NUEVAS PERSPECTIVAS SOBRE EL PATRIMONIO CULTURAL

Immunoprecipitation assays were performed to explore changes in cellular

protein levels under a range of drug inhibition and chemokine or BCR stimulation conditions.

Rap1 by GTPase activity assay

The initial Rap1 activity experiment using published conditions (237) was

performed using a fresh CLL95 sample (normal karyotype CLL) and it yielded no Western blot signal in the sample conditions, however there was a weak signal in the GTPgS positive control lane. Molecular properties of GTPgS include its

guanosine triphosphate (GTP) analogue function, G protein-activating and non- hydrolysable state which are exploited by the GTPase activity assay to provide a positive control condition. There was also no signal detected in the EPAC

positive control cells, therefore we elected to include only the GTPgS and GDP controls. A representative example of the effects of mTOR inhibitors on Rap1

activity is shown upon SDF-1 and BCR stimulation demonstrated, along with positive and negative controls (Figure 5-2). There is a clear stimulation of Rap1 activity upon SDF-1 and BCR stimulation on sample CLL152, with pronounced effects of ibrutinib to inhibit Rap1 activity in the presence and absence of with either in vitro BCR or SDF-1 stimulation. The inhibition of Rap1 activity by AZD8055 is weaker and less consistently displayed, with a mild stimulation in Rap1 activity with rapamycin treatment in the presence and absence of SDF-1 or BCR ligation.

Figure 5-2 Rap1 activity assay

CLL152 (2 x 107 cells), a normal karyotype CLL sample was serum-starved for 2 h, treated for 30 min with non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB) then stimulated with either F(ab’)2 fragment stimulation or SDF-1 for

30 min followed by immunoprecipitation for active Rap1. Rap1 levels are shown by Western blot along with positive and negative controls to confirm the success of the

immunoprecipitation procedure.

Rac1 activity in CLL cells

The GTPase Rac-1, also involved in migration and its regulation, was found to show consistent protein expression levels under drug and stimulation conditions used (Figure 5-3), highlighting the need for study of activation status of this protein in investigation of the role that GTPases play downstream of mTOR signalling.

NDC AZD RAPA IB NDC AZD RAPA IB GTPgS GDP

- - - - + + + +

NDC AZD RAPA IB NDC AZD RAPA IB GTPgS GDP

- - - - + + + + FAB 30 min

Figure 5-3 Total Rac1 protein levels after long-term mTOR inhibition and BCR stimulation Patient sample CLL18, an 11q deletion patient, was treated for 30 min with non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB) then

exposed to 48 h F(ab’)2 fragment stimulation. The Western blot antibody to Rac1 cannot

distinguish between active and inactive forms of the protein and the overall levels appear as unchanged in relation to the untreated control.

The availability of Rac1 activity assay with beads specific for active Rac1

allowed me to perform similar experiments as for Rap1 activity. The efficacy of the immunoprecipitation process was supported by the results of GTPgS and GDP controls and a representative sample result of Rac1 activity in response to either SDF-1 or BCR stimulation is shown (Figure 5-4). Inhibitors appear to have little effect in the unstimulated samples with no effect of ibrutinib overall.

Rapamycin inhibits BCR-mediated Rac1 activity and AZD8055 has no effect in the sample shown in Figure 5-4. Direct comparison of Rac1 activity in SDF1 and BCR stimulated cells on the same Western blot for CLL147 (Figure 5-5) confirmed that the overall signal generated by BCR stimulation is greater than that with SDF-1 stimulation.

Figure 5-4 Rac1 activity assay of a single normal karyotype CLL sample

CLL147, a normal karyotype CLL sample was serum-starved for 2 h, pre-treated for 30 min with non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB) then stimulated for 30 min with either F(ab’)2 fragment or 30 min SDF-1 followed by

immunoprecipitation for active Rac1.

NDC AZD RAPA IB NDC AZD RAPA IB

- - - - + + + +

GAPDH RAC1 FAB 48 H

NDC AZD RAPA IB NDC AZD RAPA IB

- - - - + + + +

FAB 30 min SDF-1 30 min

NDC AZD RAPA IB NDC AZD RAPA IB

Figure 5-5 Comparison of Rac1 activity assay SDF-1 and BCR ligation conditions

Samples of CLL147 were evaluated for levels of active Rac1 for drug conditions: non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB). Residual lysates from the active Rac1 pull-down assay were run on Western blot to repeat earlier findings.

Although protein concentrations were assumed to be constant at the outset of the experiment, based upon the use of equal aliquots of the same cell

suspension to provide individual drug and stimulation conditions and the use of equal sized aliquots of protein lysates in each lane of all Western blots

performed. Also, based upon Western blot findings shown in 3.1.6 I have shown that total GTPase levels are unaffected by drug inhibitors or in vitro stimulation. However, additional control conditions were sought to prove that there was no change in protein expression of GTPases and that the protein concentration in the protein lysates obtained by immunoprecipitation was constant. To do this, I extracted eluate from stage of sample exposure to the Rac1 beads and assessed total Rac1 content using sample CLL147. The signals in Figure 5-6 exhibit

consistency between total Rac1 levels and loading control, supportive of earlier data to demonstrate consistency in overall Rac1 levels in the presence and absence of drug inhibition and stimulation conditions. As the protein

concentrations were consistent and because original published data in CLL refer only to raw data values in relation to GTPase activity, I compared Western blot of individual samples using densitometry values for uncorrected Rap1/Rac1 activity (237, 238).

NDC AZD RAPA IB NDC AZD RAPA IB

SDF-1 FAB - + - + - + - + + - + - + - + -

Figure 5-6 Analysis of Rac1 protein expression with drug inhibitors and SDF-1 or BCR stimulation

Patient samples CLL147 lysates were obtained from the pull-down assay for analysis, from both SDF-1 and BCR stimulation experiments for drug conditions: non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB). All inactive and non-bound Rac1 and other proteins are eluted during the immunoprecipitation process and in the spin column below the filter cup the sample may be collected and maintained at -20oC for further analysis. Western blots were probed for total Rac1 protein (T-RAC) and a loading control.

The Rap1 densitometry findings most clearly demonstrate an increased activity with the addition of in vitro stimulation conditions (Figure 5-7). Both SDF-1 and BCR stimulation increase Rap1 activity, with significance in the SDF-1

experiments and a trend to significance with the addition of BCR stimulation. Ibrutinib blocks Rap1 activity more effectively than mTOR inhibitors, which appears to be overcome by SDF-1 stimulation (Figure 5-7 A) but not by BCR stimulation (Figure 5-7 B), which may reflect a greater dependence upon BTK for transduction of the BCR signal as opposed its role in chemokine signalling.

Rapamycin appears to have minimal effects upon Rap1 activity with the

inhibitory effects of AZD8055 displaying a trend to reduction in Rap1 activity in the absence of BCR or SDF-1 stimulation, suggesting that AZD8055 inhibition may be overcome by the addition of in vitro SDF-1 and BCR stimulation.

Densitometry of the active Rac1 signal also supports the effects of in vitro BCR and SDF-1 stimulation to cause increased Rac1 activity (Figure 5-8 A&B).

However, there are minimal drug effects under all 3 drug conditions. More

samples are required to be assessed by Rap1 and Rac1 activity assays, to confirm and to establish the effects of drug inhibitors.

NDC AZD RAPA IB NDC AZD RAPA IB

- - - - + + + +

GAPDH

GAPDH T-RAC

T-RAC

NDC AZD RAPA IB NDC AZD RAPA IB

- - - - + + + +

SDF-1 30 min

Figure 5-7 Rap1 activity assay Western blot densitometry

Raw mean and SEM densitometry values for Rap1 assay with n=4 samples treated for 30 min with non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB) followed by 30 min (A) SDF-1 and (B) F(ab’)2 stimulation. *=p≤0.05;

**=p≤0.01; ***=p≤0.001; ****=p≤0.0001.

Figure 5-8 Rac1 activity assay Western blot densitometry

Raw mean and SEM densitometry values for Rac1 activity assay results are also shown with 30 min exposure to non-drug control (NDC), AZD8055 100 nM (AZD), rapamycin 10 nM (RAPA) and ibrutinib 1 µM (IB) then 30 min (A) SDF-1 (n=5) and (B) F(ab’)2 stimulation (n=3).

5.3 Chemokine stimulation effects on fluorescent