ProteinâAcrylamide Copolymer Hydrogels for Array-Based Detection

Biomacromolecules 2005, 6, 2765-2775

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Protein-Acrylamide Copolymer Hydrogels for Array-Based Detection of Tyrosine Kinase Activity from Cell Lysates Shawn B. Brueggemeier,† Ding Wu,‡ Stephen J. Kron,‡ and Sean P. Palecek*,† Department of Chemical and Biological Engineering, University of WisconsinsMadison, 1415 Engineering Drive, Madison, Wisconsin 53706, and Department of Molecular Genetics and Cell Biology, University of Chicago, 924 East 57th Street, Chicago, Illinois 60637 Received April 11, 2005; Revised Manuscript Received May 26, 2005

We describe the development of an array-based assay for the molecular level detection of tyrosine kinase activity directly from cellular extracts. Glutathione S-transferase-Crkl (GST-Crkl) fusion proteins are covalently immobilized into polyacrylamide gel pads via copolymerization of acrylic monomer and acrylicfunctionalized GST-Crkl protein constructs on a polyacrylamide surface. The resulting hydrogels resist nonspecific protein adsorption, permitting quantitative and reproducible determination of Abl tyrosine kinase activity and inhibition, even in the presence of a complex cell lysate mixture. Half-maximal inhibition (IC50) values for imatinib mesylate inhibition of GST-Crkl (SH3) phosphorylation by v-Abl in a purified system and Bcr-Abl within a K562 cell lysate were determined to be 1.5 and 20 µM, respectively. Additionally, the protein-acrylamide copolymer arrays detected CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. Such a system may have direct application to the detection and treatment of cancers resulting from upregulated tyrosine kinase activity, such as chronic myeloid leukemia (CML). These findings also establish a basis for screening tyrosine kinase inhibitors and provide a framework on which protein-protein interactions in other complex systems can be studied. Introduction Many cancers are characterized by an upregulation in expression or activity of receptor or nonreceptor tyrosine kinases. For example, the majority of chronic myeloid leukemia (CML) cases, as well 15-30% of adult acute lymphoblastic leukemia (ALL) cases, are associated with a reciprocal chromosome translocation between the long arms of chromosomes 9 and 22.1,2 The resulting Philadelphia (Ph) chromosome contains the fusion of the BCR and ABL genes, which encodes the oncogenic, constitutively active tyrosine kinase Bcr-Abl.3,4 The disregulated activity of Bcr-Abl is necessary and sufficient to cause CML, and disease progression is dependent upon Bcr-Abl activity.5 Treatment with imatinib mesylate (STI571, Gleevec, Glivec) often results in complete cytogenetic remission in newly diagnosed CML patients.6 However, resistance to imatinib treatment can arise. Numerous Abl domain mutations affect the binding of imatinib to Bcr-Abl and reduce the efficacy of the drug.7-10 For example, amino acid substitutions within the ATP/imatinib binding pocket (i.e. T334I, F378V) impair imatinib binding while retaining ATP binding and kinase activity.11 Drug resistance can also arise through mutations in the activation loop (i.e. H415P) or nucleotide binding p-loop (i.e. E274K), which destabilize the inactive kinase conformation required for imatinib * To whom correspondence should be addressed. Tel: (608) 262-8931. Fax: (608) 262-5434. E-mail: [email protected]. † University of WisconsinsMadison. ‡ University of Chicago.

binding.12 Additionally, overexpression of Bcr-Abl has been implicated as a means of imatinib resistance and disease relapse.13 As a result of the likelihood of imatinib resistance, continual monitoring of the response to imatinib treatment and early diagnosis of developed resistance is desired in the clinical setting.10 Previously, Western blot-based techniques have identified that the SH2-SH3 adaptor protein Crkl binds to Abl kinases and that Crkl is highly phosphorylated in extracts from CML cells.14,15 Additionally, increased levels of in vivo Crkl phosphorylation correlate to imatinib resistance and disease progression.7,16 While mutational screening and PCR-based methods can provide similar molecular level diagnosis and disease monitoring,10 these methods in addition to the Western blot-based techniques mentioned above are too cumbersome to be widely applied in a clinical setting. A simple and robust in vitro method of measuring kinase activity and inhibition directly from white blood cell extracts would potentially provide valuable predictions of the efficacy of a particular treatment for an individual patient. Recently, protein array/microarray systems have received considerable attention due to the potential for the rapid, highthroughput, and cost-effective characterization of protein concentration and activity. Detection of protein-protein interactions and enzymatic substrate specificity was demonstrated on arrays of proteins immobilized onto aldehydecoated glass slides.17 Antibody-antigen interactions were investigated using arrays of proteins immobilized on polyL-lysine coated glass slides18 or nitrocellulose membranes.19 Protein-protein interactions have been investigated on the

10.1021/bm050257v CCC: $30.25 © 2005 American Chemical Society Published on Web 06/30/2005

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Table 1. GST-Crkl Fusion Proteins Utilized As Abl Substrates15 Crkl amino acids Crkl functional domainsa

notation GST-Crkl (full length) GST-Crkl (SH3) GST-Crkl (intra SH3)

1-303 120-303 180-245

SH2-SH3-*-SH3 SH3-*-SH3 -*-

a An asterisk (*) represents the tyrosine phosphorylation site for Abl/ Bcr-Abl.

proteome scale using arrays of yeast proteins immobilized on nickel-coated slides.20 Additional protein array systems have detected protease specificity21 and kinase activity22,23 in model systems and protein markers in serum samples.24-26 Despite these promising experimental results, significant challenges remain in the development of protein array strategies. Low surface concentration of the immobilized protein and nonspecific binding of sample components to the surface of the protein array limit signal-to-noise ratios.27 Immobilized proteins may dehydrate and denature when attached to solid surfaces, resulting in the loss of structuredependent protein activity.28,29 Also, the interactions of sample components with surface-immobilized proteins may be sterically hindered.30,31 In response to these problems, hydrogel-based protein array strategies have been investigated.26,32-34 Here we report the development of a quantitative, hydrogel-based protein array for the detection of tyrosine kinase activity directly from cell lysates. Using a protein-acrylamide copolymerization scheme, proteins containing glutathione S-transferase fused to Crkl (GST-Crkl) have been covalently immobilized onto polyacrylamide-coated glass slides. These protein substrate arrays were subsequently phosphorylated by purified v-Abl and/or Bcr-Abl containing K562 cell extracts, a Ph+ proerythroblastic cell line isolated from a CML patient.35 The GST-Crkl substrates arrays were not only able to detect v-Abl activity and inhibition in the purified kinase system but have also quantitatively described Bcr-Abl activity and inhibition in extracts from leukemic cells. Additionally, the arrays were able to detect CML cell levels as low as 15% in a background of Bcr-Abl- leukemic cells and provided the framework for the parallel evaluation of six tyrosine kinase inhibitors. We feel that this surfacebased approach to the molecular monitoring of kinase activity may have application in the diagnosis and treatment monitoring of CML and ALL and may be adaptable to the detection of other diseases caused by other disregulated kinases. Furthermore, the ability to detect protein activity in a cell lysate should allow the application of protein-acrylamide copolymer hydrogel arrays in a variety of other complex proteomic systems. Materials and Methods Preparation of Abl Substrates. GST-Crkl fusion constructs were obtained as a gift from Dr. Brian Druker (Table 1).15 Three constructs, GST-Crkl (full length), GST-Crkl (SH3), and GST-Crkl (intra SH3), were transformed into Escherichia coli BL21 cells and grown to mid-log phase at 37 °C in 2×YT (16 g of tryptone, 10 g of yeast extract, 5 g

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of NaCl, pH 6.6 in 1 L of water). Protein expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. After 3 h, cells were harvested, resuspended in lysis buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, 0.5 mM DTT, 1 mM sodium orthovandate, 1 mM PMSF, 4% 25 × complete protease inhibitor (Roche Diagnostics GmbH, Penzberg, Germany), 1% Triton X-100, pH 7.4), and lysed on ice. Cell lysate was purified by affinity chromatography following the manufacture’s instructions on a glutathione sepharose column (Amersham Biosciences, Piscataway, NJ). Briefly, following addition of cell lysate to the column, the column was washed with PBS, pH 7.3, until the flow through was protein free. The GST-Crkl fusion protein was then eluted by addition of 50 mM Tris-HCl, pH 8.0, containing 10 mM reduced glutathione. Purified protein was concentrated in a centrifugal filter (Millipore, Billerica, MA) with a 10 kDa nominal molecular weight cutoff. Preparation of K562 and HL60 Cell Lysates. K562 and HL60 cells (ATCC, Manassas, VA) were cultured at 37 °C and 5% CO2 in RPMI-1640 media (Cambrex Bio Science, Walkersville, MD) containing 100 units/mL penicillin, 100 µg/mL streptomycin, and 10% FBS. For lysis, cells were resuspended at 5 × 107 cells/mL in lysis buffer (42.3 mM HEPES, 126 mM NaCl, 1.27 mM MgCl2, 0.85 mM EDTA, 84.5 mM NaF, 8.45 mM sodium pyrophosphate, 0.169 mM sodium orthovanadate, 1 mM PMSF, 0.95% Triton X-100, 9.5% glycerol 4% 25 × complete protease inhibitor, pH 7.4) and incubated on ice for 20 min. The total cell lysate was then clarified by spinning at 1500 rpm for 10 min. Total protein concentration was determined via a Pierce BCA protein assay kit (Pierce, Rockford, IL), and cell lysates were stored at -80 °C until further use. Bead-Based Kinase Assays. SwellGel disks (Pierce) were suspended in cold 50 mM Tris, pH 7.5, so that 1 µL of bead suspension bound 1 µg of GST fusion protein. One nanomole of GST-Crkl substrate was incubated with the glutathione bead suspension for 1 h at 4 °C with constant rotation. The substrate-bound beads were washed twice with ice-cold 50 mM Tris-HCl, pH 7.5 containing 10 mM MgCl2. Substratebound beads were then incubated with either recombinant v-Abl or K562 cell lysate. The v-Abl reaction mixtures contained 20 µL of 4× buffer (200 mM Tris-HCl, 40 mM MgCl2, 4 mM DTT, pH 7.5), 20 µL of 40 µM ATP, 0.5 µL of v-Abl (EMD Biosciences, Inc., San Diego, CA), 0 or 20 µL of 400 µM imatinib, and water to a total volume of 80 µL. The K562 cell lysate reaction mixtures contained 20 µL of 4× buffer, 20 µL of 40 µM ATP, 50 µg of K562 cell lysate, 0 or 20 µL of 400 µM imatinib, and water to a total volume of 80 µL. The reactions were allowed to proceed for 1 h at 30 °C. Following the reaction, the beads were washed twice with ice-cold 50 mM Tris, pH 7.5. GST-Crkl substrates were eluted with 10 mM reduced glutathione in 50 mM Tris-HCl, pH 8.0. Kinase assay samples were loaded in a 12% SDS-PAGE gel and transferred to nitrocellulose membranes according to standard procedures. Consistent sample loading was verified using the Memcode Reversible Protein Stain Kit (Pierce). Membranes were probed with antiphosphotyrosine antibodies.

Protein-Acrylamide Copolymer Hydrogels

Fabrication of Protein-Acrylamide Copolymer Hydrogel Arrays. Glass slides were acrylic-functionalized and GST-Crkl fusion proteins were acrylic-labeled as previously described.36 Briefly, fusion proteins were labeled on primary amines by reaction with 6-((acrylo)amino)hexanoic acid, succinimidyl ester (Molecular Probes, Eugene, OR) and clean glass slides were functionalized via reaction with (3acryloxypropyl)trimethoxysilane (Gelest, Tullytown, PA). Following functionalization of the glass slides, a thin layer of 18.8% polyacrylamide gel was attached to the glass surface. Forty microliters of a mixture containing 125 µL of 1.5 M Tris (pH 8.8), 285 µL of 33% acrylamide mix [0.86 g N,N′-methylenebisacrylamide (Bis) and 32.14 g acrylamide in a total volume of 100 mL], 5 µL of 10% ammonium persulfate (APS), 75 µL of 100% glycerol, 0.5 µL of N,N,N′,N′-tetramethylethylenediamine (TEMED), and 9.5 µL of water was sandwiched between an acrylic-functionalized glass slide and a clean glass plate and allowed to polymerize in a N2 environment at room temperature for 30 min. The glass plate was then removed, and 1 µL protein spots of the following mixture were deposited via pipet on top of the 18.8% acrylamide base layer and subsequently polymerized: 6.25 µL of 1.5 M Tris (pH 8.8), 3 µL of 33% acrylamide mix, 0.5 µL of 10% APS, 3.75 µL of 100% glycerol, 0.1 µL of TEMED, 0-7.5 µL of acrylic-labeled GST-Crkl protein solution, and water to a total volume of 25 µL. Additionally, 3 µL spacer spots of the above mixture without protein were added on the exterior corners of the protein array for the subsequent creation of a reaction chamber as described below. After attachment of the proteinacrylamide copolymer spots, the slides were washed by briefly dipping into approximately 250 mL of TBST (10 mM Tris-HCl, 100 mM NaCl, 0.1% Tween-20, pH 7.5) followed by a 15-min and two 5-min washes with slight agitation in approximately 20 mL of TBST. Slides were then washed by briefly dipping into approximately 250 mL of water, followed by two 5-min washes with slight agitation in approximately 20 mL of water, before being stored overnight at 4 °C in Abl kinase assay buffer (50 mM Tris-HCl, 10 mM MgCl2, 100 µM EDTA, 1 mM DTT, 0.015% Brij 35, 100 µg/mL BSA, pH 7.5). Fabrication of Protein Arrays on Commercially Available Poly-L-lysine, Aldehyde, and Hydrogel Substrates. Protein arrays containing 165 ng/mm2 spots of immobilized GST-Crkl (SH3) were created on poly-L-lysine (CEL Associates, Pearland, TX), aldehyde (TeleChem International, Sunnyvale, CA), and hydrogel substrates (PerkinElmer, Boston, MA) using established protocols. Briefly, nonacrylic labeled GST-Crkl (SH3) fusion proteins were transferred to PBS buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.7 mM KH2PO4, pH 7.4) for subsequent immobilization. Immobilization on poly-L-lysine slides followed the procedure reported by Habb et al.18 Immobilization on aldehyde slides and hydrogel slides followed the protocols supplied by the respective manufactures. After immobilization, the protein arrays were blocked in 1% BSA in TBST for 1 h at room temperature, washed in TBST and water as described above, and stored overnight in Abl kinase assay buffer.

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Fabrication of Protein Arrays via Contact Printing. The following mixture was loaded into a pulled micropipet printing tip (fabricated in house) via capillary action and printed onto acrylic-functionalized glass slides through initiation of fluid contact between the slide and printing tip: 6.25 µL of 1.5 M Tris (pH 8.8), 3 µL of 33% acrylamide mix, 3.75 µL of freshly prepared methylene blue solution (1 mg/mL in water), 3.75 µL of 100% glycerol, 0.35 µL of TEMED, 0-2.5 µL of acrylic-labeled GST-GFP protein solution, and water to a total volume of 25 µL. After printing, protein-acrylamide copolymer spots were polymerized under UV light (254 nm) at 1500 µW/cm2 for 10 min within a nitrogen environment. After attachment of the proteinacrylamide copolymer spots, the slides were washed as above in TBST and subsequently imaged on an inverted epi fluorescence Olympus microscope (Olympus, Melville, NY).36 Array-Based Kinase Assays. Just prior to the kinase assay, the glass slides were removed from the Abl kinase assay buffer and dried under a stream of compressed air. Careful attention was paid to make sure that only the bare portions of the slide were dried and the polyacrylamide spots and polyacrylamide base layer remained hydrated. Using the 3-µL spacer spots, a reaction chamber was then created by suspending a clean glass slide (for 250 µL reactions) or a clean glass coverslip (for 100 µL reactions) over top of the 1 µL protein-acrylamide copolymer hydrogel spots. The v-Abl reaction mixtures contained 100 µL of 3× Abl kinase assay buffer (150 mM Tris-HCl, 30 mM MgCl2, 300 µM EDTA, 3 mM DTT, 0.045% Brij 35, 300 µg/mL BSA, pH 7.5), 30 µL of 1 mM ATP, 50 µL of 100% glycerol, 1.5 µL of v-Abl, 0-100 µL of 30 µM imatinib, and water to a total volume of 300 µL. The K562 cell lysate reaction mixtures contained 100 µL of 3× Abl kinase assay buffer, 3 µL of 1 mM ATP, 50 µL of 100% glycerol, 0-450 µg of K562 cell lysate, 0-450 µg of HL-60 cell lysate, 0-30 µL of 3 mM imatinib, and water to a total volume of 300 µL. A 250-µL portion of these kinase reaction mixtures was then applied to each reaction chamber, and the reactions were allowed to proceed for 30 min to 5 h at 30 °C in a saturated environment. After the reaction, the glass slide used to create the reaction chamber was removed and the protein array was washed by briefly dipping into approximately 250 mL of TBST followed by a 15-min and two 5-min washes with slight agitation in approximately 20 mL of TBST. Tyrosine kinase inhibitors AG1478, PP2, AG1296, and AG490 (Calbiochem, San Diego, CA) and PKI166 (a gift from Novartis, Basel Switzerland) were dissolved in DMSO at 50× final concentration. Experiments with these inhibitors were conducted as above and contained 50 µL of 3× Abl kinase assay buffer, 1.5 µL of 1 mM ATP, 25 µL of 100% glycerol, 225 µg of K562 cell lysate, 3 µL of 50× inhibitor in DMSO, and water to a total volume of 150 µL. A 100µL portion of this reaction mixture was applied to each reaction chamber, and the reactions were allowed to proceed for 2 h at 30 °C in a saturated environment. Chemiluminescence Detection of Phosphorylated Substrates. Slides were blocked in approximately 20 mL of TBST containing 1% BSA for 1 h under room conditions.

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Each slide was then removed from the blocking solution and incubated with 1.5 µg of monoclonal anti-phosphotyrosine antibody PY20 (Sigma, St. Louis, MO) in 1.5 mL of TBST for 1 h. Each slide was then washed by briefly dipping into approximately 250 mL of TBST followed by a 15-min and two 5-min washes with slight agitation in approximately 20 mL of TBST. Each slide was then incubated with 0.15 µg of horseradish peroxidase (HRP) conjugated goat anti-mouse secondary antibody G-21040 (Molecular Probes) in 1.5 mL of TBST for 1 h and subsequently washed in TBST as above. The slides were then stored in TBST until detection. Enhanced chemiluminescence (ECL) Western blotting detection reagent (Amersham Biosciences) was used to detect horseradish peroxidase labeled slides. Briefly, after removal from the wash buffer, each slide was treated with 1.5 mL of ECL detection reagent for 1 min. The detection reagent was then “shaken off” from the slide and the slide was placed between transparent acetate sheets for subsequent exposure to film (Amersham Biosciences). After development, the average gray value of each spot and the background signal in the near vicinity of each spot were obtained using ImageJ software (NIH, Bethesda, MD). The difference between these two signals was reported as the average gray value for the spot. Results and Discussion Abl Kinase Activity toward Bead-Immobilized Substrates. To identify a suitable substrate for assessing tyrosine kinase activity on a surface, we utilized Western blot analysis to measure phosphorylation of several GST-Crkl constructs immobilized on glutathione beads. Three GST-Crkl fusion proteins were used in this work, GST-Crkl (full length), GST-Crkl (SH3), and GST-Crkl (intra SH3). These proteins contain GST fused to full length Crkl, a Crkl fragment containing both SH3 domains, and a Crkl fragment containing only the sequence immediately surrounding the Y207 Abl phosphorylation site.15 Phosphorylation of GSTCrkl substrates by purified v-Abl and in Bcr-Abl-containing K562 cell extracts, in the presence and absence of the Abl kinase inhibitor imatinib mesylate, is shown in Figure 1. v-Abl displays tyrosine kinase activity toward GST-Crkl (SH3) and GST-Crkl (intra SH3) in the absence of imatinib, and kinase activity toward these substrates is minimal at 100 µM imatinib. Phosphorylation of GST-Crkl (full length) by v-Abl is minimal both in the presence and absence of imatinib. In the K562 cell lysate system, all GST-Crkl substrates are phosphorylated in the absence of imatinib. However, only the phosphorylation of the GST-Crkl (full length) and GST-Crkl (SH3) substrates is inhibited at 100 µM imatinib. As imatinib is a specific Abl inhibitor, the lack of inhibition of GST-Crkl (intra SH3) phosphorylation in the cell lysate system is likely due to phosphorylation by other tyrosine kinases contained within the cell lysate. Presumably, portions of the SH3 domain prevent tyrosine phosphorylation by these nonspecific kinases. Due to this decrease in specificity toward Abl kinase, the GST-Crkl (intra SH3) substrate was not used in subsequent protein array studies of the K562 cell lysate.

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Figure 1. Bead-based Abl tyrosine kinase assays. Anti-phosphotyrosine detection of GST-Crkl (full length), GST-Crkl (SH3), and GST-Crkl (intra SH3) phosphorylation by v-Abl tyrosine kinase and K562 cell lysates. The symbols + and - represent the inclusion or exclusion of 100 µM imatinib in the reaction. The relative positions of GST-Crkl (full length) 65 kDa, GST-Crkl (SH3) 50 kDa, and GSTCrkl (intra SH3) 35 kDa, are given by markers 1, 2, and 3, respectively. In the case of v-Abl, phosphotyrosine bands appearing at approximately 45 kDa correspond to phosphorylated v-Abl. In the case of the K562 cell lysate, phosphotyrosine bands appearing at approximately 58 and 75 kDa can be attributed to unknown tyrosine phosphorylated components within the K562 cell lysate that copurify with GST-Crkl in the assay.

Fabrication of GST-Crkl Arrays. Protein arrays for the surface-based detection of Abl kinase activity were created using a protein-acrylamide copolymerization attachment scheme previously developed by our group.36 Clean glass slides are treated with (3-acryloxpropyl)trimethoxysilane, forming an acrylic-functionalized surface. Free acrylamide monomer and the cross-linker Bis are then polymerized to the acrylic-functionalized surface, resulting in a polyacrylamide-coated surface (Scheme 1A). This approximately 20µm-thick polyacrylamide layer greatly reduces the background signal resulting from nonspecific binding of cell extract components to bare glass during the Bcr-Abl kinase reactions (data not shown). GST-Crkl fusion proteins are also acrylic-functionalized via reaction with 6-((acrylo)amino)hexanoic acid, succinimidyl ester (Scheme 1B). Acrylic-functionalized GST-Crkl fusion proteins are then mixed with free acrylamide monomer and cross-linker and spotted onto the polyacrylamide coated surface. Polymerization results in the incorporation of GST-Crkl into polyacrylamide gel spots, which are in turn linked to the polyacrylamide-coated glass slide (Scheme 1C). Images of typical protein arrays can be seen in Figure 2. The arrays are formed on glass slides measuring 75 mm × 25 mm, the entire surface of which is coated by the polyacrylamide base layer. Protein spots are usually deposited manually via pipet with an average 1 µL spot diameter of 2.54 mm. In the case of GST-Crkl arrays, phosphorylation reaction chambers measuring 75 mm × 25 mm or 18 mm × 18 mm are created by suspending a glass slide or glass cover slip over top of the array, respectively. In the case of the 75 mm × 25 mm reaction chamber, one protein array can be utilized to examine up to 40 substrates under identical reaction conditions (Figure 2A). When multiple reaction conditions with fewer substrates per reaction condition are desired, three 18 mm × 18 mm reaction chambers can be situated on a single protein array. In this arrangement, up to eight substrate spots can be included within each of the three reaction chambers. After reaction, phosphorylated substrates are detected via antibody-based enhanced chemiluminescence



Scheme 1. Synthesis of Abl Substrate Arrays via Protein-Acrylamide Copolymer Hydrogel Chemistrya

a (A) Clean glass slides are acrylic-functionalized by reaction with (3-acryloxypropyl)trimethoxysilane. Following functionalization, a thin layer of polyacrylamide (approximately 20 µm) is attached to the slides. (B) GST-Crkl fusion proteins are also acrylic-functionalized by reaction with 6-((acrylo)amino)hexanoic acid, succinimidyl ester. (C) The functionalized proteins are then attached to the polyacrylamide-coated surface through the copolymerization of the labeled proteins, polyacrylamide surface, free acrylamide monomer, and cross-linker. Substrate arrays are then ready for incubation with Abl and/or Bcr-Abl tyrosine kinase and subsequent detection of phosphorylated Crkl substrates.

(ECL). The coefficient of variation (CV) for separate, repeated protein array-based Abl kinase assays ranges from 0.05 to 0.20 with typical values of approximately 0.15. Roughly one-third of this value can be attributed to the variation in attachment of GST-Crkl substrate to the array and the remainder of the variation is present in the Abl kinase reaction and subsequent ECL detection (data not shown). While the GST-Crkl protein arrays described above contain spot sizes approximately 2.5 mm in diameter, the protein-acrylamide copolymerization strategy is capable of creating smaller spots. We demonstrated this by creating arrays of immobilized green fluorescent protein (GFP). By simply replacing GST-Crkl in the polymerization mixture with GST-GFP, surface-immobilized fluorescence is observed (Figure 2B,C).36 Through contact printing, proteinacrylamide copolymer arrays spots on the order of a few hundred microns in diameter can be created (Figure 2C).

Protein Array-Based Detection of Purified v-Abl Activity. Initial surface-based studies investigated the activity and inhibition of Abl in a purified system. GST-Crkl substrate arrays were incubated in the presence of 100 µM ATP and purified v-Abl, and phosphorylated Crkl was detected via anti-phosphotyrosine antibodies (Figure 3). In a 2-h reaction, v-Abl demonstrates a slight preference for the GST-Crkl (SH3) substrate relative to the GST-Crkl (intra SH3) substrate (Figure 3A). Phosphorylation of GST-Crkl (full length) is minimal (data not shown). As expected, time course data for the phosphorylation of GST-Crkl (SH3) show an increase in Crkl phosphorylation with increasing reaction time over the range of protein array substrate densities investigated (Figure 3B). Data for the inhibition of v-Abl by imatinib can be seen in Figure 4. Half-maximal inhibition (IC50) values for the inhibition of GST-Crkl (intra SH3)

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Figure 2. Images showing protein-acrylamide copolymer hydrogel arrays. (A) Typical enhanced chemiluminescence (ECL) film for the detection of phosphorylated tyrosine in GST-Crkl (intra SH3), GSTCrkl (SH3), and GST-Crkl (full length). (B) Epi fluorescence image of protein arrays in which GST-Crkl has been replaced with GSTgreen fluorescent protein (GST-GFP). (C) Demonstration of smaller spot sizes via contact printing of GST-GFP.

and GST-Crkl (SH3) phosphorylation by imatinib are 2.0 and 1.5 µM, respectively. Protein-Array-Based Detection of Bcr-Abl Activity in a Cell Lysate. After demonstrating the ability to measure v-Abl tyrosine kinase activity and inhibition in a purified system, we sought to measure Bcr-Abl activity in cell lysates. Direct kinase assays from cell lysates are very desirable from a time and cost perspective in diagnostics development; however, the complex composition of cell lysates often complicates data analysis and leads to false positives or negatives. Extracts from CML cells contain numerous phosphorylated proteins, including endogenous phosphorylated Crkl.16,37,38 Thus, minimization of the nonspecific surface binding and subsequent detection of these phosphorylated components is critical. In addition to BcrAbl, the cell lysate is also expected to contain multiple tyrosine kinases. Phosphorylation of surface-immobilized Crkl by these additional kinases could obscure the specific detection and quantification of Bcr-Abl activity. Not only will the cell lysate contain additional kinases, but it is also expected to contain phosphatases and proteases. Phosphatasecatalyzed dephosphorylation or proteolytic degradation of surface immobilized phosphorylated-Crkl would result in a lower estimation of Bcr-Abl activity than is actually present. Elimination of nonspecific binding was accomplished through the use of a polyacrylamide-coated glass surface, as discussed above. Complications due to additional tyrosine kinases, phosphatases, and proteases will be addressed below. In initial K562 cell lysate studies, GST-Crkl substrate arrays were incubated with 10 µM ATP and 375 µg of K562 cell lysate. Data from the 30-min reaction show a strong preference for phosphorylation of GST-Crkl (SH3) relative to GST-Crkl (full length) (Figure 5A). Time course data for the phosphorylation of GST-Crkl (SH3) display the

Figure 3. v-Abl activity toward Crkl constructs immobilized in polyacrylamide hydrogels. (A) Enhanced chemiluminescence (ECL) values for the detection of phosphorylated tyrosine in GST-Crkl (intra SH3) or GST-Crkl (SH3) substrates on the surface of protein arrays as a function of substrate density. (B) Phosphorylation of GST-Crkl (SH3) substrates as a function of substrate density for reaction times of 1 and 2 h. Data points are the means of four replicates and error bars represent the standard deviation of the four samples.

Figure 4. Imatinib mesylate inhibition of v-Abl activity toward Crkl constructs immobilized in polyacrylamide hydrogels. ECL values for the detection of phosphorylated tyrosine in 165 ng/mm2 spots of GST-Crkl (intra SH3) or GST-Crkl (SH3) as a function of imatinib concentration. Data points are the means of four replicates and error bars represent the standard deviation of the four samples.

expected increase in phosphorylation with an increase in reaction time; however, in contrast to v-Abl phosphorylation, the reaction appears to be nearly complete after 30 min (Figure 5B). This is likely due to a high level of Bcr-Abl activity in the cell extracts. Imatinib inhibition of GSTCrkl (SH3) phosphorylation by K562 cell lysates occurs with an IC50 value of approximately 20 µM (Figure 6). As imatinib is a relatively specific inhibitor of Bcr-Abl, these data support the notion that the measured tyrosine kinase activity


Figure 5. K562 cell extract mediated phosphorylation of Crkl constructs immobilized in polyacrylamide hydrogels. (A) ECL values for the detection of phosphorylated tyrosine in GST-Crkl (SH3) and GST-Crkl (full length) substrates on the surface of protein arrays as a function of substrate density. (B) Phosphorylation of GST-Crkl (SH3) as a function of substrate density for reaction times of 30 min and 2 h. Data points are the means of four replicates and error bars represent the standard deviation of the four samples.

Figure 6. Imatinib mesylate inhibition of K562-mediated phosphorylation of Crkl immobilized in polyacrylamide hydrogels. ECL values for the detection of phosphorylated tyrosine in 165 ng/mm2 spots of GST-Crkl (SH3) as a function of imatinib concentration. Data points are the means from three independent experiments in which each experiment contained four replicates for each data point. Error bars represent the standard error of the means.

of the cell lysate toward the immobilized Crkl constructs is indeed due to Bcr-Abl. In contrast to v-Abl inhibition, inhibition of tyrosine kinase activity within the K562 cell lysate occurs at higher concentrations of imatinib. Because imatinib is an ATPcompetitive inhibitor, this increase in the IC50 value with respect to the v-Abl system may result from a physiologic level of ATP plus supplementary ATP in the cell lysate. To investigate the relationship between imatinib IC50 and ATP concentration, bead-based solution-phase assays were conducted in which the K562 cell lysate reaction mixture was


supplemented with additional ATP (Figure 7). These data clearly demonstrate that increasing ATP concentration increased GST-Crkl (SH3) phosphorylation and increased the imatinib IC50 values. Additionally, the increased imatinib IC50 values obtained from our bead- and array-based experiments as compared to cell survival assays8,9 may be at least partially explained by the inclusion of 10 µM ATP in our cell lysate reaction mixtures. Also, imatinib concentration for half-maximal survival may differ from concentration for half-maximal Bcr-Abl activity. Given the complexity of the K562 cell extract, experiments were conducted to assess the potential influence of phosphatases and proteases within the cell lysate on the resulting level of GST-Crkl phosphorylation. Initially, protein arrays containing 165 ng/mm2 spots of GST-Crkl (SH3) were phosphorylated for 2 h in the presence of 10 µM ATP and 375 µg of K562 cell lysate. Following thorough washing, these phosphorylated protein arrays were then reexposed to the cell lysate to access dephosphorylation and degradation due to protein phosphatases and proteases. During this second exposure, the phosphorylated surfaces were incubated in the reaction buffer without K562 extract, with K562 extract, and with K562 extract and 300 µM imatinib. These reactions represent a negative control, the potential for phosphatase/ protease and Abl kinase activity in the cell extract, and the potential for only phosphatase/protease activity in the cell extract, respectively. Data indicate that the level of Crkl phosphorylation between these three samples did not significantly change with reaction times up to 2 h (Figure 8). Also, only a slight decrease in Crkl phosphorylation is observed after 5 h, suggesting minimal phosphatase and protease activity in the K562 cell lysates toward the phosphorylated, immobilized GST-Crkl substrate. To assess the lower detection limit of the proteinacrylamide copolymer arrays, Bcr-Abl activity was quantified in samples of K562 cell lysate (Bcr-Abl+) diluted into HL60 cell lysate (Bcr-Abl-). Protein arrays containing 495 ng/mm2 spots of GST-Crkl (SH3) were incubated for 2 h in reaction mixtures containing 375 µg total of cell lysate, 0-100% of that derived from K562 cells and the remainder from HL60 cells. Figure 9 demonstrates that substrate phosphorylation increases as the ratio of K562 lysate to HL60 lysate increases. At low concentrations of K562 lysate, signal varies linearly with the fraction of K562 lysate but saturates at high concentrations of K562 lysate. Bcr-Abl activity is detectable at 2.5% K562 extract, with statistically significant results obtained for levels at and above 15% K562 extract (P value of 0.02). Additionally, the level of Crkl phosphorylation in the pure HL60 cell lysate is only 3.5% of that observed in the pure K562 cell lysate. Since both K562 and HL60 cell lysates are expected to contain a variety of active tyrosine kinases but only the K562 cell lysate is expected to contain Bcr-Abl, these data again support the notion that the tyrosine kinase activity toward the immobilized GSTCrkl substrates is indeed due to Bcr-Abl. Comparison to Other Protein Array Strategies. Having established a reliable and reproducible protein array-based assay for the quantitative determination of Abl and BcrAbl tyrosine kinase activity, experiments were preformed

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Figure 7. Bead-based detection of imatinib mesylate inhibition of K562-mediated phosphorylation of GST-Crkl (SH3) as a function of the amount of ATP concentration. Position of the phosphotyrosine band corresponding to GST-Crkl (SH3) is shown by marker 1 in the upper panel. Consistent loading of GST-Crkl (SH3) is demonstrated by memcode staining in the bottom panel.

Figure 8. Assessment of phosphatase activity in K562 cell extracts toward Crkl substrates immobilized in polyacrylamide hydrogels. Initially, protein arrays containing immobilized GST-Crkl (SH3) were phosphorylated by K562 cell lysate. After washing, the protein arrays were reexposed to the reaction mixture without K562 extract, with K562 extract, and with K562 extract and 300 µM imatinib, representing a negative control, the potential for phosphatase plus protease and Abl kinase activity, and the potential for only phosphatase and protease activity, respectively. Data points are the means of four replicates and error bars represent the standard deviation of the four samples.

Figure 9. Detection limit of Bcr-Abl activity for Crkl constructs immobilized in polyacrylamide hyrogels. Protein arrays containing immobilized GST-Crkl (SH3) were incubated in reaction mixtures containing 0-100% K562 cell lysate (Bcr-Abl+) in a background of HL60 cell lysate (Bcr-Abl-). Data points are the means of four replicates and error bars represent the standard deviation of the four samples.

to compare our protein-acrylamide copolymer hydrogel array platform to other commercially available protein array technologies. GST-Crkl (SH3) substrates were immobilized on commercially available hydrogel, aldehyde, and poly-Llysine surfaces following standard procedures. These protein arrays, as well as a protein-acrylamide copolymer hydrogel array, were then incubated in the presence of K562 cell lysate

Figure 10. Comparison of protein-acrylamide copolymer hydrogel arrays to commercially available protein arrays for the detection of Bcr-Abl tyrosine kinase activity and inhibition. (A) ECL values for the detection of phosphorylated tyrosine in 165 ng/mm2 spots of GST-Crkl (SH3) immobilized on four different protein array platforms as a function of imatinib concentration. (B-E) Images of the GSTCrkl (SH3) spots phosphorylated in the presence of 25 µM imatinib on protein-acrylamide copolymer hydrogel arrays, commercially available hydrogel arrays, aldehyde arrays, and poly-L-lysine arrays, respectively. Images are 13.5 × 13.5 mm in size.

containing 0-300 µM imatinib (Figure 10). Figure 10A demonstrates that only the hydrogel-based arrays are capable of providing meaningful data. Phosphorylation signals from the aldehyde and poly-L-lysine arrays are completely obscured by the nonspecific background signal (Figure 10BE), which is likely due to the surface adsorption of tyrosine phosphorylated components contained within the cell lysate. Additionally, our protein-acrylamide copolymer hydrogel arrays provide a higher level of sensitivity and reduced spotto-spot variation as compared to the commercially available hydrogel arrays. Our arrays require an additional step to label proteins with acrylic groups, but require significantly less time for array quenching, blocking, and washing steps. Screening Chemical Inhibitors of Bcr-Abl Activity toward Arrayed Crkl Substrates. While the experiments discussed above demonstrate the ability of the proteinacrylamide copolymer hydrogel arrays to reproducibly and quantitatively detect v-Abl and Bcr-Abl activity from cellular extracts, a significant advantage of array technology


Protein-Acrylamide Copolymer Hydrogels Table 2. Tyrosine Kinase Inhibitors Investigated Using Phosphorylation of GST-Crkl Protein-Acrylamide Copolymer Hydrogel Arrays by K562 Cell Lysates inhibitor

target

mode of action

ref

imatinib PKI166 AG1478 PP2 AG1296 AG490

Abl, c-Kit, PDGFR EGFR EGFR p56lck, p59fynT PDGFR JAK-2

ATP competitive ATP competitive ATP competitive ATP competitive ATP competitive ATP competitive

40,41 42 42 43 44 39

Figure 11. Inhibition K562 cell extract mediated phosphorylation of Crkl constructs immobilized in polyacrylamide hydrogels by six different tyrosine kinase inhibitors. Protein arrays containing immobilized GST-Crkl (SH3) were incubated in K562 cell lysate reaction mixtures containing imatinib, PKI166, AG1478, PP2, AG1296, or AG490 at concentrations ranging from 100 nM to 1 mM. Data points are the means of four replicates and error bars represent the standard deviation of the four samples.

can be found in the high-throughput nature in which multiple data points can be simultaneously obtained. Thus, in addition to quantifying Bcr-Abl activity from cell extracts as a diagnostic tool, arrays of immobilized Crkl substrates may be a platform for identifying and characterizing novel inhibitors of Abl tyrosine kinase activity. To demonstrate this potential, Bcr-Abl activity was measured in the presence of varying concentrations of six different tyrosine kinase inhibitors (Table 2). Protein arrays containing 165 ng/mm2 spots of GST-Crkl (SH3) were incubated for 2 h in reaction mixtures containing K562 cell lysate and tyrosine kinase inhibitor concentrations ranging from 100 nM to 1 mM. Significant inhibition of Bcr-Abl activity can be seen with PKI166, AG1478, and PP2 at 100 µM and 1 mM concentrations (Figure 11). Thus, the resulting IC50 values for these inhibitors are in the 100 µM range. AG1296 and AG490 do not inhibit Bcr-Abl activity over the range studied (IC50 > 1 mM). In the case of AG490, these data are supported by the lack of inhibition of K562 proliferation in previous studies.39 In several respects our protein-acrylamide copolymer hydrogel system represents an in vitro extension of previous in vivo studies in which Crkl tyrosine phosphorylation was detected in CML cells and related to CML disease progression.7,16,37,38 In these previous studies, phosphorylated Crkl was identified in cellular lysates through Western blot analysis with antiphosphotyrosine or antiphospho-Crkl antibodies. We have developed a simplified, in vitro system mimicking these studies in which immobilized GST-Crkl serves as an Abl substrate. In addition to eliminating the

electrophoresis and transfer steps of the Western blot, the array format of our assay allows simultaneous detection of multiple signals. Here we have detected up to 40 GSTCrkl spots per microscope slide; however, with commercially available microarray printing techniques densities as high as thousands of spots per slide are possible.36 PCR-based methods are also capable of diagnosing CML at the molecular level by the presence of the BCR-ABL fusion gene or BCR-ABL mRNA transcript. Quantitative RT-PCR can detect residual disease at levels as low as one leukemic cell in 105-106, and increased levels of BCRABLexpressionhavebeenassociatedwithdiseaseprogression.45-49 Cytogenetic testing is also used to diagnose CML. Here, approximately 20 metaphase spreads are typically examined, corresponding to a sensitivity of one leukemic cell in 20. However, the correlation between Bcr-Abl activity and mRNA expression levels or the presence of the Bcr-Abl translocation remains poorly defined.50 Bcr-Abl tyrosine kinase activity depends on mRNA stability and translation rates, protein stability and degradation rates, and, in the case of imatinib treatment, the rate of incorporation of activating mutations. While our GST-Crkl substrate array does not have the sensitivity level of PCR-based detection, it does directly measure the tyrosine kinase activity of the oncogenic moiety. Therefore, it is not only able to directly measure imatinib resistance due to BCR-ABL transcript overexpression, but it may also be possible to measure imatinib resistance due to mutation of the Bcr-Abl protein. Additionally, the ability to detect protein activity and inhibition extends beyond the Bcr-Abl and imatinib system. By simply replacing imatinib with other inhibitors, data for multiple tyrosine kinase inhibitors at multiple concentrations can be simultaneously obtained. The ease of this transition suggests that the protein-acrylamide copolymerization strategy presented here may provide a platform on which highthroughput screening assays could be developed for other biological systems. Future development of the assay will require accounting for the heterogeneous mix of normal and malignant cells obtained from patients and determining complete vs partial kinase activity in the malignant cells. In contrast to many other protein array systems, our method takes advantage of a hydrogel-based covalent surface immobilization. Polyacrylamide and protein-acrylamide copolymer hydrogels exhibit low levels of nonspecific protein adsorption.52 Thus, the subsequent detection of nonspecifically bound proteins from cell extracts is limited. In previous studies, this reduction in nonspecific binding has been shown to produce a 6-fold increase in the signal-to-noise ratio obtained from polyacrylamide protein arrays vs poly-L-lysine protein arrays in a human serum diagnostic.25 In our system, the inclusion of a polyacrylamide layer between the glass slide and protein array spots greatly reduced the nonspecific binding of cell lysate components and minimized the detection of endogenous phosphorylated-Crkl or other phosphoproteins. These results were in contrast to experiments conducted on commercially available poly-L-lysine or aldehyde arrays in which high levels of nonspecific protein adsorption made signal discrimination from noise nearly impossible. In addition to minimizing nonspecific protein

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adsorption, our protein-acrylamide copolymerization strategy maintains the immobilized proteins within a hydrophilic environment. This environment prevents protein dehydration and minimizes denaturation due to hydrophobic and/or charged protein-surface interactions. Also, the covalent nature of the protein-acrylamide copolymerization strategy ensures stable immobilization of proteins, even over extended periods of protein array storage.36 The porous, three-dimensional shape of our proteinacrylamide copolymer array spots provides several advantages over traditional, two-dimensional protein immobilization techniques. The capacity of our hemispherical spot is greatly increased compared to strategies in which proteins are directly attached to the solid surface. The threedimensional structure of the spot also positions a majority of the immobilized proteins away from the surface. Thus, immobilized proteins are more likely to be assessable to the sample applied to the protein array. While these advantages can also be found in commercially available hydrogel substrates, experiments demonstrated that our proteinacrylamide copolymer hydrogels provided an increased level of sensitivity and reduced spot-to-spot variation. Additionally, well-established chemistry exists for controlling the porosity and pore size distribution within polyacrylamide gels. By simply changing the polymerization conditions, a proteinacrylamide copolymer hydrogel array in which each protein is immobilized within a different porosity gel spot could be created. Note that in this study we utilized polymerization conditions that provided the lowest acrylamide concentration and the least possible cross-linking while maintaining a mechanically stable hydrogel. These conditions provided maximum accessibility of the relatively large Bcr-Abl kinase (190 kDa) and detection antibodies to the immobilized substrate.36 While we have used antibody-based chemiluminescence detection of surface attached substrates due to high sensitivity, additional detection techniques should be applicable to our protein-acrylamide copolymer hydrogel system. By simply replacing HRP conjugated antibodies with fluorescently labeled antibodies, multiple protein-state specific signals could be simultaneously detected on our proteinacrylamide copolymer array. Non-antibody-based detection techniques are also conceivable. In initial solution phase studies, Abl activity was determined via radiographic detection of 32P incorporation from γ-32P ATP.53 While radiographic detection methods may provide lower detection limits and an expanded range of signal linearity, the inclusion of physiological levels of ATP in the cellular lysate may complicate their use in this system. More recently, detection of surface attached substrates via a small molecule, phosphospecific fluorescent dye has been demonstrated.54 Additionally, in gel, tryptic digestion followed by mass spectrometry26 or mass spectrometry directly from polyacrylamide gels has been reported.55 Conclusions We have developed a method by which protein tyrosine kinase activity can be quantitatively assessed in an array-

Brueggemeier et al.

based format. Using a protein-acrylamide copolymerization strategy, substrate arrays containing GST-Crkl fusion proteins immobilized within copolymer hydrogels were synthesized. Phosphorylation of the immobilized Crkl was measured in the presence of purified v-Abl or in cell extracts containing Bcr-Abl, and reproducible, quantitative kinetic data were obtained. The ability of the protein-acrylamide copolymerization strategy to create substrate arrays for detection of Bcr-Abl activity in a complex system, i.e., a cell lysate, suggests the applicability of this strategy to the detection of other disease states reliant on altered protein activity.56 Not only could a multiplexed assay be envisioned in which multiple disease states are detected on the basis of an array of multiple substrates, but the ability to modify patient treatment based on molecular level information gained from these protein-acrylamide copolymer arrays may be possible. Acknowledgment. We are grateful for the contributions of Kevin Allen this project. We thank Dr. Brian Druker and Novartis for providing GST-Crkl constructs and PKI166 inhibitor, respectively. Funding for this work was provided by National Science Foundation grant BES-0103348 and National Institutes of Health grant CA103235, to S.P.P. and S.J.K., and a National Defense Science and Engineering Graduate Fellowship, to S.B.B. S.J.K. is a Scholar of the Leukemia and Lymphoma Society. References and Notes (1) Faderl, S.; Talpaz, M.; Estrov, Z.; O’Brien, S.; Kurzrock, R.; Kantarjian, H. M. New Engl. J. Med. 1999, 341, 164-172. (2) Deininger, M. W. N.; Druker, B. J. Pharmacol. ReV. 2003, 55, 401423. (3) Konopka, J. B.; Witte, O. N. Mol. Cell. Biol. 1985, 5, 3116-3123. (4) Benneriah, Y.; Daley, G. Q.; Mesmasson, A. M.; Witte, O. N.; Baltimore, D. Science 1986, 233, 212-214. (5) Melo, J. V.; Hughes, T. P.; Apperley, J. F. Hematology 2003, 132152. (6) Kantarjian, H.; Talpaz, M.; O’Brien, S. S.; Garcia-Manero, G.; Verstovsek, S.; Giles, F.; Rios, M. B.; Shan, J. Q.; Letvak, L.; Thomas, D.; Faderl, S.; Ferrajoli, A.; Cortes, J. Blood 2004, 103, 2873-2878. (7) Hochhaus, A.; Kreil, S.; Corbin, A. S.; La Rosee, P.; Muller, M. C.; Lahaye, T.; Hanfstein, B.; Schoch, C.; Cross, N.; Berger, U.; Gschaidmeier, H.; Druker, B. J.; Hehlmann, R. Leukemia 2002, 16, 2190-2196. (8) von Bubnoff, N.; Peschel, C.; Duyster, J. Leukemia 2003, 17, 829838. (9) Shah, N. P.; Nicoll, J. M.; Nagar, B.; Gorre, M. E.; Paquette, R. L.; Kuriyan, J.; Sawyers, C. L. Cancer Cell 2002, 2, 117-125. (10) Hochhaus, A.; La Rosee, P. Leukemia 2004, 18, 1321-1331. (11) Nardi, V.; Azam, M.; Daley, G. Q. Curr. Opin. Hematol. 2004, 11, 35-43. (12) Azam, M.; Latek, R. R.; Daley, G. Q. Cell 2003, 112, 831-843. (13) Gorre, M. E.; Mohammed, M.; Ellwood, K.; Hsu, N.; Paquette, R.; Rao, P. N.; Sawyers, C. L. Science 2001, 293, 876-880. (14) Sattler, M.; Salgia, R. Leukemia 1998, 12, 637-644. (15) Heaney, C.; Kolibaba, K.; Bhat, A.; Oda, T.; Ohno, S.; Fanning, S.; Druker, B. J. Blood 1997, 89, 297-306. (16) Tenhoeve, J.; Arlinghaus, R. B.; Guo, J. Q.; Heisterkamp, N.; Groffen, J. Blood 1994, 84, 1731-1736. (17) MacBeath, G.; Schreiber, S. L. Science 2000, 289, 1760-3. (18) Haab, B. B.; Dunham, M. J.; Brown, P. O. Genome Biol. 2001, 2. (19) de Wildt, R. M.; Mundy, C. R.; Gorick, B. D.; Tomlinson, I. M. Nat. Biotechnol. 2000, 18, 989-94. (20) Zhu, H.; Bilgin, M.; Bangham, R.; Hall, D.; Casamayor, A.; Bertone, P.; Lan, N.; Jansen, R.; Bidlingmaier, S.; Houfek, T.; Mitchell, T.; Miller, P.; Dean, R. A.; Gerstein, M.; Snyder, M. Science 2001, 293, 2101-5.

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