Reverse transcription–polymerase chain reaction and western blotting analysis for detection of p63 isoforms in uterine cervical cancers

Abstract

The aim of this study was to investigate the role of TAp63 and DeltaNp63 isoforms in uterine cervical cancers. The messenger RNA (mRNA) and protein expressions of TA and DeltaN forms as well as alpha, beta, and gamma isoforms of p63 were studied in seven SiHa, ME-180, SNU17, SNU902, SNU1160, SNU703, and SNU1299 human papillomavirus (HPV)-positive uterine cervical squamous cell carcinoma (SCC) cell lines, one HT3 HPV-negative SCC cell line, and one HeLa adenocarcinoma cell line using reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Fresh nonneoplastic and neoplastic tissues of uterine cervical and endometrial cancers were also studied. RT-PCR for TA and DeltaN form and three isoforms of p63 showed positive bands for both TA and DeltaN forms and for all three isoforms in cervical cancer cell lines but weak band for alpha isoform in HPV-negative HT3 SCC cell line and no band for beta isoform in HeLa adenocarcinoma cell line. RT-PCR for TA and DeltaN and three isoforms of p63 mRNA in tissue samples showed positive bands in almost all samples, except for gamma isoform, the expression was weak or absent in nonneoplastic tissues compared with neoplastic tissues. In western blotting, cancer cell lines and both nonneoplastic and neoplastic tissue samples showed expression of TA and DeltaN, and gamma isoform but beta isoform expression with or without alpha isoform was only found in cancer cell lines and neoplastic tissues. beta isoform, possibly of DeltaNp63, may be considered as an important isoform in uterine cervical squamous cell carcinogenesis.

Late cornified envelope 1C (LCE1C), a transcriptional target of TAp63 phosphorylated at T46/T281, interacts with PRMT5

“…Wb using anti-p63 antibody detected several bands around the predicted size of endogenous p63 protein in HeLa S3 cells but little or no detectable levels in PC3 and U2OS cells (Fig. S3b ), which is consistent with previous reports 21 – 23 . Because this phospho-specific antibody was not available for western blotting and U2OS cells had little or no expression of endogenous p63, the phosphorylation of endogenous TAp63-T281 was shown by immunofluorescence (IF) using HeLa S3 cells.…”

NF-Y loss triggers p53 stabilization and apoptosis in HPV18-positive cells by affecting E6 transcription

“…The level of p63 mRNA, already low in Hela control cells, was further reduced following NF-YA loss. At the protein level, we observed a decrease in ΔNp63, the only isoform we detected by Western blot in control cells (Supplementary Figure S5) [43, 44]. This result is consistent with the established role of NF-Y as transcriptional activator of the ΔNp63 promoter [45].…”

Human Papillomavirus Infection and Its Possible Correlation with p63 Expression in Cervical Cancer in Japan, Mongolia, and Myanmar

“…ΔNp63 is highly expressed in stem cells of epithelial tissue and is required for proliferation and maintenance of the epithelial stem cell population [28, 32]. Previous studies have indicated that TAp63 promotes epithelial cell differentiation, whereas ΔNp63 favors epithelial cell proliferation [8, 18]. In the present study we found the over-expression of p63 isoforms (TA and ΔN) and the association of p63 with cell proliferation in SCC, which together may promote carcinogenesis in cervical cancer.…”