Activation of c-myc gene expression by tumor-derived p53 mutants requires a discrete C-terminal domain

Abstract

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3' end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.

FIG. 1

Mutant p53 regulates c-myc gene expression. (A) Northern blot analysis of total RNA (10 μg/sample) isolated from LAPCx3Ras (LAP Cx3), Cx3Ras (Cx3), and Cx3Ras-LAP (LAP) cells on days 1 to 3 from control (C1 to C3) and IPTG-treated (I1 to I3) samples. Northern filters were sequentially probed for expression of c-myc, histone H4, PCNA, and β-actin. (B) Western blot analysis of mutant p53 expression, using total-cell extracts (5 μg/sample) prepared on days 1 to 3 from control (C1 to C3) and IPTG-treated (I1 to I3) cell lines. Samples were analyzed by using murine monoclonal antibody DO-1 as described in Materials and Methods. The experiments were repeated at least three times, and representative data are presented. Baculo, baculovirus.

FIG. 2

Mutant p53 transactivates the c-myc promoter. (10)1 murine fibroblasts and SaOs-2 human osteosarcoma cells were transfected with 0.25 μg of BgCAT alone (C) or cotransfected with BgCAT and 1 μg of the p53 empty-vector plasmid (CMV), wild-type p53 (WT), or mutant p53-281 (281) and assayed for CAT activity as described in Materials and Methods. Transactivation function is demonstrated by conversion of unacetylated [¹⁴C]chloramphenicol (lower band) to acetylated forms (upper bands). The experiments were repeated at least three times, and representative data are presented.

FIG. 3

Panel of tumor-derived human mutant p53 alleles transactivate the c-myc promoter. SaOs-2 cells were transfected with the BgCAT reporter alone (C) and with the indicated human wild-type (WT) and mutant p53 expression vectors and then assayed for CAT activity as described in Materials and Methods. Samples were transfected in duplicate, and the experiment was repeated at least three times; representative data are presented. Similar results were obtained when the human MDR1 promoter reporter construct was used.

FIG. 4

Mutant p53 increases steady-state levels of c-myc–CAT mRNA. (10)1 cells were transiently transfected with salmon sperm DNA (ssDNA), the c-myc reporter (BgCAT), mutant p53 (281), or BgCAT and p53-281 (BgCAT + 281) as described for Fig. 2. Two days after transfection, total RNA was isolated and analyzed for c-myc expression by RNase protection assays as described in Materials and Methods. Protected fragments (expected sizes) are as follows: undigested full-length probe (1,058 nt), endogenous c-myc P1 (573 nt) and P2 (413 nt), P2CAT (639 nt), and P1CAT (799 nt). As expected (40), endogenous c-myc mRNA derived from the predominant P2 promoter (accounting for ∼90% of c-myc transcripts) and the minor P1 promoter is evident in all samples. Unspliced P1 and P2 c-myc mRNA transcripts run approximately 10 nt slower than the spliced form. Transactivation of the c-myc promoter reporter by mutant p53 is demonstrated by the appearance of c-myc–CAT mRNA in the BgCAT–p53-281 cotransfected samples but not in the ssDNA-, BgCAT-, or p53-281-only samples. The c-myc-CAT mRNA is derived from the P2 promoter, based on multiple RNase protection assays using various radiolabeled probes and RNA size standards.

FIG. 5

Mapping of the mutant p53 response region in c-myc to the 3′ end of exon 1. PvuCAT c-myc promoter reporter deletion constructs were transfected into (10)1 cells with and without mutant p53-281, and promoter activity was measured by CAT assay as described in Materials and Methods. (A) Schematic diagram of the c-myc–CAT reporter constructs. The PvuCAT reporter spans −140 to +413 and contains the P2 promoter, nontranslated exon 1, and the exon 1/intron 1 (Ex/Int) junction. The c-myc promoter 3′ deletion constructs span the regions from −140 to +358 (Δ5), +271 (Δ4), +189 (Δ3), +79 (Δ2), and +7 (Δ1). (B) Basal and p53-281-induced promoter activities are presented from two independent experiments that were each conducted in duplicate. Similar results were also obtained when the BgCAT and derivative 3′ deletion promoter-reporter constructs were used (data not shown).

FIG. 6

The mutant p53 response region in c-myc is position and orientation dependent. The c-myc-heterologous minimal promoter reporters were constructed as described in Materials and Methods and are schematically diagrammed. The heterologous reporters were transfected into (10)1 cells with and without mutant p53-281, and transactivation was measured by CAT assays as described in Materials and Methods. Results are presented as the fold induction of reporter activity with mutant p53-281 compared to basal expression of the reporter only. The basal promoter activities of p1634CAT and each of the heterologous promoters are approximately equal but close to background. c-myc exon 1 sequences spanning −22 to +413 (H fragment) (A) and c-myc exon 1 sequences from +174 to +413 (N fragment) (B) cloned into the p1634CAT minimal heterologous promoter reporter. Results of multiple independent experiments are presented. INT, tdt initiator element.

FIG. 7

The N and C termini are required for mutant p53 transactivation. (A) Schematic diagram of p53 highlights the domains for transactivation (acidic), sequence-specific DNA binding, oligomerization (oligo), single-strand DNA/RNA binding, and amino acid 392, which is covalently associated with 5.8S RNA. Point mutations in the acidic domain at amino acids 22 (L→Q), 23 (W→S), and 392 (S→A), as well as the C-terminal truncations, were constructed in wild-type and mutant p53-281 as described in Materials and Methods. Lower left, mutant p53-281 transactivation function requires the C terminus. (10)1 cells were cotransfected with BgCAT and either CMV empty vector (C), mutant p53-281, or mutant p53-281Δ360. Lower right, the C terminus is dispensable for wild-type p53 transactivation function. (10)1 cells were cotransfected with CosXCAT, which is a wild-type p53 response reporter, and either CMV empty vector (C), wild-type p53 (WT), or p53Δ360. (B and C) Amino acids 372 to 380 are essential for mutant p53-281 transactivation function. (10)1 cells were transfected with BgCAT alone and cotransfected with BgCAT and either the p53-281 or p53-281 truncation mutant. (B) Western blot analysis of full-length mutant p53-281 and truncated mutant p53 in cell extracts analyzed for CAT activity in panel C. Equal amounts of protein were analyzed by using polyclonal antibody Ab-7 as described in Materials and Methods. Baculo, purified wild-type p53 protein. Full-length mutant p53-281 and truncated proteins are as indicated. (C) BgCAT reporter activity in response to mutant p53. Data are presented as fold induction of reporter activity with mutant p53 compared to basal promoter expression. (D) Mutant p53 transactivation function requires the N terminus. (10)1 cells were cotransfected with BgCAT and either CMV empty vector, p53-281, triple mutant 22-23/281, or 281Δ360. Samples were transfected in duplicate, and data are presented as fold induction of reporter activity with mutant p53 compared to basal expression. These experiments were repeated at least three times, and representative data are presented.

FIG. 8

Inhibition of mutant p53 gain of function through a transdominant negative mechanism. (10)1 cells were transiently cotransfected with the BgCAT reporter (0.5 μg) in combination with CMV vector only (Control; 2 μg), mutant p53-281 (mtp53-281; 2 μg), wild-type p53 (wtp53; 5 μg), DD1 (5 μg), and deltaSS (5 μg) and analyzed for CAT activity as described in Materials and Methods. The experiment was repeated at least three independent times, and representative data are presented. Similar results were obtained when the MDR1-CAT reporter was used.