Supplementary Figure 4. Effects of Zeocin treatment on levels of ï�§H2AX and accumulation of cells in G2/M phase in PTEN-null Ishikawa cells after the overexpression of four different PTEN plasmids. Cells were transfected with plasmids for 24 h, treated with Zeocin for 16 h, then assayed. A-P Levles of ï�§H2AX as determined by co-immunocytostaining with anti-PTEN antibody followed by Alexa Fluor 488 conjugated secondary antibody (green) and anti-phospho H2AX (Ser 139) antibody followed by Alexa Fluor 546 conjugated secondary antibody (red). Nuclei were stained with DAPI (blue). A-D, pEGFP-EV. E-H, PTEN-WT. I-L, PTEN-NLS. M-P, PTEN-NES. Scale bar; 200 Î¼m. Q, Comparison of % ï�§H2AX+ cells as determined by flow cytometry analysis in PTEN transfected cells with or without Zeocin treatment. R, Comparison of the percentage of cells in G2/M as determined by flow cytometry analysis in PTEN transfected cells with or without Zeocin treatment. Statistical analysis performed was paired T-test for each plasmid P<0.05
ARTICLE ABSTRACTEndometrial adenocarcinoma (EndoCA) is the most common gynecologic cancer type in the United States, and its incidence is increasing. The majority of patients are disease-free after surgical resection of stage I tumors, which is often followed by radiotherapy, but most patients with advanced disease recur and have a poor prognosis, largely because the tumors become refractory to cytotoxic chemotherapies. PTEN, a commonly mutated tumor suppressor in EndoCAs, is well known for its ability to inhibit the AKT/mTOR signaling pathway. Nuclear functions for PTEN have been proposed as well, but whether those affect EndoCA development, progression, or outcomes is not well understood. Using immunohistochemistry, nuclear PTEN expression was observed in approximately half of EndoCA patient tumors, independent of grade and cytoplasmic PTEN expression. Higher levels of the DNA damage response (DDR) marker, γH2AX, were observed by immunohistochemistry and immunofluorescence in human EndoCA tumor sections that were PTEN-negative, in murine EndoCA tissues that were genetically modified to be PTEN-null, and in Ishikawa EndoCA cells, which do not express endogenous PTEN. Overexpression of exogenous PTEN-WT or PTEN-NLS, a modified PTEN with an added nuclear localization signal, significantly improved both DDR and G2–M transition in Ishikawa cells treated with a DNA-damaging agent. Whereas PARP inhibition with Olaparib was not as effective in Ishikawa cells expressing native or PTEN-NLS, inhibition with Talazoparib was not affected by PTEN overexpression. These results suggest that nuclear PTEN subcellular localization in human EndoCA could be diagnostic when considering DDR therapeutic intervention. Mol Cancer Ther; 17(9); 1995–2003. ©2018 AACR.