Disruption of p53 in human cancer cells alters the responses to therapeutic agents

Abstract

We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.

Figure 1

Nuclear morphology of cells treated with anticancer agents. Wild-type (p53^+/+), p53^–/–, and p21^–/– cells, as indicated, were treated with 0.34 μM adriamycin (ADR) for 72 hours or with 375 μM 5-FU for 60 hours, stained with HOECHST 33258, and photographed at ×40.

Figure 2

Induction of apoptosis by drug treatment. (a) HOECHST 33258–stained cells analyzed by fluorescence microscopy after 96 hours of treatment with adriamycin. (b) Time course of cell death after treatment with 0.34 μM adriamycin. (c) Cell death after 60 hours of treatment with 5-FU. Two different heterozygous cell lines (A and B) were assayed. (d) Time course of cell death after treatment with 375 μM 5-FU. Cells marked +/– had 1 allele of the indicated gene disrupted and were the parents of the cells with both alleles disrupted (–/–).

Figure 3

Cell cycle distribution of drug-treated cells with wild-type (p53^+/+) and disrupted (p53^–/–) p53 genes. HOECHST 33258–stained nuclei were assayed by flow cytometry after treatment with 0.34 μM adriamycin (ADR), 375 μM 5-FU, 10 μM Tomudex (TDX), or 25 μM methotrexate (MTX) for the indicated time periods. Positions of peaks corresponding to diploid (2N), tetraploid (4N), and subdiploid (< 2N) DNA contents are shown. The scale on the horizontal axis shown is linear.

Figure 4

Characteristics of 5-FU–induced apoptosis. (a) p53 wild-type cells incubated with the indicated concentrations of 5-FU alone (filled squares), or in combination with 400 μM thymidine (inverted filled triangles) or 400 μM uridine (filled triangles). (b) Cells were incubated with 5-FU in the absence (No CHX) or continuous presence (CHX) of 10 μg/mL cycloheximide. Alternatively, cycloheximide was added 6, 12, or 18 hours after addition of 5-FU (CHX T6, T12, and T18, respectively). All cells were harvested 60 hours after the addition of 5-FU. (c) Western blot of p53 and p21 proteins in whole-cell lysates after adriamycin (ADR) or 5-FU treatment. (d and e) Colony formation assay. After treatment with 375 μM 5-FU for 6 or 12 hours, cells were replated in drug-free medium and stained; colonies were counted 12 days later. Representative flasks are shown in d.

Figure 5

Growth of xenograft tumors. (a) Animals with 1 tumor of each genotype were treated with either 7.5 Gy or 15 Gy doses of γ-radiation. (b) Intravenous 5-FU (12.5 mg/kg) or carrier alone (No drug) was administered on 2 consecutive days, ending on day 0. Filled triangles: tumors composed of p53⁺⁺-containing cells; open triangles: tumors composed of p53^–/– cells.