Examining the biological consequences of DNA damage caused by irradiated J2-3T3 fibroblast feeder cells and HPV16: characterisation of the biological functions of Mll.
Doctoral thesis, UCL (University College London).
The study of human papillomavirus (HPV) utilises human keratinocytes as host cells that are co-cultured with lethally irradiated mouse fibroblasts. Although this co-culture system has been effective in recapitulating HPV’s life cycle, it resembles studies whereby radiation-induced bystander effect (RIBE) has been identified. We considered the possibility that Normal Immortalised Keratinocytes (NIKs) that we used as host cell for HPV16 may be in receipt of DNA damaging agents from heavily irradiated J2-3T3 fibroblasts. We report that NIKs (+/ - HPV16) cells respond to the presence of feeders by increasing DNA damage response leading to H2AX phosphorylation. NIKs receive DNA damaging agents from irradiated feeder cells primarily through direct cell-to-cell contact and secondarily through the culture media. Although ATM, ATR, and DNA-PK were activated by RIBE in NIKs, we found that feeders inhibited ATM activation in NIKs (+HPV16). Moreover, we found that expression of HPV16 E6 and E7 oncoproteins activated ATM, ATR, and DNA-PK. HPV16 also activated H2AX, 53BP1, RPA32, and NBS-1. We considered that both feeder cells and HPV16 could induce DNA damage response in NIKs. This would change the interpretations of HPV16’s interactions with host cell’s DNA damage surveillance system in NIKs (+ HPV16) cells co-cultured with feeders. We proceeded to enhance our understanding of how feeder cells and/ or HPV16 changed the way NIKs cells respond to direct DNA damage. We found that NIKs cells (+feeders or +HPV16) respond to direct gamma radiation in a broadly similar way, whereby activation of ATM was inhibited, increase in ATR was extremely small and delayed, while activation of DNA-PK was still generally responsive. We report that presence of HPV16 sensitised NIKs cells to direct gamma radiation and as such, the implications of these observations could be considered for radiotherapy of cancers with HPV. Our initial belief that feeder cells were essential in ensuring stable autonomous replication of HPV16 in NIKs cells was proven otherwise, as NIKs maintained HPV16 DNA episomal copies in the absence of feeders. Contrary to current accepted publication, ATM was found be unnecessary for maintenance of HPV16 DNA episomes and viral genome amplification.
|Title:||Examining the biological consequences of DNA damage caused by irradiated J2-3T3 fibroblast feeder cells and HPV16: characterisation of the biological functions of Mll|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Medical Sciences > Infection and Immunity (Division of)|
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