Loma Linda University

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Penelope Duerksen-Hughes, PhD
Associate Dean, Basic Sci & Translational Rsch, Associate Dean Basic Sci & Translational Rsch
School of Medicine
Chair, Basic Sciences
School of Medicine
Vice Chair, Basic Sciences, Biochemistry Division
School of Medicine
Professor, Basic Sciences
School of Medicine
Member, Biochemistry, SM, Faculty of Graduate Studies
Publications    Scholarly Journals--Published
  • Yu, Y., J. Fuscoe, C. Hao, C. Guo, M. Jia, T. Qing, D. Bannon, L. Lancashire, W. Bao, T. Du, H. Luo, Z. Su, W. Jones, C. Moland, W. Branham, F. Qian, B. Ning, Y. Li, H. Hong, L. Guo, N. Mei, T. Shi, K. Wang, Y. Nikolsy, R. Wolfinger, S. Walker, P. Duerksen-Hughes, C. Mason, W. Tong, J. Thierry-Mieg, D. Thierry-Mieg, L. Shi and C. Wang. A rat RNA-Seq transcriptomic Bodymap across eleven organs and four developmental stages”.  Nature Communications.  5:3230, 2014. PMID: 24510058.

    ( 4/2014 - 6/2014 )
  • Filippova, M., W. Evans, R. Aragon, V. Filippov, V. Williams, L. Hong, M. E. Reeves, and P. J. Duerksen-Hughes. The Small splice variant of HPV16 E6, E6*, reduces tumor formation in cervical carcinoma xenografts. Virology. 450-451:153-164, 2014. PMID: 24503078.

     

    ( 3/2014 - 6/2014 )
  • Gao, X., L. Kong, X. Lu, G. Zhang, L. Chi, Y. Jiang, Y. Wu, C. Yan, P. Duerksen-Hughes, X. Zhu and J. Yang. Paraspeckle Protein 1 (PSPC1) is Involved in the Cisplatin Induced DNA Damage Response – Role in G1/S Checkpoint. PLOS ONE. 9:e97174. 2014. ( 7/2013 - 6/2014 )
  • Whitaker, E. L., V. A. Filippov and P. J. Duerksen-Hughes. Interleukin 24:  Mechanisms and therapeutic potential of an anti-cancer gene.  Cytokine and Growth Factor Reviews, 23:323-331, 2012.

     

    ( 7/2012 - 6/2013 )

    Interleukin 24 (mda-7/IL-24) has been classified as an anti-cancer gene for its ability to selectively induce cell death in cancer cells while having little to no effect on normal cells. Although the exact mechanisms by which IL-24 functions have not been completely elucidated, several pathways have consistently been identified: endoplasmic reticulum stress, ceramide-mediated events, and the generation of reactive oxygen species. In addition to these mechanistic analyses, significant progress has also been reported regarding the clinical potential of this anti-cancer gene. For example, many groups are utilizing mda-7/IL-24 in combination with other cancer therapies. This review examines the current research and potential future of this important anti-cancer gene.

  • Gan, T. E., S. P. Xiao, Y. Jiang, H. Hu, Y. H. Wu, P. J. Duerksen-Hughes, J. T. Sheng and J. Yang.  Effects of benzo(a)pyrene on the contractile function of the thoracic aorta of Sprague-dawley rats. Biomed. Environ. Sci., 25:373-380, 2012.

    ( 7/2012 - 6/2013 )
  • C.-H. Yuan, M. Filippova and P. J. Duerksen-Hughes. Modulation of Apoptotic Pathways by HPV:  Mechanisms and Implications for Therapy. Viruses, 4:3831-3850, 2012.

    ( 7/2012 - 6/2013 )
  • Jiang, Y., X.-Y. Zhang, L. Sun, G.-L. Zhang, P. Duerksen-Hughes, X.-Q. Zhu and J. Yang.  Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a Caspase 2- and mitochondria-associated pathway.  Environmental Toxicology and Pharmacology, In Press, 2012.

    ( 7/2012 - 6/2012 )
  •  Whitaker, E. L., V. Filippov, M. Filippova, C. V. Guerrero-Jugarez, and P. J. Duerksen-Hughes.  Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells.  Cytokine 56:272-281, 2011.  PMID:  21843952 ( 7/2011 - 6/2012 )
     
  •  K. Zhang, T.-A. S. Haynes, M. Filippova, V. Filippov and P. J. Duerksen-Hughes.  Quantification of ceramide levels in mammalian cells by high performance liquid chromatography coupled to tandem mass spectrometry with multiple-reaction-monitoring mode (HPLC-MS/MS-MRM).  Analytical Methods 3:1193-1197, 2011. ( 7/2011 - 6/2012 )
     
  •  Yuan, C.-H., M. Filippova, S. S. Tungteakkhun, P. J. Duerksen-Hughes and J. L. Krstenansky. Small molecule inhibitors of the HPV16-E6 interaction with caspase 8. Bioorganic and Medicinal Chemistry Letters22:2125-2129, 2012.  PMID:  22300659                ( 7/2011 - 6/2012 )
     
  • Filippov, V., M.A. Song, K. Zhang, H. V. Vinters, S. Tung, W. M. Kirsch, J. Yang, and P. J. Duerksen-Hughes.  Increased Ceramide in brains with Alzheimer’s and other neurodegenerative diseases. Journal of Alzheimer’s Disease, 29:537-547, 2012.  PMID:  22258513

    ( 7/2011 - 6/2012 )
  • Gan, T.-E., S.-P. Xiao, Y. Jiang, H. Hu, Y.-H. Wu, P. J. Duerksen-Hughes, J.-Z. Sheng, and J. Yang.  Effects of beno(a)pyrene on contractile functions of thoracic aorta of Sprague-Dawley rats. Biomedical and Environmental Sciences, 25:549-556, 2012.  PMID: 23122312

    ( 7/2011 - 6/2012 )
  • Yan, C., Z. Chen, H. Li, G. Zhang, F. Li, P. J. Duerksen-Hughes, X. Zhu, and J. Yang.  Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells. Mutat. Res. 731:75-84, 2012.  PMID:  22138005   ( 7/2011 - 6/2012 )
     
  • Haynes, T.-A. S., V. Filippov, M. Filippova, J. Yang, K. Zhang, and P. J. Duerksen-Hughes.  DNA damage induces down-regulation of UDP-Glucose Ceramide Glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells.  BBA – Molecular and Cell Biology of Lipids, 1821:943-953, 2012.  PMID:  22349266 ( 7/2011 - 6/2012 )
     
  • Zhang, G., L. Sun, X. Lu, Z. Chen, P. J. Duerksen-Hughes, H. Hu, X. Zhu and J. Yang.  Cisplatin treatment leads to changes in nuclear protein and microRNA expression. Mutation Research/Genetic Toxicology and Environmental Mutagenesis, 746:66-77, 2012.   ( 7/2011 - 6/2012 )
     
  • Williams, V. M., M. Filippova, U. Soto and P. J. Duerksen-Hughes.  HPV-DNA Integration and Carcinogenesis:  Putative Roles for Inflammation and Oxidative Stress.  Future Virology, 6(1): 45-57, 2011.  PMID:  21318095 ( 7/2010 - 6/2011 )
     
  •  Yan, C., J. Lu, G. Zhang, T. Gan, Q. Zheng, Z. Shao, P. J. Duerksen-Hughes, and J. Yang.  Benzo[a]pyrene Induces Complex H2AX Phosphorylation Patterns by Multipole Kinases Including ATM, ATR, and DNA-PK.  Toxicology In Vitro 25:91-91, 2011.  PMID:  20888899 ( 7/2010 - 6/2011 )
     
  •  Mujumdar, P., P. J. Duerksen-Hughes, A. F. Firek and D. A. Hessinger.  Long-term, Progressive Aerobic Training Increases Adiponectin in Middle-aged, Overweight, Untrained Males and Females.  Scan. J. Clin. Lab. Invest.  71:101-7, 2011. PMID:  21271804 ( 7/2010 - 6/2011 )
     
  • Xiong, L., A. Darwanto, S. Sharma, J. Herring, S. Hu, M. Filippova, V. Filippov, Y. Wang, C.-S. Chen, P. J. Duerksen-Hughes, L. C. Sowers, K. Zhang.  Mass spectrometric studies on epigenetic interaction networks in cell differentiation.  J. Biol. Chem.  286:13657-68, 2011.    PMID:  21335548 ( 7/2010 - 6/2011 )
     
  •  Tungteakkhun, S. S., M. Filippova, N. Fodor and P. J. Duerksen-Hughes.  The Full-Length Isoform of Human Papillomavirus 16 E6 and its Splice Variant E6* Bind to Different Sites on the Procaspase 8 Death Effector Domain.  J. Virology 84:1453-1463, 2010. PMID:  19906919 ( 7/2009 - 6/2010 )
     
  •  Yan, C., W. Wu, H. Li, G. Zhang, P. J. Duerksen-Hughes, X. Zhu and J. Yang.  Benzo[a]pyrene Treatment Leads to Changes in Nuclear Protein Expression and Alternative Splicing.  Mutat. Res. 686:47-56, 2010.  PMID:  20097212 ( 7/2009 - 6/2010 ) Link...
     
  •  Wu, W., C. Yan, T. Gan, Z. Chen, X. Lu, P. J. Duerksen-Hughes, X. Zhu and J. Yang.  Nuclear Proteome Analysis of Cisplatin-Treated HeLa Cells.  Mutation Research 2010.  PMID:  20540955 ( 7/2009 - 6/2010 )
     
  •  Tungteakkhun, S. S., M. Filippova, J. W. Neidigh, N. Fodor, and Penelope J. Duerksen-Hughes.  The Interaction between Human Papillomavirus Type 16 and FADD is Mediated by a Novel E6 Binding Domain.  J. Virol. 82:9600-9614, 2008.  PMID: 18632871 ( 7/2008 - 6/2009 )
     http://www.ncbi.nlm.nih.gov/pubmed/18632871?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
  •  Filippova, M., V. A. Filippov, M. Kagoda, T. Garnett, N. Fodor and P. J. Duerksen-Hughes.  Complexes of Human Papillomavirus 16 E6 Proteins Form Pseudo-DISC Structures During TNF-Mediated Apoptosis.  J. Virology 83:210-227, 2009.  PMID:  18842714 ( 7/2008 - 6/2009 )
     
  •  Tungteakkhun, S. S. and P. J. Duerksen-Hughes.  Cellular binding partners of the human papillomavirus E6 protein.  Arch. Virol. 153:397-408, 2008.  PMID:  18172569 ( 7/2007 - 6/2008 )
     http://www.ncbi.nlm.nih.gov/pubmed/18172569?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
  • Filippov, V., M. Filippova and P. J. Duerksen-Hughes.  The Early Response to DNA Damage Can Lead to Activation of Alternative Splicing Activity Resulting in CD44 Splice Pattern Changes.   Cancer Research67:7621-7630, 2007.  PMID:  17699766 ( 7/2007 - 6/2008 )
     http://www.ncbi.nlm.nih.gov/pubmed/17699766?ordinalpos=4&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
  •  Haynes, T.-A S., P. J. Duerksen-Hughes, M. Filippova, V. Filippov and K. Zhang.  C(18) Ceramide Analysis in Mammalian Cells Employing Reversed-Phase High Performance Liquid Chromatography Tandem Mass Spectrometry.  Analytical Biochemistry378:80-86, 2008.  PMID:  18423390 ( 7/2007 - 6/2008 )
     http://www.ncbi.nlm.nih.gov/pubmed/18423390?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum
  •  Filippov, V., E. L. Schmidt, M. Filippova and P. J. Duerksen-Hughes. Splicing and splice factor SRp55 participate in the response to DNA damage by changing isoform ratios of target genes.  Gene.  420:34-41, 2008.  PMID:  18571879 ( 7/2007 - 6/2008 )
     http://www.ncbi.nlm.nih.gov/pubmed/18571879?ordinalpos=2&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum
  • Theodore O. Garnett, Maria Filippova and Penelope J. Duerksen-Hughes. "Accelerated Degradation of FADD and Procaspase 8 in Cells Expressing Human Papillomavirus 16 E6 Impairs TRAIL-mediated Apoptosis." Cell Death and Differentiation 13. (2006): 1915-1926. ( 7/2006 - 6/2007 ) Link...
    Viruses have developed sophisticated strategies to evade host defenses and facilitate the production and spread of progeny. In this study, we show that transfection of the human papillomavirus (HPV) 16 E6 oncogene into HCT116 cells provides protection from tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-mediated apoptosis. Additionally, we demonstrate that the protection provided by E6 is dose-dependent because higher levels of E6 provide greater protection. The mechanism underlying this protection involves a rapid reduction in the protein levels of both Fas-associated death domain (FADD) and procaspase 8, which results in suppression of the activation of caspase 8, 3 and 2. Interestingly, E6 does not interfere with the mitochondrial apoptotic pathway even though HCT116 cells have been classified as type II cells with regard to TRAIL signaling. These findings demonstrate that E6 has a more generalized effect on signaling by death ligands than was previously thought and support the notion that E6 can utilize p53-independent mechanisms to modulate cell survival.
  • Maria Filippova, Melyssa Johnson, Marnelli Bautista, Valery Filippov, Nadja Fodor, Sandy Tungteakkhun, Kadia Williams and Penelope Duerksen-Hughes. "The Large and Small Isoforms of HPV 16 E6 Bind to and Differentially Affect Procaspase 8 Stability and Activity." J. Virology 81. (2007): 4116-4129. ( 7/2006 - 6/2007 ) Link...
    Human papillomavirus type 16 (HPV-16) has developed numerous ways to modulate host-initiated immune mechanisms. The HPV-16 E6 oncoprotein, for example, can modulate the cellular level, and consequently the activity, of procaspase 8, thus modifying the cellular response to cytokines of the tumor necrosis factor family. E6 from HPV-16, but not E6 from the low-risk types 6b and 11, alters the cellular level of procaspase 8 in a dose-dependent manner. Both the large and small (E6*) isoforms of E6, which originate by way of alternate splicing, can modulate procaspase 8 stability. Intriguingly, although both isoforms bind to procaspase 8, the large isoform accelerates the degradation of procaspase 8 while the small isoform stabilizes it. Binding leads to a change in the ability of procaspase 8 to bind either to itself or to FADD (Fas-associated death domain), with the large version of E6 able to inhibit this binding while the small isoform does not. Consistent with this model, knockdown of the large version of E6 by small interfering RNA leads to increases in the levels of procaspase 8 and its binding to both itself and FADD. Thus, these alternatively spliced isoforms can modulate both the level and the activity of procaspase 8 in opposite directions.
  • Theodore Garnett, Maria Filippova and Penelope J. Duerksen-Hughes. "Bid is Cleaved Upstream of Caspase-8 Activation During TRAIL-Mediated Apoptosis in Human Osteosarcoma Cells." Apoptosis 12. (2007): 1299-1315. ( 7/2006 - 6/2007 ) Link...
    TRAIL induces apoptosis in many malignant cell types. In this study, we used the human papilloma virus (HPV) 16 E6 protein as a molecular tool to probe the TRAIL pathway in HCT116 colon carcinoma cells and U2OS osteosarcoma cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells expressing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAIL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8. Cathepsin B activity in U2OS cells was significantly activated shortly after exposure to TRAIL, and the cathepsin B inhibitor, CA074Me, inhibited both TRAIL- and anti-DR5-mediated apoptosis and delayed the cleavage of Bid. These findings suggest that TRAIL activates a pathway dependent on Bid, but largely independent of FADD and caspase-8, in U2OS cells.
  • Valery Filippov, Maria Filippova and Penelope J. Duerksen-Hughes. "The Early Response to DNA Damage can Lead to Activation of Alternative Splicing Activity Resulting in CD44 Splice Pattern Changes." Cancer Research 67.16 (2007): 7621-7630. ( 6/2006 - 6/2007 ) Link...
    Expression of the HPV 16 E6 oncogene interferes with several vital cellular processes, including the p53-dependent response to DNA damage. To assess the influence of E6 on the early response to DNA damage, we analyzed gene expression following mitomycin C-induced genotoxic stress in human E6-expressing U2OS cells (U2OSE64b) as well as in p53-expressing control cells (U2OSE6AS) by comparative global expression profiling. As expected, genes involved in p53-dependent pathways were activated in p53-expressing cells. In the U2OSE64b cells, however, a largely non-overlapping group of genes was identified, including two splicing factors of the SR family. Immunoblot analysis revealed increased expression of several SR proteins during the early response to DNA damage, which was accompanied by activation of alternative splicing activity. Disruption of splicing activity by treatment with siRNA directed against splicing factor SRp55 resulted in increased viability of p53-deficient cells following DNA damage. To determine whether the transient activation of splicing activity was due to E6-mediated degradation of p53 or to some other activity of E6, we compared the early response of the p53 wt and p53-/- isogenic HCT116 cell lines, and found that the increase in splicing activity was observed only in the absence of p53. Finally, both the U2OSE64b and the p53 -/- cells showed altered splicing patterns for the CD44 receptor. Together, these data show that cells lacking p53 can activate alternative splicing following DNA damage.
  • Theodore Garnett and Penelope J. Duerksen-Hughes. "Modulation of Apoptosis by HPV Oncoproteins." Archives of Virology 151. (2006): 2321-2335. ( 7/2005 - 6/2006 ) Link...
    The regulation of host-mediated apoptosis by the E6 and E7 oncoproteins has garnered attention because it is believed to be an important strategy employed by high-risk (HR)-human papillomaviruses (HPVs) to evade immune surveillance. Additionally, the revelation that E5 can protect cells from tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis suggests that it may also play a role in undermining host defense mechanisms. Cellular transformation is an unintended consequence of persistent infection by HR-HPVs, and it is therefore likely that the primary function of E5, E6 and E7 is to regulate cell survival throughout the normal viral life cycle in order to ensure viral replication and promote the spread of progeny. The purpose of this article is to review the literature on the regulation of host-mediated apoptosis by E5, E6 and E7 that describes the mechanisms employed by HR-HPVs to persist in the host and create the conditions necessary for cellular transformation.
  • Maria Filippova, Terry A. Brown-Bryan, Carlos A. Casiano, and Penelope J. Duerksen-Hughes. "The Human Papillomavirus 16 E6 Protein Can Either Protect or Further Sensitize Cells to TNF: Effect of Dose." Cell Death and Differentiation 12. (2005): 1622-1635. ( 7/2004 - 6/2005 ) Link...
    High-risk strains of human papillomavirus, including HPV 16, cause human cervical carcinomas, due in part to the activity of their E6 oncogene. E6 interacts with a number of cellular proteins involved in host-initiated apoptotic responses. Paradoxically, literature results show that E6 can both protect cells from and sensitize cells to tumor necrosis factor (TNF). To examine this apparent contradiction, E6 was transfected into U2OS cells and stable clones were treated with TNF. Intriguingly, clones with a high level of E6 expression displayed an increased sensitivity to TNF by undergoing apoptosis, while those with low expression were resistant. Furthermore, TNF treatment of cells in which the expression of E6 was regulated by the addition of doxycycline demonstrated clearly that while low levels of E6 protect cells from TNF, high levels sensitize cells. Together, these results demonstrate thata virus-host interactions can be complex and that both quantitative and qualitative aspects are important in determining outcome.
  • Jun Yang, Yingnian Yu, Shuyu Sun, and Penelope J. Duerksen-Hughes. "Ceramide and Other Sphingolipids in Cellular Responses." Cellular Biochemistry and Biophysics 40. (2004): 323-350. ( 7/2003 - 6/2004 ) Link...
    Formerly considered to serve only as structural components, sphingolipids are emerging as an important group of signaling molecules involved in many cellular revents, including cell growth, senescence, meiotic maturation, and cell death. They are also implicated in functions such as inflammation and the responses to heat shock and genotoxic stress. Defects in the metabolism of sphingolipids are related to various genetic disorders, and sphingolipids have the potential to serve as therapeutic agents for human diseases such as colon cancer and viral or bacterial infections. The best-studied member of this family, ceramide, which also serves as the structural backbone for other sphingolipids, is an important mediator in multiple cellular signaling pathways. The metabolism and functions of sphingolipids are discussed in this review, with a focus on ceramide regulation in various cellular responses.
  • Jun Yang, Zheng-Ping Xu, Yun Huang, Hope E. Hamrick, Penelope J. Duerksen-Hughes, Ying-Nian Yu. "ATM and ATR: Sensing DNA Damage." World J. Gastroenterology 10.2 (2004): 155-160. ( 7/2003 - 6/2004 ) Link...
    Cellular response to genotoxic stress is a very complex process, and it usually starts with the "sensing" or "detection" of the DNA damage, followed by a series of events that include signal transduction and activation of transcription factors. The activated transcription factors induce expression of many genes which are involved in cellular functions such as DNA repair, cell cycle arrest, and cell death. There have been extensive studies from multiple disciplines exploring the mechanisms of cellular genotoxic responses, which have resulted in the identification of many cellular components involved in this process, including the mitogen-activated protein kinases (MAPKs) cascade. Although the innitial activation of this protein kinase cascade is not fully unerstood, human protein kinases ATM (ataxia-telangiectasia, mutated) and ATR (ATM and Rad3-related) are emerging as potential sensors of DNA damage. Current progress in ATM/ATR research and related signaling pathways are discussed in this review, in an effort to facilitate a better understanding of the genotoxic stress response.
  • Maria Filippova, Lindsey Parkhurst and Penelope J. Duerksen-Hughes. "The Human Papillomavirus 16 E6 Protein Binds to Fas-associated Death Domain and Protects Cells from Fas-triggered Apoptosis." J. Biological Chemistry 279.24 (2004): 25729-25744. ( 7/2003 - 6/2004 ) Link...
    High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of the tumor suppressor p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that E6 can protect cells from tumor necrosis factor-induced apoptosis by binding to the C-terminal end of tumor necrosis factor R1, thus blocking apoptotic signal transduction. In this study, E6 was shown to also protect cells from apoptotis induced via the Fas pathway. Furthermore, use of an inducible E6 expression system demonstrated that this protection is dose-dependent, with higher levels of E6 leading to greater protection. Although E6 suppresses activation of both caspase 3 and caspase 8, it does not affect apoptotic signaling through the mitochondrial pathway. Mammalian two-hybrid and in vitro pull-down assays were then used to demonstrate that E6 binds directly to the death effector domain of Fas-associated death domain (FADD), with deletion and site-directed mutants enabling the localization of the E6-binding site to the N-terminal end of rhte FADD death effector domain. E6 is produced in two forms as follows: a full-length version of ~16 kDa and a smaller version of about half that size corresponding to the N-terminal half of the full-length protein. Pull-down and functional assays demonstrated that the full-length version, but not the small version of E6 was able to bind to FADD and to protect cells from Fas-induced apoptosis. In addition, binding to E6 leads to degradation of FADD, with the loss of cellular FADD proportional to the amount of E6 expressed. These results support a model in which E6-mediated degradation of FADD prevents transmission of apoptotic signals via the Fas pathway.
  • Jun Yang, Yingnian Yu and Penelope J. Duerksen-Hughes. "Protein Kinases and their Involvement in the Cellular Responses to Genotoxic Stress." Mutation Research 543. (2003): 31-58. ( 7/2002 - 6/2003 ) Link...
    Cells are constantly subjected to genotoxic stress, and much has been learned regarding their response to this type of stress during recent years. In general, the cellular genotoxic response can be thought to occur in three stages: 91) damage sensing; (2) activation of signal transduction pathways; (3) biological consequences and attenuation of the response. The biological consequences, in particular, induce cell cycle arrest and cell death. Although our understanding of the molecular mechanisms underlying cellular genotoxic stress responses remains incomplete, many cellular components have been identified over the years, including a group of protein kinases that appears to play a major role. Various DNA-damaging agents can activate these protein kinases, triggering a protein phosphorylation cascade that leads to the activation of transcription factors, and altering gene expression. In this review, the involvement of protein kinases, particularly the mitogen-activated protein kinases (MAPKs), at different stages of the genotoxic response is discussed.
  • Maria Filippova and Penelope J. Duerksen-Hughes. "Inorganic and Dimethylated Arsenic Species Induce Cellular p53." Chemical Research in Toxicology 16.3 (2003): 423-431. ( 7/2002 - 6/2003 ) Link...
    Arsenic compounds are known for their ability both to cause and to treat human cancers, although the molcular mechanisms underlying these actions are incompletely understood. The simplest explanation is that arsenic causes DNA damage that leads to mutations. However, the majority of scientific evidence indicates that arsenic is not a genotoxin or DNA-damaging agent. DNA damage typically leads to cellular responses designed to minimize the replication of damaged DNA, such as the induction of p53, and p53 induction has therefore been used as an indicator of DNA damage. Because this approach can be applied to human cells and does not rely on a specific, heritable mutation occurring at a particular site, it seemed possible that this method could detect DNA damage that was undetectable using other techniques. To examine the genotoxic potential of arsenic compounds, therefore, seven of these compounds (sodium aresenite, sodium aresenate, methyloxoarsine, iododimethylarsine, disodium methyl arsonate, dimethylarsinic acid, and arsenic trioxide) were tested for their ability to increase the cellular level of p53 as measured by ELISA. Of this group, arsenic trioxide was the strongest inducer of cellular p53, while dimethylarsinic acid, iododimethylarsine, and sodium arsenite also caused p53 induction in a dose- and time-dependent manner. Sodium arsenate, as well as the two monomethyl compounds tested, methyloxoarsine and disodium methyl arsonate, did not cause detectable increases in cellular p53. Our results indicate, therefore, that cells respond to several of these arsenic compounds as they do to chemicals that damage DNA, suggesting that exposure of cells to these compounds does in fact cause DNA damage. Such damage could then result in mutations and the observed development of cancer.
  • Jun Yang, Yingnian Yu, Hope E. Hamrick and Penelope J. Duerksen-Hughes. "ATM, ATR and DNA-PK: Initiators of the Cellular Genotoxic Stress Responses." Carcinogenesis 24.10 (2003): 1571-1580. ( 7/2002 - 6/2003 ) Link...
    Exposure to genotoxic agents is a major cause of human cancer, and cellular responses to genotoxic stress are important defense mechanisms. These responses are very complex, involving many cellular factors that form an extensive signal transduction network. This network includes a protein kinase cascade that connects the detection of DNA damage to the activation of transcription factors, which in turn regulate the expression of genes involved in DNA repair, cell cycle arrest and programmed cell death (apoptosis). The mitogen-activated protein kinases are the best-studied members of the kinase cascade with an acknowledged role in the genotoxic stress response. However, the initial activation of the protein kinase cascade is not fully understood, although several protein kinases, such as ataxia telangiectasia, muted (ATM), ATM- and Rad3-related (ATR), and DNA-dependent protein kinase (DNA-PK) in humans, are increasingly recognized for their potential roles in the sensing of DNA damage and initiating the subsequent protein kinase cascade. In this review, the properties of these three kinases are discussed and their functions in the initiation of the genotoxic stress response are explored.
  • Maria Filippova, Helen Song, Jodi L. Connolly, Terence S. Dermody and Penelope J. Duerksen-Hughes. "The Human Papillomavirus 16 E6 Protein Binds to Tumor Necrosis Factor (TNF) R1 and Protects Cells from TNF-induced Apoptosis." J. Biological Chemistry 277.24 (2002): 21730-21739. ( 7/2001 - 6/2002 ) Link...
    High risk strains of human papillomavirus (HPV), such as HPV 16, cause human cervical carcinoma. The E6 protein of HPV 16 mediates the rapid degradation of p53, although this is not the only function of E6 and cannot completely explain its transforming potential. Previous work in our laboratory has demonstrated that transfection of HPV 16 E6 into the tumor necrosis factor (TNF)-sensitive LM cell line protects expressing cells from TNF-induced apoptosis in a p53-independent manner, and the purpose of this study was to determine the molecular mechanism underlying this protection. Caspase 3 and caspase 8 activation were significantly reduced in E6-expressing cells, indicating that E6 acts early in the TNF apoptotic pathway. In fact, E6 binds directly to TNF R1, as shown both by co-immunoprecipitation and mammalian two-hybrid approaches. E6 requires the same C-terminal portion of TNF R1 for binding as does TNF R1-associated death domain, and TNF R1/TNF R1-associated death domain interactions are decreased in the presence of E6. HA-E6 also blocked cell death triggered by the transfection of the death domain of TNF R1. Together, these results provide strong support for a model in which HPV E6 binding to TNF R1 intereferes with formation of the death-inducing signaling complex and thus with transduction of pro-apoptotic signals. They also demonstrate that HPV, like several other viruses, has developed a method for evading the TNF-mediated host immune response.
  • Jun Yang and Penelope J. Duerksen-Hughes. "Activation of a p53-independent, Sphingolipid-mediated Cytolytic Pathway in p53-negative Mouse Fibroblast Cells Treated with N-Methyl-N-nitro-N-nitrosoguanidine." J. Biological Chemistry 276.29 (2001): 27129-27135. ( 7/2000 - 6/2001 ) Link...
    Sphingolipids such as ceramide are important mediators of apoptosis and growth arrest triggered by ligands such as tumor necrosis factor and Fas-L binding to their receptors. When LM (expressing p53) and LME6 (lacking p53) cells were exposed to the genotoxin N-methyl-N-nitro-N-nitrosoguanidine (MNNG), both cell lines underwent cytolysis in a very similar manner, suggesting the presence of a p53-independent apoptotic response to this genotoxic stress. To determine whether sphingolipids such as ceramide might serve as mediators in this system, the responses of these cells to exogenous spingolipids as well as the changes in their endogenous sphingolipid levels after DNA damage were examined. Treatment with exogenous C2-ceramide and sphingosine led to cell death in both LM and LME6, and treatment of the LME6 cells with MNNG resulted in a transient increase in intracellular ceramide of ~50 % over a period of 3 h. Finally treatment with the de novo inhibitor of ceramide synthesis ISP-1 protected LME6 cells from MNNG-triggered cell death. This MNNG-triggered induction of ceramide was not observed in the p53-expressing LM cells, suggesting that it may be down-regulated by p53. Although ceramide-mediated cell death can proceed in the absence of p53, exogenously added C2-ceramide increased the cellular p53 level in LM cells suggesting that the two pathways do intersect.
  • Penelope J. Duerksen-Hughes, Jun Yang and Ozan Ozcan. "p53 Induction as a Genotoxic Test for Twenty-Five Chemicals Undergoing in Vivo Carcinogenicity Testing." Environmental Health Perspectives 107.10 (1999): 805-812. ( 7/1998 - 6/1999 ) Link...
    In vivo carcinogenicity testing is an expensive and time-consuming process, and as a result, only a relatively small fraction of new and existing chemicals has been tested in this manner. Therefore, the development and validation of alternative approaches is desirable. We previously developed a mammalian in vitro assay for genotoxicity based on the ability of cells to increase their level of the tumor-suppressor protein p53 in response to DNA damage. Cultured cells are treated with various amounts of the test substances, and at defined times following treatment, they are harvested and lysed. The lysates are analyzed for p53 by Western blot and/or enzyme-linked immunosorbent assay analysis. An increase in cellular p53 following treatment is interpreted as evidence for DNA damage. To determine the ability of this p53-induction assay to predict carcinogenicity in rodents and to compare such results with those obtained using alternative approaches, we subjected 25 chemicals from the predictive toxicology evaluation 2 list to analysis with this method. Five substances (citral, cobalt sulfate heptahydrate, D&C Yellow No. 11, oxymetholone, and t-butyl-hydroquinone) tested positive in this assay, and three substances (emodin, phenolphthalein, and sodium xylenesulfonate) tested as possibly positive. Comparisons between the results obtained with this assay and those obtained with the in vivo protocol, the Salmonella assay, and the Syrian hamster embryo (SHE) cell assay indicate that the p53-induction assay is an excellent predictor of the limited number of genotoxic carcinogens in this set, and that its accuracy is roughly equivalent to or better than the Salmonella and SHE assays for the complete set of chemicals.
  • Penelope J. Duerksen-Hughes, Jun Yang and Stephanie B. Schwartz. "HPV 16 E6 Blocks TNF-Mediated Apoptosis in Mouse Fibroblast LM Cells." Virology 264. (1999): 55-65. ( 7/1998 - 6/1999 ) Link...
    The interaction between hosts and the viruses that infect them is a dynamic one, and a growing literature documents the fact that many viruses have developed mechanisms designed to avoid elimination by the host immune system. One of the immune strategies used by the host and targeted by virus proteins is apoptosis triggered by the cytokine tumor necrosis factor (TNF). Mouse fibroblast LM cells are spontaneously sensitive to TNF. When the wild-type E6 protein from the human papillomavirus type 16 (HPV 16) was expressed in LM cells, the cells because resistant to TNF. This resistance was examined by several means, including cell morphology, the dose- and time-dependent response to TNF in a cell death ELISA, trypan blue exclusion, and cell proliferation. The level of p53 did not rise in TNF-treated cells prior to apoptosis, suggesting a p53-independent mechanism. Significant, though not complete, resistance to TNF was also observed following transfection of a plasmid expressing a mutant E6 protein, which is unable to mediate rapid degradation of the p53 tumor suppressor. These results indicate that the HPV 16 E6 protein can protect LM cells from TNF-triggered apoptosis and likely does so by a mechanism other than mediation of p53 degradation.
  • Jun Yang and Penelope J. Duerksen-Hughes. "A New Approach to Identifying Genotoxic Carcinogens: p53 Induction as an Indicator of Genotoxic Damage." Carcinogenesis 19.6 (1998): 1117-1125. ( 7/1997 - 6/1998 ) Link...
    The tumor suppressor gene p53 encodes a nuclear phospho-protein which is critical for cell cycle control and prevention of uncontrolled cell proliferation that can lead to cancer. Previous studies have shown that cells respond to DNA damage by increasing their levels of p53, which then acts to prevent replication of damaged DNA. This study examined the effects on p53 protein levels of several different categories of chemical carcinogens. N-Methyl-N''-nitro-nitrosoguanidine and N-ethyl-N-nitrosourea, two direct-acting genotoxic (DNA-reactive) carcinogens, caused p53 induction as early as 2 h following treatment, with peak increases within 4-12 h. Aflatoxin B1 and 2-acetyl-aminofluorene, indirect-acting genotoxic carcinogens, caused a later induction of p53, with the peak increase appearing between 16 and 24 h following treatment. These observations demonstrate a correlation between p53 inducction pattern and DNA damaging mechanism of genotoxins. Phenol, diethylstilbestrol and ethylacrylate also induced increases in cellular p53. The half-life of p53 protein was increased in cells treated with genotoxic agents. On the other hand, the epigenetic (non-DNA-reactive) carcinogens azathioprine and saccharin, as well as two substances generally considered to be non-carcinogens, dimethylsulfoxide and benzethonium chloride, had no effect on p53 protein levels of treated cells. Measurement of the cytotoxic effects of each of these chemicals led to the conclusion that p53 protein induction is not a general, non-specific consequence of the cytotoxic effect of these genotoxins. These results suggest that measurement of p53 protein induction may be an effective tool to identify environmental genotoxins.
  • Ju L, Zhang G L, Zhang C, Sun L, Jiang Y, . . . Yang J. (2013). Quantum dot-related genotoxicity perturbation can be attenuated by PEG encapsulation. Mutation Research-Genetic Toxicology and Environmental Mutagenesis, 753(1), 54-64. ( 4/2013 - Present ) Link...
    Nanomaterial-biosystem interaction is emerging as a major concern hindering wide adoption of nanomaterials. Using quantum dots (Qdots) of different sizes (Qdot-440 nm and Qdot-680 nm) as a model system, we studied the effects of polyethylene glycol (PEG) thin-layer surface modification in attenuating Qdot-related cytotoxicity, genotoxicity perturbation and oxidative stress in a cellular system. We found that uncoated Qdots (U-Qdots) made of core/shell CdSe/ZnS could indeed induce cytotoxic effects, including the inhibition of cell growth. Also, both the neutral comet assay and gamma H2AX foci formation showed that U-Qdots caused significant DNA damage in a time- and dose-dependent manner. In contrast, results from cytotoxicity analysis and gamma H2AX generation indicate minimal impact on cells after exposure to PEG-coated Qdots. This lack of observed toxic effects from PEG-coated Qdots may be due to the fact that PEG-coating can inhibit ROS generation induced by U-Qdots. Based on these observations, we conclude that the genotoxicity of Qdots could be significantly decreased following proper surface modification, such as PEG encapsulation. In addition, PEG encapsulation may also serve as a general method to attenuate nanotoxicity for other nanoparticles. (C) 2013 Elsevier B.V. All rights reserved.
  • Yuan C H, Filippova M, & Duerksen-Hughes P. (2012). Modulation of Apoptotic Pathways by Human Papillomaviruses (HPV): Mechanisms and Implications for Therapy. Viruses-Basel, 4(12), 3831-3850. ( 12/2012 - Present ) Link...
    The ability of the host to trigger apoptosis in infected cells is perhaps the most powerful tool by which viruses can be cleared from the host organism. To avoid elimination by this mechanism, human papillomaviruses (HPV) have developed several mechanisms that enable the cells they infect to elude both extrinsic and intrinsic apoptosis. In this manuscript, we review the current literature regarding how HPV-infected cells avoid apoptosis and the molecular mechanisms involved in these events. In particular, we will discuss the modifications in intrinsic and extrinsic apoptotic pathways caused by proteins encoded by HPV early genes. Many of the current efforts regarding anti-cancer drug development are focused on directing tumor cells to undergo apoptosis. However, the ability of HPV-infected cells to resist apoptotic signals renders such therapies ineffective. Possible mechanisms for overcoming the resistance of HPV-infected tumor cells to anticancer drugs will be discussed.
  • Whitaker E L, Filippov V A, & Duerksen-Hughes P J. (2012). Interleukin 24: Mechanisms and therapeutic potential of an anti-cancer gene. Cytokine & Growth Factor Reviews, 23(6), 323-331. ( 12/2012 - Present ) Link...
    Interleukin 24 (mda-7/IL-24) has been classified as an anti-cancer gene for its ability to selectively induce cell death in cancer cells while having little to no effect on normal cells. Although the exact mechanisms by which IL-24 functions have not been completely elucidated, several pathways have consistently been identified: endoplasmic reticulum stress, ceramide-mediated events, and the generation of reactive oxygen species. In addition to these mechanistic analyses, significant progress has also been reported regarding the clinical potential of this anti-cancer gene. For example, many groups are utilizing mda-7/IL-24 in combination with other cancer therapies. This review examines the current research and potential future of this important anti-cancer gene. (C) 2012 Elsevier Ltd. All rights reserved.
  • Jiang Y, Zhang X Y, Sun L, Zhang G L, Duerksen-Hughes P, Zhu X Q, & Yang J. (2012). Methyl methanesulfonate induces apoptosis in p53-deficient H1299 and Hep3B cells through a caspase 2-and mitochondria-associated pathway. Environmental Toxicology and Pharmacology, 34(3), 694-704. ( 11/2012 - Present ) Link...
    Methyl methanesulfonate (MMS) has been shown to induce apoptosis in various cell types through p53-dependent pathways. Nevertheless, pharmacological and genetic blockade of p53 functions results in similar or delayed sensitivity to MMS treatment, suggesting the presence of p53-independent apoptotic mechanisms. To understand the p53-independent mechanisms that are engaged during MMS-induced apoptosis, we established MMS-induced apoptotic cell models using p53-deficient H1299 and Hep3B cells. Our results demonstrated that MMS at concentrations of 50, 100, 200, 400 and 800 mu M induced the formation of gammaH2AX foci, and that at higher concentrations, 400 and 800 mu M, MMS treatment led to apoptosis in the two cell lines. This apoptotic cell death was concurrent with the loss of mitochondrial membrane potential, nuclear-cytosolic translocation of active caspase 2, release of cytochrome c from mitochondria, and the cleavage of caspase 9, caspase 3 and PARP. However, MMS-induced DNA damage failed to stabilize the p53 family members TAp73 and DNp73. These results demonstrated a p53- and p73-independent mechanism for MMS-induced apoptosis that involves the nuclear-cytosolic translocation of active caspase 2 as well as the mitochondria-mediated pathway. (C) 2012 Elsevier B.V. All rights reserved.
  • Gan T E, Xiao S P, Jiang Y, Hu H, Wu Y H, . . . Yang J. (2012). Effects of Benzo(a)pyrene on the Contractile Function of the Thoracic Aorta of Sprague-dawley Rats. Biomedical and Environmental Sciences, 25(5), 549-556. ( 10/2012 - Present ) Link...
    Objective To evaluate the possible vascular effects of an environment carcinogen benzo(a)pyrene (BaP). Methods The cytotoxicit of BaP and rat liver 59 (0.25 mg/mL)-activated BaP were examined by MU assay. Thoracic aortic rings were dissected from Sprague-Dawley rats. Contraction of aortic rings was induced by 60 mmol/L KCl or 10(-6) mol/L phenylephrine (PE) in an ex-vivo perfusion system after BaP (100 mu mol/L) incubation for 6 h. [Ca2+](i) was measured using Fluo-4/AM. For in-vivo treatment, rats were injected with BaP for 4 weeks (10 mg/kg, weekly, i.p.). Results BaP (1-500 mu m) did not significantly affect cell viability; S9-activated BaP stimulated cell proliferation. BaP did not affect the contractile function of endothelium-intact or -denuded aortic rings. BaP did not affect ATP-induced ([Ca2+](i)) increases in human umbilical vein endothelial cells. In BaP-treated rats, heart rate and the number of circulating inflammatory cells were not affected. Body weight decreased while blood pressure increased significantly. The maximum aortic contractile responses to PE and KCl and the maximum aortic relaxation response to acetylcholine were significantly decreased by 25.0%, 34.2%, and 10.4%, respectively. Conclusion These results suggest, in accordance with its DNA-damaging properties, that metabolic activation is a prerequisite for BaP-induced cardiovascular toxicity.
  • Zhang G L, Sun L, Lu X H, Chen Z H, Duerksen-Hughes P J, . . . Yang J. (2012). Cisplatin treatment leads to changes in nuclear protein and microRNA expression. Mutation Research-Genetic Toxicology and Environmental Mutagenesis, 746(1), 66-77. ( 7/2012 - Present ) Link...
    Using a proteomic approach, we have previously shown that exposure to different concentrations of cisplatin during a 12-h period can lead to changes in nuclear protein expression and alternative splicing in HeLa cells. To further shed light on the DNA damage response (DDR) induced by cisplatin, we examined the nuclear proteome profiles of HeLa cells treated with 5 mu M cisplatin for different times (2, 12, and 24 h). Two-dimensional electrophoresis (2-DE) identified 98 differentially expressed proteins in cisplatin-treated cells as compared to control cells. Among them, 54 spots (55%) were down-regulated and 44 spots (45%) were up-regulated. 51 spots were subjected to Matrix-assisted-laser-desorption-ionization Time-of-flight/time-of-flight Mass spectrometry (MALDI-TOF/TOF MS) identification, and 40 spots were identified. Among these, 22 proteins were located in nucleus. These proteins were involved in stress response, cell cycle and division, apoptosis, mRNA processing, transport, splicing and microRNA (miRNA) maturation. The changed expression of Annexin A1 and Lamin B1 were confirmed by Western blot. The role of Annexin A1 in the response to cisplatin-induced DNA damage was further analyzed, and it was shown that after Annexin A1 knockdown, cisplatin-induced DNA damage was significantly increased. In addition, the changed expression of several miRNAs was also observed by quantitative real-time PCR (qRT-PCR). Taken together, these data indicate that cisplatin-induced DDR is a complex process, and that those proteins identified by proteomics can lead to new directions for a better understanding of this process. (C) 2012 Elsevier B.V. All rights reserved.
  • Yuan C H, Filippova M, Tungteakkhun S S, Duerksen-Hughes P J, & Krstenansky J L. (2012). Small molecule inhibitors of the HPV16-E6 interaction with caspase 8. Bioorganic & Medicinal Chemistry Letters, 22(5), 2125-2129. ( 3/2012 - Present ) Link...
    High-risk strains of human papillomaviruses (HPVs) cause nearly all cases of cervical cancer as well as a growing number of head and neck cancers. The oncogenicity of these viruses can be attributed to the activities of their two primary oncoproteins, E6 and E7. The E6 protein has among its functions the ability to prevent apoptosis of infected cells through its binding to FADD and caspase 8. A small molecule library was screened for candidates that could inhibit E6 binding to FADD and caspase 8. Flavonols were found to possess this activity with the rank order of myricetin > morin > quercetin > kaempferol = galangin >> (apigenin, 7-hydroxyflavonol, rhamnetin, isorhamnetin, geraldol, datiscetin, fisetin, 6-hydroxyflavonol). Counter screening, where the ability of these chosen flavonols to inhibit caspase 8 binding to itself was assessed, demonstrated that myricetin, morin and quercetin inhibited GST-E6 and His-caspase 8 binding in a specific manner. The structure-activity relationships suggested by these data are unique and do not match prior reports on flavonols in the literature for a variety of anticancer assays. (C) 2012 Elsevier Ltd. All rights reserved.
  • Yan C L, Chen Z J, Li H R, Zhang G L, Li F, . . . Yang J. (2012). Nuclear proteome analysis of benzo(a)pyrene-treated HeLa cells. Mutation Research-Fundamental and Molecular Mechanisms of Mutagenesis, 731(1-2), 75-84. ( 3/2012 - Present ) Link...
    Previously, we employed a proteomics-based 2-D gel electrophoresis assay to show that exposure to 10 mu M benzo(a)pyrene (BaP) during a 24 h frame can lead to changes in nuclear protein expression and alternative splicing. To further expand our knowledge about the DNA damage response (DDR) induced by BaP, we investigated the nuclear protein expression profiles in HeLa cells treated with different concentrations of BaP (0.1, 1, and 10 mu M) using this proteomics-based 2-D gel electrophoresis assay. We found 125 differentially expressed proteins in BaP-treated cells compared to control cells. Among them, 79 (63.2%) were down-regulated, 46 (36.8%) were up-regulated; 8 showed changes in the 1 mu M and 10 BaP-treated groups, 2 in the 0.1 mu M and 10 mu M BaP-treated groups, 4 in the 0.1 mu M and 1 BaP-treated groups, and only one showed changes in all three groups. Fifty protein spots were chosen for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification, and of these, 39 were identified, including subunits of the 26S proteasome and Annexin A1. The functions of some identified proteins were further examined and the results showed that they might be involved in BaP-induced DDR. Taken together, these data indicate that proteomics is a valuable approach in the study of environmental chemical-host interactions, and the identified proteins could provide new leads for better understanding BaP-induced mutagenesis and carcinogenesis. (C) 2011 Elsevier B.V. All rights reserved.
  • Haynes T A, Filippov V, Filippova M, Yang J, Zhang K, & Duerksen-Hughes P J. (2012). DNA damage induces down-regulation of UDP-glucose ceramide glucosyltransferase, increases ceramide levels and triggers apoptosis in p53-deficient cancer cells. Biochim Biophys Acta, , . ( 2/2012 - Present ) Link...
    DNA damaging agents typically induce an apoptotic cascade in which p53 plays a central role. However, absence of a p53-mediated response does not necessarily abrogate programmed cell death, due to the existence of p53-independent apoptotic pathways, such as those mediated by the pro-apoptotic molecule ceramide. We compared ceramide levels before and after DNA damage in human osteosarcoma (U2OS) and colon cancer (HCT116) cells that were either expressing or deficient in p53. When treated with mitomycin C, p53-deficient cells, but not p53-expressing cells, showed a marked increase in ceramide levels. Microarray analysis of genes involved in ceramide metabolism identified acid ceramidase (ASAH1, up-regulated), ceramide glucosyltransferase (UGCG, down-regulated), and galactosylceramidase (GALC, up-regulated) as the three genes most affected. Experiments employing pharmacological and siRNA agents revealed that inhibition of UGCG is sufficient to increase ceramide levels and induce cell death. When inhibition of UGCG and treatment with mitomycin C were combined, p53-deficient, but not p53-expressing cells, showed a significant increase in cell death, suggesting that the regulation of sphingolipid metabolism could be used to sensitize cells to chemotherapeutic drugs.
  • Filippov V, Song M A, Zhang K, Vinters H V, Tung S, . . . Duerksen-Hughes P J. (2012). Increased Ceramide in Brains with Alzheimer's and other Neurodegenerative Diseases. J Alzheimers Dis, , . ( 1/2012 - Present ) Link...
    Ceramide has been suggested to participate in the neuronal cell death that leads to Alzheimer's disease (AD), but its role is not yet well-understood. We compared the levels of six ceramide subspecies, which differ in the length of their fatty acid moieties, in brains from patients who suffered from AD, other neuropathological disorders, or both. We found elevated levels of Cer16, Cer18, Cer20, and Cer24 in brains from patients with any of the tested neural defects. Moreover, ceramide levels were highest in patients with more than one neuropathologic abnormality. Interestingly, the range of values was higher among brains with neural defects than in controls, suggesting that the regulation of ceramide synthesis is normally under tight control, and that this tight control may be lost during neurodegeneration. These changes, however, did not alter the ratio between the tested ceramide species. To explore the mechanisms underlying this dysregulation, we evaluated the expression of four genes connected to ceramide biosynthesis: ASMase, NSMase 2, GALC, and UGCG. The patterns of gene expression were complex, but overall, ASMase, NSMase 2, and GALC were upregulated in specimens from patients with neuropathologic abnormalities in comparison with age-matched controls. Such findings suggest these genes as attractive candidates both for diagnostic purposes and for intervening in neurodegenerative processes.
  • Whitaker E L, Filippov V, Filippova M, Guerrero-Juarez C F, & Duerksen-Hughes P J. (2011). Splice variants of mda-7/IL-24 differentially affect survival and induce apoptosis in U2OS cells. Cytokine, 56(2), 272-281. ( 11/2011 - Present ) Link...
    Interleukin-24 (mda-7/IL-24) is a cytokine in the IL-10 family that has received a great deal of attention for its properties as a tumor suppressor and as a potential treatment for cancer. In this study, we have identified and characterized five alternatively spliced isoforms of this gene. Several, but not all of these isoforms induce apoptosis in the osteosarcoma cell line U2OS, while none affect the survival of the non-cancerous NOK cell line. One of these isoforms, lacking three exons and encoding the N-terminal end of the mda-7/IL-24 protein sequence, caused levels of apoptosis that were higher than those caused by the full-length mda-7/IL-24 variant. Additionally, we found that the ratio of isoform expression can be modified by the splice factor SRp55. This regulation suggests that alternative splicing of mda-7/IL-24 is under tight control in the cell, and can be modified under various cellular conditions, such as DNA damage. In addition to providing new insights into the function of an important tumor suppressor gene, these findings may also point toward new avenues for cancer treatment. (C) 2011 Elsevier Ltd. All rights reserved.
  • Zhang K L, Haynes T A S, Filippova M, Filippov V, & Duerksen-Hughes P J. (2011). Quantification of ceramide levels in mammalian cells by high performance liquid chromatography coupled to tandem mass spectrometry with multiple-reaction-monitoring mode (HPLC-MS/MS-MRM). Analytical Methods, 3(5), 1193-1197. ( 5/2011 - Present ) Link...
    Ceramides play an important role in a variety of cellular functions including cell differentiation and apoptosis, responses to DNA damage and stress, and transcriptional events. Detection of ceramides in mammalian cells is required for many biological studies. Here, we report a validated method using LC-MS/MS-MRM on an Agilent 6410 triple quadrupole mass spectrometer to simultaneously quantify six ceramides extracted from mammalian cell lysates following a 5 min HPLC gradient. This method demonstrated outstanding sensitivity, accuracy, reproducibility, and speed of analysis.
  • Mujumdar P P, Duerksen-Hughes P J, Firek A F, & Hessinger D A. (2011). Long-term, progressive, aerobic training increases adiponectin in middle-aged, overweight, untrained males and females. Scandinavian Journal of Clinical & Laboratory Investigation, 71(2), 101-107. ( 4/2011 - Present ) Link...
    Adipose tissue secretes the adipokine, adiponectin (ADPN), which increases insulin sensitivity. Because some of the metabolic effects of exercise and ADPN are similar, exercise has been proposed to increase ADPN. However, most short-term (<= 3 mos) and constant-effort exercise protocols have not produced increases in ADPN. Furthermore, no direct comparisons of male and female subjects on the effect of exercise on ADPN levels have been reported. We hypothesized that long-term (6 mos), progressive training would increase ADPN levels in both males and females. We recruited middle-aged, untrained males and females to participate in an interventional study employing a marathon training regimen progressing from 9.7 to 88.5 km (6 to 55 miles) per week over 6 mos. At baseline, we matched the mean ages of the male and female groups. We collected and stored fasting plasma samples and recorded body measurements at 0 (baseline) and 6 mos. Stored samples were analysed for insulin, glucose, and ADPN. ADPN increased significantly among both males (from 5.89 +/- 2.46 (mean +/- SD) to 7.65 +/- 3.18 mu g/ml; p < 0.05) and females (from 8.48 +/- 3.22 to 10.56 +/- 4.05 mu g/ml; p < 0.05). The extent of the increase in ADPN was similar in the male (40.7 +/- 50%; median, 12.1%) and female (27.0 +/- 31.1%; median, 22.3%) groups. However, there was no significant reduction in insulin resistance as measured by the HOMA-IR scores in either group. We conclude that long-term, progressive aerobic training increases circulating ADPN levels in middle-aged, untrained males and females.
  • Xiong L, Darwanto A, Sharma S, Herring J, Hu S Y, . . . Zhang K L. (2011). Mass Spectrometric Studies on Epigenetic Interaction Networks in Cell Differentiation. Journal of Biological Chemistry, 286(15), 13657-13668. ( 4/2011 - Present ) Link...
    Arrest of cell differentiation is one of the leading causes of leukemia and other cancers. Induction of cell differentiation using pharmaceutical agents has been clinically attempted for the treatment of these cancers. Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol-12-myristate 13-acetate-induced differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin-coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were up-regulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down-regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down-regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase II-associated FACT-RTF1-PAF1 complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation.
  • Williams V M, Filippova M, Soto U, & Duerksen-Hughes P J. (2011). HPV-DNA integration and carcinogenesis: putative roles for inflammation and oxidative stress. Future Virology, 6(1), 45-57. ( 1/2011 - Present ) Link...
    HPV-DNA integration into cellular chromatin is usually a necessary event in the pathogenesis of HPV-related cancer; however, the mechanism of integration has not been clearly defined. Breaks must be created in both the host DNA and in the circular viral episome for integration to occur, and studies have shown that viral integration is indeed increased by the induction of DNA double strand breaks. Inflammation generates reactive oxygen species, which in turn have the potential to create such DNA strand breaks. It is plausible that these breaks enable a greater frequency of HPV-DNA integration, and in this way contribute to carcinogenesis. Consistent with this idea, co-infections with certain sexually transmitted diseases cause cervical inflammation, and have also been identified as cofactors in the progression to cervical cancer. This article examines the idea that inflammation facilitates HPV-DNA integration into cellular chromatin through the generation of reactive oxygen species, thereby contributing to carcinogenesis.
  • Lei Xiong, Darwanto Agus, Sharma Seema, Herring Jason, Shaoyan Hu, . . . Kangling Zhang. (2011). Mass Spectrometric Studies on Epigenetic Interaction Networks in Cell Differentiation. Journal of Biological Chemistry, 286(15), 13657-13668. ( 0/2011 - Present ) Link...
    Arrest of cell differentiation is one of the leading causes of leukemia and other cancers. Induction of cell differentiation using pharmaceutical agents has been clinically attempted for the treatment of these cancers. Epigenetic regulation may be one of the underlying molecular mechanisms controlling cell proliferation or differentiation. Here, we report on the use of proteomics-based differential protein expression analysis in conjunction with quantification of histone modifications to decipher the interconnections among epigenetic modifications, their modifying enzymes or mediators, and changes in the associated pathways/networks that occur during cell differentiation. During phorbol-12-myristate 13-acetate-indu:ed differentiation of U937 cells, fatty acid synthesis and its metabolic processing, the clathrin-coated pit endocytosis pathway, and the ubiquitin/26 S proteasome degradation pathways were up-regulated. In addition, global histone H3/H4 acetylation and H2B ubiquitination were down-regulated concomitantly with impaired chromatin remodeling machinery, RNA polymerase II complexes, and DNA replication. Differential protein expression analysis established the networks linking histone hypoacetylation to the down-regulated expression/activity of p300 and linking histone H2B ubiquitination to the RNA polymerase Il-associated FACT-RTF1PAFI complex. Collectively, our approach has provided an unprecedentedly systemic set of insights into the role of epigenetic regulation in leukemia cell differentiation. [ABSTRACT FROM AUTHOR] Copyright of Journal of Biological Chemistry is the property of American Society for Biochemistry & Molecular Biology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
  • Yan Chunlan, Lu Jing, Zhang Guanglin, Gan Tieer, Zeng Qunli, . . . Yang Jun. (2011). Benzo[a]pyrene induces complex H2AX phosphorylation patterns by multiple kinases including ATM, ATR, and DNA-PK. Toxicology in Vitro, 25(1), 91-99. ( 0/2011 - Present ) Link...
    Abstract: H2AX is phosphorylated (γH2AX) by members of the phosphatidylinositol 3-kinase (PI3K) family, including ataxia telangiectasia-mutated (ATM), ATM- and Rad3-related (ATR) and DNA-PK in response to DNA damage. While it has been reported that benzo[a]pyrene (BaP) cannot induce γH2AX alone in several cell lines, we have shown that BaP alone could induce γH2AX in human amnion FL cells. Thus, we further examined the ability of BaP to induce γH2AX in different cell systems. It was shown that BaP-induced γH2AX in HeLa cells in a time- and dose-dependent manner. BaP also induced γH2AX in ATM-/- mouse fibroblasts, DNA-PKcs-/- mouse fibroblasts, and a genetically modified human osteosarcoma U2OS cell line. PI3K inhibitors caffeine and wortmannin were then used in an effort to identify the kinase(s) responsible for BaP-induced γH2AX. Unexpectedly, in BaP-treated HeLa cells, caffeine pretreatment did not inhibit but rather increased γH2AX level. On the other hand, caffeine or wortmannin can inhibit BaP-induced γH2AX in either U2OS, DNA-PKcs-/- or ATM-/- cells. Taken together, these data suggest that BaP alone can induce H2AX phosphorylation in certain cell systems, and that members of the PI3K family, including ATM, ATR, and DNA-PK can participate in the phosphorylation of H2AX in the various cell types. [Copyright &y& Elsevier] Copyright of Toxicology in Vitro is the property of Pergamon Press - An Imprint of Elsevier Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
  • Tungteakkhun S S, Filippova M, Fodor N, & Duerksen-Hughes P J. (2010). The Full-Length Isoform of Human Papillomavirus 16 E6 and Its Splice Variant E6*Bind to Different Sites on the Procaspase 8 Death Effector Domain. Journal of Virology, 84(3), 1453-1463. ( 2/2010 - Present ) Link...
    Human papillomavirus 16 is a causative agent of most cases of cervical cancer and has also been implicated in the development of some head and neck cancers. The early viral E6 gene codes for two alternatively spliced isoforms, E6(large) and E6*. We have previously demonstrated the differential effects of E6(large) and E6* binding on the expression and stability of procaspase 8, a key mediator of the apoptotic pathway. Additionally, we have reported that E6 binds to the FADD death effector domain (DED) at a novel E6 binding domain. Sequence similarities between the FADD and procaspase 8 DEDs suggested a specific region for E6(large)/procaspase 8 binding, which was subsequently confirmed by mutational analysis as well as by the ability of peptides capable of blocking E6/FADD binding to also block E6(large)/caspase 8 binding. However, the binding of the smaller isoform, E6*, to procaspase 8 occurs at a different region, as deletion and point mutations that disrupt E6(large)/caspase 8 DED binding do not disrupt E6*/caspase 8 DED binding. In addition, peptide inhibitors that can block E6(large)/procaspase 8 binding do not affect the binding of E6* to procaspase 8. These results demonstrate that the residues that mediate E6*/procaspase 8 DED binding localize to a different region on the protein and employ a separate binding motif. This provides a molecular explanation for our initial findings that the two E6 isoforms affect procaspase 8 stability in an opposing manner.
  • Wu Wei, Yan Chunlan, Gan Tieer, Chen Zhanghui, Lu Xianghong, . . . Yang Jun. (2010). Nuclear proteome analysis of cisplatin-treated HeLa cells. Mutation Research: Fundamental & Molecular Mechanisms of Mutagenesis, 691(1/2), 1-8. ( 0/2010 - Present ) Link...
    Abstract: Cisplatin has been widely accepted as one of the most efficient anticancer drugs for decades. However, the mechanisms for the cytotoxic effects of cisplatin are still not fully understood. Cisplatin primarily targets DNA, resulting in the formation of DNA double strand breaks and eventually causing cell death. In this study, we applied two-dimensional electrophoresis coupled with LC-MS/MS to analyze the nuclear proteome of HeLa cells treated with cisplatin, in an effort to uncover new mechanistic clues regarding the cellular response to cisplatin. A total of 19 proteins were successfully identified, and these proteins are involved in a variety of basal metabolic and biological processes in cells, including biosynthesis, cell cycle, glycolysis and apoptosis. Six were related to the regulation of mRNA splicing, and we therefore asked whether the Fas gene might undergo alternative splicing following cisplatin treatment. This proved to be the case, as the splicing forms of Fas were modified in cisplatin-treated HeLa cells. This work provides novel information, from the perspective of the nuclear response, for understanding the cytotoxicity caused by cisplatin-induced DNA damage. [Copyright &y& Elsevier] Copyright of Mutation Research: Fundamental & Molecular Mechanisms of Mutagenesis is the property of Elsevier Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
  • Yan Chunlan, Wu Wei, Li Haiyan, Zhang Guanglin, Duerksen-Hughes Penelope J, Zhu Xinqiang, & Yang Jun. (2010). Benzo[a]pyrene treatment leads to changes in nuclear protein expression and alternative splicing. Mutation Research: Fundamental & Molecular Mechanisms of Mutagenesis, 686(1/2), 47-56. ( 0/2010 - Present ) Link...
    Abstract: Benzo[a]pyrene (BaP) is a potent pro-carcinogen generated from the combustion of fossil fuel and cigarette smoke. Previously, using a proteomic approach, we have shown that BaP can induce changes in the expression of many cellular proteins, including transcription regulators. In the present study, using a similar approach, we examined the nuclear protein response to BaP in HeLa cells and found that BaP treatment caused expression changes in many nuclear proteins. Twenty-four of these proteins were successfully identified, several of which are involved in the alternative splicing of mRNA, DNA replication, recombination, and repair. The changed expression levels were further confirmed by immunoblot analysis using specific antibodies for two proteins, Lamin A and mitotic checkpoint protein Bub3. The nuclear localization of these two proteins was also confirmed by confocal microscopy. To determine whether alternative splicing was activated following BaP treatment, we examined Fas and CD44, two genes previously shown to be targets of alternative splicing in respond to DNA damage. While no significant activation of alternative splicing was observed for Fas, CD44 splicing variants were found after BaP treatment. Together, these data show that DNA damage induces dramatic changes in nuclear protein expression, and that alternative splicing might be involved in the cellular response to DNA damage. [Copyright &y& Elsevier] Copyright of Mutation Research: Fundamental & Molecular Mechanisms of Mutagenesis is the property of Elsevier Science and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
  • Filippova M, Filippov V A, Kagoda M, Garnett T, Fodor N, & Duerksen-Hughes P J. (2009). Complexes of Human Papillomavirus Type 16 E6 Proteins Form Pseudo-Death-Inducing Signaling Complex Structures during Tumor Necrosis Factor-Mediated Apoptosis. Journal of Virology, 83(1), 210-227. ( 1/2009 - Present ) Link...
    High-risk strains of human papillomavirus (HPV) such as HPV type 16 (HPV16) and HPV18 are causative agents of most human cervical carcinomas. E6, one of the oncogenes encoded by HPV16, possesses a number of biological and transforming functions. We have previously shown that the binding of E6 to host apoptotic proteins such as tumor necrosis factor (TNF) R1, the adaptor protein FADD, and procaspase 8 results in a significant modification of the normal flow of apoptotic events. For example, E6 can bind to and accelerate the degradation of FADD. In addition, full-length E6 binds to the TNF R1 death domain and can also bind to and accelerate the degradation of procaspase 8. In contrast, the binding of small splice isoforms known as E6* results in the stabilization of procaspase 8. In this report, we propose a model for the ability of HPV16 E6 to both sensitize and protect cells from TNF as well as to protect cells from Fas. We demonstrate that both the level of E6 expression and the ratio between full-length E6 and E6* are important factors in the modification of the host extrinsic apoptotic pathways and show that at high levels of E6 expression, the further sensitization of U2OS, NOK, and Ca Ski cells to TNF-mediated apoptosis is most likely due to the formation of a pseudo-death-inducing signaling complex structure that includes complexes of E6 proteins.
  Scholarly Journals--Accepted
  • Hsu, H. W., R. de Necochea-Campion, V. Williams, P. J. Duerksen-Hughes, A. A. Simental, Jr., R. L. Ferris, C. S. Chen and S. Mirshahidi. Linifanib (ABT-869) enhances cytotoxicity with poly (ADP-ribose) polymerase inhibitor, veliparib (ABT-888), in head and neck carcinoma cells. Oral Oncol. In Press, 2014. PMID: 24735547.

    ( 4/2014 )
  • Ju, L., G. Zhang, C. Zhang, L. Sun, Y. Jiang, C. Yan, P. J. Duerksen-Hughes, X. Zhang, X. Zhu, F. F. Chen and J. Yang. Quantum dot-related genotoxicity perturbation can be attenuated by PEG encapsulation. Mutation Research – Genetic Toxicology and Environmental Mutagenesis. In Press, 2013.

    ( 7/2012 - 6/2013 )
  Books and Chapters
  • W. Evans, M. Filippova, R. Swensen and P. Duerksen-Hughes. Modern Molecular and Clinical Approaches to Eradicate HPV-Mediated Cervical Cancer, Chapter 11,  Human Papillomavirus and Related Diseases from Bench to Bedside: A Diagnostic and Preventative Perspective. D. Vanden Broeck, editor; ISBN 978-953-51-1072-9, 2013.

    ( 7/2012 - 6/2013 )
  Abstract
  • Zhang K L, Filippova M, Filippov V, & Duerksen-Hughes P J. (2012). Epigenetic Regulation in Leukemia Cell Differentiation. Faseb Journal, 26, . ( 4/2012 - Present )
  • Mujumdar P, Duerksen-Hughes P, Firek A, & Hessinger D. (2011). Increases in Adiponectin Levels due to Aerobic Training depend on sex and ethnicity. Faseb Journal, 25, . ( 4/2011 - Present )