Analysis of RPPA data to identify the unknown kinase responsible

To identify hits of possible kinases responsible for p53-Ser20 phosphorylation

and p53 transcriptional activity in cis-Pt resistant 2780CP/Cl-16 cell line by oxali-Pt, the

RPPA data obtained (Section 5.2.2) was subjected to different intersections. For these

studies, the intersection of interest is the one exhibiting a significant increase in the

signal obtained from A2780 treated with cis-Pt and oxali-Pt, and 2780CP/Cl-16 treated

with oxali-Pt only. This is depicted as A2780 cis-Pt up, A2780 oxali-Pt up and

2780CP/Cl-16 oxali-Pt up (Figure 34A). This stipulation is based on the premise that

the unknown kinase can be activated by either cis-Pt or oxali-Pt in sensitive cells, but

only oxali-Pt can activate it in resistant cells. Based on this intersection, five proteins

were identified: p53, BAX, MAPK pT202_Y204, Mcl-1 and Notch1 (Figure 34B). The

only positive kinase hit was MAPK pT202_Y204, which is also known as p-ERK1/2-

T202/Y204. These results provided the basis to study the involvement of the MAPK

pathway; either the ERK1/2 kinase or upstream kinase MEK1/2 in mediating p53

phosphorylation at Ser20 and p53 transcriptional activation after oxali-Pt treatment.

Validation of these RPPA results for the kinases by Western blot analysis was

employed (Figure 34C). In general, total levels of MEK1/2 and ERK1/2 remain largely

unchanged after cis-Pt and oxali-Pt treatment in A2780 and 2780CP/Cl-16 cells.

Moreover, levels of MEK1/2-S217/221 phosphorylation increased in A2780 and

2780CP/Cl-16 after cis-Pt and oxali-Pt treatment. Finally, it was observed that the level

of ERK1/2-T202/Y204 phosphorylation is lower in 2780CP/Cl-16 cells compared to

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robust in both cell lines after oxali-Pt treatment (Figure 34C). These results positively

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Figure 34. Analysis of RPPA data to identify the unknown kinase responsible for p53 phosphorylation at Ser20 and p53 transcriptional activation by oxali-Pt

A) Diagram for RPPA analysis from A2780 treated with cis-Pt 1µM or oxali-Pt 0.6 µM

and 2780CP/Cl-16 treated with cis-Pt 5 µM or oxali-Pt 3 µM for 24 hr. Only proteins significantly upregulated (up) by Pt treatment were included in the analysis. B) Output obtained from the intersection A2780 cis-Pt up, A2780 oxali-Pt up and 2780CP/Cl-16 oxali-Pt up. C) RPPA data validation through Western blot analysis in A2780 treated with cis-Pt 1µM or oxali-Pt 0.6 µM and 2780CP/Cl-16 treated with cis- Pt 5 µM or oxali-Pt 3 µM for 24 hr.

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5.2.4. Involvement of ERK1/2 in mediating p53 phosphorylation at Ser20 and p53 transcriptional activation by oxali-Pt:

To further explore the involvement of ERK1/2 as the novel kinase required by

oxali-Pt to exert its mechanism of action, the ERK1/2 SCH772984 inhibitor was used.

SCH772984 is a novel, specific inhibitor of ERK1/2 with IC50 of 4 nM and 1 nM,

respectively, which has been shown to inhibit ERK1/2 phosphorylation at T202/Y204

[163;164]. The capacity of SCH772984 to inhibit ERK1/2-T202/Y204 phosphorylation

was examined at 0.5 µM, 1.5 µM and 5 µM for 1 hr and 24 hr in 2780CP/Cl-16 (Figure

35A). The data show 1.5 µM of SCH772984 as the optimal concentration to inhibit

ERK1/2 phosphorylation at T202/Y204; thus, this concentration was selected for further

experiments involving SCH772984. The capacity of oxali-Pt to induce p53-Ser20

phosphorylation and p53 transcriptional activation in cis-Pt resistant 2780CP/Cl-16 cell

line through an ERK1/2 dependent manner was then evaluated. Thus, 2780CP/Cl-16

cells were treated with the ERK1/2 inhibitor SCH772984 or DMSO for 1 hr followed by

cis-Pt or oxali-Pt treatment for 24 hr (Figure 35B). The data shows that SCH772984

was able to inhibit ERK1/2-T202/Y204 phosphorylation. Moreover, 2780CP/Cl-16 cells

treated with cis-Pt treatment in combination with SCH772984 for 24 hr exhibited a

reduction in p53 and p53-Ser15 phosphorylation levels. In addition, induction of p53-

Ser20 phosphorylation and p21 were not observed after cis-Pt treatment in 2780CP/Cl-

16 cells treated with DMSO or SCH772984. Finally, SCH772984 did not impact the

ability of oxali-Pt to induce p53, p53-Ser15 phosphorylation, p53-S20 phosphorylation

and p21 expression in 2780CP/Cl-16 cells since high levels of these proteins were

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conclusion, these results demonstrate that ERK1/2 is not the kinase activated by oxali-

Pt to restore p53-Ser20 phosphorylation and p53 transcriptional activity in the cis-Pt

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Figure 35. Involvement of ERK1/2 in p53 phosphorylation at Ser20 and p53 transcriptional activation by oxali-Pt

A) Evaluation of ERK1/2-T202/Y204 phosphorylation levels in 2780CP/Cl-16 treated

with 0.5 µM, 1.5 µM and 5 µM of the ERK1/2 inhibitor SCH772984 for 1 hr and 24 hr.

B) Evaluation of the p53 signaling pathway in 2780CP/Cl-16 pre-treated with

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5.2.5. Involvement of MEK1/2 in p53 phosphorylation at Ser20 and p53