Caracterización del grupo de expertos y resultados del proceso de

The specificity of the HDAC4 antibody was examined by immunofluorescence technique (IF) on PEO4 transiently transfected cells with siRNA against HDAC4 (Figure 33), then untransfected cells. After 24 hours the HDAC4 antibody (Cell Signalling Technology) and Total STAT1 (T-STAT1; BD Biosciences) antibody applied. T-STAT1 was used as control because its expression supposes to be detected in both untransfected and siRNA against HDAC4 cells. Expression of HDAC4 and T-STAT1 were strong in untransfected cells. However, HDAC4 expression become weaker but T-STAT1 expression was still strong in cells with siRNA against HDAC4. This shows that the antibody is specific to HDAC4 and can be used in IF. Moreover, the formation of H2AX induced by cisplatin treatment was not HDAC4 dependant since the expression of H2AX does not change following HDAC4 knockdown.

The localization of HDAC4 and H2AX was then tested in untreated, cisplatin- sensitive PEO1 and cisplatin-resistant PEO4 cells and after 4 and 24 hours of 25uM cisplatin exposure. In untreated PEO4 cells, endogenous and abundant HDAC4 was localized mainly in the cytoplasm, whereas no detection of H2AX was evident with the absence of cisplatin. HDAC4 was detected in the nucleus after 4 hours of cisplatin treatment with no H2AX phosphorylation. The phosphorylated histone H2AX was detected in the nuclei only after 24hours cisplatin treatment with HDAC4 accumulation in the nuclei (Figure 34).

In untreated PEO1 cisplatin-sensitive cells, endogenous and scarce HDAC4 was diffused in the cytoplasm, while no detection of H2AX was evident for the absence of cisplatin (Figure 35). At 4 hours of cisplatin treatment, cytoplasmic HDAC4 translocated to the nucleus with H2AX at 4 hours. This suggests that cisplatin caused DNA damage at earlier time points in the sensitive cells than in the resistant cells. In agreement with immune-blot analysis of cisplatin time course treatment in PEO1 (Figure 32) at 24 hours post-treatment, HDAC4 was not detected. However; strong expression of H2AX was observed. In SKOV3 cisplatin-resistant cells, the cytoplasmic HDAC4 staining was present in untreated cells with no detection of H2AX. The translocation of both HDAC4 and H2AX to the nucleus was observed at 4 hours cisplatin-treatment and the co-localization of HDAC4 and H2AX to nuclear foci after double-strand DNA damage was detected in 24hours cisplatin-treated cells (Figure 36).

The expression and localization of HDAC4 and H2AX in SKOV3 cells were similar to PEO4 cells indicating that in cisplatin-resistant cells, the cytoplasmic HDAC4 is translocated to the nucleus after treatment with cisplatin and co- localises with H2AX in the nucleus 24 hours post-treatment. Cisplatin caused DNA damage at 24 hours (late time point) in PEO4 and at 4hours in SKOV3 resistant cells. In addition, the different time points of phosphorylation H2AX between PEO4 and SKOV3 might be due to different levels of resistance to platinum.

Figure 31 A: No significant change in HDAC4 expression level was observed in untreated PEO1 the sensitive cell line however, HDAC4 expression was lower at 2hours and higher at 24hours compared with 0hour in PEO4 the resistant cell line. Bands were observed at the predicted sizes for HDAC4 at 119KDa and  tubulin at 54KDa.

Figure 31 B: In PEO1 the sensitive cell line, HDAC4 levels decreased upon cisplatin exposure and were undetectable from 8 hours post treatment. In PEO4 the resistant cell line HDAC4 protein was strong and up-regulated from 4hrs after cisplatin treatment with loss of its expression at 24hours. Bands were observed at the predicted sizes for HDAC4 at 119KDa and  tubulin at 54KDa.

Figure 32: Western blot analysis showing an inverse correlation between H2AX and HDAC4 expression. H2AX level increased gradually starting from 8 hours following cisplatin treatment in PEO1 but detected at 24 hours in PEO4 cell line while HDAC4 expression decreased. Lamin A/C used as loading control. Bands were observed at the predicted sizes for lamin at 65/74KDa, H2AX at 15KDa and HDAC4 at 119KDa.

Figure 33: The specificity of the HDAC4 antibody was examined using PEO4 cells were either untransfected or transiently transfected with siRNA against HDAC4 after 2hours incubated with HDAC4 C-terminus (green) and total STAT1(red) antibodies after fixation and permeabilisation. The cells were stained with FITC to label HDAC4, Alexa 495 to label Total STAT1 and finally counterstained with DAPI to label the nucleus. The top panels show strong staining for HDAC4 and Total STAT1 in untransfected cells. In the bottom panels, Total STAT1 was detected in HDAC4 knockdown cells, however, weak HDAC4 staining was observed. Magnification, x200

Figure 34: HDAC4 cytoplasmic/nuclear shuttling after DNA damage (cisplatin). PEO4 cells were treated with 25uM cisplatin treated 4 and 24hours or without treatment then incubated with HDAC4 C-terminus (green) and H2AX (red) antibodies after fixation and permeabilisation. The cells were stained with FITC to label HDAC4, Alexa 495 to label H2AX and finally counterstained with DAPI to stain the nuclear DNA. The top panels show cytoplasmic HDAC4 staining in untreated cells and H2AX was not detected. The middle panels show detection of part of HDAC4 with detection of H2AX at 4hours. Magnification, x300. The lower panels showing co-localization of HDAC4 and H2A.X in nuclear foci after double-strand DNA damage in 24hours cisplatin-treated cells. The images in the lower panels were captured in closer magnification than the other panels to view close and clarify the co-localization of HDAC4 and H2AX. Magnification, x400

Figure 35: HDAC4 cytoplasmic/nuclear shuttling after DNA damage (cisplatin). PEO1 cells were treated with 25uM cisplatin treated 24hours or without treatment then incubated with HDAC4 C-terminus (green) and H2AX (red) antibodies after fixation and permeabilisation. The cells were stained with FITC to label HDAC4, Alexa 495 to label H2AX and finally counterstained with DAPI to label the nucleus. The top panels show diffused cytoplasmic HDAC4 staining in untreated cells and H2AX was not detected. The middle panels showing the recruitment of HDAC4 and H2AX to nuclear foci after double-strand DNA damage in 4hours cisplatin-treated cells. Magnification, x150. The lower panels show no staining for HDAC4 and the recruitment of H2AX to nucleus following 24 hours cisplatin treatment. The images in the lower panels were captured in closer magnification than the other panels to view close and clarify the co-localization of HDAC4 and H2AX. Magnification, x250

Figure 36: HDAC4 cytoplasmic/nuclear shuttling after DNA damage (cisplatin). SKOV3 cells were treated with 25uM cisplatin treated 24hours or without treatment then incubated with HDAC4 C-terminus (green) and H2AX (red) antibodies after fixation and permeabilisation. The cells were stained FITC to label HDAC4, Alexa 495 to label H2AX and finally counterstained with DAPI to label the nucleus. The top panels show cytoplasmic HDAC4 staining in untreated cells and H2AX was not detected. The middle panels are showing the translocation of both HDAC4 and H2AX to the nucleus following 24hr cisplatin treatment. The middle panels showing the co-localization of HDAC4 and H2AX to nuclear foci after double-strand DNA damage in 24hours cisplatin-treated cells. Magnification, x200.The images in the lower panels were captured at higher magnification than the other panels to view close and clarify the co-localization of HDAC4 and H2AX observed in the middle panels. Magnification, x300

3.6 Expression pattern of class I and II HDAC isoforms in