An increase in DNA double-strand breaks, induced by Ku70 depletion, is associated with human papillomavirus 16 episome loss and de novo viral integration events.
Integration of human papillomavirus type 16 (HPV16) is a common event in cervical carcinogenesis, although mechanisms of integration are poorly understood. We have tested the hypothesis that an increased number of DNA double-strand breaks (DSBs) affect HPV16 episome maintenance and integration in cervical keratinocytes. Increased DSBs were generated over prolonged periods of up to 50 population doublings in the unique polyclonal cervical keratinocyte cell line W12, which stably maintains HPV16 episomes. This was achieved using repeated treatments with short interfering RNA to obtain sustained depletion of Ku70, a key mediator of DNA non-homologous end joining. An increase in DSBs was seen shortly after commencement of Ku70 depletion. Continuous depletion was reproducibly associated with loss of HPV16 episomes and also with a new viral integration event, which was rapidly selected in outgrowing W12 cells. Despite the prolonged presence of DSBs, high-level chromosomal instability (detected by marked changes in genomic copy number) was not observed until cells containing the new integrant were almost fully selected, with no evidence of such chromosomal instability prior to integration. Our data show that increased DNA DSBs are associated with HPV16 episomal loss and integration in cervical keratinocytes. We found no evidence to support the notion that major chromosomal instability precedes HPV16 integration, although such instability is an important consequence of the integration event.
Winder
D M
DM
Medical Research Council Cancer Cell Unit, MRC/Hutchison Research Centre, Hills Road, Cambridge, CB2 0XZ, UK.
Pett
M R
MR
Foster
N
N
Shivji
M K K
MK
Herdman
M T
MT
Stanley
M A
MA
Venkitaraman
A R
AR
Coleman
N
N
D034961:Antigens, Nuclear / Q000235:genetics*; D001483:Base Sequence; D045744:Cell Line, Tumor; D043171:Chromosomal Instability; D053903:DNA Breaks, Double-Stranded; D004279:DNA, Viral / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D017353:Gene Deletion; D016679:Genome, Viral; D052162:Human papillomavirus 16 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D017403:In Situ Hybridization; D000072200:Ku Autoantigen; D008969:Molecular Sequence Data; D030361:Papillomavirus Infections / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000008:administration & dosage; D015183:Restriction Mapping; D002583:Uterine Cervical Neoplasms / Q000235:genetics / Q000821:virology; D016662:Virus Integration / Q000502:physiology*; D002578:Uterine Cervical Dysplasia / Q000235:genetics / Q000821:virology
D034961:Antigens, Nuclear; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen