A panel of isogenic human cancer cells suggests a therapeutic approach for cancers with inactivated p53

Abstract

Through targeted homologous recombination, we developed a panel of matched colorectal cancer cell lines that differ only with respect to their endogenous TP53 status. We then used these lines to define the genes whose expression was altered after DNA damage induced by ionizing radiation. Transcriptome analyses revealed a consistent up-regulation of polo-like kinase 1 (PLK1) as well as other genes controlling the G(2)/M transition in the cells whose TP53 genes were inactivated compared with those with WT TP53 genes. This led to the hypothesis that the viability of stressed cells without WT TP53 depended on PLK1. This hypothesis was validated by demonstrating that stressed cancer cells without WT TP53 alleles were highly sensitive to PLK1 inhibitors, both in vivo and in vitro.

Fig. 1.

TP53 locus before and after targeting. (A) (Top) TP53 genomic locus including exons 2, 3, and 4 (blue boxes). (Middle) Same locus after insertion of the targeting vector (62), resulting in replacement of exon 2 and surrounding intronic sequences with a cassette containing intronic sequences (INT), splice acceptor site (SA), internal ribosomal entry sequence (IRES), neomycin phosphotransferase gene (neo), stop codon (STOP), and polyadenylation site (pA). Red triangles indicate loxP recombination sites. (Bottom) The same locus after Cre-mediated excision of the targeting construct. (B) (Top) TP53 genomic locus including exons 5 to 9 (blue boxes). (Middle) Same locus after insertion of the rAAV targeting vectors, one containing the WT sequence in exon 7 and the other a mutation (R248W) in exon 7. (Bottom) The same locus after Cre-mediated excision of the knockin constructs. In both A and B, arrows indicate the position of the PCR primers used to screen clones for the desired recombination. The sizes of the PCR products generated with these primers are also indicated.

Fig. 2.

TP53 and p21 protein expression in isogenic cells lines of various TP53 genotypes. Cells were cultured in the absence or presence of 5-FU. The CypB blots were used as loading controls.

Fig. 3.

TP53 genotype-dependent effect of Nutlin-3 on isogenic cancer cell lines. The indicated lines were exposed to increasing doses of Nutlin-3 for 96 h, and their growth was evaluated by assessing cell number in a SYBR green growth assay. All values were normalized to the number of cells of untreated controls. The 2 lines marked +/SIL were independently generated clones.

Fig. 4.

G₁ arrest in cell lines containing WT TP53 genes. Flow cytometry profiles before and after exposure of the indicated lines to ionizing radiation are shown. After irradiation, the cells were treated with nocodazole to block them from undergoing mitosis and entering into a subsequent G₁. Peaks corresponding to G₁ and G₂/M are indicated.

Fig. 5.

Evaluation of BI-2536 on the growth of isogenic cell lines with or without WT TP53 genes. Growth of the indicated lines to increasing doses of the PLK1 inhibitor BI-2536 alone, in the presence of ionizing radiation, or in the presence of Nutlin-3. The growth was assessed by a SYBR green-based growth assay and was normalized to the growth of untreated controls (A and D), in the presence of irradiation (B and E) or the presence of Nutlin-3 (C and F). The 2 lines marked +/SIL were independently generated clones.

Fig. 6.

In vivo effects of BI-2536 and Nutlin-3. (A) BI-2536 treatment of nude mice bearing HCT116 TP53^−/− xenografts results in regression of the tumors. The mice were treated for 3 weeks as follows: twice a week with 100 mg/kg BI-2536 and twice a day 2 times a week with 200 mg/kg Nutlin-3 before BI-2536 treatment. The volume of the tumor was measured on the days indicated. (B) BALB/c mice were given 200 mg/kg Nutlin-3 4 and 24 h before administering a single i.v. dose of 100 mg/kg BI-2536. Neutrophils were counted before initial treatment and every 24 h thereafter just before BI-2536 administration. Means and SD of the counts from 5 mice in each treatment arm are shown.