Experiments in sections 3.3.2 and 3.3.3 show both ligand CXCL1 and receptor CXCR2 are expressed on human CD34+CD38- and CD34+CD38+ cells. The next objective was to elucidate the biological significance of this signalling pathway. First, we aimed to modulate CXCL1 expression in cell lines to optimise the techniques and to examine the resulting phenotype. We designed experiments to knock down and over express CXCL1.
Appropriate vectors were constructed and relevant techniques were optimised in HT 1080 cells.
To knock down expression of CXCL1, a lentiviral transduction approach with a shRNA vector was taken. A set of several vectors with different unique sequences against the CXCL1 protein was purchased and each vector was tested in HT 1080 cells. Several shRNA vectors against CXCL1 were shown to result in different levels of protein reduction using western blotting analysis which can be visualised in Figure 3-7.
Densitometry showed the following fold changes of 1.0, 0.99, 0.75. 1.1, 0.84 and 0.86 for untransduced, sh1, sh2, sh3, sh4 and sh5 respectively in comparison to the Scr controlled which was set to the value of 1 (Figure 3-7). The two vectors corresponding to the greatest knock down of the CXCL1 protein were cloned into the same vector (pLKO.1), but with the presence of a GFP coding sequence. Both vectors were confirmed to significantly reduce gene expression levels of CXCL1 in comparison to the control at the mRNA level (40% and 50% reduction in sh2 and sh5 in comparison to Scr control set to the value of 1, P <0.01, n = 3) (Figure 3-7).
The effect of CXCL1 reduction in HT 1080 cells was examined by assessing proliferation and viability using cell counts. CXCL1 reduction (sh2 and sh5) in HT 1080 cells resulted in a decrease in cell counts in comparison to the control (Scr) after 48 hours in culture (P
<0.05, n = 3) (Figure 3-8). The cell counts obtained using sh1 and sh2 were below the density of the input cells (10x103) indicating a loss of cells in culture after CXCL1 reduction. This would infer cells were undergoing apoptosis in response to CXCL1
reduction. Apoptosis analysis using Annexin-V and dapi staining showed no differences in apoptosis (n = 3) (Figure 3-9). However a decrease in the percentage of GFP positive cells was observed with both hairpins in comparison to the control (P <0.01 and P <0.001 sh2 and sh5 respectively) (n = 3) (Figure 3-9).
Collectively, the data shows that CXCL1 reduction reduces cell proliferation in HT 1080 cells. As the reduction in cell counts was below that of the input, it was implicated that CXCL1 reduction also reduced cell survival. However, the apoptosis data suggested there was no/very little change in apoptosis in response to CXCL1 reduction. The apoptosis assay was carried out at a later time point than the cell counts and it is therefore possible that CXCL1 reduction did reduce cell viability and at the time of the apoptosis assay, these cells had disappeared from the culture. Indeed, analysis of the cell populations showed that there was a reduction in the percentage of GFP cells in response to CXCL1 reduction.
Assuming that 100% of cells at the beginning of the assay are positive for GFP and a loss is found in response to CXCL1 reduction, this suggests positively transduced cells are being lost from the culture. It is predicted that this is due to apoptosis. To conclude this, apoptosis should be analysed at an earlier time point after transduction.
The data highlight that CXCL1 plays an important role in proliferation and survival in cell lines. To complement these experiments, an over expression vector (CXCL1-PRRL) was constructed to increase levels of CXCL1 and examine the resulting effect on HT 1080 cell properties. CXCL1-PRRL showed an increase in CXCL1 protein levels through mRNA (68.4 fold change, P <0.05) (n = 3) and protein levels (1.4 fold change increase) (n = 1) (Figure 3-10). CXCL1-PRRL was found to increase cell counts after culture for 48 hours in comparison to the control (P <0.05, n = 3) (Figure 3-11). Finally, apoptosis staining using Annexin-V and dapi staining showed an increase in the percentage of viable cells in the CXCL1-PRRL cells in comparison to the control (P <0.05, n = 3) (Figure 3-12).
Taken together, the results show that over expression of CXCL1 in cell lines increases cell proliferation and cell viability. The results in combination with the knock down studies suggest that CXCL1 is a key factor involved in proliferation and survival in HT 1080 cells.
Figure 3-7 CXCL1 reduction using shRNA mediated lentiviral reduction reduces CXCL1 protein and mRNA levels in HT 1080 cell lines.
HT 1080 cells were lentivirally transduced with 5 plasmids (pLKO.1-CXCL1 or a pLKO.1-Scr). After selection in puro, protein lysates were examined for expression of CXCL1 or housekeeping β-tubulin (A). Blot represents representative image. Densitometry analysis shows the fold reduction of density intensity/mm2 in response to CXCL1
reduction (B). A larger image of the blot with ladder can be seen in the supplementary Figure 7-3. The 2 vectors which corresponded to the best reduction in CXCL1 protein were cloned into a GFP plasmid and lentivirally transduced into HT 1080. GFP positive cells were sorted using flow cytometry, RNA was extracted, RT and analysed for CXCL1 mRNA levels. Fold change in gene expression was calculated relative to housekeeping control GAPDH according to the DeltaDeltaCT method. Data are presented as the mean fold change of expression in the knock down cells using the control (Scr) as a calibrator which is set to the value of 1 (C). Data is also presented as relative expression (2-DeltaCT) (D). A repeated measures one-way ANOVA was used with the Dunnetts’s multiple comparison test to compare the control (Scr) with each hairpin (** P <0.01)
A B
C D
Figure 3-8 CXCL1 reduction decreases proliferation in HT 1080 cell lines.
HT 1080 cells were lentivirally transduced with 2 separate plasmids pLKO.1-CXCL1 (sh2 or sh5) or a pLKO.1-Scr (Scr). After lentiviral transduction, GFP positive cells were isolated and cultured for 48 hours and cells were counted using trypan blue exclusion method and total cell count per sample was calculated. Data are presented as the mean cell count obtained. The input represents the starting number of cells seeded for all conditions.
Statistical analysis was performed using a repeated measures one-way ANOVA with Dunnett’s multiple comparison test to measure differences between the control (Scr) and each hairpin (n = 3) (* P <0.05).
Figure 3-9 CXCL1 reduction reduces the percentage of GFP+ cells in HT 1080 cell lines.
HT 1080 were lentivirally transduced with sh1, sh2 or a Scr control. After lentiviral transduction, GFP+ cells were isolated and cultured for 96 hours and cells were examined for apoptosis using Annexin-V and dapi staining. Data are presented as the mean
percentage of cells that were viable or dead (A). Cells were examined for percentage of GFP+ cells using flow cytometry. Data are presented as the mean percentage of cells that were GFP+ (B) after 96 hours in culture. Statistical analysis was performed using a repeated measures one-way ANOVA with Dunnett’s multiple comparison test to assess differences between the control column with sh2 and sh5 (B). A statistically significant difference was found between the Scr group and both sh2 and sh5 (n = 3) (**P <0.01; ***
P <0.001). A representative histogram of GFP+ cells between conditions is shown (C).
A
B C
Figure 3-10 CXCL1 over expression vector CXCL1-PRRL increases CXCL1 expression by protein and mRNA analysis.
HT 1080 cells were lentivirally transduced with CXCL1-PRRL or a PRRL empty vector control. After transduction, cells were sorted for GFP+ cells and protein lysates were made and RNA was extracted, RT and both were examined for expression of CXCL1 using western blotting or Q-PCR. Blot shows CXCL1 expression in empty vector (-) and CXCL1-PRRL transduced (+) cells using housekeeping protein β-tubulin as a loading control (A). Blot represents representative protein image and larger image of blot with molecular ladder is available in the supplementary Figure 7-4. Densitometry analysis demonstrates the fold change intensity/mm2 in CXCL1-PRRL relative to the empty vector control (B). CXCL1 gene expression was calculated relative to housekeeping control GAPDH using the DeltaDeltaCT method. Data are presented as mean fold change of
expression in the CXCL1-PRRL cells using the empty vector cells as a calibrator which is set to the value of 1 (C). Relative expression (2-DeltaCT) is shown with each dot displaying an average of technical triplicates in three independent experiments (D). Statistical analysis was performed using a two-tailed paired t test assuming equal variance (n = 3) (*P <0.05).
A B
C D
Figure 3-11 CXCL1 over expression increases proliferation in HT 1080 cell lines.
HT 1080 cells were lentivirally transduced with CXCL1-PRRL or a PRRL empty vector control. After transduction, cells were sorted for GFP+ cells and cells were cultured for 48 hours in medium. Cells were counted using trypan blue exclusion method and total cell count per sample was calculated. Data are presented as the mean cell count. The input represents the starting number of cells seeded for both conditions. Statistical analysis was performed using a paired t test assuming equal variance comparing the empty vector control with the CXCL1-PRRL group (n = 3) (*P <0.05).
Figure 3-12 CXCL1 over expression increases cell viability in HT 1080 cell lines.
HT 1080 cells were lentivirally transduced with CXCL1-PRRL or a PRRL empty vector control. After transduction, cells were sorted for GFP positive cells and cells were cultured for 96 hours in medium. Cells were analysed for levels of apoptosis using Annexin-V and dapi staining. Data are presented as the mean percentage of viable cells. Statistical analysis was performed using a paired t test assuming equal variance comparing the empty vector control with the CXCL1-PRRL group (n = 3) (*P <0.05).