Generalidades de las materias primas de la cerveza

To investigate whether HPV11 E1–E2 mediated DNA replication is arrested following DNA damage, transient DNA replication assays were carried out in 293T cells using the real- time PCR protocol developed in previously in our laboratory (Taylor and Morgan, 2003; Morgan and Taylor, 2005). Initially this protocol was used with C33A and U2OS cells however the combination of calcium phosphate transfection and drug treatment resulted in high amounts of cell death. Although 293T cells are not a cervical epithelial cell line they were chosen due to their high transfectability of the viral protein expressing plasmids and ability to withstand subsequent drug treatment. Briefly, plasmids containing HPV11 E1, HPV11 E2 and the HPV16 origin of replication were transfected into 293T cells using the calcium phosphate method of transfection (2.2.1.2). In these experiments, the pOri-16M (designed for HPV16 replication) can be used to investigate HPV11 E1-E2- mediated replication because the origin sequences are well conserved in mucosal HPVs. Cells were then treated with a DNA damaging agent or aphidicolin. In separate experiments etoposide and camptothecin were used as DNA damaging agents. Etoposide inhibits topoisomerase IIa, resulting in double strand DNA (dsDNA) breaks during the S phase of the cell cycle. To create DNA damage a relatively high concentration of etoposide was used (50μM). This concentration has been shown to produce dsDNA breaks in 293T cells (Abramson et al., 2010). Camptothecin inhibits the DNA enzyme topoisomerase I and creates dsDNA breaks. It was used at 10μM as this concentration has previously been shown to cause dsDNA breaks and is tolerated by 293T (Reinhardt et al., 2007; Fu et al., 2007). Cells were also treated with Aphidicolin (2.5μg/ml) as a control. Aphidicolin inhibits the replicative DNA polymerase (alpha/delta/epsilon) inhibitor (Ikegami et al, 1978). Therefore aphidicolin treatment should arrest both cellular and viral (HPV and SV40) DNA replication. A previous publication had used aphidicolin at a concentration of 5μg/ml to arrest U2OS cells that stabley express HPV16 E2 (Johansson et al., 2009). Concentrations slightly above and below this value were tested and it was found that 2.5μg/ml could arrest HPV replication therefore this was the concentration used in these assays. Forty eight hours later the cells were lysed and DNA extracted using HIRT extraction (2.2.2.22). DNA was purified by phenol.chloroform extraction (2.2.2.1) and ethanol precipitation (2.2.2.2). Digesting DNA with DpnI and ExoIII resulted in freshly

92 replicated origin plasmid DNA. The E.coli strains used for propagation of viral plasmid DNA cause methylation of the plasmid DNA using DAM methyltransferase. The Dpn1 digests DAM-methylated (non-replicated) DNA and ExoIII digests single stranded DNA overhangs. The amount of freshly replicated DNA which was then measured using Real Time PCR (2.2.2.4).

The results obtained with HPV11 E1 and E2 replication in vivo are shown in Figure 3.3.1. In Figure 3.1.1.(a), origin plasmid replication is not detected in the non-transfected cells (lane 1), HPV origin plasmid transfected alone (lane 2) and HPV origin plasmid with E1 expression plasmid (lane 3). Replication of the origin plasmid is detected when the E1 and E2 expression plasmid are co-transfected (lane 4). After treatment with the DNA polymerase inhibitor aphidicolin, this replication signal is greatly reduced. The small level of replication detected may be a result of origin plasmid replication prior to drug treatment (lane 6). There is a statistical difference between untreated and aphidicolin treated cells. When etoposide is added to the HPV plasmid transfected cells, there is an apparent increase in origin plasmid DNA replication (lane 5). However, there is no significant difference between HPV11 ori replication with and without etoposide treatment. In Figure 3.1.1 (b) again no DNA replication signal is detected in non- transfected cells or when a plasmid containing the HPV origin is transfected alone or with an E1 expression plasmid (lanes 1–3). DNA replication is detected when the E2 expression plasmid is also co transfected (lane 4). When the cells are treated with aphidicolin following transfection there is a minimal level of replicated origin plasmid (lane 6). As p<0.05, there is a statistical difference between untreated HPV11 E1-E2-ori transfected and aphidicolin treated cells When the transfected cells are treated with camptothecin HPV11 E1-E2 mediated replication of the origin is not inhibited (lane 5). As p>0.05, there is no statistical difference between HPV11 E1-E2-ori transfected untreated and camptothecin treated cells. This result may be ascribed to one of the repeats showing an exceptionally large increase in origin replication when treated with camptothecin.

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Figure 3.1.1 HPV11 E1-E2 mediated replication in vivo is not arrested by DNA damaging agents.

(a) HPV11 origin replication assay with etoposide treatment

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(b) HPV11 origin replication assay with camptothecin treatment

Figure3.1.1 (a) HPV11 E1-E2 mediated replication is not arrested by etoposide in vivo.

293T cells were transfected with 100pg of pOriM (lanes 2–6), 5μg of pCMV11-E1 (lanes 3– 6) and 2μg of pCMV11-E2 (lanes 4–6). 16 hours after transfection, cells were left untreated (lanes 1–4) or treated with 50µM of etoposide (lane 5) or 2.5 μg/ml of aphidicolin (lane 6). Forty-eight hours later low molecular weight DNA was harvested from the cells for transient replication assays. The results shown represent the summary of three experiments with mean and standard error of the mean shown. Results are shown as a fold difference (F.D) to lane 4 (ori, E1 &E2 with no drug treatment). Statistical significance by Student’s t-test is indicated as * (p < 0.05). (b) HPV11 E1-E2 mediated

replication is not arrested by camptothecin in vivo. The replication assay was carried out

as described in figure 3.1.1(a). 16 hours after transfection cells were treated with 10 µM of camptothecin (lane 5) instead of etoposide. The results shown represent the summary of three experiments with mean and standard error of the mean shown. Lane 4 is standardised to 1 and the results shown are a fold difference of this value. Statistical significance by Student’s t-test is indicated as * (p < 0.05).

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