Cell-Based Assays for Identification of Novel Double-Strand Break ...

ARTICLES Cell-Based Assays for Identification of Novel Double-Strand Break-Inducing Agents Heather M. Dunstan, Catherine Ludlow, Sondra Goehle, Michelle Cronk, Philippe Szankasi, David R. H. Evans, Julian A. Simon, John R. Lamb

Background: We are developing cell-based assays to identify anticancer agents that are selectively toxic to cells with defined mutations. As a test, we used a three-stage strategy to screen compounds from the National Cancer Institute’s repository for agents that are selectively toxic to doublestrand break repair-deficient yeast cells. Methods: Compounds identified in the screen were further analyzed by use of yeast and vertebrate cell-based and in vitro assays to distinguish between topoisomerase I and II poisons. Results: Of the more than 85 000 compounds screened, 126 were selectively toxic to yeast deficient in DNA double-strand break repair. Eighty-seven of these 126 compounds were structurally related to known topoisomerase poisons, and 39 were not. Twenty-eight of the 39 were characterized, and we present data for eight of the compounds. Among these eight compounds, we identified two novel topoisomerase II poisons (NSC 327929 and NSC 638432) that were equipotent to etoposide in biochemical tests and in cells, five (NSC 63599, NSC 65601, NSC 380271, NSC 651646, and NSC 668370) with topoisomerase I-dependent toxicity in yeast that induced DNA damage and toxicity in mammalian cells, and one (NSC 610898) that directly bound to DNA and induced strand breaks. Conclusions: Cell-based assays can be used to identify molecules that are selectively toxic to cells with a predetermined genetic background, including mutations in genes involved in the cell cycle and its checkpoints, for which there are currently no selectively toxic compounds. [J Natl Cancer Inst 2002;94:88–94]

The successful treatment of cancer requires therapies that selectively kill tumor cells. With this in mind, we are developing methods to identify compounds that are selectively toxic to cells with defined molecular alterations. Chemotherapeutic agents that are currently used or are under development have been identified in two ways: empirically or by target-directed drug discovery. Many agents approved by the U.S. Food and Drug Administration were identified as generally toxic compounds that were empirically found to have anticancer activity. In this way, several classes of molecules were found, including DNA-damaging agents, antimetabolites, and antimitotic compounds. However, the basis for the antitumor activity frequently is not well understood, and molecular changes of tumors that lead to drug sensitivity are not well characterized. The modern approach to anticancer drug discovery relies on selection of the most relevant targets in tumors, identification of 88 ARTICLES

inhibitory compounds, and then development of therapies using these agents. This approach has led to the development of a number of compounds, including inhibitors of farnesyltransferase, angiogenesis, and telomerase. A problem with this approach is that it is limited by our knowledge of biology, so that the viability of the chosen targets for the treatment of cancer is often not proven. In addition, this approach is limited to targets that can be used in in vitro biochemical screening assays and generally precludes targeting loss or alteration of function mutations that are often observed in tumors (e.g., p53). Often, the molecular changes that sensitize tumors to such compounds are not known, and proving that a compound is specific to the target can be a challenge. We are developing a general approach to the identification of anticancer compounds in which we directly identify compounds that are selectively toxic to cells with defined molecular changes. This approach uses matched pairs of cell lines, one line with a defined genetic alteration and the other with a corresponding wild-type gene, to identify compounds that are selectively toxic to the altered cells. The defined molecular changes include, but are not limited to, mutations that confer a loss or alteration of function that cannot be addressed by current methods (e.g., loss of p53 or pRb or activation of ras). The mechanism of action of these compounds can take advantage of any difference in the pair of cells. The biggest disadvantage of this approach is that the molecular targets of the compounds are not known and have to be determined. However, we would argue that it is better to have compounds with a known selectivity but an unknown target than to have compounds with a known target and an unknown selectivity. To test this approach, we have screened for compounds in the compound repository of the National Cancer Institute (NCI) that are selectively toxic to double-strand break-repair defective cells. We used the yeast Saccharomyces cerevisiae to screen drugs and matched pairs of vertebrate cells to retest the selected drugs. The advantages of using yeast as a model system include the high degree of conservation of DNA-repair pathways (1), the relative ease of handling, and the availability of advanced molecular genetic techniques for subsequent analysis.

Affiliation of authors: Program in Molecular Pharmacology, Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA. Correspondence to: John R. Lamb, Ph.D., Program in Molecular Pharmacology, Clinical Research Division, Fred Hutchinson Cancer Research Center, 1100 Fairview Ave., N., Seattle, WA 98109 (e-mail: [email protected]). See “Notes” following “References.” © Oxford University Press

Journal of the National Cancer Institute, Vol. 94, No. 2, January 16, 2002

MATERIALS

AND

METHODS

S. cerevisiae Strains, Screening Procedures, and Growth-Inhibition Assays All strains are derived from yeast with the A364a genetic background. Gene disruptions were generated by polymerase chain reaction (PCR) fragment-directed replacement (2). Except where noted, all mutations are deletions of the coding sequences. The mec2–1 mutation was introduced by standard genetic techniques. Growth inhibition of yeast strains was measured as described previously (3). Briefly, exponentially growing yeast strains were exposed to the compounds for six or seven generations, and relative growth was compared with solvent controls by assessing optical density at 660 nm. Three stages of screening (stages 0, 1, and 2) were used to identify selectively toxic compounds. In stage 0, compounds that produce a 70% or greater inhibition of growth relative to untreated controls at a single dose of 50 ␮M were identified and tested in stage 1; these compounds were tested at 5 and 50 ␮M. In stage 2, compounds were tested at 100, 33, 11, 3.7, and 1.2 ␮M. Compounds that produced a fivefold or greater difference between any two strains in stage 1 were tested in stage 2. After testing in stage 2, compounds were identified that were selectively toxic to rad50 and rad52 strains relative to wild-type strain and that lacked selective toxicity in rad14 or rad18 strains (specific for strains deficient in double-strand break repair). Six yeast strains were used in stages 0 and 1, and all six strains contained drug-sensitizing mutations in the erg6, pdr1, and pdr3 genes [the “epp background” (4–6)]. We reasoned that strains containing defects in unrelated pathways could act as wild-type controls for each other. For instance, a strain containing defects in a mitotic checkpoint acts as a control for DNAdamaging agents that are selectively toxic to DNA repairdefective strains. Therefore, to facilitate throughput, no wildtype strain was included in stages 0 and 1. Nine mutations in DNA repair checkpoint and cell cycle pathway genes (1,7–12) were introduced into the six strains used for stages 0 and 1 as follows: Three strains contained single mutations in rad50 (doublestrand break repair), mec2-1 (DNA damage checkpoints), or bub3 (mitotic checkpoint) genes. The remaining three strains contained one of the following pairs of mutations: rad18 and mlh1 (postreplication repair and mismatch repair); sgs1 and mgt1 (homologue of the Blooms and Werner syndrome genes and O6-alkylguaninetransferase); or rad14 and CLN2oe (nucleotide excision repair and overexpression of the G1 cyclin CLN2). The strains used in stage 2 included wild-type controls and strains with the following single mutations: bub3, CLN2oe, mec2-1, mgt1, mlh1, rad14, rad18, rad50, rad52 (double-strand break repair), and sgs1. The compounds screened were from the collection of the Developmental Therapeutics Program (DTP), NCI, Bethesda, MD. More than 85 000 compounds have been screened in stage 0, resulting in the identification of 126 compounds that are selectively toxic to rad50 and rad52 strains relative to a wild-type strain and other repair-deficient strains. Additional details of the strains and mutants and the screen are described in the website of the DTP, NCI (http://dtp.nci.nih.gov, click on “Public Data”, then “NCI Yeast Anticancer Drug Screen”). Overexpression of topoisomerase I or II was achieved by replacing the endogenous promoters with the strong constitutive glyceraldehyde-3-phosphate dehydrogenase-1 (GPD1) pro-

moter. These strains were constructed by transformation with a PCR-generated DNA fragment containing the GPD1 promoter and URA3 marker flanked by sequences homologous to the topoisomerase I or II promoters. Strains containing topoisomerase-promoter replacements were found to have increased sensitivity to standard topoisomerase I (camptothecin) or II (idarubicin) poisons as expected. Toxicity Assays in Vertebrate Cells Toxicity was measured with a modified [3H]thymidine incorporation assay as described previously (13). This assay is highly predictive of standard colony-forming assays, with a variety of cell lines and standard agents (13). Cell lines were grown at 37 °C and 5% CO2. The chicken pre-B-cell line DT40 and its derivatives (14–16) were grown in RPMI-1640 medium containing 10% fetal calf serum (FCS) and 1% chicken serum. The remaining cell lines were grown in Dulbecco’s modified Eagle medium with 10% FCS. FACS Analysis Mouse fibroblast Rat1a cells in S phase were labeled with 10 ␮M bromodeoxyuridine for 1 hour at 37 °C. After labeling, cells were incubated for 6 hours in medium without bromodeoxyuridine either with or without test compounds and subjected to FACS analysis. Topoisomerase I or II In Vitro Assays Kits and enzymes for topoisomerase I or II in vitro assays were purchased and performed according to the manufacturer’s instructions (products 1018-2 and 1009-2, respectively; Topogen, Columbus, OH). We measured the formation of topoisomerase-trapped complexes in vitro as follows: A reaction mixture containing purified topoisomerase I or II, plasmid DNA, and the test drug in the appropriate buffer was incubated at 37 °C for 30 minutes, the reaction was terminated with sodium dodecyl sulfate, topoisomerase I was digested with proteinase K, and reaction products were resolved on agarose gels containing the intercalator ethidium bromide. Bandshift Assays The ability of NSC 610898 to bind to DNA was measured by incubating various concentrations of the compound with a constant amount of plasmid DNA for 30 minutes at 37 °C in topoisomerase I drug kit buffer (Topogen). After incubation, the samples were separated by electrophoresis through 1% agarose gels. Binding of the drug to DNA was indicated by a slower mobility in the gel.

RESULTS Identification of rad50/52-Specific Compounds A three-stage process was used to identify rad50/52-specific compounds. More than 85 000 compounds were screened in stage 0, 14 300 compounds were screened in stage 1, and 795 compounds were screened in stage 2. Compounds were selected by use of the criteria described above. This procedure identified 227 compounds as selective for rad50/52. Of these 227 compounds, 126 were selective for rad50/52 but not other DNA repair-defective strains (rad14 and rad18). Eighty-seven of these compounds were known or closely related to known topoisomerase poisons, including 10 pyrazoloanthrones, nine amsacrines,


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12 ellipticines, five amonafides, six anthracyclines, 13 camptothecins, six etoposides, three mitoxanthrones, three thioxanthrones, and 20 other derivatives. The remaining 39 compounds were not related to compounds of known function. We were able to obtain quantities of 28 of the 39 compounds that were sufficient to conduct the series of experiments described below. For reasons of space, we present data for eight of the 28 compounds tested that were the most noteworthy. Structures of the eight compounds examined are in Fig. 1. Levels of Topoisomerases and Compound Toxicity The toxicity of topoisomerase poisons is directly proportional to the level of topoisomerase expression (17–20). We tested the following 28 compounds: NSC 63599, NSC 65601, NSC 67804, NSC 75503, NSC 101984, NSC 116533, NSC 124202, NSC 153625, NSC 327929, NSC 374986, NSC 380271, NSC 400244, NSC 401030, NSC 401158, NSC 402756, NSC 405361, NSC 405577, NSC 407567, NSC 408202, NSC 408478, NSC 408726, NSC 603331, NSC 610898, NSC 614579, NSC 615555, NSC 638432, NSC 651646, and NSC 668370. We also tested known poisons for topoisomerase I (camptothecin) and topoisomerase II (idarubicin) on the topoisomerase panel of strains. Results for a subset of eight compounds are shown (Table 1; Fig. 1). All eight compounds were selectively toxic to cells lacking an active rad50 gene for double-strand break repair (Table 1; Fig. 1). The toxicity of five compounds was proportional to topoisomerase I levels, that of two was proportional to topoisomerase II, and that of one was proportional to both topoisomerase I and topoisomerase II. This behavior is similar to that seen with known poisons for topoisomerase I and/or II (17–20).

Toxicity in Vertebrate Cells Defective in Double-Strand Break Repair We next measured the toxicity of these eight compounds in two lines of chicken pre-B-cell line DT40, one wild-type and the other defective in double-strand break repair (14–16). Eukaryotic cells have two pathways for double-strand break repair, homologous recombination and nonhomologous end rejoining (1,21). The latter is thought to be a major double-strand breakrepair pathway in vertebrate cells. To examine selective toxicity of the compounds in vertebrate cells, we used a derivative of the chicken pre-B-cell line DT40 that lacks the KU70 gene, an essential component of nonhomologous end-rejoining pathway (15). Initial experiments showed that topotecan was equally toxic to cells with and without KU70 (Table 1), indicating that nonhomologous end rejoining is unable to repair the double-strand breaks induced by topoisomerase I poisons. In contrast, etoposide is much more toxic to cells lacking KU70 than to cells with KU70, indicating that double-strand breaks induced by topoisomerase II poisons can be efficiently repaired by nonhomologous end rejoining. The five compounds (NSC 63599, NSC 65601, NSC 380271, NSC 651646, and NSC 668370) with toxic effects proportional to topoisomerase I levels in yeast were equally toxic to DT40 cells with and without KU70, as seen with topotecan (Table 1). The two compounds (NSC 327929 and NSC 638432) with toxic effects proportional to topoisomerase II in yeast were much more toxic to cells lacking KU70 than to cells with KU70, as seen with etoposide. NSC 610898, which had toxic effects proportional to levels of topoisomerase I or II in yeast, also was much more toxic to cells lacking KU70 than to cells with KU70.

Fig. 1. Structures of eight tested compounds that were selectively toxic to rad50/52 strains. Structures of eight of the 126 rad50/52-specific compounds identified are shown. A) NSC63599, NSC65601, NSC380271, NSC651646, and NSC668370 are responsive to topoisomerase I in yeast and induce toxicity and DNA damage in vertebrate cells in chicken DT40 and murine Rat1a cells. B) NSC327929 and NSC638432 are responsive to topoisomerase II with similar potency to etoposide in vitro and in vivo. C) NSC 610898 binds to DNA and directly induces strand breaks.

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Table 1. Analysis of eight compounds* A: Yeast strains IC50, ␮M NSC

Toxicity proportional to

wt

rad50

Top1oe

rad50 top1

TOP2oe

3.8 98.0 17.5 30.0 85.0 20.0

0.1 3.4 2.8 11.0 7.8 1.0

0.4 36.0 10.3 8.0 73.0 2.0

>100 >100 23.8 40.0 95.0 20.0

1.5 58.0 12.0 17.5 57.0 7.0

Topo I

Idarubicin 327929 638432

3.5 6.0 6.0

0.2 1.3 2.0

3.5 6.0 ND

0.1 1.0 2.0

0.2 2.0 2.0

Topo II

610898

2.8

2.3

2.3

6.5

0.5

Topo I and II

Camptothecin 63599 65601 380271 651646 668370

B: Vertebrate cells and in vitro Differential (+/– KU70)

DT40 IC50 (␮M)

Inhibition of S phase

Poison of Topo I

Poison of Topo II

Topotecan 63599 65601 380271 651646 668370

No differential No differential No differential No differential No differential No differential

0.05 12.5 6 12.5 1.5 1.5

Yes Yes Yes Yes Yes Yes

Yes No No No No No

ND ND ND ND ND ND

Topo I

Etoposide 327929 638432

Eightfold Twofold Fourfold

0.25 3 1.5

Yes Yes Yes

ND ND ND

Yes Yes Yes

Topo II

610898

艌 Fourfold

NT

No

No

No

Topo I and II

NSC

*A) Yeast strains. Eight compounds were analyzed in yeast strains with wild-type (wt) and with altered levels of topoisomerase I (Topo I) or II (Topo II), including overexpression of Topo I (TOP1oe) or II (TOP2oe) and deletion of the Topo I gene (top1). To increase the sensitivity of yeast to the test agents, we used a strain in which both rad50 and top1 had been deleted (rad50 top1). This strain, therefore, should be compared with a rad50 strain. We were unable to delete the Topo II gene (TOP2), because it is an essential gene (29,30). The sensitizing rad50 mutation was not introduced to the strains that overexpress Topo I or II because this combination produces synthetic lethality. The doses that inhibit growth by 50% (IC50) were determined by interpolation between the doses used (the doses used in yeast were 100, 33, 11, 3.7, and 1.2 ␮M). Compounds that did not inhibit growth by 50% at the highest dose used are listed as greater than 100 ␮M. ND ⳱ not done; oe ⳱ overexpression. B) Vertebrate cells. The differential fold increase in toxicity produced by the same compound in a pair of isogenic cell lines, parental wild-type (KU70 +/+) cells and a line lacking the double-strand break-repair protein KU70 (KU70 –/–). The IC50 concentration of each compound in the double-strand break-repair-proficient parental cell line DT40 is shown. NT (nontoxic) compound did not reduce viability by 50% at 50 ␮M, the highest concentration tested. IC50 values were derived from at least two experiments. NSC 610898 was found to be nontoxic as defined above but had a measurable IC50 value in the repair-deficient cell line, indicating at least a fourfold differential toxicity between the two cell lines. Inhibition of S phase was measured in Rat1a fibroblasts by flow cytometry analysis. In the absence of treatment, there are few or no labeled cells with an S-phase DNA content after the 6-hour chase period. Compounds that induce retention of high levels of labeled cells with an S-phase DNA content greater than this low background inhibit DNA synthesis. Inhibition of activity or poisoning of Topo I and II in vitro was as indicated. Inhibition of Topo I and II is also shown (Yes ⳱ compound can inhibit or poison the topoisomerase). None of the Topo I-responsive compounds behaved as poisons in vitro, despite their similarity to topotecan in vivo. The mechanisms of action of these five compounds remains unclear. Of the Topo II-responsive compounds, NSC 327929 and NSC 638432 are novel Topo II poisons with activities equivalent to etoposide.

Topoisomerase Poisons and S-Phase Arrest Topoisomerase poisons arrest cells in S phase by inducing DNA damage (Fig. 2). Cells in S phase were labeled for 1 hour with bromodeoxyuridine and then exposed to the test compounds for 6 hours (half a cell cycle). Seven of the eight compounds tested arrested cells in S phase (Table 1; Fig. 2). The eighth compound, NSC 610898, did not. In Vitro Topoisomerase I Assays We next used an in vitro system to determine whether any of the compounds had a direct effect on topoisomerase I or II. The ability to induce topoisomerase I-mediated DNA damage requires the formation of covalent complexes containing topoisomerase I and trapped DNA (22–24). We measured the formation of these complexes in vitro. Topotecan can convert

closed-circular supercoiled DNA into nicked-circular DNA, indicating that trapped complexes were formed (Fig. 3). None of the five compounds with toxic effects proportional to topoisomerase I created nicks in DNA. NSC 63599 and NSC 651646 were synthesized de novo (Simon JA: unpublished results) and were also negative when a more sensitive 32P-labeled DNA assay was used (Pourquier P, Pommier Y: personal communication). NSC 610898 created nicks in DNA, but this reaction was independent of topoisomerase I. Therefore, none of the six compounds tested in vitro behaved as topoisomerase I poisons. In Vitro Assays With Topoisomerase II Next, we tested the ability of NSC 327929 and NSC 638432 to induce double-strand breaks in vitro [Fig. 3; Table 1 (22,23)]. At the highest concentrations tested, both of the compounds


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Fig. 2. Compounds inhibit S-phase progression. The DNA of Rat1a cells was labeled for 1 hour with the nucleotide analogue bromodeoxyuridine, followed by a 6-hour chase period in the presence of dimethyl sulfoxide (DMSO) or the test compounds. Topoisomerase I- or II-responsive compounds and known topoisomerase I and II poisons inhibit S phase by inducing DNA damage. Seven of the eight test compounds also inhibit S-phase progression.

Fig. 3. In vitro assays. A) Topoisomerase I (Topo I) will relax supercoiled DNA. Topotecan at 100 ␮M converts supercoiled DNA to nicked, circular DNA (compare lanes 3 and 4). None of the five Topo I-responsive compounds produced nicked DNA (lanes 5–9). NSC 610898 induced nicked DNA species in a Topo I-independent fashion (lanes 10 and 11). Each reaction contained 2 U of Topo I and 100 ␮M of each test compound. Reactions were stopped with 2% sodium dodecyl sulfate, and Topo I was digested with proteinase K (50 ␮g/mL). Reaction products were extracted with phenol and separated by electrophoresis in a 1% agarose gel containing ethidium bromide (0.5 ␮g/mL). DMSO ⳱ dimethyl sulfoxide. B) The ability of Topo II poisons at 100 ␮M (lanes 4, 7, and 10), 10 ␮M (lanes 5, 8, and 11), and 1 ␮M (lanes 6, 9, and 12) to induce doublestrand breaks in plasmid DNA was tested. The Topo II poison etoposide, NSC 327929 (at 10 and 1 ␮M), and NSC 638432 (at 10 and 1 ␮M) produced linear plasmid DNA. However, at 100 ␮M, NSC 327929 and NSC 638432 inhibited the catalytic activity of Topo II, which inhibited the formation of linear species. Reaction products were separated by electrophoresis in 1% agarose gels containing ethidium bromide (0.5 ␮g/mL). C) Plasmid DNA was incubated with 0, 1, 10, or 100 ␮M NSC 610898 (lanes 2–5) for 30 minutes at 37 °C and then was subjected to electrophoresis through a 1% agarose gel containing ethidium bromide (0.5 ␮g/mL). NSC 610898 caused a dose-dependent retardation in the migration of the plasmid DNA, indicating that it bound to the DNA.

inhibited the catalytic activity of topoisomerase II and thus prevented formation of double-strand breaks (i.e., formation of linear DNA), as observed with other topoisomerase II poisons (8,25–27). However, at 10 and 1 ␮M, both compounds induced topoisomerase II-dependent double-strand breaks in plasmid DNA, as observed with etoposide, indicating that these compounds are topoisomerase II poisons. 92 ARTICLES

NSC 610898: DNA Binding and Strand-Break Induction NSC 610898 inhibited topoisomerases I and II and induced strand breaks in vitro, independent of topoisomerases I and II (above and data not shown). The structure of NSC 610898 suggests that it may bind to the minor groove of DNA in a manner analogous to that of netropsin (28). A band-shift assay showed


that NSC 610898 bound to DNA, but the nature of the binding was not investigated (Fig. 3).

DISCUSSION In this study, we tested the feasibility of screening for selectively toxic compounds in cell-based assays. As a test case, we identified compounds with selective toxicity in yeast cells defective in double-strand break repair and retested the compounds in vertebrate cells. Identification of Compounds That Target Double-Strand Break-Repair Defects Of more than 85 000 compounds screened, we found 126 compounds that were selectively toxic to yeast cells with defective double-strand break repair (rad50/52 specific). By use of a combination of assays, we identified two novel topoisomerase II poisons, five compounds that are topoisomerase I poisons in yeast, and one compound that directly binds to and induces topoisomerase-independent DNA strand breaks. The five compounds that were dependent on topoisomerase I in yeast induce DNA damage and were toxic in mammalian cells. However, we could not show that these five compounds nicked DNA in vitro in a topoisomerase I-dependent manner. NSC 610898 bound to DNA and induced strand breaks independent of topoisomerases or any other protein. NSC 327929 and NSC 638432 by all criteria were structurally novel topoisomerase II poisons with potency equivalent to that of etoposide. Cell-Based Assays for Identification of Selectively Toxic Small Molecules This work has demonstrated that screening large numbers of compounds in cell-based assays is feasible. The assay identifies both expected and novel compounds. These results open the possibility of screening for compounds that are selectively toxic to cells containing mutations found in tumors, including mutations in cell cycle and checkpoint proteins, for which there are currently no selectively toxic compounds.

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NOTES


Present address: H. M. Dunstan, Stratagene, La Jolla, CA. Present address: P. Szankasi, Myriad Genetics, Inc., Salt Lake City, UT. Present address: D. R. H. Evans, Curagen Corporation, New Haven, CT.

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Supported in part by Public Health Service contract N01BC65017 (to J. A. Simon) and grant 1R01CA8231101 (to J. R. Lamb) from the National Cancer Institute (NCI), National Institutes of Health, Department of Health and Human Services. We thank Lee Hartwell (Fred Hutchinson Cancer Research Center, Seattle, WA), Stephen H. Friend (Rosetta Inpharmatics, Kirkland, WA), Priscilla Cooper (Lawrence Berkeley National Laboratory, Berkeley, CA), Stephen J. Elledge (Baylor College of Medicine, Houston, TX), Jan Hoeijmakers (Erasmus University, Rotterdam, The Netherlands), Andrew Murray (Harvard University,

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Cambridge, MA), Tom Petes (University of North Carolina, Chapel Hill), and Graham C. Walker (Massachusetts Institute of Technology, Cambridge, MA) for invaluable advice and Edward Sausville (National Cancer Institute [NCI], Bethesda, MD) and members of the Developmental Therapeutics Program (DTP) of the NCI for many helpful comments and advice. We also thank Robert Schultz (DTP, NCI) for providing the compounds used in this study. Manuscript received March 27, 2001; revised October 25, 2001; accepted November 16, 2001.
