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© 1999 American Society for Clinical Oncology
Dysregulation of Apoptosis in CancerFrom the The Burnham Institute, La Jolla, CA. Address reprint requests to John C. Reed, MD, PhD, The Burnham Institute, 10901 North Torrey Pines Rd, La Jolla, CA 92037 ABSTRACT ABSTRACT: Each day, approximately 50 to 70 billion cells perish in the average adult because of programmed cell death (PCD). Cell death in self-renewing tissues, such as the skin, gut, and bone marrow, is necessary to make room for the billions of new cells produced daily. So massive is the flux of cells through our bodies that, in a typical year, each of us will produce and, in parallel, eradicate a mass of cells equal to almost our entire body weight. The morphologic ritual cells go through when experiencing PCD has been termed apoptosis and is executed by a family of intracellular proteases, called caspases. Unlike accidental cell deaths caused by infarction and trauma, these physiologic deaths culminate in fragmentation of cells into membrane-encased bodies which are cleared through phagocytosis by neighboring cells without inciting inflammatory reactions or tissue scarring. Defects in the processes controlling PCD can extend cell life span, contributing to neoplastic cell expansion independently of cell division. Moreover, failures in normal apoptosis pathways contribute to carcinogenesis by creating a permissive environment for genetic instability and accumulation of gene mutations, promoting resistance to immune-based destruction, allowing disobeyance of cell cycle checkpoints that would normally induce apoptosis, facilitating growth factor/hormone–independent cell survival, supporting anchorage-independent survival during metastasis, reducing dependence on oxygen and nutrients, and conferring resistance to cytotoxic anticancer drugs and radiation. Elucidation of the genes that constitute the core machinery of the cell death pathway has provided new insights into tumor biology, revealing novel strategies for combating cancer. LIKE THE CELL DIVISION cycle, the pathways that govern programmed cell death (PCD) are complex, with networks of cell death blockers and inducers working against each other in a delicate balance intended to achieve proper tissue homeostasis. The core machinery of the cell death pathway, however, can be reduced to a few critical proteins which are conserved across animal evolution and which are often the targets of viral exploitation. In mammalian species and humans, these core regulators typically exist as multigene families with numerous homologs that each have their own unique patterns of expression in various tissues. Numerous examples of pathologic alterations in the expression or function of cell death regulatory genes have been revealed in recent years. In this review, I will summarize some of the current knowledge about the mechanisms responsible for dysregulation of apoptosis in cancer cells, and highlight some of the ways in which aberrant regulation of cell death genes contributes to aggressive behaviors of malignant cells. THE CORE CELL DEATH MACHINERY The molecules that participate in the fundamental steps of PCD have been defined in large part through genetic studies of PCD in simple organisms such as the free-living nematode Caenorhabditis elegans.1 Three essential genes have thus been identified, Ced-3, Ced-4, and Ced-9, that control the commitment step that decides the ultimate life/death fate of individual cells (Fig 1). Homologs of each of these cell death genes have been identified in humans and other mammalian species, often occurring in families composed of multiple members. These proteins are well conserved throughout metazoan evolution, which suggests that they define the core elements of the cell death pathway. Moreover, many viruses have usurped core antiapoptotic genes from the genomes of animal cells or devised their own inhibitors of the apoptosis inducers, thus providing further evidence of the importance of these evolutionarily conserved genes for regulating cell life and death.
CED-3/Caspases Because caspases both cleave substrates at Asp residues and are themselves activated by cleavage at Asp residues, the potential for proteolytic cascades exists and indeed has been documented (reviewed in4). The concept of upstream initiator and downstream effector caspases that operate within proteolytic cascades thus has emerged.4,5 Relevant to their mechanisms of activation, all of the upstream initiator caspases contain large amino-terminal prodomains, and many of these prodomains have been shown to bind other proteins involved in triggering the cascade. The downstream caspases, which function as the ultimate effectors of apoptosis, uniformly possess small prodomains and are probably activated predominantly, if not exclusively, through cleavage by upstream caspases. The irreversible cleavage of specific protein substrates in cells by downstream effector caspases is what directly or indirectly accounts for the biochemical and morphologic changes recognized as apoptosis. When viewed microscopically, these features include characteristic chromatin condensation, nuclear fragmentation, cell shrinkage, plasma membrane blebbing, and other ultrastructural changes. Caspase activation has been documented in several types of tumor cells when they have been successfully induced to undergo apoptosis by various chemotherapeutic drugs. In this regard, it has been shown that essentially all traditional anticancer drugs use apoptosis pathways to exert their cytotoxic actions. Consequently, defects in the regulation of apoptosis can render cancer cells intrinsically resistant to therapy—not because the drugs or radiation fails to induce damage to DNA, microtubules, or other structures, but because the tumor cells remain viable after suffering the damage and hence have opportunities to subsequently undergo repair and resume their proliferative march. Although relatively little inquiry into the status of caspase gene expression in tumors has been undertaken thus far, examples of loss of expression or mutational inactivation of specific caspase genes have been found in human tumor cell lines.6-8
Tumor Necrosis Factor Family Death Receptors
Several mechanisms for tumor cell resistance to apoptosis induction by Fas and other death receptors have been reported, including (a) downregulation of the receptors, (b) mutations within the genes encoding the receptors, and (c) defects in the apoptotic signaling pathway.13-15 In this regard, DED-containing inhibitors of death receptor signaling have been identified in humans, mammals, and viruses (reviewed in16). Interestingly, one of these antiapoptotic DED family proteins, cFlip, is a homolog of caspases-8 and -10 that contains DEDs but lacks proteolytic activity (reviewed in17). Flip and other antiapoptotic DED family proteins compete with caspases for binding to Fadd/Mort-1, thus functioning as a transdominant inhibitors of these caspases involved in TNF family cytokine signaling. Elevated expression of Flip has been associated with Fas resistance in some types of tumors.18 Developing resistance within the Fas/death receptor pathway for apoptosis has several potential benefits for tumors. First, it probably allows tumor cells to avoid being killed by cytolytic T cells and natural killer cells, which rely heavily on their surface expression of Fas ligand (Fas-L) to trigger apoptosis of target cells.19 Second, it permits tumor cells to express Fas-L on their own surfaces without killing themselves via an autocrine Fas/Fas-L mechanism. This so-called Fas counterattack has been shown to be responsible for tumor-induced killing of immune cells, possibly creating holes in the T-cell repertoire by killing any activated helper T cells that may be capable of recognizing antigens on the tumor.20-22 Expression of Fas-L may also be a convenient way to clear other normal neighboring cells from the path of migrating tumor cells. Finally, in some, but certainly not all, types of tumor cells, chemotherapeutic drugs have been shown to induce apoptosis by upregulating the expression of Fas-L, Fas, or other TNF family ligands and receptors, thus delivering apoptotic signals through an autocrine mechanism (reviewed in23). Consequently, resistance to death receptor–mediated signaling can endow such tumor cells with chemoresistant phenotypes (Fig 3). Interestingly, the tumor suppressor p53 has been reported to increase transcription of Fas and DR5,24-26 thus establishing a link between death receptors and this apoptosis-inducing gene, which becomes inactivated by gene mutations and other mechanisms in more than half of human cancers. However, some studies suggest that death receptor–dependent mechanisms may be only rarely involved in tumor cell responses to anticancer drugs.27,28
CED-4/Apoptotic Protease Activating Factor-1 A single mammalian homolog of CED-4 has been identified thus far, termed apoptotic protease activating factor-1 (Apaf-1). The human Apaf-1 protein, however, is structurally more complex than the worm CED-4.31 For example, in addition to a CED-4–like domain containing a putative ATP-binding P loop, the Apaf-1 protein possesses an N-terminal domain that shares homology with the prodomains of human caspases-2 and -9 and with the prodomain of the worm CED-3 protein. This N-terminal domain, called a caspase recruitment domain, has been shown to mediate interactions with the prodomain of caspase-9, thus allowing Apaf-1 to induce autoprocessing of this particular caspase.32 In addition, however, the CED-4–like domain within Apaf-1 is flanked on its carboxyl side by 12 tandem copies of a WD domain. This C-terminal region of the protein seems to function as a negative regulatory domain that holds Apaf-1 in a latent, inactive state in cells, preventing it from binding caspase-9. The presence of this negative regulatory domain thus defines a fundamental difference between the human Apaf-1 and worm CED-4 proteins. The human protein requires an activation step to interact with caspases and induce cell death, whereas the worm CED-4 protein has constitutive caspase-binding and death-inducing activity. What then prompts the Apaf-1 protein into action? The only known mechanism for activating this protein is cytochrome c, which binds to Apaf-1, apparently relieving the repression applied by the WD domains.32 Cytochrome c is normally sequestered inside mitochondria, between the inner and outer membranes of these organelles. It becomes released into the cytosol after exposure of cells to a variety of proapoptotic stimuli, including chemotherapeutic drugs, irradiation, and growth factor withdrawal.33-35 A role for Apaf-1 in apoptosis induced by irradiation and some types of anticancer drugs has been documented by studies in which the Apaf-1 gene was knocked-out by homologous gene recombination in mice. Cells derived from these mice display marked resistance to a wide variety of apoptosis-inducing agents but remain sensitive to Fas-mediated cell death.36,37 Conversely, gene transfer–mediated overexpression of Apaf-1 in HL-60 leukemia cells reportedly increases sensitivity to apoptosis-inducing anticancer drugs.38 To date, no studies of Apaf-1 gene structure or expression in tumors have been reported, but it seems likely that defects in the expression or function of this pivotal proapoptotic protein will eventually be found in cancers.
CED-9/Bcl-2 Recently, it was reported that C elegans contains an antagonist of CED-9 called EGL-1, which binds to it and prevents it from suppressing cell death, thus functioning as a transdominant inhibitor of CED-9.49 Analogous proteins have been known in humans for sometime, including BAD, Bik, Bid, Bim, and Hrk. Dimerization of these proapoptotic proteins with Bcl-2 or Bcl-XL prevents the latter from interacting with Apaf-1 and other proteins and may also interfere with other functions of these antiapoptotic proteins (reviewed in 50). Unlike the simpler nematode, however, several proapoptotic Bcl-2 family proteins have been identified that appear to have intrinsic cell death–inducing function. Proteins such as Bax, Bak, and Mtd/Bok have autonomous cytodestructive activity,51-53 apart from their ability to bind Bcl-2, Bcl-XL, and other antiapoptotic members of the family. The structural similarity between some Bcl-2 family proteins and the pore-forming domains of certain bacterial toxins implies that these proteins may function, at least in part, by forming channels in the intracellular membranes where they typically reside, namely, the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope (reviewed in54). In this regard, Bax has been shown to induce release of cytochrome c from mitochondria,55,56 a property analogous to the structurally similar diphtheria toxin, which produces channels in endosomal/lysosomal membranes of sufficient size to allow escape into the cytosol of the toxin's adenosine 5'-diphosphate–ribosylating polypeptide A-subunit, which inhibits mRNA translation and kills cells.57 Conversely, antiapoptotic Bcl-2 family proteins prevent mitochondrial changes associated with apoptosis and block release of cytochrome c.34,35,58 Many examples exist of alterations in the expression of either apoptosis-suppressing or apoptosis-inducing members of the Bcl-2 family in human cancers (reviewed in59-61). Some of these mechanisms involve structural alterations to the genes, such as (a) t(14;18) chromosomal translocations that activate BCL-2 in most non-Hodgkin's lymphomas62; (b) single nucleotide substitution and frameshift mutations that inactivate BAX in malignancies of the colon, stomach, and hematopoietic system63-65; and (c) retrovirus gene insertions that activate the bcl-XL gene in murine leukemias.66 In most cases, however, the mechanisms responsible for aberrant levels of Bcl-2 family proteins probably reflect changes in the transcriptional and posttranscriptional regulatory networks that control the ultimate output of their genes. Imbalances in the ratios of anti- and proapoptotic Bcl-2 family members that tilt the scales toward survival can render tumor cells more resistant to a wide variety of cell death stimuli, including essentially all chemotherapeutic drugs, radiation, hypoxia, cell detachment from extracellular matrix, growth factor withdrawal, glucose deprivation, elevated cytosolic Ca2+, oxidants, tumor suppressors such as p53 that induce apoptosis, and oncogenes such as Myc and cyclin D1/Bcl-1 that drive cell division but also promote cell death (reviewed in42). The broad resistance to cell death that occurs, for instance, when Bcl-2 is overexpressed in cancers has potential relevance to a wide variety of cancer cell behaviors as well as implications for some of the newer cancer therapies, including angiogenesis inhibitors (hypoxia, hypoglycemia), mechanisms of tumor cell invasion and metastasis (cell adhesion), multistep carcinogenesis (Myc, cyclin D1), and antibodies designed to block growth factor receptors (anti-HER2). The ability of Bcl-2 to block cell death induced by anticancer drugs has established it as a novel type of multidrug resistance protein.67-70 Unlike the MDR-1 family of drug efflux pump proteins, Bcl-2 overexpression does not interfere with the entry and accumulation of drugs in tumor cells. Bcl-2 also neither obviates the initial damage induced by drugs nor alters the rates of repair. Instead, Bcl-2 simply prevents drug-induced damage from being efficiently translated into cell death.71-74 Thus, tumor cells that contain high amounts of Bcl-2 still experience the cell cycle inhibitory effects of drugs that, for example, halt DNA synthesis or interfere with the microtubule formation during mitosis but nevertheless remain viable for protracted periods of time, resulting in enhanced clonigenic survival in many cases. In general, therefore, Bcl-2 converts anticancer drugs from being cytotoxic to merely cytostatic. An additional link between Bcl-2 family proteins and chemoresponses has been found in Bax. The expression of BAX is commonly induced in tumor cells after successful induction of apoptosis with chemotherapeutic drugs or radiation.75 The induction of BAX expression after genotoxic stress injury has been attributed to p53, which binds to typical recognition elements located in the BAX gene promoter and directly transactivates this proapoptotic gene.76,77 The ability of radiation and chemotherapeutic drugs to induce BAX expression may explain why some tumors with high levels of Bcl-2 protein, such as low-grade non-Hodgkin's lymphomas and small-cell lung cancers, initially respond well to therapy. However, these clinical responses are insufficient to eradicate all tumor cells, and relapses uniformly occur, commonly in association with loss of functional p53. Several clinical correlative studies have provided support for the hypothesis that high-level expression of antiapoptotic Bcl-2 family proteins, such as Bcl-2, Bcl-XL, and Mcl-1, confers a clinically important chemoresistant phenotype on cancer cells, including studies of patients with acute myelogenous leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, non-Hodgkin's lymphomas, multiple myeloma, and prostate cancer (reviewed in70,78-80). In addition, attempts to compare the relative levels of Bcl-2 or other antiapoptotic Bcl-2 family proteins in leukemia or solid tumor cells before treatment and again at the time of relapse have demonstrated an apparent upregulation of Bcl-2 or a selective survival of those tumor cells that contain higher levels of Bcl-2 in some patients with acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, neuroblastomas, and prostate cancer.81-84 Conversely, reduced levels of proapoptotic Bcl-2 family proteins such as Bax have been associated with poor responses to chemotherapy and shorter overall survival in women with metastatic breast cancer.85 Inactivating BAX gene mutations have also been correlated with shorter overall survival in a subgroup of patients with colon and gastric cancers that arise because of problems with DNA repair enzyme function (ie, microsatellite instability; M. Perucho, personal communication, January 1999). While assessments of BCL-2 family gene activity in some types of cancer may have prognostic significance, the complexities of dealing with a large multigene family and the diverse biochemical mechanisms of action of Bcl-2 family proteins (reviewed in50,86) place limitations on our current ability to use this information clinically and to interpret its biologic significance. Probably for this reason, clinical correlative studies have not uniformly demonstrated an adverse prognostic role for higher levels of Bcl-2 protein production, and some have even shown that expression of Bcl-2 can bear a strong association with favorable clinical outcome.87-89
IAP Family Proteins Interestingly, not all caspases are targets of IAP suppression. To date, only caspases-3, -7, and -9 have been reported to be bound and inhibited by IAPs. Caspases-1, -6, -8, and -10 are not. The significance of this selective inhibition of caspases by IAPs is that the inhibited caspases operate in the distal portions of the apoptotic proteolytic cascades, with some (such as caspases-3 and -7) functioning as the ultimate effectors of apoptosis that cleave the various proteins responsible for cell death. Little is known about the expression of most IAP family proteins in either normal human tissues or tumors. However, the IAP family protein survivin is overexpressed in a large proportion of human cancers,94 providing evidence that alterations in the expression of these proteins can occur in the course of tumorigenesis. Elevated levels of survivin have been associated with a worse prognosis in children with neuroblastoma,95 as well as adults with more common types of cancer to date. MORE THAN ONE WAY TO KILL A CELL
What defines the commitment step from which the cell cannot be rescued, and how important are various apoptosis-regulatory proteins for regulating this step? Recent studies have demonstrated that blocking apoptosis induced by anticancer drugs is not necessarily synonymous with blocking cell death commitment. Although treatment of tumor cells with chemotherapeutic drugs uniformly results in proteolytic processing of certain caspases and cleavage of caspase substrates such as poly-adenosine 5'-diphosphate-ribosyl polymerase (PARP), and although inhibiting caspases with broad-specificity irreversible inhibitors such as benzyloxycarbonyl-valinyl-alaninyl-aspartyl-fluoromethylketone typically blocks all manifestations of apoptosis induced by exposure of tumor cells to anticancer drugs, in many cases, these drug-damaged cells nevertheless die because of what seems to be a delayed necrosis.96-100 This delayed nonapoptotic cell death is preceded by, and indeed probably caused by, mitochondrial damage, resulting in release of certain mitochondrial proteins, including cytochrome c, from these organelles, interrupting electron chain transport, and causing generation of free radicals, loss of the electrochemical gradient across the inner membrane (
Interestingly, although caspase inhibitors fail to prevent anticancer drug–induced mitochondrial damage and commitment to cell death, clonigenic survival studies indicate that Bcl-2 and Bcl-XL can prevent or reduce drug-induced cell death under the same circumstances (reviewed in101). Thus, at least where examined so far, Bcl-2 family proteins seem to govern a cell death commitment step upstream of anticancer drug–induced caspase activation and apoptosis (Fig 4). In this regard, Bcl-2 and Bcl-XL can prevent mitochondrial changes associated with drug-induced cell death, including cytochrome c release, loss of
BYPASSING Bcl-2 ON THE ROUTE TO APOPTOSIS While Bcl-2 clearly can govern a cell death commitment step upstream of caspase activation, it has been shown that Bcl-2–independent pathways for caspase activation and apoptosis induction also exist (reviewed in107). For example, in some (but not all) types of cells, apoptosis triggered by Fas (CD95) and other (TNF) family death receptors is not blocked by overexpression of Bcl-2 or other antiapoptotic members of the Bcl-2 family. This implies that Fas and similar TNF family receptors, which directly induce caspase activation through ligand-induced recruitment of cytosolic procaspases,9,10 can bypass a Bcl-2–dependent checkpoint that controls cell survival. The idea, then, is that in many types of cells, this "death receptor" pathway for apoptosis runs via a Bcl-2–independent pathway, circumventing the participation of mitochondria or other organelles where Bcl-2 and many of its homologs reside as integral membrane proteins (Fig 5). It is difficult to predict how important this Bcl-2–independent pathway is within the context of clinical responses to chemotherapy and radiation, since many, if not most, tumors and leukemias probably do not use a death receptor–mediated autocrine mechanism for triggering apoptosis. Further complicating our attempts to predict chemoresponses, it has been shown that even apoptosis induced via Fas can be blocked substantially by Bcl-2 or Bcl-XL overexpression in some cells but not others. Recent comparisons of cell lines in which Bcl-2 overexpression was or was not Fas-protective suggest that two types of cellular contexts can be identified: one in which Fas ligation leads to abundant amounts of pro–caspase-8 processing (which is Bcl-2–independent), and another in which Fas triggers only small amounts of caspase-8 activation (Bcl-2–suppressible).108 The basis for this difference in Bcl-2 sensitivity seems to reside in whether caspase-8 does or does not require a mitochondria-dependent amplification step to achieve sufficient activation of downstream effector caspases for apoptosis.109 Bcl-2 apparently can block caspase-8–mediated apoptosis only in cases in which a mitochondria-dependent amplification of the caspase cascade is required. It is important to recognize, however, that cross-talk between these two pathways can be extensive. For example, upstream caspases can induce irreversible mitochondrial damage and cytochrome c release.104,105,110,111 Moreover, the finding that Bcl-XL can be coimmunoprecipitated with the inactive proform of caspase-8 implies that it might be able to sequester this protease so that it cannot be recruited to liganded Fas at the plasma membrane.112 WHERE DO WE GO FROM HERE? Abundant therapeutic opportunities have been revealed by investigations of the fundamental mechanisms of apoptosis regulation and identification of the various cell survival genes that become dysregulated in tumors. For example, agents that interfere with the actions of Bcl-2 or other antiapoptotic Bcl-2 family proteins are envisioned as chemosensitizers, which would make it easier for conventional anticancer drugs and radiation to commit cells to death. Antisense DNA oligonucleotides targeted against BCL-2 mRNA have been reported to reduce Bcl-2 protein levels, thereby sensitizing cancer cells to chemotherapeutic agents in vitro and in animal models (reviewed in113). Moreover, BCL-2 antisense oligonucleotides have been tested in a phase I study of patients with relapsed and refractory lymphomas, yielding highly preliminary but promising results.114 Efforts are also underway to identify small-molecule drugs that bind to the Bcl-2 or Bcl-XL proteins, mimicking the inhibitory effects of proapoptotic transdominant inhibitory proteins such as BAD, Bik, Bim, Bid, and Hrk.115,116 Another approach centers on IAP family proteins and their interactions with caspases. Here the idea is to generate compounds that block IAP binding to caspases, thus releasing activated caspases to kill tumor cells. Suggestions that such a mechanism might be feasible come from analysis of cell death proteins in the fly Drosophila melanogaster, in which three different cell death genes (reaper, hid, and grim) have been shown to encode proteins that share a common 14–amino acid domain that binds IAP family proteins and induces cell death.117,118 To the extent that small peptides can also inactivate human IAPs, therefore, it may be possible to devise nonpeptidyl compounds that perform the same functions.
A variety of less direct approaches for altering the expression or function of apoptosis-regulating genes and their encoded proteins can also be envisioned, such as compounds that inhibit protein kinases which control signaling pathways governing the phosphorylation of apoptosis proteins or the transcriptional output of their genes (reviewed in119,120). In this regard, certain kinases linked to cell survival signaling, such as Akt, have been shown to phosphorylate and inactive particular proapoptotic Bcl-2 family proteins (reviewed in121,122). Other indirect approaches involving apoptosis include antiangiogenesis strategies, such as those that attack the alphavbeta3 ( Ultimately, the success or failure of these approaches will be dictated by the therapeutic index, relying, for instance, on exploiting differences in the relative dependence of tumor and normal cells on antiapoptotic genes. Indeed, tumor cells are potentially vulnerable if their antiapoptosis crutch can be knocked out from under them. Cancer cells, for example, often activate certain oncogenes that drive the cell division cycle in an uncontrolled manner but which also trigger cell death if not countered by overexpression of proteins such as Bcl-2.125,126 This proliferative driving force causes tumor cells with damaged DNA, for example, to disobey cell cycle checkpoint controls, which normally would trigger cell death if not for aberrant regulation of apoptosis genes such as Bcl-2. Most normal cells, in contrast, undergo cell cycle arrest when their DNA is damaged, awaiting repair before resuming their proliferation (reviewed in127). Similarly, the genetic instability that characterizes many aggressive cancers is in itself a potential drive for apoptosis. The myriad genetic lesions, chromosomal breaks, and other genomic defects seen in these tumors would trigger apoptosis in an otherwise normal cell. Another example of potential tumor cell vulnerability comes from the observation that epithelial cells that detach from the extracellular matrix in the process of metastasis rely on blocks to apoptosis so that they can survive in the absence of integrin-mediated signaling.128-130 Finally, the expression on tumor cells of mutant antigens makes them potential targets for immune-mediated eradication, provided they are not resistant to the apoptosis-inducing weapons the immune system uses for this purpose. The challenge currently facing us is to translate information gained about mechanisms of aberrant cell death control in tumors into new therapeutic opportunities that fundamentally change the course of cancer treatment as we recognize it today. The path for accomplishing this has been illuminated by basic research. The task now is to execute those strategies which hold the greatest promise. ACKNOWLEDGMENTS Supported by the National Cancer Institute, National Institute of Aging, Department of Defense, California State Breast Cancer Research Program, and CaP-CURE I thank all the members of our laboratory for their untiring dedication and T. Brown and J. Waltz for manuscript preparation. REFERENCES 1. Hengartner MO, Horvitz HR: Programmed cell death in Caenorhabditis elegans. Curr Opin Genet Dev 4:581-586, 1994[Medline] 2. Yuan J, Shaham S, Ledoux S, et al: The C. elegans cell death gene ced-3 encodes a protein similar to mammalian interleukin-1 beta-converting enzyme. Cell 75:641-652, 1993[Medline] 3. Alnemri ES, Livingston DJ, Nicholson DW, et al: Human ICE/CED-3 protease nomenclature. Cell 87:171, 1996[Medline] 4. Salvesen GS, Dixit VM: Caspases: Intracellular signaling by proteolysis. Cell 91:443-446, 1997[Medline] 5. Alnemri ES: Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 64:33-42, 1997[Medline] 6. Zapata JM, Krajewski S, Huang R-P, et al: Expression of multiple apoptosis-regulatory genes in human breast cancer cell lines and primary tumors. Breast Cancer Res Treat 47:129-140, 1998[Medline]
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Copyright © 1999 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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) functions.
). Fas-L on tumor cells can result in killing of both Fas-sensitive immune cells and normal neighboring cells.
), and presumably ATP depletion (reviewed in
vß3) integrin on endothelial cells, thereby depriving migrating endothelial cells of critical survival signals they need from this cell adhesion protein to suppress apoptosis (reviewed in123). Interestingly, 
