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. 2006 Apr 18;103(16):6344-9.
doi: 10.1073/pnas.0601418103. Epub 2006 Apr 10.

Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation

Hao Mo  1 , Marie Henriksson
Affiliations

Identification of small molecules that induce apoptosis in a Myc-dependent manner and inhibit Myc-driven transformation

Hao Mo et al. Proc Natl Acad Sci U S A. .

Abstract

The Myc transcription factor plays a central role in the regulation of cell cycle progression, apoptosis, angiogenesis, and cellular transformation. Myc is a potent oncoprotein that is deregulated in a wide variety of human tumors and is therefore an attractive target for novel cancer therapies. Using a cellular screening approach, we have identified low-molecular-weight compounds, Myc pathway response agents (MYRAs), that induce apoptosis in a c-Myc-dependent manner and inhibit Myc-driven cellular transformation. MYRA-A inhibits Myc transactivation and interferes with the DNA-binding activity of Myc family proteins but has no effect on the E-box-binding protein USF. In contrast, MYRA-B induces Myc-dependent apoptosis without affecting Myc transactivation or Myc/Max DNA binding. Our data show that cellular screening assays can be a powerful strategy for the identification of candidate substances that modulate the Myc pathway. These compounds can be useful tools for studying Myc function and may also be of therapeutic potential as leads for drug development.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
Identification of MYRA-A and MYRA-B using a cellular screening assay. (A) Tet-Myc cells were treated with doxycycline (dox) at the indicated concentrations (in μg/ml), and whole cell extracts were analyzed for Myc expression by Western blotting after 18, 42, and 66 h. β-actin was used as a loading control. (B) Chemical structures of identified compounds, MYRA-A (NSC339585) and MYRA-B (NSC45641). (C) Cell viability of Tet-Myc cells was measured by crystal violet staining after treatment for 48 h with 12.5 μM MYRA-A or 25 μM MYRA-B in the presence (open bars) or absence (filled bars) of 2 μg/ml doxycycline. The data shown represent the mean of at least three independent experiments in duplicate with SD. Whole cell extracts were analyzed for Myc expression by Western blotting after treatment with MYRA-A and MYRA-B as shown in the Inset. (D) Phase contrast images of WT and Myc-overexpressing Tet-Myc cells 48 h after treatment with MYRA-A and MYRA-B. (E) Cell viability of human p493-6 + Tet (Myc-off) and p493-6 − Tet (Myc-on) B cells treated with 0–40 μM MYRA-A and MYRA-B, respectively, was measured by the WST-1 assay. Myc expression in the presence and absence of tetracycline (tet) is shown in the Inset. (F) IC50 values for human BLs (BL-2, BL-60, and Ramos) and LCLs (IARC-139, IARC-171, and IARC-176) treated with MYRA-A and MYRA-B. The values represent the mean and SD from three independent experiments.
Fig. 2.
Fig. 2.
Induction of c-Myc-dependent apoptosis by MYRAs in Rat-1 cell lines. (A) Rat-1 cells with different c-Myc expression were treated with 0–7 μM MYRA-A or 0–80 μM MYRA-B, and cell viability was analyzed by crystal violet staining. (B) Phase contrast images of Rat-1 cells treated with 3 μM MYRA-A, 40 μM MYRA-B, or with DMSO (control). (C) Hoechst staining of Rat-1 cells treated with MYRA-A, MYRA-B, or with DMSO. Apoptotic nuclei are indicated by arrows. (D) Apoptosis was analyzed by using the Cell Death Detection ELISAPLUS kit after treatment with 3 μM MYRA-A, 40 μM MYRA-B, or with DMSO. Data represent the mean and SD of three independent experiments.
Fig. 3.
Fig. 3.
Interference with DNA binding of Myc/Max and Mnt/Max by MYRA-A. Five micrograms of total cell extracts from HL60 cells (A), Myc/Max transfected COS cells (B), or Mnt/Max transfected COS cells (C) were preincubated with 100, 50, 25, or 12.5 μM MYRA-A or 400, 200, or 100 μM MYRA-B before addition of the labeled CMD-oligonucleotide. The Mnt/Max, USF, Myc/Max, and Max/Max DNA–protein complexes were identified by antibody supershifts and are indicated to the left.
Fig. 4.
Fig. 4.
Effects on luciferase activity by the treatment with MYRAs. CV1 cells were transiently transfected with pSP-Myc or pSP-vector together with minM4Luc (A), or were transfected either with CMV-p53 or CMV-vector and the p53-reporter (B), or with CMV-reporter alone (C). pCMV-β-galactosidase was included in all transfections. Twenty-four hours posttransfection, the cells were treated with the indicated concentrations of MYRA-A and MYRA-B. The luciferase activities were normalized for β-galactosidase as a control of transfection efficiencies, and the mean values with SD of at least three independent experiments are shown. (D) Myc immunoprecipitation was performed by using cell extracts from Tet-Myc cells treated with MYRA-A in vivo (Left), or in vitro (Center) and from Myc/Max transfected COS cells (Right) that had been incubated with MYRA-A in vitro. The inhibitor of Myc/Max dimerization 10058-F4 was used as control for the in vitro experiment. Precipitated protein complexes were separated by SDS/PAGE and analyzed by Western blotting for Myc and Max.
Fig. 5.
Fig. 5.
Inhibition of soft agar colony formation by MYRAs. Rat-1a-myc (A) and MR (B) cells were seeded in soft agar and treated with different concentrations of MYRA-A and MYRA-B. For each compound, two of the concentrations used (in μM) are shown. Representative plates from two independent experiments are presented.
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