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Molecular targeting: new therapeutic strategies to improve tumour apoptosis
Annals of Oncology 15 (Supplement 4): iv229 – iv231, 2004 doi:10.1093/annonc/mdh931
Molecular targeting: new therapeutic strategies to improve tumour apoptosis S. Marsoni & G. Damia SENDO Foundation, Milan, Italy
Introduction
Major apoptotic pathways Many different mechanisms of apoptosis have been discovered, but schematically there are two major pathways [6]. The intrinsic (mitochondrial –apoptosome) pathway is triggered by chemical or physical stress signals arising within the cell, while the extrinsic or death receptor-induced pathway is initiated by the binding of extracellular molecules, the death activators [tumour necrosis factor (TNF)-a, lymphotoxin, Fas ligand (FASL)] to specific death receptors at the cell surface. Ultimately, both pathways are dependent upon a family of cysteine proteases, the caspases, which are generally kept inactive in the cytosol by being maintained as zymogen, and can cleave several hundred cellular substrates. Caspases can be classified as ‘executioners’ (caspases-3, -6 and -7) and ‘initiators’ (caspases-8 and -9). The former can attack their q 2004 European Society for Medical Oncology
Downloaded from http://annonc.oxfordjournals.org/ by guest on August 19, 2015
There are two ways for cells to die: death by injury and death by suicide. The pattern of events in death by suicide is called apoptosis, the ancient Greek term for the falling of leaves, and is a genetically regulated mechanism of programmed cell death [1]. Why should a cell commit suicide? Programmed cell death is needed whenever tissue modelling is required, during embryogenesis, for the removal of old cells and to prevent overgrowth after repair of cell loss by injury [2]. Programmed cell death is also needed to destroy cells that represent a threat to the integrity of the organism, such as virus-infected cells, effectors cells of the immune system that are no longer necessary and cells with DNA damage that can become cancerous. The mechanism of apoptosis is switched on by the imbalance between proapoptotic (death signals) and antiapoptotic (survival signals) factors. This happens in both normal and cancer cells. Dysregulation of apoptosis occurs in cancer cells and has not only been implicated in tumour progression and pathogenesis, but also plays an important role in response to therapy [3– 5]. In fact, cancer treatments, radiation and chemotherapeutic agents can destroy tumours by triggering cancer cell apoptosis. On the other hand, cancer cells can develop numerous mechanisms to evade apoptosis through either inactivation of proapoptotic or up-regulation of antiapoptotic factors.
cellular substrates if their zymogens are proteolytically cleaved by the latter. The death receptor pathway, through the binding between ligands and their receptors (such as Fas, TNF-a and TRAIL) activates caspase-8 and caspase-10 via the adaptor protein Fas-associated death domain (FADD) [7]. The death receptor family includes the receptors for TNF-a, FASL and Apo2L/ TRAIL. These receptors are characterised by an 80 amino acid death domain in their cytoplasmic tail that, when activated by agonistic antibodies or their cognate ligands, recruits signalling components that initiate apoptosis. In some cell types, activation of caspase-8 is sufficient to trigger apoptosis, while in other cell types amplification of the extrinsic pathway through the intrinsic pathway is necessary to commit cells to apoptosis. In fact, the cleavage of Bid by caspase-8 and the translocation of the cleaved Bid to mitochondria have been shown to be the link between the two pathways. Yet to be defined are the molecular mechanisms that regulate and control the expression of death receptors, their ligands and the cytoplasmic adapter molecules both in normal and tumour cells. The intrinsic pathways is induced by different kinds of stresses, and caspase-9 is typically activated by the scaffold protein Apaf-1, which undergoes a conformational change induced by cytochrome c released by the damaged mitochondria. Such conformational change allows the recruitment of pro-caspase-9 and its activation. Bcl2 family proteins have a central role in the control of mitochondria-induced apoptosis [8]. More than 20 proteins have been identified, including proteins that suppress apoptosis (Bcl2, BclXl, MCL1) and proteins that suppress apoptosis (Bax, Bad, Bid, Bik). It has been proposed that the Bcl2 members form channels that facilitate protein transport and interaction with other mitochondrial proteins, and also induce rupture of the outer mitochondrial membrane [9]. While the Bcl2 family proteins regulate the mitochondrial pathway before caspase activation, the inhibitors of apoptosis (IAP) proteins regulate apoptosis after caspase activation. The unravelling of the molecular mechanisms at the base of apoptosis and the better understanding of the apoptosis mechanisms relevant for tumour development and for tumour chemo- and radiosensitivity have brought up new strategies for restoring apoptosis sensitivity in tumour cells. Indeed, in solid tumours, the low propensity to undergo apoptosis has
iv230 been associated with resistance to treatment. Experimental evidence has suggested that it is theoretically possible to drive cells to apoptosis (rather than cell cycle arrest) with a dramatic increase in the sensitivity of anticancer agents [10].
Targeting apoptosis
References 1. Adams JM. Ways of dying: multiple pathways to apoptosis. Genes Dev 2003; 17: 2481–2495. 2. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257. 3. Townson JL, Naumov GN, Chambers AF. The role of apoptosis in tumor progression and metastasis. Curr Mol Med 2003; 3: 631– 642. 4. Stenner-Liewen F, Reed JC. Apoptosis and cancer: basic mechanisms and therapeutic opportunities in the postgenomic era. Cancer Res 2003; 63: 263– 268. 5. Kaufmann SH, Vaux DL. Alterations in the apoptotic machinery and their potential role in anticancer drug resistance. Oncogene 2003; 22: 7414–7430. 6. Hu W, Kavanagh JJ. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol 2003; 4: 721 –729. 7. Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNFfamily death receptors. Oncogene 2003; 22: 8628– 8633. 8. Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 2003; 22: 8590–8607.
Downloaded from http://annonc.oxfordjournals.org/ by guest on August 19, 2015
How the apoptotic machinery can be targeted for therapeutic benefits using inhibitors of apoptosis protein (IAP inhibitors, anti Bcl2), direct inducers of apoptosis (TRAIL agonists) and modulators of apoptosis through the targeting of protein kinases, phosphatases and transcriptional factors will be discussed. IAP genes constitute a highly conserved family coding for proteins, including XIAP, c-IAP1, c-IPA2 and survivin, that directly bind and inhibit caspases. IAP overexpression has been described in cancer cells and in primary tumour samples. The potential advantages of IAPs as drug targets are represented by the fact that they operate at a downstream point in the apoptosis pathways (after the activation of caspases), potentially bypassing the many upstream defects in apoptosisregulatory mechanisms in tumours. The discovery that the peptidyl motifs in their protein sequence are necessary to overcome apoptosis suppression will lead to the synthesis and production of nonpeptidyl small molecule antagonists to be used in the treatment of tumours. Recently, small molecule inhibitors of XIAP have indeed been shown to block the XIAP-induced inhibition on caspase-3 and to be effective in inducing apoptosis in cancer cells with limited toxicity against normal cells [11]. Interestingly, these XIAP antagonists have also been shown to suppress the in vivo growth of tumour xenograft models as single agents and to sensitise cancer cells to chemotherapy and TRAIL treatment in vitro. Antisense therapy aimed at decreasing the expression of different antiapoptotic genes (Bcl2, XIAP) is something that has been pursued and evaluated at the preclinical and clinical level [12]. An example of this class of products is G3139, an antisense phosphorothioate oligodeoxynucleotide suppressing bcl-2 expression, which is now in phase III clinical trials. Indeed, preclinical studies had shown the ability of G3139 to increase the antitumour activity of different cytotoxic agents [13]. The possibility of killing cancer cells by binding to death receptor has been investigated for a long time. Indeed, TNF-a and FASL are effective inducers of apoptosis, but they have very severe side-effects, precluding their systemic use [14]. In contrast, the family of TRAIL proteins and receptors has attracted many laboratories, based on the observation that TRAIL preferentially induce apoptosis in cancer cells compared with normal cells. The presence of decoy receptors for TRAIL, as well as the role of intracellular proteins such as FLIP, relevant for signal transduction, account for the specificity of TRAIL in preferentially inducing apoptosis in cancer cells [15]. Administration of soluble recombinant TRAIL has been shown in animal experiments to induce significant tumour regression with no systemic toxicity [16]. Current approaches for the development of TRAIL-R1 and/or TRAIL-R2 agonists
as anticancer agents include antibody targeting of the receptor(s) and the use of soluble forms of TRAIL. Experimental in vitro and in vivo data would suggest that the combined treatment of recombinant TRAIL and chemotherapeutic agents exert an antitumour activity superior to that found when the two regimens are given separately. Both p53 and nuclear factor (NF)-kB transcriptional factors can modulate the apoptotic process [17, 18]. Activated p53, which generally occurs after cellular damage, can positively regulate apoptotic-related genes, including Bax, and has also been shown to trigger the expression of death receptors. As most human tumours have mutations in the p53 gene, restoration of p53 function by the delivery of its gene in tumour cells has been shown to suppress the growth of human xenografts through different effects, including an increased susceptibility to undergoing apoptosis. Even though the possibility to use such gene therapy-based approaches in humans needs additional improvements in their delivery systems, clinical trials exploring adenovirus-mediated transfer of wild-type p53 in human cancer have been initiated [19]. NF-kB has been demonstrated to be able to regulate the transcription of different anti-apoptotic genes, including c-IAPs, Bcl2 and BclX, and elevated levels have been described in several types of human malignancies, making it an attractive target. NF-kB is kept inactive through the binding with different inhibitory molecules (Ika, Ikb, p105 and p100) that sequester NF-kB in the cytoplasm. Its activation involves the degradation of its inhibitory molecules with subsequent translocation to the nucleus, where it can induce transcription. The proteasome inhibitor Velcade (PS341), the first compound of this class investigated in clinical trails, has been shown to inhibit NF-kB activity through the degradation of Ikb [20]. A better knowledge of the cellular mechanisms and the regulation of apoptosis induced by the extrinsic and intrinsic stimuli including anticancer agents will help in an individualisation of the chemotherapy regimens to be administered.
iv231 9. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770–776. 10. De Feudis P, Vignati S, Rossi C et al. Driving p53 response to Bax activation greatly enhances sensitivity to taxol by inducing massive apoptosis. Neoplasia 2000; 2: 202–207. 11. Schimmer AD, Welsh K, Pinilla C et al. Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity. Cancer Cell 2004; 5: 25– 35. 12. Nahta R, Esteva FJ. Bcl-2 antisense oligonucleotides: a potential novel strategy for the treatment of breast cancer. Semin Oncol 2003; 30: 143– 149. 13. Leung S, Miyake H, Zellweger T et al. Synergistic chemosensitization and inhibition of progression to androgen independence by antisense Bcl-2 oligodeoxynucleotide and paclitaxel in the LNCaP prostate tumor model. Int J Cancer 2001; 91: 846–850. 14. Walczak H, Miller RE, Ariail K et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157–163.
15. Zhang XD, Nguyen T, Thomas WD et al. Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types. FEBS Lett 2000; 482: 193 –199. 16. Nakamoto T, Inagawa H, Takagi K, Soma G. A new method of antitumor therapy with a high dose of TNF perfusion for unresectable liver tumors. Anticancer Res 2000; 20: 4087–4096. 17. Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene 2003; 22: 9030–9040. 18. Kucharczak J, Simmons MJ, Fan Y, Gelinas C. To be, or not to be: NF-kappaB is the answer—role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene 2003; 22: 8961–8982. 19. Ahn WS, Bae SM, Lee KH et al. Recombinant adenovirus-p53 gene transfer and cell-specific growth suppression of human cervical cancer cells in vitro and in vivo. Gynecol Oncol 2004; 92: 611– 621. 20. Yang HH, Ma MH, Vescio RA, Berenson JR. Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis. J Clin Oncol 2003; 21: 4239–4247.
Downloaded from http://annonc.oxfordjournals.org/ by guest on August 19, 2015
Molecular targeting: new therapeutic strategies to improve tumour apoptosis S. Marsoni & G. Damia SENDO Foundation, Milan, Italy
Introduction
Major apoptotic pathways Many different mechanisms of apoptosis have been discovered, but schematically there are two major pathways [6]. The intrinsic (mitochondrial –apoptosome) pathway is triggered by chemical or physical stress signals arising within the cell, while the extrinsic or death receptor-induced pathway is initiated by the binding of extracellular molecules, the death activators [tumour necrosis factor (TNF)-a, lymphotoxin, Fas ligand (FASL)] to specific death receptors at the cell surface. Ultimately, both pathways are dependent upon a family of cysteine proteases, the caspases, which are generally kept inactive in the cytosol by being maintained as zymogen, and can cleave several hundred cellular substrates. Caspases can be classified as ‘executioners’ (caspases-3, -6 and -7) and ‘initiators’ (caspases-8 and -9). The former can attack their q 2004 European Society for Medical Oncology
Downloaded from http://annonc.oxfordjournals.org/ by guest on August 19, 2015
There are two ways for cells to die: death by injury and death by suicide. The pattern of events in death by suicide is called apoptosis, the ancient Greek term for the falling of leaves, and is a genetically regulated mechanism of programmed cell death [1]. Why should a cell commit suicide? Programmed cell death is needed whenever tissue modelling is required, during embryogenesis, for the removal of old cells and to prevent overgrowth after repair of cell loss by injury [2]. Programmed cell death is also needed to destroy cells that represent a threat to the integrity of the organism, such as virus-infected cells, effectors cells of the immune system that are no longer necessary and cells with DNA damage that can become cancerous. The mechanism of apoptosis is switched on by the imbalance between proapoptotic (death signals) and antiapoptotic (survival signals) factors. This happens in both normal and cancer cells. Dysregulation of apoptosis occurs in cancer cells and has not only been implicated in tumour progression and pathogenesis, but also plays an important role in response to therapy [3– 5]. In fact, cancer treatments, radiation and chemotherapeutic agents can destroy tumours by triggering cancer cell apoptosis. On the other hand, cancer cells can develop numerous mechanisms to evade apoptosis through either inactivation of proapoptotic or up-regulation of antiapoptotic factors.
cellular substrates if their zymogens are proteolytically cleaved by the latter. The death receptor pathway, through the binding between ligands and their receptors (such as Fas, TNF-a and TRAIL) activates caspase-8 and caspase-10 via the adaptor protein Fas-associated death domain (FADD) [7]. The death receptor family includes the receptors for TNF-a, FASL and Apo2L/ TRAIL. These receptors are characterised by an 80 amino acid death domain in their cytoplasmic tail that, when activated by agonistic antibodies or their cognate ligands, recruits signalling components that initiate apoptosis. In some cell types, activation of caspase-8 is sufficient to trigger apoptosis, while in other cell types amplification of the extrinsic pathway through the intrinsic pathway is necessary to commit cells to apoptosis. In fact, the cleavage of Bid by caspase-8 and the translocation of the cleaved Bid to mitochondria have been shown to be the link between the two pathways. Yet to be defined are the molecular mechanisms that regulate and control the expression of death receptors, their ligands and the cytoplasmic adapter molecules both in normal and tumour cells. The intrinsic pathways is induced by different kinds of stresses, and caspase-9 is typically activated by the scaffold protein Apaf-1, which undergoes a conformational change induced by cytochrome c released by the damaged mitochondria. Such conformational change allows the recruitment of pro-caspase-9 and its activation. Bcl2 family proteins have a central role in the control of mitochondria-induced apoptosis [8]. More than 20 proteins have been identified, including proteins that suppress apoptosis (Bcl2, BclXl, MCL1) and proteins that suppress apoptosis (Bax, Bad, Bid, Bik). It has been proposed that the Bcl2 members form channels that facilitate protein transport and interaction with other mitochondrial proteins, and also induce rupture of the outer mitochondrial membrane [9]. While the Bcl2 family proteins regulate the mitochondrial pathway before caspase activation, the inhibitors of apoptosis (IAP) proteins regulate apoptosis after caspase activation. The unravelling of the molecular mechanisms at the base of apoptosis and the better understanding of the apoptosis mechanisms relevant for tumour development and for tumour chemo- and radiosensitivity have brought up new strategies for restoring apoptosis sensitivity in tumour cells. Indeed, in solid tumours, the low propensity to undergo apoptosis has
iv230 been associated with resistance to treatment. Experimental evidence has suggested that it is theoretically possible to drive cells to apoptosis (rather than cell cycle arrest) with a dramatic increase in the sensitivity of anticancer agents [10].
Targeting apoptosis
References 1. Adams JM. Ways of dying: multiple pathways to apoptosis. Genes Dev 2003; 17: 2481–2495. 2. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 1972; 26: 239–257. 3. Townson JL, Naumov GN, Chambers AF. The role of apoptosis in tumor progression and metastasis. Curr Mol Med 2003; 3: 631– 642. 4. Stenner-Liewen F, Reed JC. Apoptosis and cancer: basic mechanisms and therapeutic opportunities in the postgenomic era. Cancer Res 2003; 63: 263– 268. 5. Kaufmann SH, Vaux DL. Alterations in the apoptotic machinery and their potential role in anticancer drug resistance. Oncogene 2003; 22: 7414–7430. 6. Hu W, Kavanagh JJ. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol 2003; 4: 721 –729. 7. Wang S, El-Deiry WS. TRAIL and apoptosis induction by TNFfamily death receptors. Oncogene 2003; 22: 8628– 8633. 8. Cory S, Huang DC, Adams JM. The Bcl-2 family: roles in cell survival and oncogenesis. Oncogene 2003; 22: 8590–8607.
Downloaded from http://annonc.oxfordjournals.org/ by guest on August 19, 2015
How the apoptotic machinery can be targeted for therapeutic benefits using inhibitors of apoptosis protein (IAP inhibitors, anti Bcl2), direct inducers of apoptosis (TRAIL agonists) and modulators of apoptosis through the targeting of protein kinases, phosphatases and transcriptional factors will be discussed. IAP genes constitute a highly conserved family coding for proteins, including XIAP, c-IAP1, c-IPA2 and survivin, that directly bind and inhibit caspases. IAP overexpression has been described in cancer cells and in primary tumour samples. The potential advantages of IAPs as drug targets are represented by the fact that they operate at a downstream point in the apoptosis pathways (after the activation of caspases), potentially bypassing the many upstream defects in apoptosisregulatory mechanisms in tumours. The discovery that the peptidyl motifs in their protein sequence are necessary to overcome apoptosis suppression will lead to the synthesis and production of nonpeptidyl small molecule antagonists to be used in the treatment of tumours. Recently, small molecule inhibitors of XIAP have indeed been shown to block the XIAP-induced inhibition on caspase-3 and to be effective in inducing apoptosis in cancer cells with limited toxicity against normal cells [11]. Interestingly, these XIAP antagonists have also been shown to suppress the in vivo growth of tumour xenograft models as single agents and to sensitise cancer cells to chemotherapy and TRAIL treatment in vitro. Antisense therapy aimed at decreasing the expression of different antiapoptotic genes (Bcl2, XIAP) is something that has been pursued and evaluated at the preclinical and clinical level [12]. An example of this class of products is G3139, an antisense phosphorothioate oligodeoxynucleotide suppressing bcl-2 expression, which is now in phase III clinical trials. Indeed, preclinical studies had shown the ability of G3139 to increase the antitumour activity of different cytotoxic agents [13]. The possibility of killing cancer cells by binding to death receptor has been investigated for a long time. Indeed, TNF-a and FASL are effective inducers of apoptosis, but they have very severe side-effects, precluding their systemic use [14]. In contrast, the family of TRAIL proteins and receptors has attracted many laboratories, based on the observation that TRAIL preferentially induce apoptosis in cancer cells compared with normal cells. The presence of decoy receptors for TRAIL, as well as the role of intracellular proteins such as FLIP, relevant for signal transduction, account for the specificity of TRAIL in preferentially inducing apoptosis in cancer cells [15]. Administration of soluble recombinant TRAIL has been shown in animal experiments to induce significant tumour regression with no systemic toxicity [16]. Current approaches for the development of TRAIL-R1 and/or TRAIL-R2 agonists
as anticancer agents include antibody targeting of the receptor(s) and the use of soluble forms of TRAIL. Experimental in vitro and in vivo data would suggest that the combined treatment of recombinant TRAIL and chemotherapeutic agents exert an antitumour activity superior to that found when the two regimens are given separately. Both p53 and nuclear factor (NF)-kB transcriptional factors can modulate the apoptotic process [17, 18]. Activated p53, which generally occurs after cellular damage, can positively regulate apoptotic-related genes, including Bax, and has also been shown to trigger the expression of death receptors. As most human tumours have mutations in the p53 gene, restoration of p53 function by the delivery of its gene in tumour cells has been shown to suppress the growth of human xenografts through different effects, including an increased susceptibility to undergoing apoptosis. Even though the possibility to use such gene therapy-based approaches in humans needs additional improvements in their delivery systems, clinical trials exploring adenovirus-mediated transfer of wild-type p53 in human cancer have been initiated [19]. NF-kB has been demonstrated to be able to regulate the transcription of different anti-apoptotic genes, including c-IAPs, Bcl2 and BclX, and elevated levels have been described in several types of human malignancies, making it an attractive target. NF-kB is kept inactive through the binding with different inhibitory molecules (Ika, Ikb, p105 and p100) that sequester NF-kB in the cytoplasm. Its activation involves the degradation of its inhibitory molecules with subsequent translocation to the nucleus, where it can induce transcription. The proteasome inhibitor Velcade (PS341), the first compound of this class investigated in clinical trails, has been shown to inhibit NF-kB activity through the degradation of Ikb [20]. A better knowledge of the cellular mechanisms and the regulation of apoptosis induced by the extrinsic and intrinsic stimuli including anticancer agents will help in an individualisation of the chemotherapy regimens to be administered.
iv231 9. Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407: 770–776. 10. De Feudis P, Vignati S, Rossi C et al. Driving p53 response to Bax activation greatly enhances sensitivity to taxol by inducing massive apoptosis. Neoplasia 2000; 2: 202–207. 11. Schimmer AD, Welsh K, Pinilla C et al. Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity. Cancer Cell 2004; 5: 25– 35. 12. Nahta R, Esteva FJ. Bcl-2 antisense oligonucleotides: a potential novel strategy for the treatment of breast cancer. Semin Oncol 2003; 30: 143– 149. 13. Leung S, Miyake H, Zellweger T et al. Synergistic chemosensitization and inhibition of progression to androgen independence by antisense Bcl-2 oligodeoxynucleotide and paclitaxel in the LNCaP prostate tumor model. Int J Cancer 2001; 91: 846–850. 14. Walczak H, Miller RE, Ariail K et al. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 1999; 5: 157–163.
15. Zhang XD, Nguyen T, Thomas WD et al. Mechanisms of resistance of normal cells to TRAIL induced apoptosis vary between different cell types. FEBS Lett 2000; 482: 193 –199. 16. Nakamoto T, Inagawa H, Takagi K, Soma G. A new method of antitumor therapy with a high dose of TNF perfusion for unresectable liver tumors. Anticancer Res 2000; 20: 4087–4096. 17. Fridman JS, Lowe SW. Control of apoptosis by p53. Oncogene 2003; 22: 9030–9040. 18. Kucharczak J, Simmons MJ, Fan Y, Gelinas C. To be, or not to be: NF-kappaB is the answer—role of Rel/NF-kappaB in the regulation of apoptosis. Oncogene 2003; 22: 8961–8982. 19. Ahn WS, Bae SM, Lee KH et al. Recombinant adenovirus-p53 gene transfer and cell-specific growth suppression of human cervical cancer cells in vitro and in vivo. Gynecol Oncol 2004; 92: 611– 621. 20. Yang HH, Ma MH, Vescio RA, Berenson JR. Overcoming drug resistance in multiple myeloma: the emergence of therapeutic approaches to induce apoptosis. J Clin Oncol 2003; 21: 4239–4247.
Downloaded from http://annonc.oxfordjournals.org/ by guest on August 19, 2015