DENSIDAD Y FORMA URBANA
3.4 La densidad percibida: actitud personal y experiencia espacial
109 As established in Section 1.10 (Page 39) the phosphorylation status of the GR is a crucial determinant of its activity. It is known that ligand-binding ultimately leads to altered phosphorylation status, and that GC-sensitive and GC-resistant cells exhibit different patterns of phosphorylation.
Due to the widespread effects of the microenvironment and the key role of the GR in numerous physiological pathways, it was hypothesised that the microenvironment (and CM) could alter GR levels or post-translational status, which in turn could affect response of leukaemia cells to dexamethasone or other chemotherapeutic agents.
To investigate this, Western blot analysis (complemented by densitometry) was employed on C1-15 and C7-14 cells under CM, dexamethasone and etoposide treatment in varying combinations. The proteins analysed were Total GR (H300), S211- phosphorylated GR, S226-phosphorylated GR, with Actin being used as a loading control:
Figure 3.3.1: Western blot analysis of the GR and its phosphoisoforms.
C1-15 and C7-14 cells were treated with CM for 48 hours, 1μM dexamethasone (D) for 24 hours or 10μMetoposide(E)for24hoursindividuallyorincombination.CellswerelysedandtheGRandtwo phosphoisoforms were detected by Western blot analysis. Actin was used as a loading control. Data is representative of at least three independent experiments.
Although the Westerns above provide a visual analysis, densitometry allows for a semi- quantitative approach to the Western data. Each antibody above will be analysed in the following sections.
110 3.3.1 Analysis of Total GR
Figure 3.3.2: Densitometric analysis of GR protein levels.
C1-15 and C7-14 cells were treated with CM for 48 hours, 1μM dexamethasone (D) for 24 hours or 10μMetoposide(E)for24hoursindividuallyorincombination. Blots were analysed with ImageJ. GR band readings were normalised to the corresponding actin band reading and then expressed relative to untreated cells. This Figure contains quantification of blot data generated by both the author of this thesis and an additional researcher. Data is representative of at least three independent experiments +/- SEM. P- value <0.05 is indicated by *.
In Figure 3.3.2 above, CM trends towards reducing the GR in C1-15 cells, but increasing the GR in C7-14 cells (lane 2, compare dark bars to light bars). The difference in cellular response to hormone can be observed under dexamethasone treatment, with C7-14 cells showing tendency towards increased GR levels under hormone treatment (lane 3, compare dark to light bars). Etoposide trended towards reducing GR levels, whilst dexamethasone and etoposide combination led to a statistically significant reduction in GR levels (compare light bars of lanes 1 and 7). This is consistent with established knowledge, as it has been shown previously that GR and TP53 (activated by etoposide) may exhibit negative crosstalk (Sengupta et al., 2000).
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Figure 3.3.3: Densitometric analysis of S226-phosphorlated GR.
C1-15 and C7-14 cells were treated with CM for 48 hours, 1μM dexamethasone (D) for 24 hours or 10μMetoposide(E)for24hours individually or in combination. Blots were analysed with ImageJ and S226 values were normalised to the corresponding actin-normalised total GR values. Values were then expressed relative to untreated cells. This Figure contains quantification of blot data generated by both the author of this thesis and an additional researcher. Data is representative of at least three independent experiments +/- SEM. P-value <0.05 is indicated by * (black asterisk for comparison of treatment to control). Red asterisks indicate statistical significance at p<0.05 between other groups.
As shown in Figure 3.3.3, differential effects of CM between C1-15 and C7-14 cells are still observed. In C1-15 cells, CM induces a statistically significant increase in S226- phosphorylated GR, whilst this effect is not seen in C7-14 cells (lane 2, compare dark to light bars). This correlates with the trend towards reduction of T-GR by CM in Figure 3.3.2, given that phosphorylation at S226 is associated with nuclear export and reduced GR activity (Galliher-Beckley and Cidlowski, 2009).
Opposing effects of dexamethasone on phosphorylation at S226 were observed between C1-15 and C7-14 cells, with C1-15 trending towards an increase whilst C7-14 cells trended towards a decrease (lane 3, compare dark to light bars of Figure 3.3.3). However, addition of CM to this led to a decrease in S226-phosphorylated GR levels (compare dark bar of lane 4 to dark bar of lanes 3 and 1 of Figure 3.3.3). In C1-15 cells, etoposide induced a statistically significant downregulation of S226-phosphorylated
112 GR, which was inhibited by the addition of CM (compare dark bars of lane 5 to lane 6 of Figure 3.3.3).
A similar effect was observed with dexamethasone and etoposide combination treatment; although etoposide lead to a statistically significant decrease in S226- phosphorylated GR, this was lost following the addition of dexamethasone (compare dark bars of lane 5 to lane 7 of Figure 3.3.3). This provides further evidence for the role of dexamethasone in increasing S226 phosphorylation in C1-15 cells. However, addition of CM to dexamethasone and etoposide combination reduced levels of S226- phosphorylated GR, though there was no change in statistical significance. Thus, it appears CM exerts different effects depending on the treatment combinations; most effects in C1-15 cells appear to be increasing S226-phosphorylated GR, with the exception of when dexamethasone is present, at which point CM appears to exert a negative effect, potentially interfering with the hormone response.
Although etoposide lead to a statistically significant reduction of S226-phosphorylated GR in C7-14 cells, this effect was not inhibited by CM, indicating a cell-specific difference in response to CM (compare light bars of lanes 5 and 6 of Figure 3.3.3). Although dexamethasone and etoposide lead to a slight loss of the inhibition of S226- phosphorylated GR (compare light bars of lanes 5 and 7 of Figure 3.3.3). Lastly, addition of CM to dexamethasone and etoposide treatment led to a statistically significant reduction in S226 levels relative to dexamethasone and etoposide alone in C7-14 cells (compare light bars of lanes 7 and 8 of Figure 3.3.3). Thus, CM appears to have a generally negative effect on S226 expression in C7-14 cells, while its effects in C1-15 cells may be slightly more complex.
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Figure 3.3.4: Densitometric analysis of S211-phosphorylated GR.
C1-15 and C7-14 cells were treated with CM for 48 hours, 1μM dexamethasone (D) for 24 hours or 10μMetoposide(E)for24hoursindividuallyor in combination. Blots were analysed with ImageJ and S211 values were normalised to the corresponding actin-normalised total GR values. Values were then expressed relative to untreated cells. This Figure contains quantification of blot data generated by both the author of this thesis and an additional researcher. Data is representative of at least three independent experiments +/- SEM. P-value <0.05 is indicated by *.
Analysis of phosphorylation at S211 indicates a trend for CM to increase its expression in both C1-15 and C7-14 cells. Other than CM treatment, levels of S211-phosphorylated GR were largely unchanged for C1-15 cells, whilst C7-14 showed increase in S211- phosphorylated GR across all treatments.