Human papillomavirus type 16 E6-enhanced susceptibility of L929 cells to tumor necrosis factor alpha correlates with increased accumulation of reactive oxygen species.
Human papillomavirus type 16 (HPV-16) E6 has been shown to prevent or enhance apoptosis depending on the stimulus and cell type. Here we present evidence that HPV-16 E6 sensitized murine fibrosarcoma L929 cells to tumor necrosis factor alpha (TNF)-induced cytolysis. The E6-enhanced cytolysis correlated with a precedent increase in reactive oxygen species (ROS) level and antioxidant treatment could completely block the E6-dependent sensitization. These findings represent the first demonstration of a link between a viral oncogene-sensitized cytolysis and ROS. Previous studies have shown conflicting results regarding whether TNF-induced cytolysis of L929 cells is through necrosis or apoptosis. Here we report that, although L929 cells underwent DNA fragmentation after exposure to TNF, they retained the morphology of intact nuclei while gaining permeability to propidium iodide, features characteristic of necrosis rather than apoptosis. We confirmed that the broad spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone markedly increased the susceptibility of L929 cells to TNF, and further demonstrated that E6 enhanced this susceptibility, which again correlated with increased ROS accumulation. We showed that the expression of E6 in L929 cells did not alter the stability of p53, and the cells retained a p53 response to actinomycin D. Furthermore, two E6 mutants defective for p53 degradation in other systems exhibited differential effects on TNF sensitization. These results suggest that the enhancement of TNF-induced L929 cytolysis by E6 is independent of p53 degradation. We also found that TNF-induced activation of NF-kappaB did not account for the enhanced TNF susceptibility by E6.
Liu
Y
Y
Department of Dermatology, New England Medical Center and Tufts University School of Medicine, Boston, Massachusetts 02111, USA. yliu1@opa1.tufts.edu
Tergaonkar
V
V
Krishna
S
S
Androphy
E J
EJ
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D005434:Flow Cytometry; D017930:Genes, Reporter; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008856:Microscopy, Fluorescence; D016328:NF-kappa B / Q000235:genetics; D009336:Necrosis; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012097:Repressor Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
D000590:Amino Acid Chloromethyl Ketones; C068651:E6 protein, Human papillomavirus type 16; D016328:NF-kappa B; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha; D016159:Tumor Suppressor Protein p53; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone