Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation - PubMed (original) (raw)

Ecteinascidin 743, a transcription-targeted chemotherapeutic that inhibits MDR1 activation

S Jin et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Ecteinascidin 743 (ET-743), a highly promising marine-based antitumor agent presently in phase II clinical trials, has been shown to interfere with the binding of minor-groove-interacting transcription factors, particularly NF-Y, with their cognate promoter elements in vitro. We have shown that NF-Y is a central mediator of activation of transcription of the human P glycoprotein gene (MDR1) by a variety of inducers and that NF-Y functions by recruiting the histone acetyltransferase PCAF to the MDR1 promoter. In the present study, we tested whether ET-743 could block activation of the MDR1 promoter by agents that mediate their effect through the NF-Y/PCAF complex. We report that physiologically relevant concentrations of ET-743 abrogate transcriptional activation of both the endogenous MDR1 gene and MDR1 reporter constructs by the histone deacetylase inhibitors as well as by UV light, with minimal effect on constitutive MDR1 transcription. Notably, this inhibition does not alter the promoter-associated histone hyperacetylation induced by histone deacetylase inhibitors, suggesting an in vivo molecular target downstream of NF-Y/PCAF binding. ET-743 is therefore the prototype for a distinct class of transcription-targeted chemotherapeutic agents and may be an efficacious adjuvant to the treatment of multidrug-resistant tumors.

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Figures

Figure 1

Figure 1

ET-743 inhibits activation of the transfected and endogenous MDR1 promoter by multiple inducers. (A) Structure of ET-743. (B–D) SW620 cells stably transfected with an MDR1 promoter/luciferase construct were treated with 100 ng/ml trichostatin A (TSA; B), 2 mM sodium butyrate (C), or 10 J/m2 UV irradiation (D) without (white bars) or with (black bars) 50 nM ET-743. The stably transfected cells were treated as indicated for 24 h, and luciferase activity was determined. The data represent the results of three independent experiments performed in triplicate. (E) Nuclease protection analysis of MDR1 RNA from untreated SW620 cells (lane 1) or cells treated with 100 ng/ml TSA (lane 2), 2 mM sodium butyrate (lane 3), 50 ng/ml ET-743 (lane 4), 50 nM TSA and 50 ng/ml ET-743 (lane 5), or 2 mM sodium butyrate and 50 ng/ml ET-743 (lane 6). Total RNA was extracted from cells, and nuclease protection assays were performed with MDR1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene-specific ribonucleotide probes (6) by using 20 μg and 0.66 μg RNA, respectively.

Figure 2

Figure 2

ET-743 does not affect TSA-induced global histone hyperacetylation or accumulation of MDR1 promoter-associated acetylated histone H4. Stably transfected SW620 cells were incubated for 24 h in the presence of TSA alone (100 ng), ET-743 alone (50 nM), or a combination of both. (A) After incubation, total histones were isolated and subjected to Western analysis with an anti-acetylated histone H4 antibody. The two bands observed represent different acetylated forms of histone H4. (B and C) For chromatin immunoprecipitation assays, cells were treated as above, and then chromatin was immunoprecipitated with mouse IgG, anti-acetylated H4 antibody, or no antibody (negative control). MDR1 promoter DNA associated with hyperacetylated H4 was visualized after PCR amplification. The arrow indicates the specific MDR1 promoter PCR product, and the asterisk denotes the input primers. (B) Input DNA. (C) Immunoprecipitated DNA. ChIP, chromatin immunoprecipitation; NTC, negative control; UT, untreated; M, marker.

Figure 3

Figure 3

ET-743 abrogates TSA-induced cell-cycle arrest. Cell nuclei from untreated SW620 cells (A) or cells treated with 100 ng/ml TSA (B), 50 nM ET-743 (C), or TSA and ET-743 (D) were prepared and subjected to flow cytometric cell-cycle analysis (17).

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