Eileen White
Professor

Rutgers University
Molecular Biology and
Biochemistry
CABM-Room 140
679 Hoes Lane
Piscataway. NJ 08854
(732) 235-5329
FAX - 5795
ewhite@cabm.rutgers.edu

Adenovirus. p53. apoptosis. oncogenes. programmed cell death. tumor suppressor genes. cancer. E1B. Bcl-2. cell cycle. caspase

Regulation of Apoptosis by Viral Oncogenes Primary epithelial cells become transformed as a result of the combined action of deregulation of cell cycle control and inhibition of programmed cell death (apoptosis). Expression of the human adenovirus (Ad2/5) E1A oncogene releases normal restrictions on cell cycle progression through interactions with the retinoblastoma tumor suppressor protein and its relatives. and with the p300 and CBP transcriptional co-activators. The cellular response to this deregulation of cell growth control by E1A is the stabilization of the p53 tumor suppressor protein and the induction of p53-dependent apoptosis. The activation of this apoptotic program prevents transformation and can diminish virus replication. The adenonvirus E1B oncogene encodes two proteins (E1B 19K and 55K) that function to inhibit apoptosis. which thereby sustains transformation and productive infection. E1B 55K functions by interacting with and inhibiting p53. whereas E1B 19K is a viral Bcl-2 homologue which functions as a general apoptosis inhibitor by binding to and inhibiting components of the apoptotic machinery. The long-term focus of the White laboratory has been to determine the mechanism by which E1A induces. and E1B 19K inhibits. apoptosis. As deregulation of apoptosis is a common feature of many disease states. knowledge gained by this pursuit should provide new opportunities for the development of novel therapies. particularly for cancer treatment.

Induction of apoptosis the by the adenovirus E1A oncogene

A genetic approach was taken to determine the mechanism by which E1A stabilizes p53 and induces apoptosis. The interaction of E1A with p300 disrupts the ability of the p53 tumor suppressor protein to transactivate mdm-2. the product of which binds to p53 and promotes p53 degradation. Thus. E1A disengages the negative feedback loop to down-regulate p53 levels. causing a dramatic increase in the stability of p53. Apoptosis requires the transcription regulatory activity of p53. indicating that p53 is either repressing the transcription of survival genes or activating the transcription of death genes. Indeed. one transcriptional target of p53 is the pro-apoptotic bax gene. However. bax induction by p53 is not sufficient for apoptosis. which led us to identify other p53 target genes that play a role in apoptosis. Once such gene is that encoding the GTP-binding protein Cdc42. Expression of wild-type or a constitutively active mutant of Cdc42 results in caspase activation and apoptosis. whereas expression of a dominant-negative mutant of Cdc42 diminishes p53-dependent apoptosis. As Cdc42 is known to activate protein kinase signaling pathways. we are presently identifying these pathways and how they regulate caspase activation and apoptosis.

Inhibition of apoptosis by the adenovirus E1B oncogene

The E1B 19K protein functions as an apoptotic inhibitor by binding directly to pro-apoptotic proteins via the BH3 interaction domain of proapoptotic proteins. For example. 19K binds to BH3 of Bax and thereby prevents the release of mitochondrial of cytochrome c. which activates caspases causing apoptosis. The role of 19K-Bax binding in the inhibition of p53-dependent apoptosis by 19K is currently under investigation. Furthermore. analysis of the 19K-Bax structure and its regulation will provide a unique and valuable insight into novel approaches to apoptosis control in normal development and disease.

Mechanism of inhibition of TNF-a -mediated apoptosis by E1B 19K

The adenovirus E1B 19K gene product is a potent inhibitor of death receptor-mediated apoptosis. including that mediated by tumor necrosis factor-a TNF-a ) during viral infection. TNF-a receptor 1 (TNFR-1) activation by TNF-a recruits adaptor molecules that facilitate caspase-8 activation. Caspase-8 cleaves the Bcl-2 family member Bid to truncated Bid (tBid). which translocates to mitochondria facilitating cytochrome c release and caspase-9 activation. and thereby caspase-3 activation and apoptosis. We have discovered that TNF-a signaling induces a Bid-dependent conformational change in Bax which leads to cytochrome c release and downstream caspase-9 and -3 activation. E1B 19K inhibits neither caspase-8 activation nor caspase-8-dependent Bid cleavage. TNF-a did induce an interaction between E1B 19K and Bax. and translocation of E1B 19K to mitochondria. Following E1B 19K binding to a conformationally altered Bax. cytochrome c release and caspase-9 activation were completely inhibited. Interestingly. E1B 19K expression interrupted caspase-3 processing. permitting cleavage to remove the p12 subunit but not the pro-domain. consistent with caspase-8. and not caspase-9. enzymatic activity. Thus. E1B 19K blocks TNF-a -mediated death signaling by targeting and inhibiting a specific form of Bax which blocks caspase activation at a specific point.

Mechanism of inhibition of TNF-a -mediated apoptosis by the adenovirus E1B 19K transforming protein. TNF-a activates caspase-8. which cleaves caspase-3 to remove the carboxyl terminus. and cleaves Bid to generate truncated Bid (tBid). tBid interacts with. and induces a conformational change in. Bax. which enables 19K (orange) to bind to Bax. By binding to Bax. 19K blocks cytochrome c release from mitochondria and thereby caspase-9 activation. This in turn prevents the final processing and activation of caspase-3 and hence. apoptosis. Thus. inhibition of Bax by 19K is a means to prevent apoptototic signaling by TNF-a.

Selected Publications

Andrianasolo EH, Haramaty L, Vardi A, White E, Lutz R, Falkowski P. (2008) Apoptosis-Inducing Galactolipids from a Cultured Marine Diatom, Phaeodactylum tricornutum. J Nat Prod. Jun 21. [Epub ahead of print]

Karp CM, Tan TT, Mathew R, Nelson D, Mukherjee C, Degenhardt K, Karantza-Wadsworth V, White E. (2008) Role of the polarity determinant crumbs in suppressing Mammalian epithelial tumor progression. Cancer Res. 68(11):4105-15.

Prives C, White E. (2008) Does control of mutant p53 by Mdm2 complicate cancer therapy?
Genes Dev. 22(10):1259-64.

Tan TT, White E. (2008) Therapeutic targeting of death pathways in cancer: mechanisms for activating cell death in cancer cells. Adv Exp Med Biol. 615:81-104. Review.

White E. (2008) Autophagic cell death unraveled: Pharmacological inhibition of apoptosis and autophagy enables necrosis. Autophagy. 4(4):399-401.

Jin S, White E.(2008) Tumor suppression by autophagy through the management of metabolic stress. Autophagy. 4(5):563-6.

White E. (2007) Entosis: it's a cell-eat-cell world. Cell. 131(5):840-2.

Mathew R, Karantza-Wadsworth V, White E. (2007) Role of autophagy in cancer. Nat Rev Cancer. 7(12):961-7.

Andrianasolo EH, Haramaty L, Degenhardt K, Mathew R, White E, Lutz R, Falkowski P. (2007)
Induction of apoptosis by diterpenes from the soft coral Xenia elongata. J Nat Prod. 70(10):1551-7.

Karantza-Wadsworth V, White E. (2007) Role of autophagy in breast cancer. Autophagy. 3(6):610-3.

Mathew R, White E. (2007) Why sick cells produce tumors: the protective role of autophagy.
Autophagy. 3(5):502-5.

Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S, White E. (2007) Autophagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev. 21(13):1621-35.

Ewings KE, Hadfield-Moorhouse K, Wiggins CM, Wickenden JA, Balmanno K, Gilley R, Degenhardt K, White E, Cook SJ. (2007) ERK1/2-dependent phosphorylation of BimEL promotes its rapid dissociation from Mcl-1 and Bcl-xL. EMBO J. 26(12):2856-67.

Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K, Chen G, Jin S, White E. (2007) Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev. 21(11):1367-81. Epub 2007 May 17. Erratum in: Genes Dev.

Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K, Chen G, Jin S, White E. (2007) Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev. 21(11):1367-81.

Shimazu T, Degenhardt K, Nur-E-Kamal A, Zhang J, Yoshida T, Zhang Y, Mathew R, White E, Inouye M. (2007) NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition. Genes Dev. 21(8):929-41.

Jin S, DiPaola RS, Mathew R, White E. (2007) Metabolic catastrophe as a means to cancer cell death. J Cell Sci. 120(Pt 3):379-83. Review.

Jiang M, Pabla N, Murphy RF, Yang T, Yin XM, Degenhardt K, White E, Dong Z. (2007) Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation. J Biol Chem. 282(4):2636-45.

Degenhardt K, White E. (2006) A mouse model system to genetically dissect the molecular mechanisms regulating tumorigenesis. Clin Cancer Res. 12(18):5298-304. Review.

Jin S, White E. (2007) Role of autophagy in cancer: management of metabolic stress. Autophagy. 3(1):28-31.

Degenhardt K, Mathew R, Beaudoin B, Bray K, Anderson D, Chen G, Mukherjee C, Shi Y, Gelinas C, Fan Y, Nelson DA, Jin S, White E. (2006) Autophagy promotes tumor cell survival and restricts necrosis, inflammation, and tumorigenesis. Cancer Cell. 10(1):51-64.

Mathew R. White E. (2006) FLIPping the balance between apoptosis and proliferation in thyroid cancer. Clin Cancer Res. 12(12):3648-51.

Marin YE. Namkoong J. Shin SS. Raines J. Degenhardt K. White E. Chen S. (2005) Grm5 expression is not required for the oncogenic role of Grm1 in melanocytes. Neuropharmacology. 49 Suppl 1:70-9.

Gelinas C.. White E. (2005) BH3-only proteins in control: specificity regulates MCL-1 and BAK-mediated apoptosis. Genes Dev. 19(11):1263-8.

Lesne S. Gabriel C. Nelson DA. White E. Mackenzie ET. Vivien D. Buisson A. (In press). Akt-dependent expression of NAIP-1 protects neurons against amyloid-beta toxicity. J Biol Chem. 280(26):24941-7.

Sundararajan R. Chen G. Mukherjee C. White E. (In press). Caspase-dependent processing activates the proapoptotic activity of deleted in breast cancer-1 during tumor necrosis actor-alpha-mediated death signaling. Oncogene. 24(31):4908-20.

Tan T.T.. Degenhardt K.. Nelson D.A.. Beaudoin B.. Nieves-Neira W.. Bouillet P.. Villunger A.. Adams J.M.. White E. (2005) Key roles of BIM-driven apoptosis in epithelial tumors and rational chemotherapy. Cancer Cell. 3:227-38.

Nelson. D. A.. Tan. T.-T.. Rabson. A. B.. Degenhardt. K.. and White. E. (2004) Hypoxia and defective apoptosis drive genomic instability and tumorigenesis. Genes & Dev. 18:2095-2107.

Nelson. D.A. and White. E. (2004) Exploiting different ways to die. Genes & Dev. 18:1223-1226.

Ioffe. M.L.. White. E.. Nelson. D.. Dvorzhinski. D.. and DiPaola. R.S. (2004) Epothilone induced cytotoxicity is dependent on p53 status in prostate cells. The Prostate. 9999: 1-5.

Cuconati. A.. Mukherjee. C.. Perez. D.. and White. E. (2003) DNA damage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells. Genes & Dev. 17: 2922-2932.

White. E. (2003) The pims and outs of survival signaling: Role for the Pim-2 protein kinase in the suppression of apoptosis by cytokines. Genes & Dev. 17(15): 1813-6.

Schwerk. C.. Prasad. J.. Degenhardt. K.. Erdjument-Bromage. H.. Tempst. P.. Kidd. V. J.. White. E.. Manley. J. L.. Lahti. J. M.. and Reinberg. D. (2003) ASAP. a novel protein complex involved in RNA processing and apoptosis. Mol. Cell. Biol. 23: 2981-2990.

Mikhailov. V.. Mikhailova. M.. Degenhardt. K.. Venkatachalam. A.. White. E.. and Saikumar. P. (2003) Association of Bax and Bak homo-oligomers in mitochondria. Bax requirement for Bak reorganization and cytochrome c release. J. Biol. Chem. 278: 5367-5376.