High-level expression of wild-type p53 in melanoma cells is frequently associated with inactivity in p53 reporter gene assays

Abstract

Background: Inactivation of the p53 pathway that controls cell cycle progression, apoptosis and senescence, has been proposed to occur in virtually all human tumors and p53 is the protein most frequently mutated in human cancer. However, the mutational status of p53 in melanoma is still controversial; to clarify this notion we analysed the largest series of melanoma samples reported to date.

Methodology/principal findings: Immunohistochemical analysis of more than 180 melanoma specimens demonstrated that high levels of p53 are expressed in the vast majority of cases. Subsequent sequencing of the p53 exons 5-8, however, revealed only in one case the presence of a mutation. Nevertheless, by means of two different p53 reporter constructs we demonstrate transcriptional inactivity of wild type p53 in 6 out of 10 melanoma cell lines; the 4 other p53 wild type melanoma cell lines exhibit p53 reporter gene activity, which can be blocked by shRNA knock down of p53.

Conclusions/significance: In melanomas expressing high levels of wild type p53 this tumor suppressor is frequently inactivated at transcriptional level.

Figure 1. p53 expression in melanoma tissues.

(A) Immunohistochemical detection of p53 in a primary melanoma. (B) 104 primary cutaneous and 81 metastatic melanoma samples were stained for p53. The percentage of positively stained tumor cells is given as bar graph.

Figure 2. Reproducibility of p53 sequencing.

Sequencing chromatograms depicting the only reproducible p53 mutation and an example of an apparent mutation which could not be reproduced in an independent PCR amplification from the same sample.

Figure 3. Functional inactivity in 4 out of 6 melanoma cell lines expressing wild type p53 measured by the p53 reporter construct PathDetect®.

The p53 reporter construct PathDetect® encoding luciferase under the control of a p53 responsive element (14× TGCCTGGACTTGCCTGG) was transfected into the indicated melanoma cell lines. Following selection for a co-transfected puromycin resistence integration of the luciferase gene was confirmed by real time PCR and the relative copy numbers are indicated (top row). The cells were then infected with lentiviral supernatants carrying the shRNA expression construct KH1 containing either a scrambled or a sequence targeting p53 . On Day 4 following infection the cells were harvested and lysates were subjected to a luciferase assay (upper part) or were analysed for expression of the indicated proteins by western blot (lower part). Sequence analysis of all p53 coding exons revealed that four cell lines carried the indicated p53 amino acid substitutions.

Figure 4. Functional inactivity in 4 out of 8 melanoma cell lines expressing wild type p53 measured by the p53 reporter construct pGreenFire®.

Human epidermal melanocytes and the indicated melanoma cell lines were stably transduced with a lentiviral pGreenFire reporter construct encoding luciferase and GFP under the control of a p53 responsive element (4× CGACATGCCCGGGCATGT). The cells were then infected with lentiviral supernatants carrying the shRNA expression construct KH1 containing either a scrambled or a sequence targeting p53 . On Day 4 following infection total cell lysates were analysed for p53 expression by immunoblotting (lower part). Equal loading was controlled by Ponceau S staining of the blot. In the upper part the corresponding luciferase activity is depicted, normalized to the relative luciferase load of the cell lines determined by Real time PCR.

Figure 5. Determination of p53 reporter activities associated with cell cycle arrest.

The indicated cells stably transduced with the pGreenFire reporter construct encoding luciferase and GFP under the control of a p53 reporter were cultured in the absence or presence of the mdm-2 inhibitor Nutlin 3a. After 24 hours p53 expression (C), luciferase acitvity (A) and GFP fluorescence (B) were analyzed. Cell cycle analysis was performed on day 2 of treatment and the percentage of cells in S-phase are depicted (D).