Analysis of p53-regulated gene expression patterns using oligonucleotide arrays

Abstract

Oligonucleotide microarrays were employed to quantitate mRNA levels from a large number of genes regulated by the p53 transcription factor. Responses to DNA damage and to zinc-inducible p53 were compared for their transcription patterns in cell culture. A cluster analysis of these data demonstrates that genes induced by gamma radiation, UV radiation, and the zinc-induced p53 form distinct sets and subsets with a few genes in common to all these treatments. Cell type- or cell line-specific p53 responses were detected. When p53 proteins were induced with zinc, the kinetics of induction or repression of mRNAs from p53-responsive genes fell into eight distinct classes, five different kinetics of induction, and three different kinetics of repression. In addition, low levels of p53 in a cell induced or repressed only a subset of genes observed at higher p53 levels. The results of this study demonstrate that the nature of the p53 response in diverse mRNA species depends on the levels of p53 protein in a cell, the type of inducing agent or event, and the cell type employed. Of 6000 genes examined for p53 regulatory responses, 107 induced and 54 repressed genes fell into categories of apoptosis and growth arrest, cytoskeletal functions, growth factors and their inhibitors, extracellular matrix, and adhesion genes.

Figure 1

Inducible p53 in colon carcimona cell line EB-1. p53 and human MDM2 were detected by Western blots using monoclonal antibodies 1801 and 2A9, respectively. A small GTPase Ran was used as control. (A) EB-1 cells were stimulated with 100 μm zinc chloride starting at time 0 hr, and the level of p53 protein was elevated as early as 2 hr. Maximum induction of p53 was seen between 4 and 8 hr. Human MDM2 protein was also induced in these cells with a delay compared with p53 induction. No induction of p53 or hMDM2 in parental EB cells was observed following the same stimulation. The amount of 10 μg of total protein was loaded in each lane, and the same blot was probed by anti-p53, anti-hMDM2, and anti-Ran antibodies. (B) The levels of induced p53 protein in EB-1 cells depend on the zinc concentration in the culture media. EB-1 cells were cultured in presence of 0, 1, 10, 50, 100, and 200 μm of zinc chloride for 8 hr. A minimum of 50 μm of zinc was required to induce p53 proteins in these cells, and 100 and 200 μm zinc induced p53 to much higher levels in a dose-dependent manner. The amount of 20 μg of total protein was loaded in each lane. (C) Induction of p53 by UV and γ irradiation in different cancer-derived cell lines. Each cell line was treated with radiation as indicated, and cell lysates were prepared after 3–6 hr (see Materials and Methods). p53 was induced following both UV and γ irradiation in all the cell lines tested. The amount of 65 μg of total protein was loaded in each lane.

Figure 2

Functional grouping of p53-induced genes (open bars) compared with the same grouping of 100 randomly selected genes (solid bars). Significant differences (P < 0.02) are indicated by an asterisk. p53-induced genes were enriched for genes involved in apoptosis, growth arrest, the cytoskeleton, growth factors, and cell adhesion. Genes involved in cell metabolism were less abundant compared with a randomly selected gene pool.

Figure 3

Cluster analysis of gene expression profiles following p53 induction. A total of 69 genes selected for this analysis were clustered into several groups on the basis of the similarity of their expression profiles by a reported procedure (Eisen et al. 1998). The expression level of each gene is represented by the ratio of gene intensity in EB-1 over that in EB and is shown in a horizontal strip. The degree of redness represents level of induction, whereas that of greenness represents level of repression.

Figure 4

Average expression profiles for each gene cluster. The expression profile of each gene in a cluster was normalized onto a 0–10 scale and then averaged. Eight clusters were derived from the cluster analysis, showing different induction kinetics.

Figure 5

Independent measurements of selected gene expression levels by RT–PCR (left) and p53-spotted oligonucleotide arrays (right). Expression levels measured by RT–PCR were normalized to GAPDH and plotted relative to the level in control cells. Both methods gave very similar results in the gene expression profiles.

Figure 6

Gene regulation by different levels of p53. (A) Genes in this group were responsive to p53 at both high and low levels. (B) Genes in this group only responded to high level of p53. Selected gene expression profiles from each group are shown for both 100 and 50 μm zinc-induced cells.

Figure 7

Cluster analysis of gene expression patterns after UV or γ irradiation. A total of 112 genes and 5 different cell lines following indicated radiation were included in this analysis. This analysis was performed in two dimensions, with one for cell lines and radiation, the other one for 112 genes. Cells with wild-type p53 fall into distinct clusters following different irradiation. The 112 genes were clustered into 9 groups with different expression patterns in cells with wild-type p53 following different types of radiation.