Additional work

3.3.1

Improving molecular tools for imaging: HIF-1α-GFP

stable cell line

Most of the studies into the dynamics of the HIF-α subunits performed so far have required transient transfection of plasmids encoding HIF-1α / HIF-2α fused with fluorescent proteins such as EGFP or dsRed. Transient transfection has many disadvantages such as heterogeneity in expression levels, the protocol can be unreliable and the reagents can be toxic to the cells. In addition to this, the gene is under the control of a promoter that permits constitutive expression, the CMV promoter, which can lead to high levels of expression that are not necessarily representative of those observed for the endogenous promoter, and expression can vary between cell types (Smith et al., 2000). Therefore, to improve the quality of our HIF dynamic studies, we aimed to create stably transfected cell lines for both HIF-1α and HIF-2α.

The initial plan was to create three stable cell lines via lenti-viral transduction, however creating cell lines stably expressing HIF-1α-EGFP and EGFP-HIF-2α proved difficult and we only had success with the ODD-EGFP construct (mentioned earlier). With this cell line, although similar dynamics to HIF-1α-EGFP (ectopically expressed in WT HeLa) in hypoxia were observed, the degradation appeared to be slower. This is could be explained by the fact that the ODD-EGFP is lacking the DNA binding domain and so is not transcriptionally active, resulting in an increase of substrate (ODD domain) in the system with no additional activation of PHD2 transcription to compensate. In addition to this, the ODD-EGFP only has one prolyl residue for hydroxylation which may result in reduced affinity of pVHL binding, thus less efficient targeting of the ODD-EGFP for proteasomal degradation. Although the ODD-EGFP cell line provides some valuable information, there was a need for a cell line stably expressing fluorescently-labelled full-length HIF-1α and HIF-2α. Given the lack of success with the lenti-virus approach to achieve this, I directed my efforts towards alternative strategies. I tried to generate a cell line stably expressing fluorescently labelled HIF-2α using several methods including zinc finger nucleases (ZFNs), but all were unsuccessful (work not shown) and so all work with HIF-2α-EGFP presented in this thesis was carried out using transient transfection. However, I had the opportunity to use a Bacterial Artificial Chromosome (BAC) for HIF-1α-GFP, thanks to the kind gift from Prof B

van de Water’s laboratory (University of Leiden), and was able to create a stable cell line expressing HIF-1α-GFP.

3.3.1.1

HIF-1α-GFP BAC stable cell line

A HIF-1α-EGFP cell line was established via stable transfection of HeLa with a BAC encoding the fusion protein. In comparison to the ODD-EGFP cell line, this gene is under the control of its endogenous promoter and BACs can hold large pieces of mammalian DNA (up to 350 Kb, compared to 15 Kb for plasmids and 7 Kb for lenti-viral vectors) resulting in the inclusion of lengthy flanking regions. Therefore a large proportion of the regulatory elements are incorporated and so utilising a BAC provides a more true reflection of endogenous expression (Adamson et al., 2011;Casali, 2003). Another advantage of the BAC cell line is that it was established from a single cell and is therefore a clonal population and so could provide the opportunity to determine whether the heterogeneity observed in Bagnall et al. (2014) is a feature of the HIF signalling system or an artefact of transient transfection.

The HIF-1α-GFP BAC cell line was validated via western blot and imaging. Figure 3.1A shows a strong induction of wild type HIF-1α following treatment with 0.5 mM DMOG (6 h) and a transient induction of endogenous HIF-1α in hypoxia. A second band, approximately 40 kDa higher, follows the same pattern of induction and is presumed to be HIF-1α-GFP. The cells were imaged on a confocal microscope before and after (6 h) treatment with the hypoxia mimic and nuclear accumulation of fluorescent protein was observed (Figure 3.1B). Following these validation experiments, this cell line was utilised to repeat time-lapse experiments that were performed in Bagnall et al. (2014).

Figure 3.1 ǀ Validation of HIF-1α-GFP stable cell line. A) Western blot analysis of HIF-1α-GFP HeLa incubated in different conditions (D = 0.5 mM DMOG, 6 h, N = normoxia, H = hypoxia, number indicates number of hours incubated in hypoxia).Immunoblot was probed with anti-HIF-1α (top) and anti-β-actin (bottom) antibodies. B) HIF-1α-GFP HeLa were imaged on a Zeiss LSM780, before and after treatment with 0.5 mM DMOG (i = brightfield, ii = GFP, iii = merge of i and ii). Scale bar = 10 μ. In pre-treatment cells nuclei are outlined in white.

Figure 3.2 ǀ HIF-1α-GFP BAC time-lapse experiments.A) Confocal images of HIF-1α-GFP BAC cells in 0.5 % O2. Cells were subjected to hypoxic conditions from the second time point onwards. Nuclear boundary highlighted by white dashed line. White arrow indicates perinuclear autofluorescence. B) Quantification of fluorescence signal. Each data series represents the fluorescence measured in the nucleus of a single cell over time.

Upon incubation in hypoxia, the cells showed a transient induction of HIF-1-GFP. After approximately 1 h the levels of fluorescence started to increase, peaked at 3-3.5 h and then started to decrease, reaching levels similar to those at the start of the time-lapse by 6-8 h (Figure 3.2B, M1 on supplemental CD).

Figure 3.3 ǀ Comparison of HIF-1α dynamics in hypoxia Example traces from single HeLa cell ectopically expressing CMV-HIF-1α-EGFP (top) and single HIF-1α-GFP BAC cell line (bottom) in hypoxia. Hypoxic incubation starts at 0 h.

Figure 3.3 compares the typical shape of HIF-1α accumulation in hypoxia in a single cell ectopically expressing CMV-HIF-1α-EGFP (taken from James Bagnall’s data set) and one from the HIF-1α-GFP BAC experiments. In both systems the levels of HIF-1α start to increase at around 1-2 hours and peaks around 3-4 hours. Compared to the stable cell line, the cell transiently transfected with HIF-1α-EGFP shows a sharper increase in fluorescence, reaching much higher levels that then decrease more rapidly. The stable cell line exhibits stabilisation of much lower levels and a softer decline in HIF-1α, similar to what was observed with the ODD-EGFP cell line. The difference in the accumulation pattern might be explained by the difference in HIF-1α levels. The very high HIF levels obtained with transient transfection will induce a stronger negative feedback response compared to the 2 fold increase obtained with the BAC cell line.

Although the BAC cell line should be closer to physiological HIF expression at the mRNA levels as the transgene is under the control of the endogenous promoter and other regulatory elements, the dynamic range of HIF-1α-GFP accumulation in hypoxia is poor i.e. the expression of the transgene are much lower than the endogenous protein in the western-blot (Figure 3.1A). Therefore the accumulation pattern observed with this cell line is unlikely to actually represent the reality any more than transient transfection.