pubmed.ncbi.nlm.nih.gov

p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs - PubMed

  • ️Thu Jan 01 1998

p53 activates the CD95 (APO-1/Fas) gene in response to DNA damage by anticancer drugs

M Müller et al. J Exp Med. 1998.

Abstract

Chemotherapeutic drugs cause DNA damage and kill cancer cells mainly by apoptosis. p53 mediates apoptosis after DNA damage. To explore the pathway of p53-dependent cell death, we investigated if p53-dependent apoptosis after DNA damage is mediated by the CD95 (APO-1/Fas) receptor/ligand system. We investigated hepatoma, gastric cancer, colon cancer, and breast cancer cell lines upon treatment with different anticancer agents known to act via p53 accumulation. Cisplatin, mitomycin, methotrexate, mitoxantrone, doxorubicin, and bleomycin at concentrations present in the sera of patients during therapy led to an upregulation of both CD95 receptor and CD95 ligand. Induction of the CD95 ligand occurred in p53 wild-type (wt), p53 mutant (mt), and p53 deficient (p53(-/-)) cell lines and at wt and mt conformation of temperature-sensitive p53 mutants. In contrast, upregulation of the CD95 receptor was observed only in cells with wt p53, not in cells with mt or without any p53. Restitution of inducible wt p53 function restored the ability of p53(-/-) Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. This rendered the cells sensitive to CD95-mediated apoptosis. In an attempt to understand how CD95 expression is regulated by p53, we identified a p53-responsive element within the first intron of the CD95 gene, as well as three putative elements within the promoter. The intronic element conferred transcriptional activation by p53 and cooperated with p53-responsive elements in the promoter of the CD95 gene. wt p53 bound to and transactivated the CD95 gene, whereas mt p53 failed to induce apoptosis via activation of the CD95 gene. These observations provide a mechanistic explanation for the ability of p53 to contribute to tumor progression and to resistance of cancer cells to chemotherapy.

PubMed Disclaimer

Figures

Figure 2
Figure 2

Quantitative flow cytometry analysis of CD95 receptor expression in different solid human tumor cell lines after treatment with diverse anticancer drugs. *Clinically relevant concentrations of the cytostatic drugs. Percent CD95 expression was calculated as % CD95+ cells − % Quantum Red+ cells. Only cell lines with wt p53 expression, HepG2, AGS, HS746T, and MCF-7 ( filled symbols), displayed upregulated CD95 receptor expression up to >80% positive cells in response to drug treatment. In contrast, neither Hep3B cells lacking p53 nor Huh7 and HT29 cells (open symbols) with mt p53 responded to cytotoxic treatment with upregulation of the CD95 receptor.

Figure 1
Figure 1

FACS® analysis of CD95 receptor expression in AGS (wt p53) and HT29 cells (mt p53). Clinically relevant concentrations of diverse anticancer drugs with different mechanisms of action induce CD95 receptor expression (dependent on the p53 status). As an example for the investigated cell lines expressing either wt or mt p53, AGS colon cancer cells and HT29 colon cancer cells are shown here. Enhanced CD95 receptor expression was observed in hepatoma, gastric, colon, and breast cancer cell lines with wt p53 only. Percent enhanced CD95 expression was calculated as (% CD95+ treated cells – % Quantum Red+ treated cells) – (% CD95+ control cells – % Quantum Red+ control cells). AGS cells, for example, displayed enhanced CD95 receptor expression: up to 60% positive cells in response to mitomycin, up to 20% in response to 5-fluorouracil, and up to 50% positive cells in response to bleomycin.

Figure 3
Figure 3

Increased responsiveness towards induction of apoptosis by CD95 receptor stimulation after treatment with anticancer drugs. Cytotoxicity assay with MTT staining of viable cells. HepG2 cells (wt p53, inverted triangles), Huh7 cells (mt p53, squares), and Hep3B cells (p53−/−, diamonds) were treated with different doses of 5-fluorouracil, methotrexate, mitomycin, cisplatin, mitoxantrone, doxorubicin, etoposide, and cyclophosphamide alone for 48 h and in combination with or without IgG3 anti–APO-1, 100 ng/ml, for an additional 24 h. Data are expressed as the fraction of living cells treated with specific anticancer drug only (mean ± SD, n = 6 wells). Only HepG2 cells with wt p53 exhibited an increased responsiveness towards induction of apoptosis by agonistic anti–APO-1 antibodies after cytostatic treatment. Anti–APO-1 treatment did not induce further toxicity in Huh7 cells (mt p53) or Hep3B cells (p53−/−). * By MANOVA, between-subject effect P < 0.0001 compared with Hep3B, P < 0.0001 compared with Huh7.

Figure 4
Figure 4

Induction of the CD95L by cytostatic drugs with different mechanisms of action. Semiquantitative PCR analysis of CD95L mRNA expression in HepG2, Hep3B, and Huh7 cells upon treatment with cyclophosphamide (cyclo), 5-fluorouracil (5-FU), doxorubicin (doxo), mitomycin (mitom), mitoxantrone (mitox), and actinomycin (actino). CD95L mRNA expression was induced in HepG2 (wt p53), Hep3B (p53−/−), and Huh7 cells (mt p53). Densitometry was performed to analyze CD95L expression in relation to β-actin expression. CD95L mRNA induction was independent of the p53 status of the cells. The extent of CD95L expression upon chemotherapeutic treatment showed variability dependent on the agent and the cell line tested.

Figure 5
Figure 5

Induction of the CD95L by bleomycin (bleo) in the temperature-sensitive (ts) mutants p53ala143 and p53val135. Semiquantitative PCR analysis of CD95L mRNA expression in Hep3B cells stably expressing the temperature-sensitive mutant p53ala143 or p53val135. Densitometry was performed to quantify CD95L expression in relation to β-actin expression. Bleomycin treatment induced CD95L mRNA expression in the temperature-sensitive mutants p53ala143 and p53val135 at both temperatures, independent of their p53 mutational status.

Figure 6
Figure 6

Restitution of a wt p53 status restores the ability of the p53−/− cell line Hep3B to increase the CD95 receptor upon anticancer treatment (FACS® analysis). Transient transfection of p53−/− Hep3B cells with wt p53. Either a wt p53 expression plasmid in which expression of the p53 gene is under the control of the CMV promoter, or the corresponding control plasmid without the p53 insert (mock) was transiently transfected into Hep3B cells. Upon transient transfection with wt p53 cDNA and bleomycin treatment, p53−/− Hep3B cells increased CD95 receptor expression.

Figure 7
Figure 7

Induction of the CD95 receptor by tamoxifen activation of a p53–estrogen receptor fusion protein in stably transfected p53−/− Hep3B cells (FACS® analysis). The p53−/− cell line Hep3B was stably transfected with either puromycin resistance alone (control cell line: BT-2E) or with a p53 (modified)–estrogen receptor chimera (cell line: BT-4P). In BT-4P cells, p53 activity is induced by addition of the specific ligand 4-OH tamoxifen. The tamoxifen-activated p53 induces CD95 receptor expression in BT-4P cells. A further increase in CD95 receptor expression up to 30% of the cells is induced by bleomycin treatment (A). BT-2E did not exhibit CD95 receptor expression either before or after tamoxifen treatment (B). The functionality of the upregulated CD95 receptor in BT-4P cells is evident upon treatment of BT-4P with IgG3 anti–APO-1. Stimulation of the CD95 receptor by IgG3 anti–APO-1 and concurrent bleomycin treatment lead to an increased rate of apoptosis, after tamoxifen activation of p53 in BT-4P cells (C, + tamo + anti-APO).

Figure 8
Figure 8

Restitution of inducible wt p53 function restores the ability of p53−/− Hep3B cells to upregulate the CD95 receptor in response to anticancer drugs. FACS® analysis of CD95 receptor expression in Hep3B clones stably expressing the temperature-sensitive (ts) mutant p53val135 (A) or p53ala143 (B). These temperature-sensitive mutants show mt p53 conformation at 37°C; at 32°C, the permissive temperature, they regain wt p53 characteristics. The CD95 receptor is inducible only at the permissive temperature, 32°C. In the temperature-sensitive mutant p53val135, temperature down-shift induces the CD95 receptor in up to 40% of the cells; additional treatment with bleomycin leads to CD95 expression in up to 70% of the cells (A). Likewise, in the temperature-sensitive mutant p53ala143, CD95 receptor expression increased to 30% of the cells upon temperature down-shift and additional bleomycin treatment (B).

Figure 9
Figure 9

(A) Map of the human CD95 gene. Exons 1–9 are numbered and represented by black boxes. (B) Position of the putative p53 binding elements (p53-BE) within the CD95 gene promoter, suggested by computer analysis. Numbering is according to Wada et al. (reference 57). Shown also is a comparison between each element and the consensus p53-binding site (bottom [reference 63]). R, purine; Y, pyrimidine; W, A or T. *Putative. Lower case letters indicate deviations from the consensus.

Figure 10
Figure 10

The intronic p53-binding site within the CD95 gene. (A) Sequence of the first 266 nucleotides of the Sau3A1 fragment, which contains the p53-binding site in the first intron of the CD95 gene (sequence data available from EMBL/GenBank/DDBJ under accession no. AJ011034). Numbering is according to Wada et al. (reference 57). p53BE, the putative p53-binding element. (B) Position of the putative p53-binding element within the CD95 gene. Numbering is as in Wada et al. (reference 57); the first ATG of the CD95 protein coding region is indicated. (C) Comparison between the CD95 intronic putative p53-binding element (top line) and the consensus p53-binding site (bottom line; reference 63). R, purine; Y, pyrimidine; W, A or T. Lower case letters indicate deviations from the consensus.

Figure 11
Figure 11

The p53-binding intronic CD95 region confers p53-dependent transcriptional activation. (Left) Schematic diagram of relevant luciferase reporter constructs used for transcriptional analysis. Ps, 1.43-kb CD95 promoter region (reference 57); P, 1.9-kb CD95 promoter region with the three putative (computer-identified) p53-binding sites (reference 58); I, CD95 0.7-kb intronic region selected for p53 binding; SV, SV40 minimal promoter; Luc, luciferase gene; p53-BE, p53-binding element. (Right) Analysis of p53-dependent luciferase activity. Hep3B cells were transfected with 1 μg of each of the indicated reporter plasmids together with 100 ng of either a wt p53 expression plasmid, pCMVp53wt, or an equivalent amount of empty vector. Shown is the fold p53-dependent activation of each reporter plasmid, calculated relative to the value obtained with the same reporter in the absence of p53. These results were supported in H1299 human lung cancer carcinoma cells: the CD95 promoter alone was only minimally stimulated by wt p53, highly activated (34-fold) in conjunction with the p53-binding intronic region in the long (Ps+I) or short (Ps+Is) version downstream of the CD95 promoter. The effect was CD95 promoter specific, not seen with the RSV promoter (devoid of a p53- responsive element, 0.95-fold activation), and comparable to activation (36-fold) with a natural p53-responsive promoter of the cyclin G gene.

Similar articles

Cited by

References

    1. Fisher DE. Apoptosis in cancer therapy: crossing the threshold. Cell. 1994;78:539–542. - PubMed
    1. Fung CY, Fisher DE. Shifting the cancer paradigm: must we kill to cure? . J Clin Oncol. 1995;13:801–807. - PubMed
    1. Friesen C, Herr I, Krammer PH, Debatin KM. Involvement of the CD95 (APO-1/Fas) receptor/ ligand system in drug-induced apoptosis in leukemia cells. Nat Med. 1996;2:574–580. - PubMed
    1. Micheau O, Solary E, Hammann A, Martin F, Dimanche BM. Sensitization of cancer cells treated with cytotoxic drugs to fas-mediated cytotoxicity. J Natl Cancer Inst. 1997;89:783–789. - PubMed
    1. Fulda S, Sieverts H, Friesen C, Herr I, Debatin KM. The CD95 (APO-1/Fas) system mediates drug- induced apoptosis in neuroblastoma cells. Cancer Res. 1997;57:3823–3829. - PubMed

Publication types

MeSH terms

Substances