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

Abstract

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.

Characterization of Naturally Occurring HPV16 Integration Sites Isolated from Cervical Keratinocytes under Noncompetitive Conditions

“…In the first group (n = 8), the host sequence directly adjoined the viral sequence with no homology, suggesting a role for nonhomologous end joining (NHEJ) in the integration process (Table 2). In the second group (n = 7), regions of microhomology (1-10 bp) were seen between the host and viral sequences, suggesting microhomology-mediated NHEJ (6,27,28), whereas in the third group (n = 9), stretches of unrecognized ''orphan'' DNA (1-36 bp) were present between the host and viral sequences.…”

“…S1). Although we could not exclude that these other integrants were present in an extremely small number of cells in the starting population, we considered it more likely that the relevant integration events occurred de novo after cell cloning in a background of E2-expressing episomes, as has been observed previously in W12 (6).…”

“…We also determined the nearest host gene [coding gene, pseudogene, or microRNAs (miR)] to each integration site in the direction of transcription using the Ensembl BIOMART function 5 and the miR-base website. 6 Twenty of the 24 different integration events (83%) occurred within the same chromosomal band as a cancer-associated gene, which included C-MYC, TP73L(TP63), MAPK10, MYCN, RASSF6, and MDM2 (Table 1). However, the cancer-associated genes were generally a considerable distance from the integration site (median, 2,379 kb; range, 0-12,343 kb) and often in the opposite orientation to that of transcription from the viral promoter.…”

“…Cervical neoplasms are clonal, usually with a single integration site (5), suggesting that particular integration events are selected during carcinogenesis. Mechanisms of integration are not well understood, but the process most likely involves double-strand breaks (DSB) in viral and host genomes, followed by DNA ligation by host proteins (6).…”

Hepatitis B Surface Antigen-based Vaccines for Human Neoplastic Disease

“…The mechanisms behind HPV integration are unknown and it appears to occur at random sites within the host's genome (Winder et al, 2007), however the integrants do have a predisposition for insertion into chromosome bands coding cancer associated genes or microRNAs (Dall et al, 2008). A review examining a number of studies concluded that of the 192 integrants studied, 38% were found within known common fragile sites (CFSs) but more recent studies indicate that CFSs and integration events did not appear to co-localise (Dall et al, 2008).…”