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, 101 (16), 5862-6

Identification of GAPDH as a Protein Target of the Saframycin Antiproliferative Agents

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Identification of GAPDH as a Protein Target of the Saframycin Antiproliferative Agents

Chengguo Xing et al. Proc Natl Acad Sci U S A.

Abstract

Saframycin A (SafA) is a member of a class of natural products with potent antiproliferative effects in leukemia- and tumor-derived cells. This activity is frequently conjectured to derive from the ability of saframycins to covalently modify duplex DNA. We used a DNA-linked affinity purification technique to identify GAPDH as a protein target of DNA-small molecule adducts of several members of the saframycin class. Nuclear translocation of GAPDH occurs upon treatment of cancer cells with saframycins, and depletion of cellular GAPDH levels by small interfering RNA transfection confers drug resistance. Roeder and coworkers have recently suggested that GAPDH is a key transcriptional coactivator necessary for entry into S phase. Our data suggest that GAPDH is also capable of forming a ternary complex with saframycin-related compounds and DNA that induces a toxic response in cells. These studies implicate a previously unknown molecular mechanism of antiproliferative activity and, given that one member of the saframycin class has shown efficacy in cancer treatment, suggest that GAPDH may be a potential target for chemotherapeutic intervention.

Figures

Fig. 1.
Fig. 1.
Molecular structures of SafA, Et743, the quinaldic acid derivatives (QAD) of SafA (QADCN, X = CN; QADOH, X = OH), and phthalascidin (Pt650), an analog of Et743.
Scheme 1.
Scheme 1.
DNA-linked affinity reagents for the isolation of proteins that bind small molecule-DNA adducts. The resin-bound 26-mer ss-oligonucleotide (5′-CCTTGGCCCGAGCCCGGTTCCTATTT-3′) was prepared by automated synthesis on Toyopearl Oligo-Affinity Resin. Complementary-strand annealing with the 21-mer ss-oligonucleotide 5′-GGAACCGGGCTCGGGCCAAGG-3′ followed by covalent modification of the resulting DNA duplex with DNA-reactive small molecules (represented in yellow) provided affinity reagents used to identify specific binding proteins within a pool derived from tissue lysate.
Fig. 2.
Fig. 2.
Identification of proteins recognizing duplex DNA-small molecule adducts by a DNA-linked affinity chromatography technique and confirmation of a direct binding interaction of the protein GAPDH by Southwestern blot analysis. (A) SDS/PAGE of proteins bound in affinity chromatography experiments using a protein pool derived from bovine-brain lysate. Lane 1, dsDNA resin with no drug modification: control matrix to lanes 2–4; lane 2, QADCN-alkylated dsDNA resin plus 40 equivalents (equiv) poly(dI-dC); lane 3, QADCN-alkylated dsDNA resin plus 40 equiv dsDNA-QADCN complex; lane 4, Pt650-alkylated dsDNA resin plus 40 equiv poly(dI-dC); lane 5, dsDNA resin with no drug modification, control matrix to lane 6; lane 6, SafA-alkylated dsDNA resin plus 40 equiv poly(dI-dC); lane 7, dsDNA resin with no drug modification, control matrix to lane 8; lane 8, MMC-alkylated dsDNA resin plus 40 equiv poly(dI-dC); lane 9, dsDNA resin with no drug modification, control matrix to lanes 10 and 11; lane 10, cisplatin-alkylated dsDNA resin plus 40 equiv poly(dI-dC); lane 11, transplatin-alkylated dsDNA resin plus 40 equiv poly(dI-dC). (B) Results of Southwestern blotting experiments. Purified human GAPDH or HMG1 was resolved by SDS/PAGE, electroblotted onto nitrocellulose, and then renatured (except for lane 6) on the membrane. The renatured blots were probed with a 32P-labeled 21-mer dsDNA (sequence: Scheme 1) unmodified (lane 1) or alkylated with the various compounds of study (as indicated, lanes 2–9).
Fig. 3.
Fig. 3.
The role of GAPDH in the response of eukaryotic cells to treatment with saframycin analogs (QAD). (A) Transcription-profiling studies performed in a SafA-sensitive yeast strain show that all three isoforms of GAPDH (TDH1, TDH2, and TDH3) are substantially up-regulated after treatment with QADCN or SafA, whereas the gene transcript for phosphfructokinase, pfk2, catalyzing the principle, rate-limiting step of glycolysis is down-regulated >2-fold. Transcripts for each of the histones were down-regulated >2-fold (14). (B) Western blots for the determination of GAPDH levels in the nuclear and cytoplasmic fractions of HeLa-S3 cells treated with QADOH for the indicated times. (C) Confocal microscopy of HeLa-S3 cells treated (b) or untreated (a) with QADOH (17.5 nM, 48 h) and visualized by using a standard FITC-conjugated antibody immunostaining protocol. Cell nuclei were visualized separately by Hoechst dye 33340 staining (not shown). (D) GAPDH levels in A549 cells that had been transfected with GAPDH siRNA or control siRNA, respectively, as determined by immunoblotting. (E) Percentage of viable cells transfected with GAPDH siRNA (filled circle) or control siRNA (filled square) after 72-h treatment with varying concentrations of QADCN. (F)Asin E, but treated with cisplatin in lieu of QADCN.

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