Características nutricionales del calafate

The in vitro results in Figure 3.1.4 suggest that ATR is responsible for regulating LT replication. This result was extended in the in vivo co-immunoprecipitation assay where LT was shown to be targeted for phosphorylation by ATR/ATM in response to etoposide treatment (Figure 3.1.5). Attempts were made to generate protein extracts lacking in ATR activity. The ATR-deficient extract could then be used during in vitro replication studies to investigate LT-mediated replication during DNA damage without ATR activation. This would validate the hypothesis that ATR is the protein kinase that regulates LT mediated replication. Two proteins that were targeted for knockdown were ATR and TopBP1. TopBP1 plays the role of a sensor protein in the DNA damage response that is essential for ATR activation (Kumagai et al., 2006). TopBP1 coordinates activation of the DNA damage checkpoint response by coupling the interaction of the 9-1-1 checkpoint clamp at sites of ssDNA. In return this activates the ATR-ATRIP checkpoint kinase complex (Chapter 1.9.2). The activation of ATR triggers a cascade of phosphorylation events which ultimately leads to cell cycle arrest. Owing to the fact that previous studies have demonstrated the essential nature of ATR (making it unlikely to generate sufficient amounts of ATR depleted cell extract for replication assays) the dual approach of TopBP1

124 and ATR knockdown offered a greater chance of success. The phenotype of TopBP1 depleted cells is well defined.

Oligonucleotides were designed against ATR and TopBP1 (siATR and siTopBP1). The siRNA oligonucleotides were transfected using the Lipofectamine 2000 protocol (2.2.1.4) into 293T cells that were harvested forty eight hours later for western blot analysis (Figure 3.2.1 (a)). Levels of knockdown were measured by probing the membranes for ATR and TopBP1. In lane 1 and 4, 293T cells were mock transfected. A control oligonucleotide (siLuciferase) was transfected into the cells in order to control for any protein knockdown due to the presence of an oligonucleotide targeting a non essential gene (lanes 2 and 3). In lane 3, ATR protein levels have greatly reduced confirming the knockdown by the siATR oligonucleotide. TopBP1 protein levels are also significantly reduced after transfection with siTopBP1 (lane 6). Transfecting siRNA oligonucleotides into cells can cause off target effects such as the silencing of non specific target genes and reduced protein levels (Jackson et al., 2003; Snove & Holen, 2004; Jackson & Linsley, 2004). Studies have demonstrated that the knock down of TopBP1 reduces the activation of ATR (Kumagai et al., 2006; Pedram et al., 2009). However due to the off target effects or siRNA oligonucleotides, ATR and TopBP1 protein levels should have been determined after knockdown of TopBP1 and ATR (respectively). This would have been useful to determine whether knock down of TopBP1 could reduce DNA damage signalling independently from ATR.

The effect of ATR and TopBP1 knockdown on the activation of the DNA damage response was investigated by examining Chk1 phosphorylation. TopBP1 was knocked down as described above. Twenty four hours later cells were treated with hydroxyurea (HU) (Figure 3.2.1 (b)). Hydroxyurea has previously been shown to activate ATR DNA damage pathways (Hammond et al., 2003). After HU treatment, the cells were harvested for western blot analysis. In lanes 1 and 2, cells were mock treated. In lane 2 Chk1 is phosphorylated at S317 in response to HU treatment, indicating an activated checkpoint response. In lanes 3 and 4, cells were transfected with the control oligonucleotide, siLuciferase. In lane 4, Chk1 is phosphorylated when cells are treated with HU. In lanes 5 and 6 the cells were transfected with siTopBP1. Levels of phosphorylated Chk1 protein are activated upon HU treatment in lane 6, however these levels are much lower than

125 those seen in lanes 2 and 4. This may be a result of a partial knockdown of TopBP1. A pan-Chk1 antibody that detects the total amount of Chk1 in each protein extraction was used as a control for Chk1 levels, and gamma tubulin antibody was used as a loading control. These results indicate that TopBP1 knock down in 293T cells results in reduced Chk1 phosphorylation. ATR was knocked down and cells treated with hydroxyurea in a similar way to TopBP1 (Figure 3.2.1 (c)). In lane 1 and 2, cells were mock treated. In lane 2 Chk1 is phosphorylated at S317 in response to HU treatment. In lanes 3 and 4, cells were transfected with the control oligo, siLuciferase. In lane 4, Chk1 is phosphorylated in response to HU treatment. In lanes 5 and 6 the cells were transfected with siATR. In lane 6, siATR transfected-HU treated cells do not appear to contain any phosphorylated Chk1 protein. A pan-Chk1 antibody that detects the total amount of Chk1 is each extraction was used as a control for Chk1 levels, and gamma tubulin antibody was used as a loading control. These results indicate that when ATR or TopBP1 are knocked down, DNA damage signalling through Chk1 kinase is significantly inhibited indicating a reduction in DNA damage signalling.

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Figure 3.2.1 ATR and TopBP1 are essential for activation of Chk1 protein kinase.

(a) (b) (c)

Figure 3.2.1 ATR and TopBP1 are essential for activation of Chk1 protein kinase. (a) 293T

cells were mock transfected or transfected with oligonucleotides against Luciferase/ATR/TopBP1 genes. The oligonucleotide efficiency for ATR/TopBP1 knockdown was analysed by western blotting using antibodies against ATR and TopBP1. (b) 293T cells were mock transfected (lanes 1 and 2), transfected with siLuciferase (lanes 3 and 4) or

1 2 3 4 5 6

127 siATR (lanes 5 and 6). Lanes 2, 4, and 6 are samples that were treated with 3mM of HU for 4 hours. Cells were then harvested and analysed for western blotting for antibodies against phospho-Chk1 S317, pan-Chk1 and gamma tubulin. (c) cells were mock transfected (lanes 1 and 2), transfected with siLuciferase (lanes 3 and 4) or siTopBP1 (lanes 5 and 6). Lanes 2, 4, and 6 are samples that were treated with 3mM of HU for 4 hours. Cells were then harvested and analysed by western blotting for antibodies against phospho-Chk1 S317, pan-Chk1 and gamma tubulin.

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