Translational control in stress and apoptosis

Nature Reviews Molecular Cell Biology

Volumn 6, Issue 4, 2005, Pages 318-327

  Holcik, Martin ^a   Sonenberg, Nahum ^b  

^a CHILDREN S HOSPITAL OF EASTERN ONTARIO   (Canada)

^b MCGILL UNIVERSITY   (Canada)

Author keywords

[No Author keywords available]

Indexed keywords

INITIATION FACTOR 2ALPHA;

APOPTOSIS; CELL SURVIVAL; EUKARYOTE; GENE EXPRESSION; INTERNAL RIBOSOME ENTRY SITE; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; REVIEW; STRESS; TRANSLATION INITIATION; TRANSLATION REGULATION;

ANIMALS; APOPTOSIS; APOPTOTIC PROTEASE-ACTIVATING FACTOR 1; DNA-BINDING PROTEINS; EUKARYOTIC INITIATION FACTOR-2; HUMANS; PHOSPHORYLATION; PROTEIN BIOSYNTHESIS; PROTEIN KINASES; PROTEINS; RIBOSOMES; RNA, MESSENGER; SACCHAROMYCES CEREVISIAE PROTEINS; STRESS; X-LINKED INHIBITOR OF APOPTOSIS PROTEIN;

EUKARYOTA;

EID: 17144424622     PISSN: 14710072     EISSN: None     Source Type: Journal    
DOI: 10.1038/nrm1618     Document Type: Review

References (111)

1. Nishizuka, S. et al. Proteomic profiling of the NCI-60 cancer cell lines using new high-density reverse-phase lysate microarrays. Proc. Natl Acad. Sci. USA 100, 14229-14234 (2003).
2. Gygi, S. P., Rochon, Y., Franza, B. R. & Aebersold, R. Correlation between protein and mRNA abundance in yeast. Mol. Cell. Biol. 19, 1720-1730 (1999).
3. Rajasekhar, V. K. et al. Oncogenic Ras and Akt signaling contribute to glioblastoma formation by differential recruitment of existing mRNAs to polysomes. Mol. Cell 12, 889-901 (2003).
4. Rajasekhar, V. K. & Holland, E. C. Postgenomic global analysis of translational control induced by oncogenic signaling. Oncogene 23, 3248-3264 (2004).
5. Celis, J. E. et al. Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics. FEBS Lett. 480, 2-16 (2000).
6. Le Naour, F. et al. Profiling changes in gene expression during differentiation and maturation of monocyte-derived dendritic cells using both oligonucleotide microarrays and proteomics. J. Biol. Chem. 276, 17920-17931 (2001).
7. Hanash, S. M. et al. Integrating cancer genomics and proteomics in the post-genome era. Proteomics 2, 69-75 (2002).
8. Ideker, T. et al. Integrated genomic and proteomic analyses of a systematically perturbed metabolic network. Science 292, 929-934 (2001).
9. Mathews, M. B., Sonenberg, N. & Hershey, J. W. B. in Translational Control of Gene Expression (eds Sonenberg, N., Hershey, J. W. B. & Mathews, M. B.) 1-32 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, 2000).
10. Hershey, J. W. B. & Merrick, W. C. in Translation Control of Gene Expression (eds Sonenberg, N., Hershey, J. W. B. & Mathews, M. B.) 33-88 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, 2000).
11. Gebauer, F. & Hentze, M. W. Molecular mechanisms of translational control. Nature Rev. Mol. Cell Biol. 5, 827-835 (2004).
12. Johannes, G., Carter, M. S., Eisen, M. B., Brown, P. O. & Sarnow, P. Identification of eukaryotic mRNAs that are translated at reduced cap binding complex elF4F concentrations using a cDNA microarray. Proc. Natl Acad. Sci. USA 96, 13118-13123 (1999). This was the first work to indicate that cap-independent translation was more common than had been previously assumed.
13. Hellen, C. U. & Sarnow, P. Internal ribosome entry sites in eukaryotic mRNA molecules. Genes Dev. 15, 1593-1612 (2001).
14. Johannes, G. & Sarnow, P. Cap-independent polysomal association of natural mRNAs encoding c-myc, BiP, and elF4G conferred by internal ribosome entry sites. RNA 4, 1500-1513 (1998).
15. Pelletier, J. & Sonenberg, N. Internal initiation of translation of eukaryotic mRNA directed by a sequence derived from poliovirus RNA. Nature 334, 320-325 (1988).
16. Jang, S. K. et al. A segment of the 5′ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J. Virol. 62, 2636-2643 (1988). References 15 and 16 presented the first evidence of an IRES in a eukaryotic mRNA.
17. Verge, V., Vonlanthen, M., Masson, J. M., Trachsel, H. & Altmann, M. Localization of a promoter in the putative internal ribosome entry site of the Saccharomyces cerevisiae TIF4631 gene. RNA 10, 277-286 (2004).
18. Man, B. & Zhang, J. T. Regulation of gene expression by internal ribosome entry sites or cryptic promoters: the elF4G story. Mol. Cell. Biol. 22, 7372-7384 (2002).
19. Van Eden, M. E., Byrd, M. P., Sherrill, K. W. & Lloyd, R. E. Demonstrating internal ribosome entry sites in eukaryotic mRNAs using stringent RNA test procedures. RNA 10, 720-730 (2004).
20. Hennecke, M. et al. Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs. Nucleic Acids Res. 29, 3327-3334 (2001).
21. Merrick, W. C. Cap-dependent and cap-independent translation in eukaryotic systems. Gene 332, 1-11 (2004).
22. Holcik, M., Sonenberg, N. & Korneluk, R. G. Internal ribosome initiation of translation and the control of cell death. Trends Genet. 16, 469-473 (2000).
23. Stoneley, M. & Willis, A. E. Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression. Oncogene 23, 3200-3207 (2004).
24. Martinez-Salas, E., Ramos, R., Lafuente, E. & Lopez De Quinto, S. Functional interactions in internal translation initiation directed by viral and cellular IRES elements. J. Gen. Virol. 82, 973-984 (2001).
25. Mitchell, S. A., Brown, E. C., Coldwell, M. J., Jackson, R. J. & Willis, A. E. Protein factor requirements of the Apaf-1 internal ribosome entry segment: rotes of polypyrimidine tract binding protein and upstream of N-ras. Mol. Cell. Biol. 21, 3364-3374 (2001).
26. Holcik, M. & Komeluk, R. G. Functional characterization of the X-linked inhibitor of apoptosis (XIAP) internal ribosome entry site element: role of La autoantigen in XIAP translation. Mol. Cell. Biol. 20, 4648-4657 (2000).
27. Kim, Y. K., Back, S. H., Rho, J., Lee, S. H. & Jang, S. K. La autoantigen enhances translation of BiP mRNA. Nucleic Acids Res. 29, 5009-5016 (2001).
28. Holcik, M., Gordon, B. W. & Komeluk, R. G. The internal ribosome entry site-mediated translation of antiapoptotic protein XIAP is modulated by the heterogeneous nuclear ribonucleoproteins C1 and C2. Mol. Cell. Biol. 23, 280-288 (2003).
29. Sella, O., Gerlitz, G., Le, S. Y. & Elroy-Stein, O. Differentiation-induced internal translation of c-sis mRNA: analysis of the as elements and their differentiation-linked binding to the hnRNP C protein. Mol. Cell. Biol. 19, 5429-5440 (1999).
30. Nevins, T. A., Harder, Z. M., Korneluk, R. G. & Holcik, M. Distinct regulation of internal ribosome entry site-mediated translation following cellular stress is mediated by apoptotic fragments of elF4G translation initiation factor family members elF4Gl and p97/DAP5/NAT1. J. Biol. Chem. 278, 3572-3579 (2003).
31. Henis-Korenblit, S. et al. The caspase-cleaved DAP5 protein supports internal ribosome entry site-mediated translation of death proteins. Proc. Natl Acad. Sci. USA 99, 5400-5405 (2002). References 30 and 31 presented the first evidence that p97/DAP5 could function as an apoptosis-specific translation-initiation factor.
32. Henis-Korenblit, S., Strumpf, N. L., Goldstaub, D. & Kimchi, A. A novel form of DAP5 protein accumulates in apoptotic cells as a result of caspase cleavage and internal ribosome entry site-mediated translation. Mol. Cell. Biol. 20, 495-506 (2000).
33. Kullmann, M., Gopfert, U., Siewe, B. & Hengst, L. ELAV/Hu proteins inhibit p27 translation via an IRES element in the p27 5′UTR. Genes Dev. 16, 3087-3099 (2002).
34. Wolin, S. L. & Cedervall, T. The La protein. Annu. Rev. Biochem. 71, 375-403 (2002).
35. Creancier, L., Mercier, P., Prats, A. C. & Morello, D. c-myc internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice. Mol. Cell. Biol. 21, 1833-1840 (2001).
36. Fernandez, J., Yaman, I., Sarnow, P., Snider, M. D. & Hatzogiou, M. Regulation of internal ribosomal entry site-mediated translation by phosphorylation of the translation initiation factor elF2α. J. Biol. Chem. 277, 19198-19205 (2002).
37. Gerlitz, G., Jagus, R. & Elroy-Stein, O. Phosphorylation of initiation factor-2α is required for activation of internal translation initiation during cell differentiation. Eur. J. Biochem. 269, 2810-2819 (2002).
38. Warnakulasuriyarachchi, D., Cerquozzi, S., Cheung, H. H. & Holcik, M. Translational induction of the inhibitor of apoptosis protein HIAP2 during endoplasmic reticulum stress attenuates cell death and is mediated via an inducible internal ribosome entry site element. J. Biol. Chem. 279, 17148-17157 (2004).
39. Pearce, A. K. & Humphrey, T. C. Integrating stress-response and cell-cycle checkpoint pathways. Trends Cell Biol. 11, 426-433 (2001).
40. Warner, J. R. The economics of ribosome biosynthesis in yeast. Trends Biochem. Sci. 24, 437-440 (1999).
41. Rudra, D. & Warner, J. R. What better measure than ribosome synthesis? Genes Dev. 18, 2431-2436 (2004).
42. Holcik, M., Yeh, C., Korneluk, R. G. & Chow, T. Translational upregulation of X-linked inhibitor of apoptosis (XIAP) increases resistance to radiation induced cell death. Oncogene 19, 4174-4177 (2000).
43. Harding, H. P., Zhang, Y., Bertolotti, A., Zeng, H. & Ron, D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 5, 897-904 (2000).
44. Hinnebusch, A. G. in Translation Control of Gene Expression (eds Sonenberg, N., Hershey, J. W. B. & Mathews, M. B.) 185-243 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, 2000). This comprehensive review summarizes the literature on the translational activation of GCN4 mRNA by the GCN2 kinase.
45. Dever, T. E. et al. Phosphorylation of initiation factor 2α by protein kinase GCN2 mediates gene-specific translational control of GCN4 in yeast. Cell 68, 585-596 (1992).
46. Clemens, M. J. Targets and mechanisms for the regulation of translation in malignant transformation. Oncogene 23, 3180-3188 (2004).
47. Chen, J. J. in Translation Control of Gene Expression (eds Sonenberg, N., Hershey, J. W. B. & Mathews, M. B.) 529-546 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, 2000).
48. Kimball, S. R. Regulation of translation initiation by amino acids in eukaryotic cells. Prog. Mol. Subcell. Biol. 26, 155-184 (2001).
49. Deng, J. et al. Activation of GCN2 in UV-irradiated cells inhibits translation. Curr Biol. 12, 1279-1286 (2002).
50. Jiang, H. Y. & Wek, R. C. Gcn2 phosphorylation of elF2α activates NF-κB in response to UV irradiation. Biochem. J. 385, 371-380 (2005).
51. Kaufman, R. J. in Translation Control of Gene Expression (eds Sonenberg, N., Hershey, J. W. B. & Mathews, M. B.) 503-527 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, New York, 2000).
52. Ron, D. Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 110, 1383-1388 (2002).
53. Hinnebusch, A. G. Evidence for translational regulation of the activator of general amino acid control in yeast. Proc. Natl Acad. Sci. USA 81, 6442-6446 (1984).
54. Mueller, P. P. & Hinnebusch, A. G. Multiple upstream AUG codons mediate translational control of GCN4. Cell 45, 201-207 (1986). This report was the first to describe both positive and negative roles of upstream AUG codons in translational upregulation.
55. Kaufman, R. J. Regulation of mRNA translation by protein folding in the endoplasmic reticulum. Trends Biochem. Sci. 29, 152-158 (2004).
56. Danial, N. N. & Korsmeyer, S. J. Cell death: critical control points. Cell 116, 205-219 (2004).
57. Nakagawa, T. et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-β. Nature 403, 98-103 (2000).
58. Morishima, N., Nakanishi, K., Takenouchi, H., Shibata, T. & Yasuhiko, Y. An endoplasmic reticulum stress-specific caspase cascade in apoptosis. Cytochrome c-independent activation of caspase-9 by caspase-12. J. Biol. Chem. 277, 34287-34294 (2002).
59. Saleh, M. et al. Differential modulation of endotoxin responsiveness by human caspase-12 polymorphisms. Nature 429, 75-79 (2004).
60. Harding, H. P., Zhang, Y. & Ron, D. Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase. Nature 397, 271-274 (1999). This was the first description of PERK and its activation by the UPH.
61. Bertolotti, A., Zhang, Y., Hendershot, L. M., Harding, H. P. & Ron, D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nature Cell Biol. 2, 326-332 (2000).
62. Harding, H. P. et al. Regulated translation initiation controls stress-induced gene expression in mammalian cells. Mol. Cell 6, 1099-1108 (2000).
63. Harding, H. P. et al. An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. Mol. Cell 11, 619-633 (2003).
64. Vattem, K. M. & Wek, R. C. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc. Natl Acad. Sci. USA 101, 11269-11274 (2004).
65. Lu, P. D., Harding, H. P. & Ron, D. Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response. J. Cell Biol. 167, 27-33 (2004).
66. Scheuner, D. et al. Translational control is required for the unfolded protein response and in vivo glucose homeostasis. Mol. Cell 7, 1165-1176 (2001).
67. -/- mice reveals a role for translational control in secretory cell survival. Mol. Cell 7, 1153-1163 (2001). References 66 and 67 presented the first evidence of the role of PERK in the UPR and glucose metabolism in vivo.
68. Zhang, P. et al. The PERK eukaryotic initiation factor 2α kinase is required for the development of the skeletal system, postnatal growth, and the function and viability of the pancreas. Mol. Cell. Biol. 22, 3864-3874 (2002).
69. Kaufman, R. J. et al. The unfolded protein response in nutrient sensing and differentiation. Nature Rev. Mol. Cell Biol. 3, 411-421 (2002).
70. Delepine, M. et al. EIF2AK3, encoding translation initiation factor 2-α kinase 3, is mutated in patients with Wolcott-Rallison syndrome. Nature Genet. 25, 406-409 (2000).
71. Nicholson, D. W. Caspase structure, proteolytic substrates, and function during apoptotic cell death. Cell Death Differ. 6, 1028-1042 (1999).
72. Clemens, M. J.. Bushell, M., Jeffrey, I. W., Pain, V. M. & Morley, S. J. Translation initiation factor modifications and the regulation of protein synthesis in apoptotic cells. Cell Death Differ. 7, 603-615 (2000).
73. Coldwell, M. J., Mitchell, S. A., Stoneley, M., MacFarlane, M. & Willis, A. E. Initiation of Apaf-1 translation by internal ribosome entry. Oncogene 19, 899-905 (2000).
74. Holcik, M., Lefebvre, C. A., Yeh, C., Chow, T. & Korneluk, R. G. A new internal-ribosome-entry-site motif potentiates XIAP-mediated cytoprotection. Nature Cell Biol. 1, 190-192 (1999).
75. Adams, J. M. & Cory, S. Apoptosomes: engines for caspase activation. Curr. Opin. Cell Biol. 14, 715-720 (2002).
76. Liston, P., Fong, W. G. & Korneluk, R. G. The inhibitors of apoptosis: there is more to life than Bcl2. Oncogene 22, 8568-8580 (2003).
77. Budihardjo, I., Oliver, H., Lutter, M., Luo, X. & Wang, X. Biochemical pathways of caspase activation during apoptosis. Annu. Rev. Cell Dev. Biol. 15, 269-290 (1999).
78. Zou, H., Henzel, W. J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90, 405-413 (1997).
79. Rodriguez, J. & Lazebnik, Y. Caspase-9 and APAF-1 form an active holoenzyme. Genes Dev. 13, 3179-3184 (1999).
80. Cain, K. et al. Apaf-1 oligomerizes into biologically active approximately 700-kDa and inactive approximately 1.4-MDa apoptosome complexes. J. Biol. Chem. 275, 6067-6070 (2000).
81. Holcik, M. & Korneluk, R. G. XIAR, the guardian angel. Nature Rev. Mol. Cell Biol. 2, 550-556 (2001).
82. Yamagiwa, Y., Marienfeld, C., Meng, F., Holcik, M. & Patel, T. Translational regulation of X-linked inhibitor of apoptosis protein by interleukin-6: a novel mechanism of tumor cell survival. Cancer Res. 64, 1293-1298 (2004).
83. Pardo, O. E. et al. Fibroblast growth factor 2-mediated translational control of IAPs blocks mitochondrial release of Smac/DIABLO and apoptosis in small cell lung cancer cells. Mol. Cell. Biol. 23, 7600-7610 (2003).
84. Brockstedt, E. et al. Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J. Biol. Chem. 273, 28057-28064 (1998).
85. Rutjes, S. A. et al. The La (SS-B) autoantigen, a key protein in RNA biogenesis, is dephosphorylated and cleaved early during apoptosis. Cell Death Differ. 6, 976-986 (1999).
86. Schwartz, E. I., Intine, R. V. & Maraia, R. J. CK2 is responsible for phosphorylation of human La protein serine-366 and can modulate rpL37 5′-terminal oligopyrimidine mRNA metabolism. Mol. Cell. Biol. 24, 9580-9591 (2004).
87. Marienfeld, C. et al. Translational regulation of XIAP expression and cell survival during hypoxia in human cholangiocarcinoma. Gastroenterology 127, 1787-1797 (2004).
88. Mitchell, S. A., Spriggs, K. A., Coldwell, M. J., Jackson, R. J. & Willis, A. E. The Apaf-1 internal ribosome entry segment attains the correct structural conformation for function via interactions with PTB and unr. Mol. Cell 11, 757-771 (2003).
89. Lassus, P., Opitz-Araya, X. & Lazebnik, Y. Requirement for caspase-2 in stress-induced apoptosis before mitochondrial permeabilization. Science 297, 1352-1354 (2002).
90. Gingras, A. C., Raught, B. & Sonenberg, N. elF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation, Annu. Rev. Biochem. 68, 913-963 (1999).
91. Clemens, M. J., Bushell, M. & Morley, S. J. Degradation of eukaryotic polypeptide chain initiation factor (elF) 4G in response to induction of apoptosis in human lymphoma cell lines. Oncogene 17, 2921-2931 (1998).
92. Ohlmann, T., Rau, M., Pain, V. M. & Morley, S. J. The C-terminal domain of eukaryotic protein synthesis initiation factor (elF) 4G is sufficient to support cap-independent translation in the absence of elF4E. EMBO J. 15, 1371-1382 (1996).
93. Borman, A. M. et al. elF4G and its proteolytic cleavage products: effect on initiation of protein synthesis from capped, uncapped, and IRES-containing mRNAs. RNA 3, 186-196 (1997).
94. Nanbru, C. et al. Alternative translation of the proto-oncogene c-myc by an internal ribosome entry site. J. Biol. Chem. 272, 32061-32066 (1997).
95. Stoneley, M., Paulin, F. E., Le Quesne, J. P., Chappell, S. A. & Willis, A. E. c-Myc 5′ untranslated region contains an internal ribosome entry segment. Oncogens 16, 423-428 (1998).
96. Stoneley, M. et al. c-Myc protein synthesis is initiated from the internal ribosome entry segment during apoptosis. Mol. Cell. Biol. 20, 1162-1169 (2000).
97. Subkhankulova, T., Mitchell, S. A. & Willis, A. E. Internal ribosome entry segment-mediated initiation of c-Myc protein synthesis following genotoxic stress. Biochem. J. 359, 183-192 (2001).
98. Warnakulasuriyarachchi, D., Ungureanu, N. H. & Holcik, M. The translation of an antiapoptotic protein HIAP2 is regulated by an upstream open reading frame. Cell Death Differ. 10, 899-904 (2003).
99. Semenza, G. L. HIF-1 and human disease: one highly involved factor. Genes Dev. 14, 1983-1991 (2000).
100. Koumenis, C. et al. Regulation of protein synthesis by hypoxia via activation of the endoplasmic reticulum kinase PERK and phosphorylation of the translation initiation factor elF2α. Mol. Cell. Biol. 22, 7405-7416 (2002).
101. Blais, J. D. et al. Activating transcription factor 4 is translationally regulated by hypoxic stress. Mol. Cell. Biol. 24, 7469-7482 (2004).
102. Tinton, S. A. & Buc-Calderon, P. M. Hypoxia increases the association of 4E-binding protein 1 with the initiation factor 4E in isolated rat hepatocytes. FEBS Lett. 446, 55-59 (1999).
103. Lang, K. J. D., Kappel, A. & Goodall, G. J. Hypoxia-inducible factor-1α MRNA contains an internal ribosome entry site that allows efficient translation during normoxia and hypoxia. Mol. Biol. Cell 13, 1792-1801 (2002).
104. Huez, I. et al. Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA. Mol. Cell. Biol. 18, 6178-6190 (1998).
105. Plate, K. H., Breier, G., Weich, H. A. & Risau, W. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359, 845-848 (1992).
106. Baek, J. H. et al. Hypoxia-induced VEGF enhances tumor survivability via suppression of serum deprivation-induced apoptosis. Oncogene 19, 4621-4631 (2000).
107. Stein, I. et al. Translation of vascular endothelial growth factor mRNA by internal ribosome entry: implications for translation under hypoxia. Mol. Cell. Biol. 18, 3112-3119 (1998).
108. Akin, G. et al. Regulation of vascular endothelial growth factor (VEGF) expression is mediated by internal initiation of translation and alternative initiation of transcription. Oncogene 17, 227-236 (1998).
109. Vagner, S. et al. Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes. Mol. Cell. Biol. 15, 35-44 (1995).
110. Macejak, D. G. & Sarnow, P. Internal initiation of translation mediated by the 5′ leader of a cellular mRNA. Nature 353, 90-94 (1991).
111. Holcik, M. Targeting endogenous inhibitors of apoptosis for treatment of cancer, stroke and multiple sclerosis. Expert Opin. Ther. Targets 8, 241-253 (2004).