Carcinogenesis vol.23 no.6 pp.949–957, 2002
High-throughput measurement of the Tp53 response to anticancer drugs and random compounds using a stably integrated
Taylor A.Sohn1, Ravi Bansal3, Gloria H.Su2,3, Kathleen M.Murphy2 and Scott E.Kern2,4 Departments of Surgery1, Pathology2, and Oncology3, The Johns Hopkins Medical Institutions, Baltimore, MD, USA 4To
whom correspondence should be addressed Email:
[email protected].
Human Tp53 is normally a short-lived protein. Tp53 protein is stabilized and levels are increased in response to a variety of cellular stresses, including those induced by genotoxic anticancer drugs and environmental exposures. To engineer an efficient assay based on this property, we constructed and integrated a Tp53-specific reporter system into human cancer cells, termed p53R cells. We tested a range of conventional chemotherapeutic agents as well as over 16 000 diverse small compounds. Ionizing radiation and two-thirds of conventional chemotherapeutic agents, but only 0.2% of diverse compounds activated Tp53 activity by two-fold or greater, consistent with the presumptive genotoxic activation of Tp53 function. Cytotoxicity was independent of TP53 genetic status when paired, syngeneic wild-type TP53 and TP53-null cells in culture were treated with compounds that activated Tp53. From the unbiased survey of random compounds, Tp53 activation was strongly induced by an analog of AMSA, an investigational anti-cancer agent. Tp53 was also strongly induced by an N-oxide of quinoline and by dabequine, an experimental antimalarial evaluated in humans; dabequine was reported to be negative in other screens of mutagenicity and clastogenicity but carcinogenic in animal studies. Further exploration of antimalarial compounds identified the common medicinals chloroquine, quinacrine, and amodiaquine as Tp53-inducers. Flavonoids are known to have DNA topoisomerase activity, a Tp53-inducing activity that is confirmed in the assay. A reported clinical association of Tp53 immunopositive colorectal cancers with use of the antihypertensive agents was extended by the demonstration of hydralazine and nifedipine as Tp53-inducers. p53R cells represent an efficient Tp53 functional assay to identify chemicals and other agents with interesting biologic properties, including genotoxicity. This assay may have utility in the identification of novel chemotherapeutic agents, as an adjunct in the pharmaceutical optimization of lead compounds, in the exploration of environmental exposures, and in chemical probing of the Tp53 pathway.
Introduction The TP53 gene is among the most commonly mutated genes in human cancers (1,2). The TP53 gene encodes for a tumor-suppressor protein, which functions as a short-lived transcription factor. Tp53 protein is stabilized in response to a wide variety of cellular stresses including DNA damage, hypoxia, metabolic changes, mitotic spindle disruption, hyp© Oxford University Press
oxia, and activated oncogenes (3,4). The Tp53 protein is often thought of as a central point between stressful stimuli and the ultimate fate of the cell, with suppression of cellular proliferation either through cell cycle arrest or apoptosis (3,5,6). Under normal conditions, cells contain low levels of Tp53 proteins that are quickly degraded. Stressful or genotoxic stimuli trigger upstream events that lead to the accumulation of Tp53 which, in turn, triggers downstream pathways. The DNA damage-related inputs are currently the best understood. Upstream mediators of Tp53 responses include the human Rad3 homologues Atm (ataxia-telangiectasia mutated) and Atr, which regulates entry into mitosis (7–12). Upon exposure to DNA-damaging agents, the Atm and Atr proteins redistribute to intranuclear foci that may be the sites of DNA synthesis and repair (13,14). Atm and Atr proteins are necessary for the G2/M checkpoint that is activated upon irradiation and other causes of DNA damage (14,15). The checkpoint kinase Chk2 is activated upon phosphorylation by the Atm kinase (16). Chk2 in turn phosphorylates Tp53, preventing its binding to the Mdm2 protein. Complexation with Mdm2 blocks the activity of Tp53 and targets Tp53 for ubiquitin-mediated degradation in the proteosome. Downstream, Tp53 is known to transcriptionally activate the MDM2 gene, representing a negative feedback loop (17–19). Other downstream targets include CDKN1A, BAX, GADD45, and SFN (14–3–3σ), all of which function in cell cycle arrest or apoptosis (20). A minimal consensus DNA-binding site for Tp53 comprising two copies of an internally symmetric 10-base pair motif has been identified (21–23). Reporter genes, such as luciferase or chloramphenicol acetyltransferase, when placed downstream of this DNA-binding site in an engineered construct, express the reporter protein in levels that correlate to the levels of active Tp53 (24). Such reporters are means to measure the Tp53 stabilization in response to the activation of the upstream kinases of the Tp53 pathway. The above model is not without experimental exceptions, but in confirmation of this model, Tp53 stabilization in response to genotoxic agents does not occur when the Atm and Atr kinases are blocked by caffeine (8,25,26), and ATM- and ATR-deficient cells lack the G2/M checkpoint response (14,27). The detection of the Tp53 response has been proposed as a facile survey for genotoxic compounds and potentially useful anticancer agents (28–32). In support of this concept, there is evidence that the Tp53 response is not attributable to a general, non-specific effect of the cytotoxicity of these particular compounds (31), although classes of non-genotoxic stresses that activate Tp53 function are known (4). The current study analyzes a luciferase-based, Tp53-specific reporter system stably integrated into the genome of cancer cells (p53R cells) for use as a high-throughput compound screen. This system could be used to screen for specific biologic activities in human cells caused by environmental substances, dietary constituents, food additives, and the commercial pharmacopoeia. It also has potential application in 949
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probing the upstream origins of the critical Tp53 tumorsuppressive pathway and in the identification of novel chemotherapeutic agents (those to be genotoxic), as an adjunct in the pharmaceutical optimization of lead compounds, and in the evaluation of chemopreventatives intended to interfere with genotoxicity and carcinogenicity. Materials and methods Reporter constructs Concatemers of the internally symmetric Tp53 consensus DNA-binding site (p53bs-luc) (21–23) or a mutant binding site (p53ms-luc) were inserted upstream of the SV40 minimal promoter in the pGL3-promoter vector (Promega, Madison, WI). The p53bs-luc construct contained four copies of the 10-bp motif (5⬘-PuPuPuC(A/T)(T/A)GPyPyPy-3⬘) and p53ms-luc contained six copies with critical C and G residues mutated at residues 4 and 7 of the 10 bp motif shown above. Cell lines The Hs766T pancreatic cancer and the RKO colon cancer cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). Hs766T has a wild-type TP53gene. The HCT116 colon cancer cell line (wildtype TP53, HCT116 p53⫹/⫹) and daughter TP53-knockout cells (HCT116 p53–/–) were gifts of Bert Vogelstein (Johns Hopkins University, Baltimore, MD) (33). Hs776T cells were grown in Dulbecco’s modified Eagle media (DMEM) supplemented with 10% FBS, L-glutamine, and pencillin/ streptomycin. RKO cells were grown in RPMI with similar supplementation. HCT116 p53⫹/⫹ and HCT116 p53–/– cells were grown in McCoy’s modified 5A media supplemented with penicillin/streptomycin. Panc-1 cells with a stably transfected SBE (Smad-Binding Element) reporter were used as controls (34,35). Transfection Stable transfectants were generated by cotransfection of pcDNA3.1 (Invitrogen, Carlsbad, CA) and p53bs-luc into Hs766T cells using Lipofectamine (Life Technologies) for 5 h. Limiting dilutions of transfected cells were selected in 96-well plates in the presence of 0.75 mg/ml G418 (Geneticin, Life Technologies). Single clones were expanded and tested for basal luciferase activity and for the inducibility of reporter activity upon the addition of etoposide. One clone, termed p53R, was chosen on the basis of 3.0- to 5.0-fold induction of luciferase expression with low concentrations of etoposide (3 µg/ml) and 10.0- to 12.0-fold induction at high concentrations (12 µg/ml) of etoposide (34). Stable transfectants of the p53mut-bs were obtained in similar fashion (p53Rm). Transfectants containing the desired mutant construct were identified by lack of luciferase induction in response to etoposide in the presence of a robust luciferase response to Scriptaid, a general transcriptional activator. Transient transfections of p53bs-luc and p53ms-luc in all cell lines were done using the Lipofectamine protocol (Life Technologies). All transient transfections were performed as cotransfections with pCMVβ, and β-gal values were used to normalize for the efficiency of transfection. Compound screening Conventional chemotherapeutic agents, select additional pharmaceuticals and flavonoids, and trichostatin A were purchased from Sigma-Aldrich (St Louis, MO) or obtained from The Johns Hopkins Hospital pharmacy. High-throughput diverse compound screening was performed with the commercially available DIVERSet compound library (ChemBridge, San Diego, CA, compound descriptions available at http://pathology2.jhu.edu/skern/E-set.sdf and viewable with ChemFinder software, CambridgeSoft, Cambridge, MA). Each compound of the library was dissolved and diluted in DMSO at 1 mg/ml. Scriptaid was obtained from ChemBridge (San Diego, CA). Additional pharmaceutical agents were purchased from Prestwick Chemical (Washington, DC). Stably transfected Hs766T cells were plated in 96-well cluster plates (Corning, Cambridge, MA) and incubated with each compound at a final concentration of 2 µg/ml for 20–24 h. Cytotoxicity was not microscopically evident at this time point and concentration, although cell death was seen with some compounds at longer exposures and at higher concentrations. Luciferase activity was measured in a Wallac Trilux photodetector (Wallac, Gaithersburg, MD) after the addition of Steady-Glo luciferase substrate (Promega, Madison, WI). Measurements from each experiment were compared with control wells on the same plate, and a ratio reflecting the relative increase in luciferase activity was calculated for each chemical using an Excel (Microsoft, Redmond, WA) spreadsheet. Some compounds that activated the reporter were retested over a thousand-fold range of concentration in a two-fold dilution series from 1000 to below 1 µg/ml. Compounds of interest were also tested in the presence of caffeine, an Atm and Atr inhibitor (8,25,26).
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Immunoblots For those compounds that activated the reporter, p53R, HCT116 p53⫹/⫹ or HCT116 p53–/– cells were treated at the concentration of maximal activation for the particular drug and were incubated for 20–24 h. Treated and untreated cells were harvested with trypsin-EDTA, washed with PBS and resuspended in protein sample buffer. A dot blot was stained with Naphthol Blue Black for protein quantification. 200 µg protein from each sample was separated electrophoretically on a 10% SDS-poylacrylamide gel and subsequently transferred to a nylon membrane (Imobilon P, Millipore, Burlington, MA) using a Milliblot-Graphite Electroblotter I (Millipore). The membranes were then probed with mouse antibodies against human Tp53 (monoclonal Tp53 pantropic (Ab-2), Oncogene Research Products, Boston, MA), followed by goat anti-mouse antibody coupled to horseradish peroxidase (Pierce Chemical Co., Rockford, IL). Reactive proteins were viewed using enhanced chemiluminescence (Pierce). Cytotoxicity assays Matched syngeneic cells (HCT116 p53⫹/⫹ and HCT116 p53–/– cells) (36) were cultured in 96-well plates and treated with varying concentrations of chemotherapeutic agents in media and incubated for 72 h. Concentrations initially followed a two-fold dilution series from 1000 to below 1 mg/ml, and the study was repeated upon rescaling with a two-fold dilution series starting from the highest concentration that had resulted in observable cell survival. After rinsing with PBS, cells were lysed with water and 10 µl of the cell lysate was diluted in 95 µl of TE. 100 µl of 0.5% PicoGreen (Molecular Probes, Eugene, OR) was added to 100 µl of each sample. DNA-induced fluorescence was measured in a fluorometer and relative cellular DNA (1 – cytotoxicity) was calculated. Wells having no cells and no compound were used as blanks, and wells containing cells but no compound served as controls.
Results Development of p53R cells A luciferase-based Tp53 reporter system was developed. The Hs776T cell line (ATCC, Manassas, VA), which has a wildtype TP53gene (37), was stably transfected with the Tp53 reporter to form the p53R cell line (35). p53R cells were used to screen conventional chemotherapeutic agents, a subset of which are known to be directly genotoxic. Novel compounds were then screened to test the general ability of p53R screening for large numbers of chemical compounds. A similar strategy was used to develop p53Rm cells, Hs766T cells stably transfected with a Tp53-specific binding element mutated at C or G residues critical for Tp53 binding (21). Gamma radiation (137Cs, GammaCell 40), a known inducer of Tp53 stabilization, was used to introduce uniform and graded DNA damage; 1.1fold, 1.4-fold, and 2.3-fold responses were seen 24 h after doses of 1, 5, and 11.4 Gy, respectively, in comparison to an unirradiated plate. In radiated microtiter plates containing 96 wells of p53R cells, the standard deviation was 5.6–6.7% of the average measured luciferase values among the four plates. Based on these parameters (from a replicate set of nonindependent samples), a positive test result could conservatively be defined at three standard deviations from the mean, or an average fold-increase of 1.3 or greater in single assays. Positive results were verified in replicate assays. Compound screening – conventional chemotherapeutic agents An initial screen of 13 conventional chemotherapeutic agents from five different effector classes were evaluated for their induction of Tp53 activity in p53R cells. Classes of chemotherapeutic agents tested included the alkylating agents, intercalating agents, antimetabolites, topoisomerase inhibitors, and antimicrotubule agents (Table I). This was chosen as a useful initial panel for assay validation because the compounds represent those agents that are engineered and selected to be genotoxic to a broad range of human cell types. For many, the precise doses that can result in carcinogenesis (secondary
Tp53 response to anticancer drugs
Table I. Conventional chemotherapeutic agents Compound
Alkylating agents Cyclophosphamide (nitrogen mustard) Streptozotocin (nitrosourea) Mitomycin C (aziridene) Intercalating agents Cis-platin Antimetabolites Methotrexate (anti-folate) 5-Fluorouracil (nucleotide analog) Cytarabine (nucleotide analog) Gemcitabine (nucleotide analog) Topoisomerase inhibitors Etoposide (epidophyllotoxin) Camptothecin Doxorubicin (anthracycline) m-AMSA Antimicrotubule agents Vincristine (Vinca alkaloids) Paclitaxel (taxane) aTp53 response was measured in p53R cells. bHCT116 p53⫹/⫹ and HCT116 p53–/– syngeneic cSimilar levels of Tp53 response were seen over
Tp53 response at 2 µg/mla
Maximal Tp53 responsea
Concentration at maximal Tp53 responsea
Classification of cell deathb
0.9 1 3.4
1.1 1.1 9.8
N/A N/A 15 µg/ml
p53-independent p53-independent p53-independent
2
5.7
8 µg/ml
p53-independent
0.6 1.6 2 2.2
0.6 11.9 2 2.5
2.2 2.6 10.1 1.7
17.6 3.9 16.7 13.4
1 3
1.1 3.2
N/A 250 µg/ml 2 µg/ml 1 µg/ml–1 mg/mlc
p53-independent p53-independent p53-independent p53-independent
µg/ml µg/ml µg/ml µg/ml
p53-independent p53-independent p53-independent ND
N/A 3 µg/ml
p53-independent p53-independent
62 31 1 16
cells were used. a wide concentration range.
ND, no data.
malignancies) are known. The compounds were simultaneously tested using a previously reported stably transfected SmadBinding Element (SBE) reporter system as a control to rule out effects from general transcriptional activation (34,35). The use of this comparison and a brief report of the construction of p53R cells has been published (35). All anticancer agents were tested in duplicate over a concentration range of 1 µg/ml to 1000 µg/ml. Nine of the 13 compounds (69%) were found to increase Tp53 function by two- to 40-fold over untreated cells (Figure 1) whereas four compounds produced no increase in reporter activity. When the same 13 therapeutic agents were tested in the SBE reporter system, only three drugs were found to increase reporter activity (Figure 1), as did the two control compounds, trichostatin A and Scriptaid, which were known to be general transcriptional activators capable of reporter activation in both systems. Doxorubicin activated both reporters, but the activation was 15- to 16-fold with the Tp53 reporter and only two-fold with the SBE reporter. Mitomycin C, cis-platinum, 5-fluorouracil (5-FU), cytarabine, gemcitabine, etoposide, camptothecin, and paclitaxel were specific for the Tp53 element, while cyclophosphamide and vincristine only activated the SBE response element. Streptozotocin produced no response. All agents that activated Tp53 were tested in transient transfections of the wild-type TP53-containing cell lines, HCT116 and RKO, with consistent results, suggesting that the activity was not cell line-dependent. p53Rm cells, a stable transfectant of the Hs766T cell line containing a mutant reporter, demonstrated marked blunting of the reporter activity with etoposide over a wide concentration range (1 µg/ml to 250 µg/ml), with a maximal activation of 1.4-fold compared with 17.6-fold in p53R cells stimulated with etoposide. Additional pharmaceuticals and flavonoids were tested similarly. A panel of hormones and growth factors were tested over a 1000-fold range of concentration, greater and less
Fig. 1. Comparison of Tp53 transcriptional activity, measured with the integrated reporter, with that of an unrelated response element. Concentrations generating the highest induction of Tp53 activity are represented here. Gemcitabine showed constant Tp53 reporter activation over a thousand-fold range of concentrations and data obtained at 2 µg/ml is shown. Tp53 responses were determined in p53R cells. SBE responses were determined in Panc-1 cells stably transfected with 6SBE-luc (SBE cells). Stable transfectants of the mutant binding site, p53ms-luc, abolished the Tp53 response. Induction of Tp53 activity (black bars) or SBE activity (open bars) were relative to reporter activity in untreated cells (activity ⫽ 1.0). *Scriptaid data at 2 µg/ml, not at concentration of maximal activation, to maintain the scale of the graph (see Table I for maximal activation).
than physiologic concentrations. These included insulin, EGF (epidermal growth factor), glucagon, hydrocortisone, progesterone, somatostatin, VIP (vasoactive intestinal polypeptide), secretin, and TGFβ (transforming growth factor β); none produced a Tp53 response in p53R cells over this range of concentrations. A panel of antihypertensives was also 951
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Table II. Novel Tp53 activators and known analogs Compound
Novel compounds m-AMSA and analogs (Figure 2) m-AMSA Quinacrine 175328 175437 Benzo[g]quinolines (Figure 3) 175308 (dabequine) 175310 175323 175324 Quinolines (Figure 4) Chloroquine Amodiaquin 175136 108437 Quinoline N-oxide (Figure 4) 175195
Tp53 at response 2 µg/mla
Maximal Tp53 responsea
Concentration at maximal Tp53 responsea
Classification of cell deathb
1.7 2.4 5.5 2.7
13.4 6.8 ND ND
16 µg/ml 1.3 µg/ml ND ND
ND ND ND ND
5.7 3.0 14.3 3.9
18.7 12.9 14.3 7.8
3 µg/ml 1 µg/ml 2 µg/ml 0.3 µg/ml
1.1 1.0 2.0 2.3
7.2 2.7 ND ND
32 µg/ml 10 µg/ml ND ND
ND ND ND ND
5.0
ND
ND
ND
p53-independent p53-independent p53-independent p53-independent
aTp53
response was measured in p53R cells. For some compounds, optimal responses were seen at a concentration lower than the screening concentration, presumably due to cytotoxicity. For compound 175308, the Tp53 response at 2 µg/ml was variable, ranging from no response, to 5.7-fold response. Tp53 activation was more robust and consistent at 3 µg/ml. bHCT116 p53⫹/⫹ and HCT116 p53–/– syngeneic cells differing in TP53 genotype were used. ND, no data.
studied at 2–20 µg/ml, including acetozolamide, hydrochlorothiazide, captopril, minoxodil, diltiazem, verapamil, labetolol, bumetanide, lisinopril, enalopril, nifedipine, and hydralazine. Hydralazine and nifedipine produced activation of 1.5-fold and 1.8-fold, respectively, at 10 µg/ml, while other antihypertensives produced no response. Among the flavonoid compounds, apigenin produced responses of 1.7-fold at 2 µg/ml and 2.9-fold at 10 µg/ml. Hesperetin produced responses of 1.5-fold at 2 µg/ml and 2-fold at 20 µg/ml. Myricetin caused a response of 1.5-fold at 10 µg/ml, while quercetin produced no response at these concentrations. Compound screening – diverse compounds A screen of the ChemBridge DIVERSet chemical library, consisting of 16 320 diverse compounds, was then performed. The use of this library is well-described in compound screening (34,35,38–40). The mean response among the 16 320 compounds (a set of independent samples) was 1.0 and the standard deviation was 0.2. Thirty-seven compounds (0.2%) tested at 2 µg/ml had at least 2.0-fold activation of the reporter gene (five standard deviations above the mean of the independent sample set and nearly 15 times the standard deviation of the replicate set described above). All were additionally confirmed by at least three measurements. Twenty-six compounds had a 2.0- to 2.9-fold increase in luciferase activity; six, 3.0- to 4.9-fold increase; four, 5.0- to 9.9-fold increase; and one, ⬎10.0-fold increase in reporter activity. The remaining compounds had an average luciferase activation of 0.98-fold. The same library, when screened with the SBE reporter system, identified 12 compounds (0.07%) that activated DPC4 function ⬎2.0-fold.(34,35) Five compounds (0.03%) activated both reporters, suggesting that they were general transcriptional activators. The remaining compounds had specificity for the Tp53 system. Among the Tp53-activating compounds identified in this unbiased survey, two were 9-substituted acridine analogs (compound 175328 and 175437; Table II and Figure 2) of 952
Fig. 2. The chemical structures and molecular weights of two novel 9-substituted acridine analogs (compound 175328 and 175437) identified in the DIVERSet chemical library and m-AMSA, an investigational anti-cancer agent. The levels of Tp53 activation are shown in Tables I and II.
m-AMSA (amsidine, amsine, or 4⬘-(9-acridinylamino)-3⬘methoxymethanesulfonanilide), an investigational anti-cancer topoisomerase II inhibitor. Other 9-substituted acridine and azacridine derivatives of AMSA were previously evaluated as potential anti-cancer agents, demonstrating cytotoxicity against cells grown in culture (41–43). Compound 175328 produced a 5.5-fold Tp53 activation while compound 175437 had 2.7-fold activation at a concentration of 2 µg/ml. m-AMSA (Figure 2) was procured and tested. It activated the reporter 1.7-fold at 2 µg/ml to a maximum of 13.4-fold at 16 µg/ml. Tp53 activation was present among all of the topoisomerase inhibitors tested, including etoposide, camptothecin, and doxorubicin. Compound 175323 (Table II and Figure 3), a benzo[g]quinoline, activated Tp53 by 14.0-fold at 2 mg/ml. Three additional benzo[g]quinolines were represented in library, compounds
Tp53 response to anticancer drugs
Fig. 3. The chemical structures and molecular weights of four novel benzo[g]quinolines identified in the diverse compound screen of p53R cells. Compound 175308 proved to be identical to dabequine, an antimalarial known to be carcinogenic in rats. The level of Tp53 activation is shown in Table II.
175324, 175308, and 175310 (Figure 3). These compounds activated Tp53 by 8.0- to 19.0-fold at concentrations ranging from 0.3–3 µg/ml (see Table II). Compound 175308 was identical to dabequine, an antimalarial developed in the Soviet Union and then found to be carcinogenic in rats (44). In addition to benzo[g]quinolines, two quinolines activated Tp53 by 2.0- to 2.3-fold, compounds 175136 and 108437 (Figure 4). Upon identification of these compounds, two chlorinated quinolines (chloroquine and amodiaquine, Figure 4), an acridine (quinacrine), and two non-quinoline antimalarials (artemisinin and pentamidine; Figure 4) were tested. Chloroquine, amodiaquine, and quinacrine (an acridine) demonstrated moderate to strong activation, while the other compounds did not activate Tp53 (Table II). The behavior of the antimalarial chlorinated quinolines is consistent with evidence showing that quinolines may be carcinogenic, likely through the production of quinoline N-oxide as a metabolite (45,46). Interestingly, a third quinoline compound, compound 175195 (Figure 4), activated Tp53 by 5.0-fold in the screen. This compound is a quinoline N-oxide. Similar to other conventional chemotherapeutic agents, the chloroquine response was blunted when p53Rm cells were used. Fifteen compounds (0.1%) were found to inhibit Tp53 function 2.0-fold or greater when cells maximally stimulated with etoposide were treated with the compounds. All of these compounds demonstrated suppression of reporter activity in the SBE system, suggesting that they functioned either as general transcriptional inhibitors, general metabolic toxins, or cytotoxic agents that reduced the overall cell numbers. Pifthrin-α, a compound identified as a Tp53 inhibitor using a related reporter and compound screening strategy in mouse cells (39), was tested multiple times in our reporter system with no inhibition seen over a 10-fold range of concentration (mean Tp53-activation, 1.0-fold).
Fig. 4. The chemical structures and molecular weights of two novel quinolines identified in the screening of p53R cells (compounds 175136 and 108437), as well as chloroquine (a medicinal quinoline), compound 175195 (a novel quinoline N-oxide), and pentamidine and artemisinin (two nonquinoline antimalarials). Pentamidine and artemisinin did not activate Tp53. The levels of Tp53 activation for all other compounds are given in Tables I and II.
Inhibition by caffeine Caffeine is an inhibitor of Atm and Atr, known upstream mediators of the Tp53 response (8,25,26,28). Use of caffeine to block the Tp53 activation should provide a facile confirmation of the role of the Tp53 upstream pathway in a highthroughput format. Caffeine (2 mM) blunted the Tp53 response to etoposide, doxorubicin, 5-fluorouracil, compound 175323, and chloroquine all of which strongly activated Tp53 (Table I). Cells were also stimulated with etoposide, 5-FU, doxorubicin, or compound 175323 at concentrations which maximally stimulated the reporter. Cells were then treated over a range of concentration with caffeine [0.25 mM (50 µg/ml) – 130 mM (6 mg/ml)] or remained untreated. After 20–24 h, inhibition of the Tp53 response was determined. Inhibition was dosedependent. At concentrations of 4 mM, there was 50–80% inhibition of the reporter relative to cells not treated with caffeine. Upon microscopic examination, no cell death was observed at the time of assay. Immunoblots Immunoblots to quantitate the Tp53 were used to further confirm the stabilization of Tp53 protein. Identical quantities of extracts from treated p53R cells were evaluated in parallel immunoblots to compare relative Tp53 and Mdm2 levels. All conventional compounds which activated Tp53 in the initial screen were tested. Tp53 levels were increased by all compounds which activated Tp53 in the initial screen (Figure 5). Vincristine, which did not increase reporter activity in the 953
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Fig. 5. Tp53 immunoblot. Tp53 was increased relative to untreated cells when cells were treated with mitomycin C, 5-fluorouracil, cytarabine, gemcitabine, etoposide, doxorubicin, vincristine, paclitaxel, camptothecin, and cis-platin. The compounds which strongly activated (⫹), equivocally activated (⫾), or failed to activate the Tp53 reporter (–) in p53R cells are indicated below the immunoblot.
initial screen, also increased Tp53 levels on immunoblot. It was noted that vincristine killed a high proportion of cells and so may have been falsely missed in the initial screen due to cytotoxicity. Assay of an earlier time point or other assay modifications might circumvent this problem. TP53 status and cytotoxicity The output of the p53R assay is in part dependent on the proportion of cells that remain viable in the presence of a given compound. Although it was rare to observe microscopic evidence of cytotoxicity at the time point and concentrations chosen for the standard assay, it was certain that under modified conditions (such as longer incubations with compound and the use of higher concentrations of compound), cytotoxicity would affect the assay results. Indeed, the dose–response studies of conventional agents often produced a dose-dependent falloff in luciferase values seen in concentrations ⬎16 µg/ml (data not shown). In particular, because Tp53 under some conditions can be strongly pro-apoptotic (5,6), we wished to further characterize this relationship (a theoretical confounder) under the general conditions of our assay. Syngeneic HCT116 cells, wild-type (HCT116 p53⫹/⫹) and null for TP53 (HCT116 p53–/–), were used since they are the only human pair of syngeneic cells matched for the presence or absence of the wild-type native TP53 gene (36). A Tp53 immunoblot was used to confirm the TP53 status of the HCT116 cells (data not shown). The syngeneic cells were then treated with etoposide, cyclophosphamide, gemcitabine, mitomycin C, cytarabine, doxorubicin, vincristine, camptothecin, paclitaxel, streptozotocin, 5-FU, and methotrexate as well as four Tp53 activators identified in the library (175308, 175310, 175323, and 175324; Tables I and II; Figure 3). Cells were incubated for 72 h with each compound over a concentration range (0.09 µg/ml to 1000 µg/ml) to maximize the chance to observe a Tp53-dependent toxicity. For all compounds tested, ⬎90% cytotoxicity was observed at all concentrations above 6 µg/ml. The PicoGreen assay is affected by both the proportion of cells surviving and by their proliferation (population growth) over the period of the assay. For this reason, wells containing 954
Fig. 6. Cytotoxicity of conventional and novel compounds in HCT116 p53⫹/⫹ and HCT116 p53–/– syngeneic cells differing in TP53 genotype. Matched cell pairs were treated with etoposide, doxorubicin, 5-fluoracil, and 175323 and assayed at 72 h for cytotoxicity. Cytotoxicity was independent of TP53 genetic status under the conditions tested. Data points represent averages of three trials. Standard deviations for three determinations was ⬍5% within an experiment. A representative experiment is shown.
untreated cells were used as controls and to reflect the absence of cytolethality. For HCT116 p53⫹/⫹ and HCT116 p53–/– cells treated with etoposide, doxorubicin, 5-fluorouracil, and 175323, cell survival curves spanning the concentration range of 0.09 µg/ml to 6 µg/ml were generated. Cytotoxicity was dosedependent and there were no differences in the proportion of cells killed relative to untreated cells upon comparison of the wild-type TP53 cells and the TP53-null cells (Figure 6). Under these conditions, cytotoxicity was Tp53-independent for compounds shown to activate Tp53 function in p53R cells. Thus while various forms of cytotoxicity will affect the p53R assay, these effects can be taken into account by the use of varied chemical concentration (Figure 6 and Table I) and optimal timing of the measurements, and are not necessarily due to the Tp53 response per se. Discussion p53R cells offer an inexpensive, high-throughput assay for Tp53 activation in human cells. Upon screening of p53R cells, two-thirds of conventional chemotherapeutic agents but only 0.2% of diverse compounds activated Tp53, and the identities of these compounds were consistent with the known genotoxic stabilization of the Tp53 protein. Among the compounds identified in the screen of diverse compounds were analogs of m-AMSA, quinolines, benzo[g]quinolines, and quinoline N-oxides, which have been shown or suggested to be genotoxic (41–46), supporting the use of p53R cells as useful screening assay, at the low cost and robotic capabilities of microtiter luciferase assays. The responses of p53R cells to many of the chemicals tested in the present work are attributable to a caffeine-sensitive stabilization of the Tp53 protein, in line with the known Tp53 response pathway and the ability of caffeine to inhibit the upstream DNA-damage-responsive kinases, the Rad3 homologs Atm and Atr (8,25,26,28,31). Additional forms of cellular stress and input pathways are also expected to
Tp53 response to anticancer drugs
effect Tp53 responses (4), although they were not specifically evaluated in the present work. We therefore offer support to the suggestion (30–32) that facile assays for Tp53 induction in mammalian cells could contribute an additional and useful tool for the predictive toxicology of novel chemicals, environmental substances, dietary constituents, food additives, and the commercial pharmacopoeia. Although a number of bacterial and other cell-based toxicity assays are available, a number of carcinogens lack mutagenicity in common assays, and false positive results can occur among non-carcinogens (47). It would be beneficial to identify additional inexpensive, reproducible, high-throughput systems that could be used to quantify specific toxicities to human cells. We confirmed a potential deficiency in the Tp53based screen in the detection of certain DNA-damaging agents (28), as only one of the three chemotherapeutic alkylating agents tested were positive in the p53R cell assay, and the anti-folate methotrexate was negative. It is known that not all cells and tissues exhibit an increase in Tp53 in response to cyclophosphamide (48,49). For the alkylating agents, the induction of strand breaks by such agents is a secondary event that would depend upon both their dose and the rate of DNA repair. It is known that the Tp53 response is especially sensitive to DNA strand breaks, and damaged bases may not efficiently activate the pathway (50). Further improvements in the Tp53 response assay would be envisioned to improve upon the sensitivity to alkylating agents, to utilize the cytochrome p450mediated conversion of promutagens, to employ a panel of cell lines that harbor differing sensitivities to various cellular or genetic toxins, and to incorporate appropriate controls for potential cytotoxicity. Tp53 response assays could be an additional tool to survey the predictive toxicology of health food supplements, a multibillion dollar annual industry which is not currently controlled by the US Food and Drug Administration (FDA). As an example, flavonoids are often suggested as safe antineoplastic dietary constituents; enriched sources are sold over-the-counter, even though they have a spectrum of activities to inhibit DNA topoisomerases I and II, a property of many conventional anticancer chemotherapeutic agents (51). Indeed, it has been suggested that the antineoplastic effects of flavonoids might be attributable to their function as topoisomerase inhibitors, and a caution has been offered for their further study prior to recommending their use as a supplement (52). Apigenin, a major flavonoid of chamomile tea, causes a G2/M arrest in cells (53), an event accompanied by the induction of Tp53 (54). We found three of the four flavonoids tested to produce moderate responses in the p53R cell assay. Most flavonoids are inefficiently absorbed after oral intake. A different situation occurs with the administration of pharmaceuticals designed for efficient absorption, bypass of liver detoxification, and widespread tissue distribution. In an unbiased molecular epidemiology study, Tp53 immunopositivity in colorectal cancers was unexpectedly found to be most highly associated with the use of antihypertensives containing hydralazine (55). Separately, hydralazine was shown to inhibit the cell division cycle with highest susceptibility for cells in S phase (56). Hydralazine is a widely used drug, including common use in pregnancy-associated hypertension. We found hydralazine and nifedipine moderately active in induction of Tp53 activity in the p53R cell assay, a property not shared by ten additional antihypertensives and nine growth factors and hormones tested.
The ability of dabequine and chloroquine to induce Tp53, while not directly anticipated in the prior literature, can be rationalized. Quinolone compounds are the basis of a wide array of human pharmaceuticals, including the broad spectrum antibiotics ciprofloxacin and ofloxacin and a number of antimalarials. In the development of quinolone congeners, toxicity related to the inhibition of human gyrases and DNA topoisomerases must be minimized, often while maintaining such activities against the microbial forms of these enzymes (57). Indeed, there have been efforts to devise antineoplastic agents from quinolines (57), although as yet, potentially useful agents whose topoisomerase inhibitory potency or cytotoxicity would exceed that of conventional agents such as etoposide, have not been reported. The screening of random compounds in p53R cells uncovered a number of quinolone congeners with very high assay results, including dabequine as the most potent in our screen. Dabequine was developed in the Soviet Union as a novel antimalarial. It was well absorbed upon oral dosing, exhibited slow elimination, distributed well in tissues (58), and was negative in salmonella-based engineered mutational screens (Ames assay with and without metabolic activation) (59). It produced no excess of chromosomal aberrations in human lymphocyte studies, no abnormalities of mouse spermatogenesis, and no changes in mouse fertility or embryo deaths or in studies of mouse bone marrow (59). Its use in malarial epidemics in the Sudan and in Tanzania are reported, where dabequine was better tolerated than chloroquine in its association with side effects when given to children (60). A sole report of a carcinogenesis study in animals hints at a significant toxicity (44). We found dabequine to be 20-fold more effective than etoposide in generating responses in p53R cells, and a similar quinolone tested was highly effective at killing cancer cells in culture. We thereupon initiated a survey of common antimalarials; we identified chloroquine, amodiaquine, and quinacrine to produce responses in p53R cells. Further potential applications of efficient Tp53-stabilization assays might be found in the identification of novel chemotherapeutic agents for use as anti-cancer agents, as an adjunct in the pharmaceutical optimization of lead compounds, and in the probing of the upstream origins of the Tp53 protein stabilization system(s). Acknowledgements We would like to thank Kenneth W. Kinzler and Christopher Torrance for access to the compound library, Bert Vogelstein and Shibin Zhou for contributing the HCT116 p53⫹/⫹ and HCT116 p53–/– cells. We also thank Thanos Halazonetis, John Groopman, and Theresa Shapiro for helpful discussions and advice. We reserve special thanks for Ivan Litvinov for his translation of the Russian language literature regarding dabequine. This work was supported by the NIH SPORE (Specialized Program of Research Excellence) in Gastrointestinal Cancer grant CA 62924 and NIH grant CA68228, the Niarchos Foundation, and the American Hepato– Pancreato–Biliary Association/Ethicon.
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