MARCH/APRIL 2002 Volume 13, Number 2 © Copyright 2002 by the American Chemical Society
COMMUNICATIONS Total Analysis and Purification of Cellular Proteins Binding to Cisplatin-Damaged DNA Using Submicron Beads Takenori Tomohiro,*,† Jun-ichi Sawada,‡ Chika Sawa,‡ Hironori Nakura,‡ Shuhei Yoshida,† Masato Kodaka,† Mamoru Hatakeyama,§ Haruma Kawaguchi,§ Hiroshi Handa,‡ and Hiroaki Okuno† Institute of Molecular and Cell Biology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8566, Japan, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama 226-8501, Japan, and Faculty of Science and Technology, Keio University, Yokohama 223-8522, Japan. Received September 27, 2001
A high-performance affinity purification technique has been developed for cisplatin (CDDP)-damaged DNA binding proteins directly from crude nuclear extracts of HeLaS3 cell using novel submicron beads synthesized by copolymerization of styrene and glycidyl methacrylate (GMA). The beads dramatically decreased both nonspecific protein adsorption on solid surfaces and elution volume and simplified the handling procedure. Preparation of the beads for purification was carried out by immobilization of telomeric repeats, (TTAGGG)n, on the surface after the reaction with CDDP. At least nine proteins clearly showed higher affinity to CDDP-DNA and were identified by amino acid sequence analysis including HMGB (high mobility group), hUBF (human upstream binding factor), and Ku autoantigen, which were previously reported to be components of CDDP-damaged DNA binding proteins.
INTRODUCTION
Cisplatin (cis-diamminedichloroplatinum(II), CDDP) has been widely used as a chemotherapeutic agent in several types of cancer including ovarian, testicular, bladder, small-cell lung, cervical, and head and neck carcinomas. One key step in its mechanism is the binding * To whom correspondence should be addressed. Phone: +81298-61-6124. Fax: +81-298-61-6123. E-mail: t.tomohiro@ aist.go.jp. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ Tokyo Institute of Technology. § Keio University.
to DNA, which induces transcriptional interruption (1, 2), cell cycle arrest (3), DNA repair (4), programmed cell death (apoptosis) via phosphorylation of tumor suppressor protein p53 (5), etc. The coordination binding of CDDP unwinds and bends the double helical DNA, especially in the case of intrastrand cross-links to G-rich sequences such as GG and AG (6). Various anticancer phenomena based on DNA-damage are attributable to the interactions between CDDP-DNA and some cellular proteins such as HMG-domain proteins acting as triggers (7, 8). Though the purification of proteins binding to CDDP-DNA complexes was performed using affinity chromatography on cellulose (9), the efficiency was not
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164 Bioconjugate Chem., Vol. 13, No. 2, 2002
Tomohiro et al.
Figure 1. Preparation scheme of CDDP-DNA fixed beads.
satisfactory because of the low binding ability. To solve this problem, we have developed novel submicron latex beads synthesized by copolymerization of styrene and GMA in a soap-free aqueous solution (10), with which drug receptors (11) and transcription factors (12) were successfully purified with high efficiency. In the present paper, we show for the first time that affinity proteins of CDDP-DNA can be totally analyzed in a very simple way by using whole protein extracts. The human telomere, a G-rich region as tandem repeats of TTAGGG located at the ends of chromosomes, is important for stabilization of chromosomes, cell aging, and cancer growth (13). As the telomere length is known to be shortened by the treatment of CDDP in HeLa cells (14, 15), there is a high possibility that the complex formation of CDDP and telomere is closely related to anticancer activity. Our attention was, therefore, focused on the interaction between the CDDP-telomere and cellular affinity proteins.
Figure 2. Affinity purification of CDDP-DNA binding proteins by salt elution. +, CDDP-DNA fixed beads; -, DNA fixed beads; N, DNA free beads; lane 1-5, proteins in the first elution from the beads with the buffer containing 1.0 M KCl; lane 1, DNAfree beads using NE; lane 2, DNA-fixed beads using NE; lane 3, CDDP-DNA-fixed beads using NE; lane 4, DNA-fixed beads using WCE; lane 5, CDDP-DNA-fixed beads using WCE. Arrows indicate the proteins that were identified this time.
RESULTS AND DISCUSSION
Two complementary oligonucleotides, 5′- GGTTAGGGTTAG-3′ and 5′-CCCTAACCCTAA-3′, were chemically synthesized, annealed, 5′-phosphorylated, and ligated to give, on the average, several hundred base pairs of telomeric repeats, followed by gel filtration on Sephadex G-50. The telomeric repeats were reacted with CDDP in 10 mM phosphate buffer in the dark at 37 °C for 1 day according to the ref 15. The platinated DNA showed a gel mobility shift, and the platination reaction was pursued by gel electrophoresis using RI-labeled DNA. The platinum contents were determined by atomic absorption spectroscopy (AA) on a Varian spectrAA-400 spectrophotometer to get the bound drug-to-nucleotide (D/N)b of 0.079-0.083. A 100 mg amount of the resultant CDDPDNA complex was reacted with the latex beads through the reaction between the surface epoxy groups and the amino groups of nucleic bases at the extruding ends of the double-strand DNA at 50 °C for 1 day, followed by masking the unreacted epoxy groups with ethanolamine (Figure 1). The yield of CDDP-DNA fixed on the beads (2-3 µg per mg of beads) was calculated from the amount of the unreacted DNA, and those beads showed a minimum amount of nonspecific protein absorption compared with the case of beads with a large amount of DNA on the surface. The DNA-fixed beads without CDDP and the ethanolamine-treated beads without DNA were also prepared as control samples. Nuclear extracts (NE) and whole cell extracts (WCE) of HeLaS3 cells were prepared according to the methods of Dignam (16) and Manley (17), respectively. The DNAfixed beads (2 mg) were suspended in 0.5 mL of NE or
Figure 3. Competition analysis of CDDP-DNA binding proteins. +, CDDP-DNA fixed beads; -, DNA fixed beads; lane 1-5, proteins eluted from CDDP-DNA fixed beads using WCE; lane 1, without competitor (free CDDP-DNA); lane 2-4, including certain amount of competitor; lane 5, using DNA-fixed beads as a control. Arrows indicate the proteins whose amount was changed by addition of free CDDP-DNA as a competitor.
WCE including 20 µg of single-strand DNA and 2 µg of poly dI-dC in a 1.5 mL centrifuge tube. After incubation at 0 °C for 1 h, proteins were purified by the salt elution method as follows. The beads were spun down by brief centrifugation and washed three times with 0.1 mL of HEPES buffer containing 20 mM HEPES (pH 7.9), 10% glycerol, 5 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol (DTT), and 0.1 M KCl and then washed five times with 0.1 mL of the buffer solution containing 0.4 M KCl. The proteins adsorbed on the beads were eluted three times with 0.1 mL of the buffer solution containing 1.0 M KCl. The proteins in each buffer solution were subjected to electrophoresis by SDS-PAGE, whose separated bands were detected by staining with silver (Figure 2). The affinity of eluted proteins to CDDP-DNA was confirmed by competition experiments in which certain amounts of free CDDP-DNA were added to the protein solution before incubation at 0 °C (Figure 3).
Communications
Figure 2 clearly shows the high-performance purification of CDDP-DNA affinity proteins. Compared with CDDP-free DNA beads (lane 2 or lane 4), some bands (especially 26.5, 28, 94, and 97 kDa) were recognized in the eluants from CDDP-DNA beads (lane 3 or lane 5) by silver staining. By using beads without DNA, adsorption of proteins was little observed under these conditions (lane 1). At least nine proteins had higher affinity to CDDP-DNA than to DNA without CDDP. Most of these proteins were identified by amino acid sequence analysis to be HMGB proteins (26.5 and 28 kDa), hUBF (human upstream binding factor, heterodimer of 94 and 97 kDa) proteins, and Ku autoantigen (heterodimer of 70 and 80 kDa), which were reported to interact with both telomeric repeat and CDDP-DNA as a part of DNA-dependent protein kinase (18, 19). We further found mtTF1 (mitochondrial transcription factor 1, 24 kDa) as a new affinity protein and two other proteins whose biological functions are under investigation. Recent papers reported some relationship between telomere and DNA damage by CDDP in telomerase activity (20) and p53 dependent apoptosis (21), while the telomere is well-known as a transcriptional silent part of the genome. It should be emphasized that the present technique is exceedingly simple (one-pot purification) and efficient despite using crude whole protein extracts in comparison with the ordinary affinity chromatography on cellulose or agarose that are more laborious procedures. Recently, affinity purification by Sepharose column chromatography was reported for CDDP-DNA (22). The new latex beads have the following characteristics superior to the conventional materials. Since the beads can be essentially treated as hard spheres with fairly monodisperse diameter (200 nm on average), chemical and physical reactions are not affected by diffusion-controlled processes inside the beads, which provide a homogeneous environment for binding between DNA and proteins. Subsequent polymerization of GMA after the styrene-GMA copolymerization makes the surface of the beads rather hydrophilic, which significantly reduces nonspecific adsorption of proteins on the beads. Easy removal of proteins from the beads through washing without requiring stringent conditions (e.g., high salt concentration) made it possible to isolate CDDP-DNA affinity proteins with binding constants relatively lower than those of sequence-specific DNA binding proteins. In addition, large surface area per weight (20 m2 per 1 g) provides sufficient capacity to purify proteins in a milligram scale. Degree of the dilution of eluted proteins depends on the volume of the solution used for elution from the beads. Usually, only 50-100 µL of buffer is used for 1-10 mg of beads. Therefore, a precipitation procedure of the eluted proteins was not necessary to detect them by ordinary staining. CONCLUSIONS
In the present method, an extensive analysis of binding proteins is generally possible under various conditions even when crude whole protein solutions are used. In addition, this simplified technique for affinity purification may be useful for screening chemical libraries to create new drugs and their receptor library in a short time directly from crude cell extracts. ACKNOWLEDGMENT
Financial support from the New Energy and Industrial Technology Development Organization (NEDO) is gratefully acknowledged.
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