Identification and classification of p53-regulated genes

Abstract

Sequence-specific transactivation by p53 is essential to its role as a tumor suppressor. A modified tetracycline-inducible system was established to search for transcripts that were activated soon after p53 induction. Among 9,954 unique transcripts identified by serial analysis of gene expression, 34 were increased more than 10-fold; 31 of these had not previously been known to be regulated by p53. The transcription patterns of these genes, as well as previously described p53-regulated genes, were evaluated and classified in a panel of widely studied colorectal cancer cell lines. "Class I" genes were uniformly induced by p53 in all cell lines; "class II" genes were induced in a subset of the lines; and "class III" genes were not induced in any of the lines. These genes were also distinguished by the timing of their induction, their induction by clinically relevant chemotherapeutic agents, the absolute requirement for p53 in this induction, and their inducibility by p73, a p53 homolog. The results revealed substantial heterogeneity in the transcriptional responses to p53, even in cells derived from a single epithelial cell type, and pave the way to a deeper understanding of p53 tumor suppressor action.

Figure 1

Modified tet-inducible expression system. (A) Diagram of the modified tet-off system constructs. *Neo denotes a optimized Neo-resistance gene (18). (B) Induction of p53 in p53 wtA cells. Cells were induced by removing doxycycline for the indicated times and assayed by Western blot analysis for p53 and p21 expression. β-Catenin served as the loading control. (C) Inducible expression of p53 and p73 resulted in induction of p21, but mutant forms of p53 and p73 did not. A second clone expressing mutant p53 and p73 yielded results identical to those shown.

Figure 2

Effects of p53 expression. (A) Mutant or wt p53- and p73-inducible clones were assayed for nuclear morphology by fluorescence microscopy at the indicated time points. Red bars represent p53- or p73-induced cells, and blue bars represent uninduced cells. At least 200 cells were counted for each determination. (B) Morphology after p53 induction. The same fields are shown as viewed under phase contrast or fluorescence microscopy. The four panels at Left were p53 wtA cells, and the four panels at Right were p53 mut cells grown in induction (Induced) or normal medium (Uninduced) for 24 hr.

Figure 3

Confirmation of SAGE results by Northern blot analysis. Representative Northern blots are shown with RNA prepared from cells induced (“+”) for 9 hr or uninduced (“−”). (A) RNA from two independent p53-inducible clones was analyzed to confirm induction of transcripts by p53. No induction of these transcripts was observed in RNA prepared from cells with inducible mutant p53 genes (data not shown). (B) RNA from two independent p73-inducible clones was used to assay induction of transcripts by p73.

Figure 4

Heterogeneity in the induction of p53-responsive genes. (A) Time course of induction. The p21, Jun-B, Caveolin, and DR-5 genes were detectably induced by 3 hr after doxycycline withdrawal, whereas Gadd45 was induced 12 hr later. RNA was prepared from wtA cells at the indicated time points. (B) Induction by p53 adenoviruses. Class I genes included p21, Bax, Caveolin (Cav), DR5, Htk, and Gpc, whereas RTP, Jun-B, and Fas were class II genes, and thrombospondin (Tsp) was a class III gene. “W” and “M” refer to the adenoviruses expressing wt p53 and mutant p53, respectively. (C) Induction by chemotherapeutic agents. RNA from the indicated colorectal cancer cells lines treated with adriamycin (Adr) or 5-FU for 24 hr was analyzed. RNA from untreated cells was used as control (con). The expression of p21 was strongly induced by both drugs in both cell lines expressing wt p53 but was not induced in DLD-1 cells containing endogenous mutant p53 or in p53 KO cells. In contrast, Bax was induced by adriamycin but not by 5-FU. The induction of Bax by adriamycin was considerably stronger in the two lines with wt p53 than in DLD-1 or p53 KO cells. Another pattern was observed with Fas, in which little induction was observed in any of the lines studied.