Quantitative analysis of p53-targeted gene expression and visualization of p53 transcriptional activity following intratumoral administration of adenoviral p53 in vivo

Abstract

To analyze the mechanism of the antitumor effect of an adenoviral vector expressing the p53 tumor suppressor (Ad-p53) in vivo, we quantitatively assessed p53-targeted gene expression and visualized transcriptional activity of p53 in tumors in nude mice treated with Ad-p53. Human lung cancer (H1299) xenografts established in nude mice were treated by intratumoral administration of Ad-p53. The levels of expression of exogenous p53 and p53-targeted genes p21, MDM2, Noxa, and p53AIP1 were quantified by real-time reverse transcription-PCR (RT-PCR) and induction of apoptosis was observed histochemically on days 1–3, 7, and 14 after treatment. Expression of mRNA of exogenous p53 and p53-targeted genes (except p53AIP1) was at its maximum 1 day after Ad-p53 treatment and then decreased rapidly; apoptosis was evident in situ 2–3 days after treatment. We developed a noninvasive and simple method for monitoring the transcriptional activity of exogenous p53 following intratumoral administration of Ad-p53 in nude mice. We established H1299 cells that express the green fluorescent protein (GFP) reporter gene under the control of p53-responsive p21 promoter (i.e., the p53R-GFP reporter system). Xenografts of these cells in nude mice were treated by intratumoral administration of Ad-p53, and the transcriptional activity of exogenous p53 could be visualized as intratumoral GFP expression in real time by 3-CCD camera. Expression of GFP was maximal 3 days after treatment and decreased remarkably by 7 days after treatment. We demonstrated that Ad-p53 treatment rapidly induced p53-targeted genes and apoptosis in tumors and succeeded in visualizing p53 transcriptional activity in vivo. We also found that Ad-p53 infection induced phosphorylation of p53 at Ser46 in p53-sensitive H1299 cells in vitro but not in p53-resistant H226Br cells, suggesting that phosphorylation of Ser46 is involved in p53-dependent apoptosis. Our data indicate that quantitative analysis of p53-targeted gene expression by real-time quantitative RT-PCR and visualization of p53 transcriptional activity in fresh xenografts by using the p53R-GFP reporter system may be useful in assessing the mechanisms of the antitumor effects of Ad-p53 and novel therapeutic approaches.

Vasohibin‐1 as a potential predictor of aggressive behavior of ductal carcinoma in situ of the breast

“…Quantitative normalization of each target genes in each tissue sample was performed using the expression of RPL13A mRNA as an internal control and evaluated as a ratio compared with the average expression ratio of CD31 and vasohibin-1 in DCIS cases (the averages of CD31 ⁄ RPL13A and vasohibin-1 ⁄ RPL13A in DCIS cases), respectively. (18)(19)(20) PCR was set up at 2 mM Mgcl2, (Vasohibin,CD31), 3 mM (RPL13A) and 10 pmol ⁄ lL of each primer. The information of primers used in this study is summarized in Table 2.…”

Structure-Based Discovery of MDM2/4 Dual Inhibitors that Exert Antitumor Activities against MDM4-Overexpressing Cancer Cells

“…To further examine the cellular mechanism of 31, a quantitative polymerase chain reaction (qPCR) assay analyzing the expression of p53-targeted genes (Figure 10) was performed in RKO cells in the presence of increasing concentrations of 31. The data in Figure 10 show that 31 dose-dependently increased the expression of p53-targeted genes, including p21, MDM2, and PUMA genes, 59,60 with the exception of MDM4 and TP53 (SI, Figure S1). It is worth noting that 31 increased the gene expressions of p21 and MDM2 at 0.31 μM, which is lower than 0.62 μM for RG7388, indicating a higher cellular potency of 31.…”

Isotretinoin treatment upregulates the expression of p53 in the skin and sebaceous glands of patients with acne vulgaris

“…Total RNA was extracted from 10 mg skin tissue after lysis and homogenization, using silicate gel technique provided by the RNeasy Mini Kit (Qiagen) [ 65 ]. The concentration and purity of RNA were measured at 260, 280 and 230 nm using Nano Drop 2000c spectrophotometer (Thermo Scientific, USA).…”