upstream of proteasomes in the cervical cancer setting. Our results show that, when probed for their capacity to interfere with degradation of poly-ubiquitinated proteins, RAMB treatment is associated with increased levels of poly-ubiquitinated proteins (occurring as early as two hours from exposure) but unaltered 20S proteasomal catalytic activity when tested in vitro and in living cells. Notably, while not statistically significant, RAMB1 treatment led to some stabilization of the Ub-FL degron. This may reflect a feedback inhibition of proteasome function due to increasing levels of poly-ubiquitinated protein content in RAMB1 exposed cultures. Importantly, when tested against a panel of cervical cancer cell lines versus keratinocytes, RAMBs showed a preferential killing for the malignant versus the normal phenotype. This appears to be consistent with the notion that due to their endogenously high levels of UPS stress cancer cells are more sensitive than normal cells to perturbation of ubiquitin-mediated protein degradation [4]. Activation of p53 via its stabilization is a potential therapeutic approach for treatment of those cancers, including cervical cancer, expressing wild-type p53. E6 oncoprotein transforms cervical cancer cells by targeting p53 for ubiquitin-dependent proteasomal degradation resulting in reduction of the levels of this and other tumor-suppressor proteins. Therefore, stabilization of p53 by preventing its degradation could recover sufficient levels of wild- type p53 to trigger apoptotic cell death as a response to the abnormal growth of the cancer cell. We show here that RAMB1 treatment causes dose-dependent accumulation/stabilization of p53. Because stabilization of the levels of p53 occurs concomi- tantly with accumulation of high-molecular weight poly-ubiquiti- nated species and as early as 2 hours post-treatment (well before cell death is observed, not shown), this suggests that stabilization of p53 is the cause rather then the consequence of the decrease in cell viability in cervical cancer cells. Induction of p53 via stabilization of its steady state levels correlates with decrease in the levels of cyclin D1 possibly via repression of its transcription [40]. RAMB1 treatment also results in a dose- and time-dependent reduction in the steady state levels of cyclin D1 which could at least partially account for the decrease in cell viability observed in the cervical cancer cell lines. Interestingly, because the decrease in the cyclin D1 expression levels are seen as early as 30 minutes following References 1. Ciechanover A (1994) The ubiquitin-proteasome proteolytic pathway. Cell 79: 13–21. 2. Rolen U, Kobzeva V, Gasparjan N, Ovaa H, Winberg G, et al. (2006) Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines. Mol Carcinog 45: 260–269. 3. Sun Y (2006) E3 ubiquitin ligases as cancer targets and biomarkers. Neoplasia 8: 645–654. 4. Bazzaro M, Lee MK, Zoso A, Stirling WL, Santillan A, et al. (2006) Ubiquitin- proteasome system stress sensitizes ovarian cancer to proteasome inhibitor- induced apoptosis. Cancer Res 66: 3754–3763. 5. Chen D, Frezza M, Schmitt S, Kanwar J, Dou QP (2011) Bortezomib as the First Proteasome Inhibitor Anticancer Drug: Current Status and Future Perspectives. Curr Cancer Drug Targets 11: 239–253. 6. Bazzaro M, Lin Z, Santillan A, Lee MK, Wang MC, et al. (2008) Ubiquitin proteasome system stress underlies synergistic killing of ovarian cancer cells by bortezomib and a novel HDAC6 inhibitor. Clin Cancer Res 14: 7340–7347. 7. Lin Z, Bazzaro M, Wang MC, Chan KC, Peng S, et al. (2009) Combination of proteasome and HDAC inhibitors for uterine cervical cancer treatment. Clin Cancer Res 15: 570–577. 8. Parkin DM, Bray F (2006) Chapter 2: The burden of HPV-related cancers. Vaccine 24 Suppl 3: S3/11–25. 9. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, et al. (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189: 12–19. 10. Hawley-Nelson P, Vousden KH, Hubbert NL, Lowy DR, Schiller JT (1989) HPV16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO J 8: 3905–3910. 11. Horner SM, DeFilippis RA, Manuelidis L, DiMaio D (2004) Repression of the human papillomavirus E6 gene initiates p53-dependent, telomerase-independent Causing UPS Stress for Cervical Cancer Treatment RAMB1 exposure and before detectable stabilization of p53 expression levels, it is possible that cyclin D1 down-regulation is occurring in response to UPS stress rather to p53 activity. In this scenario an arrest in S-phase would precede the onset of apoptosis in RAMBs exposed cervical cancer cells as confirmed by Annexin V staining and PARP-cleavage. This is likely the same mechanism responsible for reduction in colony number observed in the panel of cervical cancer cell lines exposed to RAMB1 treatment. Interestingly, because the reduction in colony number is accompanied by reduction in colony size we conclude that RAMB1 has an effect in slowing the proliferation rate of cervical cancer cells that are initially resistant to its action. Inhibition of conventional (proteasomal-mediated) ubiquitin- dependent protein degradation triggers activation of alternative pathways to proteasomal degradation as a mechanism to compensate for abnormally increased levels of UPS stress. The increase in the levels of poly-ubiquitinated proteins following RAMB1 exposure corresponds to their sub-cellular localization in vimentin-encaged structures consistent with the formation of aggresomes and occurs concomitantly with up-regulation of Hsp90 protein expression levels. This indicates activation of the lysosomal protein degradation pathway as an attempt to ‘‘isolate’’ mis-folded and oxidized proteins and subsequent recycling of the aggresomes via autophagy to cope with increasing levels of UPS stress [6,39]. In this scenario we show that a combinatorial approach of using RAMB1 and the lysosome inhibitor Chloroquine reduce the viability of cervical cancer cells significantly better than either treatment alone. This synergism presumably reflects blockade of the proteasomal and compensatory lysosomal degradation path- ways by RAMB1 and Chloroquine respectively, and provides a rational to further explore this new therapeutic approach for the treatment of cervical cancer. Author Contributions Conceived and designed the experiments: RKA SRK RBSR MB. Performed the experiments: RKA TS AF YI FD SP MB. Analyzed the data: RKA TS AF YI RG RIV SP RBSR MB. Contributed reagents/ materials/analysis tools: SRK RBSR MB. Wrote the paper: MB. senescence and apoptosis in HeLa cervical carcinoma cells. J Virol 78: 4063–4073. 12. Scheffner M, Huibregtse JM, Vierstra RD, Howley PM (1993) The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53. Cell 75: 495–505. 13. Hengstermann A, Linares LK, Ciechanover A, Whitaker NJ, Scheffner M (2001) Complete switch from Mdm2 to human papillomavirus E6-mediated degrada- tion of p53 in cervical cancer cells. Proc Natl Acad Sci U S A 98: 1218–1223. 14. Bazzaro M, Anchoori RK, Mudiam MK, Issaenko O, Kumar S, et al. (2011) alpha,beta-Unsaturated carbonyl system of chalcone-based derivatives is respon- sible for broad inhibition of proteasomal activity and preferential killing of human papilloma virus (HPV) positive cervical cancer cells. J Med Chem 54: 449–456. 15. Milacic V, Banerjee S, Landis-Piwowar KR, Sarkar FH, Majumdar AP, et al. (2008) Curcumin inhibits the proteasome activity in human colon cancer cells in vitro and in vivo. Cancer Res 68: 7283–7292. 16. Achanta G, Modzelewska A, Feng L, Khan SR, Huang P (2006) A boronic- chalcone derivative exhibits potent anticancer activity through inhibition of the proteasome. Mol Pharmacol 70: 426–433. 17. O’Sullivan-Coyne G, O’Sullivan GC, O’Donovan TR, Piwocka K, McKenna SL (2009) Curcumin induces apoptosis-independent death in oesophageal cancer cells. Br J Cancer 101: 1585–1595. 18. Bisht S, Mizuma M, Feldmann G, Ottenhof NA, Hong SM, et al. (2010) Systemic administration of polymeric nanoparticle-encapsulated curcumin (NanoCurc) blocks tumor growth and metastases in preclinical models of pancreatic cancer. Mol Cancer Ther 9: 2255–2264. 19. Yang H, Chen D, Cui QC, Yuan X, Dou QP (2006) Celastrol, a triterpene extracted from the Chinese ‘‘Thunder of God Vine,’’ is a potent proteasome inhibitor and suppresses human prostate cancer growth in nude mice. Cancer Res 66: 4758–4765. PLoS ONE | www.plosone.org 9 August 2011 | Volume 6 | Issue 8 | e23888