Generation and analysis of ANXA3 and SATB1 over expressing PC3 clones

As the microarray results were approved by qPCR, the particular involvement in chemoresistance of two distinct genes was checked. 1) SATB1, a transcription factor, which

A. B.

Results

31 was reported to play a role in resistance upon cisplatin resistance in hepatocellular carcinoma [64] and 2) ANXA3, a protein previously found in a proteomic study to be more abundant in resistant PC3 clones on protein level [13]. In cooperation with Mark Laible, Stephanie Blaich and Melanie Hudler (DKFZ, Heidelberg) PC3 clones were stably transfected with SATB1 (PC3 SATB1), ANXA3 (PC3 ANXA3) or an empty vector construct (PC3 ev). The target genes are expressed under the regulation of a doxycycline dependent promoter. To ensure a stable over-expression a system which uses homologous recombination was applied (see dissertation of Stephanie Blaich, Ruprecht - Karls – Universität Heidelberg, 2007 for details). To control the correct expression of the target genes mRNA and protein expression was determined by qPCR and western blot analysis. PC3 SATB1 and PC3 ev (control) were seeded. 24 hours later the expression was induced with 50 nM doxycycline for another 24 hours. Induction of the target gene expression was analyzed by qPCR. Results are shown in Figure 3-3 A. SATB1 expression was strongly induced by doxycycline in PC3 SATB1 but not in the empty vector control. Notably, PC3 SATB1 already expressed minor amounts of SATB1 mRNA without induction of doxycycline. In a next step the correct translation of the protein was analyzed by western blot. As shown in Figure 3-3 B detectable amounts of SATB1 were only expressed by induced PC3 SATB1 cells.

Figure 3-3 Validation of PC3-SATB1 by qPCR and western blot

A. qPCR of RNA B. western blot of protein lysates isolated from induced (24 hours, 50 nM doxycycline (+dox)) and not induced (-dox) PC3 SATB1 cells and PC3 ev (control).

To evaluate the correct localization of SATB1 inside the cell, immuno-fluorescence imaging was performed. As shown in Figure 3-4 SATB1 protein was localized in the nucleus of doxycycline induced PC3 SATB1 cells.

Results

32

Figure 3-4 Immuno-fluorescence imaging of PC3-SATB1

PC3-SATB1 cells were cultivated on coverslips in 6-well plates and A. not induced (control) or B. induced using 50 nM doxycycline for 24 hours; blue = nuclei (Hoechst) green = SATB1 (alexa 488)

Subsequent to validation of the correct inducible over-expression of SATB1 in PC3 SATB1 cells its influence on the chemo-sensitivity in vitro was evaluated. CPA is a prodrug which is only active in its metabolized form (4-HOO-CPA). After activation in the liver 4-HOO-CPA is a strong alkylating drug, targeting proliferating cancer cells (reviewed by Fenselau [65]). In the in vitro experiments CPA was substituted by melphalan another alkylating drug which does not have to be metabolically activated. Additionally, cell death induction by cisplatin was analyzed, as SATB1 earlier had been shown to induce cisplatin resistance in hepatocellular carcinoma [64]. Cells were seeded in 96-well plates and expression of SATB1 was induced by 50 nM doxycycline for 24 hours. Subsequently melphalan or cisplatin treatment was carried out. As shown in Figure 3-5 A SATB1 overexpression had no influence on the chemo- sensitivity to melphalan in vitro. Cell death was provoked under induced and not induced conditions at the same level. In contrast, SATB1 overexpression altered the chemo-sensitivity against cisplatin. As shown in Figure 3-5 B SATB1 overexpressing cells were less sensitive to the cisplatin treatment than not induced cells.

Results

33

Figure 3-5 Functional analysis of SATB1 overexpression PC3 SATB1 cell line

Cells were seeded in 96-well plates. One day after seeding STAB1 expression was induced by 50 nM doxycycline for 24 hours. Subsequently chemotherapy, melphalan A. or cisplatin B., was applied for 72 hours. Cytotoxicity was determined by CellTiterGlo assay. Data is presented as percent of untreated control cells. *p < 0.05, **p< 0.005, ***p<0.0005 (t-test)

Similar experiments were performed using the PC3 ANXA3 cell line. Again the empty vector cell line (PC3 ev = empty vector) served as control.

As shown in Figure 3-6 A the endogenous expression of ANXA3 mRNA and protein was already high. The induction by doxycycline increased the mRNA expression about 1.75-fold. In concordance, the expression on protein level was only increased marginally (Figure 3-6 B). Immuno-fluorescence imaging showed that ANXA3 was located in the cytoplasm of both ANXA3 induced and not induced cells (Figure 3-6 C).

The functional analysis showed no alteration in chemo-sensitivity to neither melphalan nor cisplatin of ANXA3 overexpressing cells compared to not induced control cells (Figure 3-6 D and E).

Figure 3-6 continued

A. B.

Results

34

Figure 3-6 Analysis of ANXA3 overexpression PC3 ANXA3 cell line

A. qPCR of RNA B. western blot of protein lysates isolated from induced (24 hours, 50 nM doxycycline (+dox)) and not induced (control (-dox)) PC3 ANXA3 cells and PC3 ev (control) C. ANXA3 expression was induced using 50 nM doxycycline and not induced (control) for 24 hours; blue = nuclei (Hoechst) green = ANXA3 (alexa 488).

D. + E. ANXA3 expression was induced by 50 nM doxycycline for 24 hours. Thereafter, chemotherapy, melphalan or cisplatin, was applied for 72 hours. Cytotoxicity was determined by CellTiterGlo assay. Data is presented as percent of untreated control cells.

C.

Results

35 Taken together neither ANXA3 nor SATB1 alone were able to induce an in vitro resistance to the alkylating drug melphalan. Notably, a potential influence of ANXA3 or SATB1 overexpression in vivo was not evaluated within the framework of this thesis.

Further analysis was no longer focused on one special gene but more on the involvement of whole pathways and microenvironmental factors.