Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53.
After treatment with ultraviolet radiation (UV), human fibroblasts that express the HPV type 16 E6 oncoprotein display defects in repair of cyclobutane pyrimidine dimers, hypersensitivity to inactivation of clonogenic survival and an inability to sustain DNA replication. To determine whether these effects are specific to depletion of p53 or inactivation of its function, fibroblast lines were constructed with ectopic expression of a dominant-negative p53 allele (p53-H179Q) to inactivate function or a short-hairpin RNA (p53-RNAi) to deplete expression of p53. Only the expression of HPV16E6 sensitized fibroblasts to UV or the chemical carcinogen, benzo[a]pyrene diolepoxide I (BPDE). Carcinogen-treated cells expressing p53-H179Q or p53-RNAi were resistant to inactivation of colony formation and did not suffer replication arrest. CHK1 is a key checkpoint kinase in the response to carcinogen-induced DNA damage. Control and p53-RNAi-expressing fibroblasts displayed phosphorylation of Ser345 on CHK1 45-120 min after carcinogen treatment with a return to near baseline phosphorylation by 6 h after treatment. HPV16E6-expressing fibroblasts displayed enhanced and sustained phosphorylation of CHK1. This was associated with enhanced phosphorylation of Thr68 on CHK2 and Ser139 on H2AX, both markers of severe replication stress and DNA double strand breaks. Incubation with the phosphatase inhibitor okadaic acid produced more phosphorylation of CHK1 in UV-treated HPV16E6-expressing cells than in p53-H179Q-expressing cells suggesting that HPV16E6 may interfere with the recovery of coupled DNA replication at replication forks that are stalled at [6-4]pyrimidine-pyrimidone photoproducts and BPDE-DNA adducts. The results indicate that HPV16E6 targets a protein or proteins other than p53 to deregulate the activity of CHK1 in carcinogen-damaged cells.
Department of Nutrition Health and Food Safety, School of Public Health, Fudan University, Shanghai, China.
Bo
B
Chen
Dennis A
DA
Simpson
Yingchun
Y
Zhou
Amritava
A
Mitra
David L
DL
Mitchell
Marila
M
Cordeiro-Stone
William K
WK
Kaufmann
D001580:Benzopyrenes / Q000494:pharmacology; D002273:Carcinogens / Q000494:pharmacology*; D000071877:Checkpoint Kinase 1; D018736:DNA Adducts / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D005347:Fibroblasts / Q000187:drug effects*; D006801:Humans; D019319:Okadaic Acid / Q000494:pharmacology; D009856:Oncogene Proteins, Viral / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D034622:RNA Interference; D012097:Repressor Proteins / Q000378:metabolism*; D015398:Signal Transduction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D014466:Ultraviolet Rays
D001580:Benzopyrenes; D002273:Carcinogens; D018736:DNA Adducts; C068651:E6 protein, Human papillomavirus type 16; D009856:Oncogene Proteins, Viral; D012097:Repressor Proteins; D016159:Tumor Suppressor Protein p53; D019319:Okadaic Acid; C072019:benzo(a)pyrene diolepoxide I; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1