposomes were prepared with dioleoylphosphatidylcholme and dioleoylphosphatidic acid at 70% and 30% respectively. The lipids were hydrated, sonicated and extruded as descπbed in Schlesmger et al, Proc Natl Acad Sci USA 94:11357-11362, 1997 The liposomes were prepared m the presence of either carboxyfluorescein, 20 mg/rnl, FITC-dextran, 10 mg/ml, or FITC-cytochrome c, 10 mg/ml (FITC=fluorescem isothiocyanate). The liposomes were then purified by gel filtration chromatography to remove extravesicular fluorescent molecules At these concentrations the fluorescence of mtravesicular fluorescein is concentrations quenched so that the introduction of a pore m the Hposome membrane can be followed. If the pore is of sufficient size to permit the passage of the contained molecules, they pass out of the hposome and are diluted m the surrounding media. The increasing fluorescence provides a quantitative assay of the pore formation. This "dequenchmg" assay has been previously used in the characteπzation of pore forming peptides (Rex et al. Biochemistry 37:2336-2345,1998, We stem et al. Science 795:489-492, 1977). Using liposomes containing carboxyfluorescein the kinetics of Bax pore formation was determined (Figure 12). The carboxyfluorescein releasing pores appear in a concentration dependent process with an exponential time course. The number of Bax molecules involved in the active pore, as determined by Hill coefficients, is dependent upon the protein concentration. At low concentrations (50-200 Bax molecules per vesicle), the pores involve dimers of Bax At higher concentrations (200-500 Bax molecules per vesicle), the pore formation involves four Bax molecules. The Bax interaction with liposomes is restricted to the formation of these well defined pores and does not include the formation of lipid disks and lysis of multilamellar vesicles as reported for melittm (Dufourcq et al, Bwchem Biophys Ada 559:33-48, 1986). At higher, non-physiologic concentrations of Bax higher order molecular aggregates are formed but were not further characterized. When pore formation was studied using liposomes containing cytochrome c, the protein was found to be released, as was the similarly size FITC-dextran. The kinetics of pore activation in carboxyfluorescein and cytochrome c containing liposomes is shown m Figure 1. The concentration of Bax per vesicle is equivalent in these two preparations and the kinetics of release show that although both carboxyfluorescein and FITC-cytochrome c exit the hposome m a Bax dependent manner, pore activation in the presence of cytochrome c is accelerated. The ability of the Bax pore to release cytochrome c from liposomes demonstrates that, independent of interactions with any other protein, the pro-apoptotic molecule is capable of releasing cytochrome c from mitochondria and generating the cytoplasmic programmed cell death cascade. To verify that this was not the result of vesicle lysis, the size of the Bax pore was determined with FITC-dextran molecules of defined dimensions. The molecular diameters were determined by dynamic light scattering measurements and are given with their standard deviations in Table 1 Using these molecules the efflux of CF via BAX pores was inhibited in a size dependent manner with the greatest inhibition occurπng as the blocking dextran approached but did not exceed the diameter of the BAX pore. The theory of this technique has been well developed in the permeability of membrane channels, the characterization of gel permeation and m the application of coulter counting devices (Levitt, Curr. Top Membr Transport 21 :181-197 , 1984; Pappenheirner et al. Am. J. Physiol. 167:13- 23, 1951; Cruickshank et al, Biochem. J. 73:1925-1931, 1997; Bunville, Modern Methods of Particle Size Analysis, Wiley, 1984). The measurement of τ (time constants for dequenchmg) in the presence of dextran provided the estimation of pore size shown in Figure 2. From this data several characteristics of the Bax pore are apparent. The rate of dequenchmg and size of the pore are concentration dependent. At 5 nM Bax, equivalent to 125 Bax molecules per vesicle, the dominant pore is 10 A in diameter, while at 9 nM (250 Bax molecules per vesicle) and greater, the dominant pore is 20 A in diameter. Thus, Bax forms a pore that is large enough to permit the observed transport of cytochrome c. Increasing the concentration of Bax from 9 to 20 nM does not generate larger pores, showing that the Bax pores are well defined and not the result of aggregation in the membrane. Furthermore, as shown m Figure 2, the inhibition of dequenchmg decreases with very large dextran molecules that are too large to enter the respective pore, again confirming that the Bax pores are defined in structure and that dequenchmg is not a consequence of Hposome lysis. Table 1. Diffusion Parameters determined by Dynamic Light Scattering Dextran MW Diameter - A ± S.D. D x 10 "y cmVsec 1,500 9 + 2 1714 6,000 15 + 5 970 9,000 19 + 4 872 19,500 26 ± 9 637 39,400 36 + 9 481 66,900 52 ± 19 320 148,000 85 + 33 194 Cytochrome C 17 + 3 1181 FITC-cytochrome C 21 + 7 969 FITC-cytochrome C, 2nd preparation 20 + 5 1038 These data indicate that by itself Bax forms pores in Hposome bilayers that are of sufficient size to permit the release of cytochrome c. This is consistent with a Bax or other pro-apoptotic pore being the basis of cytochrome c release in vivo and serving as the key mitochondrial decision point in apoptosis. To further correlate the ability of a pro-apoptotic pore to allow passage of cytochrome c with the presence of a Cl " selective pore and apoptosis (and conversely, the inhibition of cytochrome c release with K + selective anti-apoptotic pore), it is noted that the K + selective pore created by the Bax loss-of-function (anti-apoptotic) variant ScBax G67R was markedly reduced in its ability to allow passage of cytochrome c. In view of the above, it will be seen that the several advantages of the invention are achieved and other advantageous results attained. As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense. All references cited in this specification either supra or infra are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by their authors and no admission is made as to the accuracy or pertinence of the references or that any reference is material to patentability. SEQ IDs SEQ ID NO: 1 - 11 mer pro-apoptotic peptide from positions 61-71 of Bax (SEQ ID NO:3) 1 10 ECLKRIGDELD SEQ ID NO:2 - 11 mer anti-apoptotic peptide from positions 95-105 of Bcl-2 (SEQ ID NO:4) 1 10 LTLRQAGDDFS SEQ ID NO:3 - Human Bax 10 20 30 40 50 MDGSGEQPRGGGPTSSEQIMKTGALLLQGFIQDRAGRMGGEAPELALDPV 60 70 80 90 100 PQDASTKKLSECLKRIGDELDSNMELQRMIAAVDTDSPREVFFRVAADMF 110 120 130 140 150 SDGNFNWGRWALFYFASKLVLKALCTKVPELIRTIMGWTLDFLRERLLG 160 170 180 190 WIQDQGGWDGLLSYFGTPTWQTVTIFVAGVLTASLTIWKKMG SEQ ID NO:4 - Human Bcl-2 10 20 30 40 50 MAHAGRSGYDNREIVNKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGFFS 60 70 80 90 100 SQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPWHLTLRQA 110 120 130 140 150 GDDFSRRYRRDFAEMSSQLHLTPFTARGCFATVVEELFRDGVNWGRIVAF 160 170 180 190 200 FEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVE 210 220 230 LYGPSMRPLFDFSWLSLKTLLSLALVGACITLGAYLGHK

Claims: What is Claimed is: 1. A method of inducing apoptosis in a cell comprising: (a) selecting a compound which induces the formation of a pore capable of transporting cytochrome c out of mitochondria, wherein said selecting comprises testing the compound for the induction of said pore formation; and (b) administering the compound to the cell. 2. The method of claim 1, wherein the compound is a polypeptide or derivative thereof. 3. The method of claim 2, wherein the polypeptide comprises a BH3 domain of a pro-apoptotic BCL-2 family member or a fragment thereof. 4. The method of claim 3, wherein the pro-apoptotic BCL-2 family member is selected from the group consisting of Bax, Bak, Bcl-x s , Bad, Bik, and Bid. 5. The method of claim 4, wherein the polypeptide comprises SEQ ID NO:l. 6. The method of claim 1, wherein the cell is a cell of a patient with a condition mediated by excessive down-regulation of apoptosis. 7. The method of claim 6, wherein the patient is a human and the condition is selected from the group consisting of neoplasias, diseases caused by Epstein-Barr virus, African swine fever virus, and adenovirus, lymphoproliferative conditions, cancer, arthritis, Crohn's disease, inflammation, and autoimmune disease. 8. A method of inhibiting apoptosis in a cell comprising: (a) selecting a compound which inhibits the formation of a pore capable of transporting cytochrome c out of mitochondria wherein said selecting comprises testing the compound for the inhibition of said pore formation; and (b) administering the compound to the cell. 9. The method of claim 8, wherein the compound is a polypeptide or derivative thereof. 10. The method of claim 9, wherein the polypeptide comprises a BH3 domain of a anti-apoptotic BCL-2 family member or a fragment thereof. 11. The method of claim 10, wherein the anti-apoptotic BCL-2 family member is selected from the group consisting of Bcl-2, Bcl-x L , Mcl-1, and Al. 12. The method of claim 11, wherein the polypeptide comprises SEQ ID NO:2. 13. The method of claim 8, wherein the cell is a cell of a patient with a condition mediated by excessive apoptosis. 14. The method of claim 13, wherein the patient is a human and the condition is selected from the group consisting of immunodeficiency diseases, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and a wound. 15. A method of treating a patient having a condition mediated by excessive down- regulation of apoptosis comprising: (a) selecting a compound which induces the formation of a pore capable of transporting cytochrome c out of mitochondria, wherein said selecting comprises testing the compound for the induction of said pore formation; and (b) administering the compound to the patient. 16. The method of claim 15, wherein the compound is a polypeptide or derivative thereof. 17. The method of claim 16, wherein the polypeptide comprises a BH3 domain of a pro-apoptotic BCL-2 family member or a fragment thereof. 18. The method of claim 17, wherein the polypeptide comprises SEQ ID NO:l. 19. The method of claim 15, wherein the patient is a human and the disease condition is selected from the group consisting of neoplasias, diseases caused by Epstein-Barr virus, African swine fever virus, and adenovirus, lymphoproliferative conditions, cancer, arthritis, Crohn's disease, inflammation, and autoimmune disease. 20. A method of treating a patient having a condition mediated by excessive apoptosis comprising: (a) selecting a compound which inhibits the formation of a pore capable of transporting cytochrome c out of mitochondria wherein said selecting comprises testing the compound for the inhibition of said pore formation; and (b) administering the compound to the patient. 21. The method of claim 20, wherein the compound is a polypeptide or derivative thereof. 22. The method of claim 21, wherein the polypeptide comprises a BH3 domain of a anti-apoptotic BCL-2 family member or a fragment thereof. 23. The method of claim 22, wherein the polypeptide comprises SEQ ID NO:2. 24. The method of claim 20, wherein the patient is a human and the condition is selected from the group consisting of immunodeficiency diseases, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and a wound. 25. A method of inducing apoptosis in a cell comprising: (a) selecting a compound which changes the ion selectivity of an anti-apoptotic BCL- 2 family member from K + selective to Cl " selective, wherein said selecting comprises testing the compound for activity in converting the K + selectivity of the anti-apoptotic BCL-2 family member to Cl " selectivity; and (b) administering the compound to the cell. 26. The method of claim 25, wherein the compound is a polypeptide or a derivative thereof. 27. The method of claim 26, wherein the polypeptide comprises a BH3 domain of a pro-apoptotic BCL-2 family member or a fragment thereof. 28. The method of claim 27, wherein the pro-apoptotic family member is selected from the group consisting of Bax, Bak, Bcl-x s , Bad, Bik, and Bid. 29. The method of claim 28, wherein the polypeptide comprises SEQ ID NO:l. 30. The method of claim 25, wherein the anti-apoptotic BCL-2 family member is Bcl-2. 31. The method of claim 25, wherein the cell is a cell of a patient with a condition mediated by excessive down-regulation of apoptosis. 32. The method of claim 31 , wherein the patient is a human and the condition is selected from the group consisting of neoplasias, diseases caused by Epstein-Barr virus, African swine fever virus, and adenovirus, lymphoproliferative conditions, cancer, arthritis, Crohn's disease, inflammation, and autoimmune disease. 33. A method of inhibiting apoptosis in a cell comprising: (a) selecting a compound which changes the ion selectivity of a pro-apoptotic BCL-2 family member in the cell from Cl " selective to K + selective, wherein said selecting comprises testing the compound for activity in converting the Cl " selectivity of the pro-apoptotic BCL-2 family member to K + selectivity; and (b) administering the compound to the cell. 34. The method of claim 33, wherein the compound is a polypeptide or derivative thereof. 35. The method of claim 34, wherein the compound comprises a BH3 domain of a anti-apoptotic BCL-2 family member of fragment thereof. 36. The method of claim 35, wherein the anti-apoptotic BCL-2 family member is selected from the group consisting of Bcl-2, Bcl-x L , Mcl-1, and Al. 37 The method of claim 36, wherein the polypeptide comprises SEQ ID NO:2. 38. The method of claim 33, wherein the pro-apoptotic BCL-2 family member is selected from the group consisting of Bax, Bad, and Bid. 39. The method of claim 38, wherein the pro-apoptotic BCL-2 family member is Bax. 40. The method of claim 33, wherein the cell is a cell of a patient with a condition mediated by excessive apoptosis. 41. The method of claim 39, wherein the patient is a human and the condition is selected from the group consisting of immunodeficiency diseases, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and a wound. 42. A method of treating a patient having a condition mediated by excessive down- regulation of apoptosis comprising: (a) selecting a compound which changes the ion selectivity of an anti-apoptotic BCL- 2 family member from K + selective to Cl " selective wherein said selecting comprises testing the compound for activity in converting the K + selectivity of the anti-apoptotic BCL-2 family member to Cl " selectivity; and (b) administering the compound to the patient. 43. The method of claim 42, wherein the compound is a polypeptide or derivative thereof. 44. The method of claim 43, wherein the polypeptide comprises a BH3 domain of a pro-apoptotic BCL-2 family member or a fragment thereof. 45. The method of claim 44, wherein the polypeptide comprises SEQ ID NO: 1. 46. The method of claim 42, wherein the anti-apoptotic BCL-2 family member is Bcl-2. 47. The method of claim 42, wherein the patient is a human and the disease condition is selected from the group consisting of neoplasias, diseases caused by Epstein-Barr virus, African swine fever virus, and adenovirus, lymphoproliferative conditions, cancer, arthritis, Crohn's disease, inflammation, and autoimmune disease. 48. A method of treating a patient having a condition mediated by excessive apoptosis comprising: (a) selecting a compound which changes the ion selectivity of a pro-apoptotic BCL-2 family member from Cl " selective to K + selective wherein said selecting comprises testing the compound for activity in converting the Cl " selectivity of the pro-apoptotic BCL-2 family member to K + selectivity; and (b) administering the compound to the patient. 49. The method of claim 48, wherein the compound is a polypeptide or derivative thereof. 50. The method of claim 49, wherein the polypeptide comprises a BH3 domain of a anti-apoptotic BCL-2 family member or a fragment thereof. 51. The method of claim 50, wherein the polypeptide comprises SEQ ID NO:2. 52. The method of claim 48, wherein the pro-apoptotic BCL-2 family member is selected from the group consisting of Bax, Bak, Bcl-x s , Bad, Bik, and Bid. 53. The method of claim 48, wherein the patient is a human and the condition is selected from the group consisting of immunodeficiency diseases, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and a wound. 54. A method for identifying apoptosis-modulating compounds, the method comprising: (a) providing a lipid bilayer comprising a K + or a Cl " selective channel comprised of an anti-apoptotic or pro-apoptotic polypeptide of the BCL-2 family, respectively; (b) contacting a compound of interest with the bilayer; and (c) determining ion-selectivity of the channel, wherein a change from K + selective to Cl " selective indicates the compound is a death agonist, and a change from Cl " selective to K + selective indicates the compound is a death antagonist. 55. The method of claim 54, wherein the lipid bilayer comprises a planar bilayer in the presence of a concentration gradient and the determining ion selectivity step comprises measuring the reversal potential of the channel. 56. The method of claim 55, wherein the channel comprises an anti-apoptotic polypeptide of the BCL-2 family. 57. The method of claim 56, wherein the anti-apoptotic polypeptide is Bcl-2ΔTM. 58. The method of claim 57, wherein the channel comprises a pro-apoptotic polypeptide of the BCL-2 family. 59. The method of claim 58, wherein the pro-apoptotic polypeptide is BaxΔTM. 60. The method of claim 54, wherein the lipid bilayer comprises a proteoliposome loaded with a mixture of K + and Cl " in known amounts and wherein determining ion selectivity comprises measuring the relative rates of K + and Cl " efflux from the proteoliposome. 61. The method of claim 60, wherein the channel comprises an anti-apoptotic polypeptide of the BCL-2 family. 62. The method of claim 61, wherein the anti-apoptotic polypeptide is Bcl-2ΔTM. 63. The method of claim 60, wherein the channel comprises a pro-apoptotic polypeptide of the BCL-2 family. 64. The method of claim 63, wherein the pro-apoptotic polypeptide is BaxΔTM. 65. A method for identifying apoptosis-modulating compounds, the method comprising: (a) providing a lipid bilayer comprising a K + or a Cl " selective channel comprised of an anti-apoptotic or pro-apoptotic polypeptide of the BCL-2 family, respectively; (b) contacting a compound of interest with the bilayer; (c) determining ion-selectivity of the channel, and (d) assessing pH dependence of channel formation by the test compound, which comprises (1) providing lipid vesicles m separate solutions at pH values of 3.5, pH 4.0, pH 5.0, and pH 7.0, the vesicles containing a channel indicator, (2) contacting the test compound with the vesicles m each of the solutions, and (3) assaying for release of the channel indicator from the vesicles, wherein if the test compound forms an Cl selective channel m the lipid bilayer compπsmg the anti-apoptotic polypeptide, shows a ratio of channel indicator release at pH 3.5 and pH 4 0, (R 3 5 4 o) of less than 1 , and shows a ratio of indicator release at pH 4.0 and 5 0 (R t 0 50 ) of between 1 and 2, the test compound is a death agonist and if the test compound forms a K + selective channel in the lipid bilayer comprising the pro-apoptotic polypeptide and produces a R 3 5/ o of about 1.0 and a R, 0 5 0 of greater than 7.0, the test compound is a death antagonist. 66. The method of claim 65, wherein the lipid bilayer comprises a planar bilayer m the presence of a concentration gradient and the determining ion selectivity step comprises measuring the reversal potential of the channel 67. The method of claim 66, wherein the channel compπses an anti-apoptotic polypeptide of the BCL-2 family. 68. The method of claim 67, wherein the anti-apoptotic polypeptide is Bcl-2ΔTM. 69. The method of claim 65, wherein the channel compπses a pro-apoptotic polypeptide of the BCL-2 family. 70. The method of claim 69, wherein the pro-apoptotic polypeptide is BaxΔTM. 71. The method of claim 70, wherein the channel indicator is carboxyfluorescein or fluorescem-dextran. 72. A method for identifying apoptosis-modulating compounds, the method comprising: (a) evaluating ion selectivity of a channel formed by a test compound, which compπses providing a lipid bilayer, contacting the bilayer with the test compound, and assaying for ion-selective channel formation, and (b) comparing release of a channel indicator from artificial liposomes in the presence of the test compound over a pH range between 3.5 and 7.0, which comprises (1) providing lipid vesicles m separate solutions at pH values of 3.5, pH 4.0, pH 5.0, and pH 7.0, the vesicles containing a channel indicator, (2) contacting the test compound with the vesicles m each of the solutions, and (3) assaying for release of the channel indicator from the vesicles, wherein if the test compound forms an Cl " selective channel, shows a ratio of channel indicator release at pH 3.5 and pH 4.0, (R 3 5/40 ) of less than 1, and shows a ratio of indicator release at pH 4.0 and 5.0 (R 4 0/50) of between 1 and 2, the test compound will have apoptotic activity, and if the test compound forms a K + selective channel and produces a R 3 5/40 of about 1.0 and a R» 0/50 of greater than 7.0, the test compound will have anti-apoptotic activity. 73. The method of claim 72, wherein the lipid bilayer compπses a planar bilayer m the presence of a concentration gradient and assaying for ion selective channel formation comprises measuring the reversal potential of the channel. 74. The method of claim 72, wherein the lipid bilayer compπses a lipid vesicle loaded with a mixture of K + and Cl " m known amounts and assaying for ion-selective channel formation comprises determining the relative rates of K + and Cl " efflux from the lipid vesicle. 75. The method of claim 72, wherein the channel indicator is carboxyfluorescein or fluorescein-dextran. 76. A composition comprising a polypeptide of no more than 50 contiguous ammo acids containing SEQ ID NO:l, or a derivative of said polypeptide. 77. The composition of claim 76, compπsmg a conservatively substituted derivative of SEQ ID NO: 1. 78. The composition of claim 76, consisting of SEQ ID NO: 1 in a pharmaceutically acceptable preparation. 79. A method of inducing apoptosis m a cell comprising admmisteπng the composition of claim 76 to the cell. 80. The method of claim 79, wherein the cell is a cell of a patient with a condition mediated by excessive down-regulation of apoptosis. 81. The method of claim 80, wherein the patient is a human and the condition is selected from the group consisting of neoplasias, diseases caused by Epstem-Barr virus, African swine fever virus, and adenovirus, lymphoproliferative conditions, cancer, arthπtis, Crohn's disease, inflammation, and autoimmune disease. 82. A composition compπsmg a polypeptide of no more than 50 contiguous ammo acids containing SEQ ID NO:2, or a deπvative of said polypeptide. 83. The composition of claim 82, comprising a conservatively substituted deπvative of SEQ ID NO:2. 84. The composition of claim 82, consisting of SEQ ID NO:2 in a pharmaceutically acceptable preparation. 85. A method of inhibiting apoptosis in a cell comprising administering the composition of claim 82 to the cell. 86. The method of claim 85, wherein the cell is a cell of a patient with a condition mediated by excessive apoptosis. 87. The method of claim 86, wherein the patient is a human and the condition is selected from the group consisting of immunodeficiency diseases, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, and a wound.

Inventors: KORSMEYER STANLEY J, SCHLESINGER PAUL H, KORSMEYER, STANLEY, J., SCHLESINGER, PAUL, H.
Applicants: KORSMEYER STANLEY J, SCHLESINGER PAUL H, UNIV WASHINGTON, KORSMEYER, STANLEY, J., SCHLESINGER, PAUL, H., WASHINGTON UNIVERSITY
Application year: 1999
Main classification: A61K38
IPC codes: A61K38, A61K45, A61K48, A61P9, A61P15, A61P17, A61P19, A61P25, A61P29, A61P31, A61P35, A61P37, A61P43, C07K14, C12N5, G01N33