This page contains links to sites that require the plug-in Chime for optimal viewing. Chime may be downloaded free from MDL Information Systems, Inc.
This page was created and is maintained by Jim Mullally for PHTX 6500 Biochemical Mechanisms of Signal Transduction Coursemaster: Don Blumenthal, Ph.D., Associate Professor of Pharmacology and Toxicology Please send your comments and suggestions to:
Tumorigenesis can be the result of any number of insults, such as DNA alterations (via UV radiation, covalent modification with carcinogens, and other environmental factors), genetic predisposition (i.e. inherited mutations which may or may not cause immediate neoplasia), or random mutations (a result of imperfect DNA replication). On a molecular basis, the modes of carcinogenesis are just as varied, such as tumor suppresser inactivation (e.g. p53), constitutive activation of various oncoproteins such as Ras, the splicing together of various genes which activate transcription (e.g. EWS-Fli1), etc. If exposed to any of these insults, an important mode by which a cell may circumvent transformation to a neoplastic phenotype is via apoptosis, or programmed cell death. Upon exposure to a variety of stimuli, both naturally occurring (i.e. extracellular signals, etc.) and man-made (i.e. genotoxins and microtubule toxins, etc.), a number of apoptotic pathways may induce cell death. Bcl-2 is a key member in one of these pathways, for in its active form it inhibits apoptosis. This protein has been found to be altered, and thus constitutively active, or up-regulated in a subset of neoplasms. In these altered or up-regulated states, the cell may not undergo apoptosis, and thus tumorigenesis may occur. As mentioned above, apoptosis may be induced by man-made stimuli. Such stimuli, therefore, can make very useful drugs in the treatment of various cancers. One of these man-made stimuli, the microtubule stabilizing drug Taxol (Paclitaxel, See 2D structure or Chime generated 3D structure [Click for Chime tips]), has been known for some time to induce apoptosis in certain cancer cells. Taxol has been shown to have a distinct effect on Bcl-2, in which it induces Bcl-2 phosphorylation and inactivation, followed, subsequently, by activation of the Bcl-2 regulated apoptotic pathway. Although not all of the details of Taxol induced Bcl-2 inactivation are known, the intent of this paper is illustrate how Taxol may, in part, convey its anti-neoplastic effects via the Bcl-2 regulated apoptotic pathway. In doing so, this paper will also demonstrate how Taxol can be used to treat neoplasms in which Bcl-2 has been up-regulated.
In 1984, B cells were isolated from a young patient with lymphoblastic leukemia and were found to have 8;14 and 14;18 chromosomal translocations (Pegoraro et al., 1984). They located a putative oncogene at the breakpoint of chromosome 18 which they termed B cell lymphoma 2 (Bcl-2). It had been previously observed in 1982, that in patients with one of the most common forms of B-cell neoplasia, follicular lymphoma, a 14;18 chromosomal translocation was commonly found. (Yunis et al., 1982) Also, Liu et al. found that the frequency of this translocation increased with population age, coincident with increased risk for lymphoma (Liu et al., 1994). It is believed that this translocation results in up-regulation of the bcl-2 gene product. In 1989, it was discovered that B-cells, transfected with a vector which overexpressed Bcl-2, had a distinct growth advantage over non-transfected cells, further suggesting Bcl-2 involvement in follicular lymphoma (Tsujimoto, Y., 1989). Similarly, it was observed that Bcl-2 overexpression inhibited apoptosis in the photoreceptor cells of transgenic mice with various retinal degenerations (Chen et al., 1996). Furthermore, in 1993, it was shown that T and B-cells disappeared from Bcl-2 knockout mice by 4 weeks of age, indicating that Bcl-2 is indispensable for a functional immune system (Nakayama et al., 1993). This evidence illustrates Bcl-2's role in cell survival when expressed normally, as well as it's role in carcinogenesis when it is up-regulated.
The Bcl-2 family members consist of proteins interacting with or having homology to Bcl-2, which also contain so called BH (Bcl-2 homology) domains. Among these family members are the anti-apoptotic proteins Bcl-2, Bcl-XL, Bfl-1, Mcl-1 and A1, which contain the anti-apoptotic BH-1 and BH-2 domains (some of which also contain regulated BH-3 domains). Also included are the pro-apoptotic family members Bax, Bak, Bad, Bcl-Xs, Nbk/Bik, Hrk, and Bid, which contain the pro-apoptotic BH-3 domain (Allen et al., 1998). Interaction between the BH domains of various members is believed to be responsible for the differential activities (i.e. apoptosis inhibition/promotion) of the Bcl-2 family (Diaz et al., 1997; Kelekar et al., 1997). A dramatic illustration of this is the interaction between Bcl-XL and a 16 residue peptide representing the BH3 domain of Bak (see 1D structure or see below for Chime generated image), which is sufficient for promotion of apoptosis (Sattler et al., 1997).
Click the following buttons in sequence: Turn Bak peptide into wireframe structure Show Bak Van der Waals radii Return Bak to cartoon structure Display Bcl-XL backbone Show hydrogen bonding interactions IV. Cellular location of Bcl-2 family members Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999). V. Apoptosis via the Bcl-2 pathway In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999). VI.Taxol discovery and development Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree. VII. Taxol induced microtubule stabilization Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999). VIII. Taxol induced apoptosis via Bcl-2 As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis. IX. Conclusions The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers. X. Useful Links PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page XI. References Allen et al. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998 May;54(5):427-45. ( MEDLINE ) Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE ) Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE ) Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE ) Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE ) Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE ) Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE ) Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE ) Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE ) Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE ) Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE ) Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE ) Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE ) Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE ) Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE ) Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE ) Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE ) Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE ) Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE ) Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE ) Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE ) Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE ) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE ) Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE ) Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE ) Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE ) Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE ) Chime Tips: Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
Turn Bak peptide into wireframe structure Show Bak Van der Waals radii Return Bak to cartoon structure Display Bcl-XL backbone Show hydrogen bonding interactions IV. Cellular location of Bcl-2 family members Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999). V. Apoptosis via the Bcl-2 pathway In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999). VI.Taxol discovery and development Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree. VII. Taxol induced microtubule stabilization Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999). VIII. Taxol induced apoptosis via Bcl-2 As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis. IX. Conclusions The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers. X. Useful Links PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page XI. References Allen et al. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998 May;54(5):427-45. ( MEDLINE ) Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE ) Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE ) Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE ) Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE ) Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE ) Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE ) Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE ) Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE ) Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE ) Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE ) Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE ) Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE ) Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE ) Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE ) Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE ) Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE ) Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE ) Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE ) Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE ) Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE ) Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE ) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE ) Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE ) Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE ) Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE ) Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE ) Chime Tips: Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
Show Bak Van der Waals radii Return Bak to cartoon structure Display Bcl-XL backbone Show hydrogen bonding interactions IV. Cellular location of Bcl-2 family members Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999). V. Apoptosis via the Bcl-2 pathway In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999). VI.Taxol discovery and development Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree. VII. Taxol induced microtubule stabilization Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999). VIII. Taxol induced apoptosis via Bcl-2 As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis. IX. Conclusions The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers. X. Useful Links PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page XI. References Allen et al. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998 May;54(5):427-45. ( MEDLINE ) Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE ) Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE ) Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE ) Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE ) Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE ) Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE ) Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE ) Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE ) Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE ) Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE ) Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE ) Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE ) Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE ) Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE ) Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE ) Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE ) Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE ) Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE ) Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE ) Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE ) Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE ) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE ) Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE ) Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE ) Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE ) Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE ) Chime Tips: Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
Return Bak to cartoon structure Display Bcl-XL backbone Show hydrogen bonding interactions IV. Cellular location of Bcl-2 family members Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999). V. Apoptosis via the Bcl-2 pathway In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999). VI.Taxol discovery and development Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree. VII. Taxol induced microtubule stabilization Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999). VIII. Taxol induced apoptosis via Bcl-2 As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis. IX. Conclusions The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers. X. Useful Links PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page XI. References Allen et al. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998 May;54(5):427-45. ( MEDLINE ) Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE ) Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE ) Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE ) Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE ) Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE ) Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE ) Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE ) Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE ) Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE ) Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE ) Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE ) Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE ) Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE ) Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE ) Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE ) Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE ) Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE ) Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE ) Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE ) Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE ) Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE ) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE ) Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE ) Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE ) Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE ) Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE ) Chime Tips: Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
Display Bcl-XL backbone Show hydrogen bonding interactions IV. Cellular location of Bcl-2 family members Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999). V. Apoptosis via the Bcl-2 pathway In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999). VI.Taxol discovery and development Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree. VII. Taxol induced microtubule stabilization Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999). VIII. Taxol induced apoptosis via Bcl-2 As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis. IX. Conclusions The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers. X. Useful Links PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page XI. References Allen et al. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998 May;54(5):427-45. ( MEDLINE ) Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE ) Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE ) Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE ) Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE ) Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE ) Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE ) Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE ) Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE ) Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE ) Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE ) Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE ) Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE ) Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE ) Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE ) Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE ) Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE ) Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE ) Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE ) Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE ) Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE ) Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE ) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE ) Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE ) Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE ) Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE ) Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE ) Chime Tips: Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
Show hydrogen bonding interactions IV. Cellular location of Bcl-2 family members Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999). V. Apoptosis via the Bcl-2 pathway In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999). VI.Taxol discovery and development Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree. VII. Taxol induced microtubule stabilization Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999). VIII. Taxol induced apoptosis via Bcl-2 As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis. IX. Conclusions The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers. X. Useful Links PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page XI. References Allen et al. Mechanisms controlling cellular suicide: role of Bcl-2 and caspases. Cell Mol Life Sci 1998 May;54(5):427-45. ( MEDLINE ) Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE ) Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE ) Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE ) Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE ) Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE ) Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE ) Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE ) Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE ) Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE ) Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE ) Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE ) Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE ) Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE ) Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE ) Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE ) Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE ) Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE ) Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE ) Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE ) Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE ) Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE ) Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE ) Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE ) Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE ) Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE ) Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE ) Chime Tips: Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
IV. Cellular location of Bcl-2 family members
Several of the Bcl-2 family members, including Bcl-2, Bcl-XL, Bcl-Xs (Bcl-XL and Bcl-Xs are alternatively spliced variants of the same gene, bcl-x, where Bcl-Xs lacks the regulatory BH-1 and BH-2 domains), and Bax, have a putative transmembrane domain, which is believed to localize them to membranes. Bad, Bax and Bcl-Xs have been found to be mainly cytosolic proteins, even though Bax and Bcl-Xs have the putative transmembrane domain (Jia et al., 1999). However, as will be illustrated, cellular events may occur which can dramatically alter the distribution of these proteins. In the resting state, Bcl-2 is mainly localized to mitochondria, but it is also located to some extent in the endoplasmic reticulum, nuclear envelope, and golgi complex. Bcl-XL was found to be located both in the outer mitochondrial membrane and in soluble cytosolic fractions. The pro-apoptotic proteins, Bcl-Xs, Bad, and Bax, were found to be located in the cytoplasmic fractions of resting cells (Jia et al., 1999). However, upon TNF-alpha stimulation, there is a shift in the cellular location of some of these proteins. For instance, there is a clear translocation of the pro-apoptotic members Bcl-Xs, Bad, and Bax from the cytoplasm to the mitochondrial outer membrane. Under these conditions, Bcl-2 and Bcl-XL remained on the outer mitochondrial membrane (Jia et al., 1999).
In order to have a clear understanding of the biochemical events leading to apoptosis, it is efficacious to first understand the role of the molecules in the Bcl-2 apoptotic pathway which inhibit apoptosis. As mentioned above, one of the roles of the inhibitory proteins in this pathway is the sequestration of the pro-apoptotic members. For instance, as was dramatically illustrated in the crystal structure shown in (Figure 1), Bcl-XL has been found to form a heterodimer with Bak, thus promoting cell survival (Yang et al., 1995). Bcl-2 has been shown to heterodimerize with Bax and Bak. Also, it has been found that disruption of these interactions, via site directed mutagenesis of Bcl-2 BH domains, leads to formation of Bak homodimers and apoptosis (Yin et al., 1994). This has lead to the hypothesis that the tissue specific ratio of free Bak to Bcl-2 bound bak is an important determinant of whether or not a cell will undergo apoptosis upon exposure to a variety of stimuli (Oltvai et al., 1994). Several groups have demonstrated a correlation between apoptosis inducing stimuli and Bcl-2 phosphorylation (Haldar et al., 1995; Poruchynsky et al., 1998; Srivastava et al., 1998). This phosphorylation has further been shown to disrupt Bcl-2 heterodimerization with Bax and Bak. The phosphorylation of Bcl-2 has been more definitively demonstrated to induce apoptosis in experiments utilizing site mutagenesis. These studies have demonstrated the requirement of a disordered loop region in Bcl-2 for phosphorylation, cytochrome c release, and subsequent entry into apoptosis (Fang et al., 1998; Burri et al., 1999). Recently, mutagenesis experiments have demonstrated that the expression of a Ser70Ala Bcl-2 mutant, in cells lacking endogenous Bcl-2 expression, inhibited JNK (c-terminal Jun kinase) induced apoptosis by almost 50 percent (Srivastava et al., 1999). As far as an overall view of the apoptotic pathway involving Bcl-2 is involved (Figure 2), these studies and others have led to the proposal that, upon stimulation with various trophic factors or anti-microtubule agents, JNK is activated by JNKK (c-Jun N-terminal kinase kinase) which results in Bcl-2 is phosphorylation and inactivation and release of free Bax. This inactivation also leads to the release of cytochrome c from mitochondria, which then may interact with APAF-1 (apoptosis protease-activating factor-1). This in turn, in the presence of dATP, activates caspase-9 (cysteine protease-9), which activates caspase-3 (and other caspases), resulting in PARP cleavage, DNA fragmentation and apoptosis (Srivastava et al., 1999).
Taxol is the registered trademark name for the anti-cancer compound Paclitaxel. This compound was extracted from the bark of the Pacific yew tree (Taxus brevifolia) in the 1960’s during an extensive natural product search conducted by the National Cancer Institute (NCI). At this time it was identified as having anti-cancer properties, however the mechanism of action was unknown. Not until the late 1970’s did this mechanism become clear: microtubule stabilization (Fuchs et al., 1978). In 1983, the NCI began human clinical trials with Taxol against several different types of cancer. The result of these trials underscored Taxol’s efficacy in treating, among others, ovarian cancer. This research lead to the FDA approval of Taxol for the treatment of ovarian cancer (1992) and then breast cancer (1994). However, increased demand for Taxol identified a major problem…supply. Not only does the Pacific yew have a very low abundance of Taxol in the bark, but also, the area that has the highest population of these trees is the natural habitat of the endangered spotted owl. The solution to this problem has been the utilization of semi-synthetic methods in the production of Taxol, which make this compound from derivatives found in the needles of the Pacific yew, thus eliminating the need to sacrifice the tree.
Microtubules are essential structural molecules needed for cell division. However, once the cell begins to divide (upon entering M phase) these microtubules need to be disassembled to allow separation of the daughter cells. Taxol’s anti-tumorigenic characteristics are derived, in part, from it’s ability to bind tubulin subunits, thus disallowing microtubule degradation (Arnal et al., 1995), resulting in cell cycle arrest in the G2/M phase (Schiff et al., 1980). In fact there are several anti-microtubule compounds (both stabilizing and destabilizing) which have been shown to result in cell cycle arrest and apoptosis including colchicine, colcemid, podophyllotoxin (Haldar et al., 1998), vincristine, vinblastine, nocodazole, taxotere (Poruchynsky et al., 1998), and others. Recently, it was demonstrated that the microtubule stabilizing compounds may have a common pharmacophore characteristic of their mode of action (Ojima et al., 1999).
As was illustrated in the preceding section, Taxol has been shown to stabilize microtubules resulting in cell cycle arrest in the G2/M phase. However, it is believed that it is apoptosis, and not microtubule stabilization, that leads to the ultimate demise of the cell. This is where the Bcl-2 apoptotic pathway is believed to be important. It has been demonstrated by several groups that Bcl-XL (Poruchynsky et al., 1998) and Bcl-2 become phosphorylated in response to cell treatment with Taxol, as well as many other microtubule active agents, in a variety of human cancer cells, including prostate (Haldar et al., 1996), ovarian (Wang et al., 1999), and leukemia (Hu et al., 1998). It has also been demonstrated that expression of mutant forms of Bcl-2, either lacking Ser70 or the disordered loop region altogether, eliminate Bcl-2 phosphorylation, cytochrome c release, PARP cleavage, DNA fragmentation, and apoptosis, in cells exposed to Taxol (Srivastava et al., 1999). This suggests that the disruption of microtubule dynamics leads to the phosphorylation and inactivation of Bcl-2, which then may no longer promote cell survival, resulting in apoptosis.
The purpose of this paper was to demonstrate that certain types of cancer are the result of Bcl-2 overexpression, including lymphoblastic leukemia, as well as certain breast and prostate cancers, and that Taxol's anti-tumorigenic effects may, in some part, be the result of Bcl-2 phosphorylation and inactivation in these cells. In order to achieve this goal, evidence was offered that Bcl-2 phosphorylation leads to it's inactivation and apoptosis. Also, it was demonstrated that exposure to Taxol results in the phosphorylation of Bcl-2 in a number of cancers. Furthermore, it was illustrated that expression of mutant forms of Bcl-2, abrogate Taxol induced phosphorylation and apoptosis, without disrupting microtubule stabilization. Thus, this and other evidence serve to implicate apoptosis, via a Bcl-2 mediated pathway, in Taxol’s anti-neoplastic effects in a number of cancers.
PubMed National Cancer Institute CancerNet NCBI Chime Plug-in Signal Transduction Home Page
Arnal et al. How does taxol stabilize microtubules? Curr Biol 1995 Aug 1;5(8):900-8. ( MEDLINE )
Burri et al. 'Loop' domain deletional mutant of Bcl-xL is as effective as p29Bcl-xL in inhibiting radiation-induced cytosolic accumulation of cytochrome c (cyt c), caspase-3 activity, and apoptosis. Int J Radiat Oncol Biol Phys 1999 Jan 15;43(2):423-30. ( MEDLINE )
Chen et al. Bcl-2 overexpression reduces apoptotic photoreceptor cell death in three different retinal degenerations. Proc Natl Acad Sci U S A 1996 Jul 9;93(14):7042-7. ( MEDLINE )
Diaz et al. A common binding site mediates heterodimerization and homodimerization of Bcl-2 family members. J Biol Chem 1997 Apr 25;272(17):11350-5. ( MEDLINE )
Fang et al. "Loop" domain is necessary for taxol-induced mobility shift and phosphorylation of Bcl-2 as well as for inhibiting taxol-induced cytosolic accumulation of cytochrome c and apoptosis. Cancer Res 1998 Aug 1;58(15):3202-8. ( MEDLINE )
Fuchs et al. Cytologic evidence that taxol, an antineoplastic agent from Taxus brevifolia, acts as a mitotic spindle poison. Cancer Treat Rep 1978 Aug;62(8):1219-22. ( MEDLINE )
Haldar et al. Inactivation of Bcl-2 by phosphorylation. Proc Natl Acad Sci U S A 1995 May 9;92(10):4507-11. ( MEDLINE )
Haldar et al. Taxol induces bcl-2 phosphorylation and death of prostate cancer cells. Cancer Res 1996 Mar 15;56(6):1253-5. ( MEDLINE )
Hu et al. Phosphorylation of BCL-2 after exposure of human leukemic cells to retinoic acid. Blood 1998 Sep 1;92(5):1768-75. ( MEDLINE )
Jia et al. Subcellular distribution and redistribution of Bcl-2 family proteins in human leukemia cells undergoing apoptosis. Blood 1999 Apr 1;93(7):2353-9. ( MEDLINE )
Kelekar et al. Bad is a BH3 domain-containing protein that forms an inactivating dimer with Bcl-XL. Mol Cell Biol 1997 Dec;17(12):7040-6. ( MEDLINE )
Liu et al. Bcl-2 translocation frequency rises with age in humans. Proc Natl Acad Sci U S A 1994 Sep 13;91(19):8910-4. ( MEDLINE )
Nakayama et al. Disappearance of the lymphoid system in Bcl-2 homozygous mutant chimeric mice. Science 1993 Sep 17;261(5128):1584-8. ( MEDLINE )
Ojima et al. A common pharmacophore for cytotoxic natural products that stabilize microtubules. Proc Natl Acad Sci U S A 1999 Apr 13;96(8):4256-61. ( MEDLINE )
Oltvai et al. Checkpoints of dueling dimers foil death wishes. Cell 1994 Oct 21;79(2):189-92. ( MEDLINE )
Pegoraro et al. A 14;18 and an 8;14 chromosome translocation in a cell line derived from an acute B-cell leukemia. Proc Natl Acad Sci U S A 1984 Nov;81(22):7166-70. ( MEDLINE )
Poruchynsky et al. Bcl-xL is phosphorylated in malignant cells following microtubule disruption. Cancer Res 1998 Aug 1;58(15):3331-8. ( MEDLINE )
Sattler et al. Structure of Bcl-xL-Bak peptide complex: recognition between regulators of apoptosis. Science 1997 Feb 14;275(5302):983-6. ( MEDLINE )
Schiff et al. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci U S A 1980 Mar;77(3):1561-5. ( MEDLINE )
Srivastava et al. Involvement of microtubules in the regulation of Bcl2 phosphorylation and apoptosis through cyclic AMP-dependent protein kinase. Mol Cell Biol 1998 Jun;18(6):3509-17. ( MEDLINE )
Srivastava et al. Deletion of the loop region of bcl-2 completely blocks paclitaxel-induced apoptosis. Proc Natl Acad Sci U S A 1999 Mar 30;96(7):3775-80. ( MEDLINE )
Tsujimoto, Y. Overexpression of the human BCL-2 gene product results in growth enhancement of Epstein-Barr virus-immortalized B cells. Proc Natl Acad Sci U S A 1989 Mar;86(6):1958-62. ( MEDLINE )
Wang et al. Microtubule dysfunction induced by paclitaxel initiates apoptosis through both c-Jun N-terminal kinase (JNK)-dependent and -independent pathways in ovarian cancer cells. J Biol Chem 1999 Mar 19;274(12):8208-16. ( MEDLINE )
Yang et al. Bad, a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death. Cell 1995 Jan 27;80(2):285-91. ( MEDLINE )
Yin et al. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994 May 26;369(6478):321-3. ( MEDLINE )
Yunis et al. Distinctive chromosomal abnormalities in histologic subtypes of non-Hodgkin's lymploma. N Engl J Med. 1982 Nov 11;307(20):1231-6. ( MEDLINE )
Chime Tips:
Use your Control key and right mouse button in combination while rolling the mouse to translate the molecule across the screen. Use your Shift button and left mouse button in combination while rolling the mouse to zoom in and out. Use your right mouse button on the Chime generated image to obtain additional options.
I.
Introduction
