PUMA mediates the apoptotic response to p53 in colorectal cancer cells

Abstract

Although several genes that might mediate p53-induced apoptosis have been proposed, none have previously been shown to play an essential role in this process through a rigorous gene disruption approach. We used a gene-targeting approach to evaluate p53-mediated death in human colorectal cancer cells. Expression of p53 in these cells induces growth arrest through transcriptional activation of the cyclin-dependent kinase inhibitor p21. If p21 is disrupted via gene targeting, the cells die through apoptosis. If the PUMA gene is also disrupted in such cells, apoptosis is prevented. The effects of PUMA on apoptosis were observed after exogenous overexpression of p53 as well as after exposure to hypoxia, a physiologic activator of p53, and DNA damage. The PUMA protein interacts with Bcl-X(L) and promotes mitochondrial translocation and multimerization of Bax. Accordingly, genetic disruption of BAX makes cells resistant to the apoptosis resulting from PUMA expression. These results suggest that the balance between PUMA and p21 is pivotal in determining the responses to p53 activation and provide a model for understanding the basis of p53 mutations in human cancer.

Figure 1

Targeted deletion of PUMA. (A) Two targeting constructs were designed, each containing a homologous arm and an overlapping fragment of the neomycin-resistant gene. The SphI sites within the PUMA gene, the targeting construct, and the position of the probe used for Southern blotting are shown. PCR screening primers P1 and P2 were described in Materials and Methods. Homologous recombination results in the deletion of exon 3, which contains the BH3 domain. Filled boxes represent PUMA exons 1a–4. The same constructs were used in the second round of targeting after excision of the neomycin-resistance gene by Cre recombinase. (B Upper) A Southern blot obtained with a PUMA probe after SphI digestion of genomic DNA. (Lower) The blot obtained with a p21 probe after BglII digestion. Clone 3 (PUMA^−/−, Upper) was used for targeting of the p21 locus, generating clones 5 and 6 (p21^+/−). A second round of targeting generated clones 7 and 8, which were p21^−/−. (C) Lysates from cells with the indicated genotypes were analyzed by immunoblotting with antibodies against PUMA, p21, p53, Bax, Bak, and α-tubulin. Cells were infected with Ad-p53 24 h before preparation of the lysates. Uninfected parental HCT116 cells (“Un”) show the basal level of p53-induced proteins in the absence of Ad-p53.

Figure 2

Apoptosis induced by p53 and hypoxia. (A) Cells of the indicated genotypes were infected with Ad-p53 for the times shown on the x axis. The fraction of apoptotic cells was assessed by fluorescence microscopy of DAPI-stained cells. The means and standard errors of the mean are illustrated. (B) Cells were infected with Ads encoding WT (Ad-p53) or mutant p53 (Ad-R175H) and then seeded in six-well plates. Attached cells were stained with crystal violet 14 days later. (C) Cells of the indicated genotypes were exposed to adriamycin for various periods and scored as in A. (D) Cells of the indicated genotypes were exposed to hypoxic conditions for various periods and scored as in A.

Figure 3

Protein domains required for PUMA function. (A) GFP-PUMA expression constructs. GFP was fused to the N terminus of the indicated fragments of PUMA cDNA. (B) GFP-PUMA constructs were transfected into 911 cells and visualized through the fluorescence of GFP. MitoTracker red dye was used to visualize mitochondria. The N150 construct devoid of the C-terminal 43 residues had a diffuse staining pattern that was distinct from GFP-PUMA or GFP-PUMAΔBH3. (C) DLD-1 cells were transfected with the indicated constructs and selected with geneticin for 2 weeks before staining with crystal violet. There was no difference in colony formation after transfection with PUMA-ΔBH3 compared with transfection with the empty vector (data not shown).

Figure 4

Biochemical changes during PUMA-induced apoptosis are Bax-dependent. (A) Cells with the indicated genotype were infected with Ad-PUMA or Ad-PUMAΔBH3 viruses, harvested at the indicated times, stained with CMXRos, and analyzed by flow cytometry to measure changes in mitochondrial membrane potential (Δψ). FL2 on the x axis reflects the intensity of the CMXRos signal. (B) Lysates from cells indicated genotypes infected with Ad-PUMA (wt) or Ad-PUMAΔBH3 (Δ) were analyzed by immunoblotting, using antibodies specific for the indicated proteins. The intact caspase 9 polypeptide is 46 kDa, whereas a degraded fragment of 37 kDa is detected in cells undergoing apoptosis. Tagged PUMA and PUMAΔBH3 proteins were detected by an antibody to HA. (C) DLD-1 cells were induced to express either PUMA or p53 in the presence or absence of the caspase inhibitor Z-VAD. At the indicated times, cells were assayed for apoptosis by fluorescence microscopy after staining with DAPI.

Figure 5

BAX deficiency rescues PUMA-induced apoptosis. (A) Mitochondrial and cytosolic fractions were prepared from DLD-1 cells in which PUMA had been induced by the removal of doxycycline. The fractions were analyzed by immunoblotting with antibodies to the indicated proteins. Cox IV and α-tubulin provided controls for the fractionation. (B) DLD-1 cells in which PUMA had been induced were cross-linked before subcellular fractionation and subjected to immunoblotting with an antibody to Bax. Bax monomers (1X) and multimers (2X–4X) are indicated. (C) Clones with various BAX genotypes were infected with Ad-PUMA (wt) or Ad-PUMAΔBH3 (Δ) for 48 h and the fraction of apoptosis was assessed by fluorescence microscopy of DAPI-stained cells. (D) HCT116 clones with the indicated BAX genotypes were infected with Ad-PUMA (wt) or Ad-PUMAΔBH3 (Δ) for 48 h then diluted into 12-well plates. Colonies were stained with crystal violet 10–14 days later. (E) Model of p53 function during tumorigenesis. As tumors progress to malignancy, they accumulate DNA damage (1). They also outgrow their blood supply, resulting in hypoxia of large regions of the tumors (31). The DNA damage and/or hypoxia activate p53, which transcriptionally activates several genes, including p21, 14-3-3σ, PUMA, and, in some cases, BAX. The cells either arrest or enter into apoptosis, depending on which of the two arms of the pathway is predominant in the particular cell type and microenvironment. Cells with a mutant p53 gene would have a selective growth advantage in either case, because both growth arrest and apoptosis would be diminished in such mutant cells. Although many other genes are induced by p53 (7), the ones included in the model have all been shown to be essential for the indicated downstream effects through gene-targeting experiments in mammalian cells.