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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Biomol Screen. Author manuscript; available in PMC 2009 September 1.
Published in final edited form as:
PMCID: PMC2693415
NIHMSID: NIHMS114890

Novel Dual-Reporter Preclinical Screen for Anti-Astrocytoma Agents Identifies Cytostatic and Cytotoxic Compounds

Abstract

Astrocytoma/glioblastoma is the most common malignant form of brain cancer and is often unresponsive to current pharmacological therapies and surgical interventions. Despite several potential therapeutic agents against astrocytoma and glioblastoma (1), there are currently no effective therapies for astrocytoma, creating a great need for the identification of effective anti-tumor agents. We have developed a novel dual-reporter system in Trp53/Nf1-null astrocytoma cells to simultaneously and rapidly assay cell viability and cell cycle progression as evidenced by activity of the human E2F1 promoter in vitro. The dual-reporter high-throughput assay was used to screen experimental therapeutics for activity in Trp53/Nf1-null astrocytoma. Several compounds were identified demonstrating selectivity for astrocytoma over primary astrocytes. The dual-reporter system described here may be a valuable tool for identifying potential anti-tumor treatments that specifically target astrocytoma.

Keywords: astrocytoma, Nf1, p53, E2F1, luciferase

Introduction

Astrocytic gliomas, including astrocytomas and glioblastoma multiforme (GBM), are the most common malignant form of brain cancer and are often unresponsive to surgical intervention and current pharmacological therapy. The best current treatment option is surgical removal; however, astrocytomas/GBM diffusely infiltrate the central nervous system rendering resection difficult or impossible, leading to poor patient prognosis (2, 3). Consequently, the five-year survival rate for GBM is less than 5% (4). Although a finite list of pharmacological agents have been reported as potential therapeutic agents against astrocytoma and GBM (1), the cure rate is still very low, demonstrating the tremendous need for identification of more effective anti-tumor agents.

One of the earliest and most common genetic alterations in astrocytoma is loss of heterozygosity on chromosome 17p (57). This chromosomal region encompasses the p53 gene, which plays a primary role in the progression of multiple types of tumors, including astrocytoma. Nearly 50% of astrocytomas include loss of heterozygosity at 17p and/or mutation of p53, with up to 90% of GBMs displaying alterations in the p53 pathway (8). The p53 protein serves as a key component of the cell-cycle checkpoint by halting proliferation in response to DNA damage and as a transcription factor that induces genes responsible for growth arrest and apoptosis (9). Furthermore, tumors known to contain mutations in p53 are more resistant to radiation and many anti-tumor agents are inactive in p53-null tumors, making these tumors also resistant to chemotherapy (10).

The familial cancer syndrome neurofibromatosis type 1 (NF1) is an autosomal dominant syndrome that predisposes individuals to developing multiple tumors including astrocytoma and GBM (11) NF1 patients carry a mutation in the NF1 gene (the Nf1 gene in mice) that encodes for the protein neurofibromin. Neurofibromin is a tumor suppressor rasGAP protein that downregulates the ras signaling pathway linking growth factor signals to cellular proliferation (12). Consequently, loss of neurofibromin plays a key role in the induction of tumorigenesis leading to overactivation of the oncogenic ras pathway.

Since many human astrocytomas contain mutations in Tp53 (Trp53 in mice), encoding the p53 protein, and upregulation of ras signaling is critical for astrocytoma tumorigenesis and maintanence, preclinical models that reflect these alterations may be ideal for characterizing and identifying potential astrocytoma therapeutics. Mice carrying mutations in Nf1 and Trp53 on the same chromosome (Nf1−/+;Trp53−/+cis: NPcis) have been characterized as a mouse model of NF1 (13, 14) and astrocytoma (14, 15). NPcis mice undergo spontaneous loss of heterozygosity at the wild-type copies of Nf1 and Trp53 resulting in the development of brain tumors with high penetrance and close similarity to human astrocytomas (15). NPcis brain tumors range from low-grade astrocytomas to high-grade GBMs, forming diffusely infiltrative tumors (13, 15) Primary tumor cells isolated from NPcis astrocytomas show loss of the wild-type copies of Nf1 and Trp53 and maintain tumor cell characteristics similar to human astrocytoma in vitro (15). Thus, NPcis astrocytoma cells can be used to build an in vitro assay for identifying novel anti-astrocytoma therapeutic candidates.

KR158 tumor cells from a grade III NPcis aggressive anaplastic astrocytoma (15) were used to generate a green and red luciferase (G/R-luc) dual-reporter system that simultaneously assesses activity of the human E2F1 promoter and cellular cytotoxicity in a high-throughput assay. The G/R-luc dual-reporter system was used to screen chemically diverse compounds to identify agents with anti-proliferative activity in astrocytoma cells. This system distinguishes cytostatic compounds from cytotoxic agents during the initial screening, discriminating cytotoxic agents from inhibitors of proliferation. Thus, the G/R-luc dual-reporter system system could significantly decrease the time and cost required to screen compound libraries. This system was also used to examine the pharmacology of identified anti-tumor agents. The G/R-luc dual-reporter system is a valuable tool in the identification and characterization of potential anti-tumor treatments specifically targeting astrocytoma.

Methods

G/R- luc cell line

For construction of the pEf-CBGluc plasmid expressing the green luciferase gene under control of the human E2F1 promoter, green click beetle luciferase from pCBG68luc (Promega, Madison, WI) was subcloned in place of the firefly luciferase gene into pEf-luc (gift from Dr. Eric Holland) (16). The hygromycin resistance gene was PCR cloned into pEf-CBGlu upstream of the E2F1 promoter for clonal selection. pHygro-Ef-CBGluc was stably transfected into grade III KR158 astrocytoma cells (15) using Fugene (Roche Applied Science, Indianapolis, IN) to generate G-luc astrocytoma cells. For construction of pCMV-CBRluc, the CMV promoter was cloned in place of the SV40 promoter into pCBR-Basic (Promega, Madison, WI) that contains the modified click beetle red-emitting luciferase. The PKG promoter and puromycin resistance pac gene were cloned upstream of the CMV promoter for clonal selection. pPuro-CMV-CBRluc was stably transfected into G-luc astrocytoma cells to generate the G/R-luc astrocytoma dual-reporter cell line. All cell lines were maintained as described previously (15).

Dual Luciferase Assay

At the time of assay, growth media was replaced with 50 μl fresh media immediately followed by 50 μl Chroma-Glo (Promega, Madison, WI) lysis and luciferase reagent and incubated at room temperature. At 15 and 30 minutes after lysis, green (537 nm) and red (613 nm) luminescence were detected with a Fluorostar (BMG Technologies, Durham, NC) microplate reader by quantitating photon emissions passing through 540nm and 615nm filters.

Luciferase Induction Assay

G/R-luc cells were plated in 96-well black optical bottom plates at a density of 15,000 cells per well. Six hours after plating, the media was changed to starving media (SV) lacking serum or to fresh growth media (GM), incubated for 24 hours at 37 °C, and changed again to SV or GM for an additional 24 hours at 37 °C. For time induction experiments, SV was replaced with GM at 4, 8, 18, 24, and 30-hour time points prior to the luciferase assay.

G/R-luc Dual-reporter Validation

Serial dilutions of either U0126 (Calbiochem, San Diego, CA) or nocodazole (Sigma-Aldrich, St. Louis, MO) were added to G/R-luc cells 6 hours after plating. Approximately 40 hours after compound addition, green and red luciferase expression was determined using a dual-luciferase assay. Cells treated with growth media containing DMSO vehicle alone (V) were used as positive controls for cell proliferation and cells treated with SV were used as negative controls. Green luminescence values for the compound of interest (GλC) and vehicle positive controls (GλV) were used to determine growth inhibition (GI) values at each compound concentration, Eq. (1). Lethal concentration (LC) values were determined using red luminescence for the compound of interest (RλC) values and SV negative controls (RλSV) Eq. (2).

equation M1
Eq.(1)
equation M2
Eq.(2)

The concentration of compound at the GI50 value (GI = 50), representing a 50% reduction in green luminescence, and the LC50 value (LC = −50), representing a 50% reduction in red luminescence, was determined by plotting the sigmoidal dose response curves using GraphPad Prism (GraphPad Software Inc., San Diego, CA). The half maximal inhibitory concentration (IC50) was also determined for nocodazole using the sigmoidal dose response curve and GraphPad Prism.

High-Throughput Screen (HTS)

G/R-luc cells were plated in 96-well black optical bottom plates with each well receiving identical numbers of cells. Depending on the experimental run, cells were plated at a density of 3000 to 5000 cells per well. The plating density was chosen to be higher than the lower limit of detection of 2500 cells, and low enough to allow 2 cell doublings over the course of the experiment in uninhibited controls. Six hours after plating, the media was changed to fresh media containing controls or 10 μM of compounds from the NCI Diversity set library (Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, Frederick, MD). Two sets of negative controls, SV containing DMSO (vehicle) or 100 ng/ml nocodazole, were included in duplicate. GM containing DMSO was included in duplicate as a positive control for cell growth and a negative control for growth inhibition. Ten μM of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY; Calbiochem, San Diego, CA) was included as a positive control for inhibition of growth signaling, and was also used to assess plate-to-plate variability. Two wells of each control were included in the first and last columns of each plate for a total of 4 wells. By separating the controls into the first and last columns, the controls are the first and last samples read on each plate and the consistency between both these groups controls for the stability of the reaction over the period of reading the plate. Approximately 40 hours after addition of compounds, green and red luminescence was determined using a dual-luciferase assay. Vehicle alone internal controls were used to determine the raw activity threshold (RAT) for normalization of individual 96-well assays based on the inhibition of cell proliferation. The RAT is the level of green luminescence corresponding to 50% growth inhibition compared to the total growth of the vehicle alone control. Green luminescence of the compound of interest (GλC) and of the vehicle alone controls (GλV) were used to calculate the growth inhibition (GI) for screened compounds, Eq. (1). Compounds resulting in less than 50% inhibition of green luminescence, GI < 50, such that their green luminescence measurement exceeded the RAT, were excluded as inactive compounds. Red luminescence of nocodazole-arrested controls (RλN) was used to identify cytotoxic compounds with negative LC values, Eq. (2), where RλC is the red luminescence of the compound of interest. Positive hits from the initial screen were assayed again to confirm reproducibility of anti-proliferative activity and to eliminate false positives.

Dual-luciferase assays were also used to generate drug response curves and calculate GI50 values for active compounds identified from the screen. G/R-luc dual-reporter cells were treated with 4-fold logarithmic dilutions of compound for approximately 40 hours, followed by a dual-luciferase assay. IC50 values were determined for cytostatic compounds using the sigmoidal dose response curve and GraphPad Prism.

Washout Experiments

G/R-luc astrocytoma cells were grown in 96-well plates as described above and treated with 100 ng/ml nocodazole, 1μM NSC#676693, 1μM deoxybouvardin, or DMSO 6 hours after plating. After 24 hours, the media was replaced in all wells and half of the cells were allowed to recover in media without inhibitors and half of the cells were maintained in the presence of inhibitors. Cells were allowed to recover for 32 hours and green luminescence was determined with Chroma-Glo luciferase assays described above. Green luminescence was calculated as percent control of vehicle DMSO controls. Significance was determined using repeated-measures ANOVA and Tukey’s HSD and Bonferroni post hoc tests with p<0.05 considered to be significant.

Alamar Blue assay

G/R-luc, KR158, primary astrocytes, or SF295 (human grade IV astrocytoma/GBM) cells were plated as described for the HTS screen in 96-well tissue culture plates. Compounds were added to the cells at logarithmic dilutions in triplicate and incubated at 37 °C. After approximately 40 hours, GM containing 30% alamar blue (Invitrogen, Carlsbad, CA) was added to the cells to a final concentration of 10% alamar blue and incubated at 37 °C for 4 hours. Fluorescence was measured with 560 nm excitation and 590 nm emission filters using a Novostar (BMG Technologies, Durham, NC) microplate reader.

Primary Astrocytes

Primary astrocytes were harvested as previously described (17) with minor modifications. Neocortex was dissected from the brains of wild type C57BL/6J pups at P1. Neocortical cells were dissociated by mechanical dissociation in DMEM/20% FBS, pelleted by centrifugation, and plated in at a density of 2×106 cells per 10 cm plate in DMEM/20% FBS and incubated at 37 °C for 14 days. Surviving astrocytes were plated at 5000 cells per well in 96-well plates for alamar blue assays.

Phalloidin staining

Astrocytoma cells were plated on glass coverslips in 12-well plates at a density of 100,000 cells per well. Six hours after plating, 1 μM of NSC#676693 or DMSO (vehicle control) were added to the cells and incubated at 37 °C for 48 hours. Cells were fixed in 4% paraformaldehyde, stained with Texas Red-X Phalloidin conjugated antibody (Molecular Probes, Eugene, OR) and mounted onto slides with Prolong gold anti-fade reagent with DAPI (Invitrogen, Carlsbad, CA).

Results and Discussion

The G/R-luc dual-reporter system was generated by stably expressing green and red click beetle luciferase reporters in previously characterized grade III mouse astrocytoma cells (15), where expression of green luciferase is under control of the human E2F1 promoter and expression of red luciferase is driven by the constitutively active human cytomegalovirus immediate-early (CMV) promoter. Therefore, green luciferase expression is controlled by active cellular proliferation and red luciferase expression is actively expressed in all healthy cells. Since the peak luminescence for the green (537 nm) and red (613 nm) click beetle luciferases are well separated, it is possible to simultaneously assay both using filtered luminescence with little spectra overlap (18, 19). Although filtered luminescence decreases the total luminescence signal intensity by up to 90%, background signal is extremely low. The G/R-luc reporter system is sensitive enough to detect significant green filtered luminescence over background in as few as 2500 cells (Fig. 1A). Furthermore, green luciferase expression in rapidly dividing cells is linear with respect to cell number up to 40,000 cells, when cell proliferation begins to be inhibited by increasing cell densities and cell contact inhibition. The threshold for red luminescence sensitivity is slightly higher than green luminescence (Fig. 1B). Red luminescence is linear with cell number up to very high cell density (160,000 cells per well), suggesting that red luciferase expression correlates with cell number regardless of the proliferation rate or E2F1 promoter activity.

Figure 1
Characterization of the G/R-luc dual-reporter system. Green filtered luminescence (A) is sensitive at low cell densities and is linear in rapidly dividing populations. Red filtered luminescence (B) is linear with respect to cell number even at high cell ...

Green luciferase expression reflects active cellular proliferation and is significantly lower when cellular proliferation is slowed in the absence of serum (Fig. 1C). When growth media containing serum is added back to serum-starved cells, green luminescence increases. Green luminescence is significantly increased 6 hours after the addition of serum and rises rapidly after 20 hours (Fig. 1D). The increase in green luciferase expression at 6 hours coincides with activation of transcription and protein translation downstream of the E2F1 promoter. The rapid rise in green luciferase expression after 20 hours is consistent with the timing of the cell cycle, requiring approximately 24 hours to complete. These data suggest that the human E2F1 promoter drives the green luciferase expression in the G/R-luc astrocytoma cell line in an efficient and proliferation dependent manner.

Well-characterized growth inhibitors were used to validate the G/R-luc dual-reporter system for determining LC50, GI50 or IC50 values in a 96-well dual luciferase assay. Since green luciferase expression correlates with activity of the E2F1 promoter and active cell cycle, green luminescence was used to calculate GI50 values, the concentration of compound at which cell proliferation is inhibited by 50% as compared to controls. Since red luciferase expression correlates with total cell number, red luminescence values were used to calculate LC50 values, the lethal concentration of compound at which the total cell number is decreased by 50% as compared to nocodazole-arrested controls. The MEK inhibitor U0126 restricts cell growth in multiple cell lines, including astrocytoma (20). In G/R-luc astrocytoma cells, the GI50 value for U0126 was found to be 5 μM (Fig. 2A) and the LC50 value for U0126 was determined to be 96 μM using dual-luciferase assays (Fig. 2B), which are consistent with previously reported data (21). Nocodazole is a cell cycle inhibitor that depolymerizes microtubules and arrests cells during mitosis or in G2. Nocodazole treatment reaches Emax at about 55% growth inhibition with a threshold dose of 0.33 μM (100 ng/ml) (Fig. 2C), which is consistent with previous reports where nocodazole was used at this dose to induce complete growth arrest (22, 23). Since nocodazole is cytostatic and Emax does not reach 100% growth inhibition, the IC50 value is more informative to compound activity than the GI50 value. Therefore the G/R-luc dual-reporter assay was used to determine the IC50 value for nocodazole as being 0.06 μM in astrocytoma cells (Fig. 2C). The U0126 and nocodazole pharmacology analyzed with the G/R-luc dual-reporter system is congruent with known pharmacology of these compounds and validates this system for determining LC50, GI50 and IC50 values.

Figure 2
G/R-luc dual-reporter validation. Sigmoidal dose response curves for U0126 (A) and nocodazole (C) used to calculate GI50 values. Concentration response curves used to calculate LC50 values for U0126 (B) and nocodazole (D). Screening window coefficients ...

To verify the potential of the G/R-luc dual-reporter assay for high-throughput screens (HTS), the screening window coefficient (Z′-factor) was determined for both the green and red filtered luminescence, Eq. (3). The Z′-factor takes into account the experimental standard deviation and the signal to noise ratio to determine the quality and potential of HTS assays (24). The Z′-factor was determined using raw luminescence data values from duplicate positive and baseline controls on each plate used in the HTS as follows. Cells grown in 10% FBS growth media in the presence of DMSO vehicle (GM) were used as positive growth controls and cells cultured in starving media (SV) in the presence of 100 ng/ml nocodazole to arrest cell growth were used as baseline controls. The two positive controls and two baseline controls were used to determine the Z′-factor for each plate and Z′factors from 6 independent runs were averaged to determine the Z′-factor for the HTS. The Z′-factor equation was modified to fit the dual-luciferase model (C), such that σV = standard deviation in GM controls, σN = standard deviation in Noc controls, μV = average of the raw luminescence data for GM controls, and μN = average of the raw luminescence data for Noc controls. The Z′-factor for the green luminescence is consistently within the range for an excellent HTS assay, 0.5 ≤ Z′ < 1, in both 96-well format and 384-well formats (Fig. 2E). Although red luminescence exhibits higher standard deviation between replicates, the Z′-factor for red luminescence is consistently within the range of a good HTS assay, 0.2 ≤ Z′ < 0.5, in both 96-well and 384-well formats. Thus, the G/R-luc dual-reporter assay has a high potential to discriminate changes in red and green luminescence compared to positive and negative controls in HTS assays.

equation M3
Eq.(3)

Ten μM of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY; Calbiochem, San Diego, CA) was used to conduct validation tests as outlined by the Assay Guidance Manual (25). In KR158 astrocytoma cells, 10 μM LY acts as a cytostatic compound near the RAT by inhibiting E2F activity by 40 to 50%. During a potency analysis, the Minimum Significant Ratio (MSR) between multiple HTS runs is 1.26 (MSR<3), demonstrating that there is good individual agreement between the multiple runs and the assay passes the reproducibility test. The upper and lower Limits of Agreement (LsA) fall between 0.94 and 1.49, demonstrating that the assay also passes the LsA criterion (0.33<LsA<3.0) and equivalence test. Ten μM LY was included in all HTS assays as the primary internal control and to monitor assay drift between runs. The overall MSR across the diversity set HTS screen (described below) is 7.13, demonstrating that the assay reproducibility is stable over time (MSR<7.5).

The G/R-luc dual-reporter assay was used to screen the NCI diversity set for compounds with anti-astrocytoma activity (Fig. 3A,B). The NCI diversity set is composed of 1,982 chemically diverse compounds chosen to represent the various chemical structure groups found in of the larger set of almost 140,000 compounds. G/R-luc cells were treated with 10 μM of each compound for 40 hours, followed by a dual-luciferase HTS assay. Vehicle alone controls were used to normalize the individual assays (see Methods Section for details) (Fig. 3A) and calculate the RAT as the level of luminescence corresponding to a 50% reduction in the E2F1 promoter activity relative to vehicle alone. Thus, raw data can be quickly screened to find active compounds that show luminescence measurements below RAT for individual plates. Compounds resulting in a decrease in red luminescence, LC < 0, as compared nocodazole-arrested controls were identified as cytotoxic compounds (Fig. 3B). Compounds that do not result in a decrease in red luminescence as compared to arrested controls, such that LC ≥ 0, represent non-cytotoxic compounds. Thus, positive 50% growth inhibition, GI > 50, and positive LC values, LC ≥ 0, represent cytostatic compounds that inhibit activity of the E2F1 promoter but do not generally inhibit transcription factors, as evidenced by the activity of the CMV promoter, or kill the cells.

Figure 3
Representative data plots from the dual-luciferase HTS assay (A, B). GI values from green luciferase data are normalized to cells treated with growth media + vehicle (GM) (A) and LC values from red luciferase data are normalized to nocodazole-treated ...

Compounds with GI > 50 in the initial screen were rescreened to confirm reproducibility and eliminate false positives. During rescreening, cells were also examined briefly under a light microscope to determine the frequency of false positives where compound treatment inhibited both green and red luminescence, but did not kill the cells. These compounds are likely to inhibit activity of the E2F1 and CMV promoters indiscriminately or activity of the luciferase enzymes, but are not specific for the E2F1 promoter. The false positive rate was found to be less than 0.2%. The remaining compounds were further divided into three groups; cytotoxic, cytostatic and cytotoxic/cytostatic. Ninety-seven cytotoxic compounds were identified (4.9% of tested compounds). The cytostatic group consisted of 68 compounds (3.4% of tested compounds) that reproducibly inhibited cellular proliferation without cytotoxicity. The third group, cytotoxic/cytostatic, consisted of 9 compounds that were cytotoxic in one screen and cytostatic in another. These compounds are likely to be cytotoxic, such that the concentration used at screening, 10 μM, is at a threshold concentration level near the LC50.

To further refine the list of compounds with activity against brain tumors, the compounds identified from the compound screen in the G/R-luc astrocytoma cell line were compared to the NCI-60 data that was available through the NCI DTP program (http://www.dtp.nci.nih.gov/index.html). Thirty-seven of the identified compounds have activity in at least one human CNS cell line and 14 showed significant activity in at least 3 or more CNS cell lines. The G/R-luc astrocytoma cells were then used to determine the GI50 value for the top candidates in a 96-well dual-reporter assay.

Camptothecin and several of its derivatives, including topotecan, were identified as cytotoxic agents in the G/R-luc astrocytoma cell line with GI50 values in the nanomolar range (Table 1A). Although camptothecin failed clinical trials in the 1970s, topotecan was approved by the FDA in 1996 as a secondary treatment for ovarian and small-cell lung cancers and is currently being investigated for the treatment of astrocytoma (26). Although these compounds are not novel, their identification from the compound library serves as positive controls for hit identification in the HTS assay and further validates the use of the G/R-luc dual-reporter assay as a therapeutic-screening tool to identify active compounds. Bouvardin and deoxybouvardin were found to have cytotoxic activity in the 28–41 nM range. The bouvardins have also been reported as potential anti-tumor agents (27, 28). Eight other compounds were identified from the NCI diversity set as having cytotoxic activity in the p53/NF1-null G/R-luc dual-reporter astrocytoma cells (Table 1A). Four of these compounds have potent cytotoxic activity in the nanomolar range.

Washout experiments were used to confirm that the G/R-luc dual-reporter system differentiates between cytostatic and cytotoxic activities. Green luminescence is inhibited in the presence of cytostatic compounds, nocodazole and NSC#676693 (Table 1B,C, Fig. 4), and the cytotoxic compound, deoxybouvardin (Table 1A,C, Fig. 4). When inhibitors are removed from the media, green luminescence significantly increases in cells pre-treated with nocodazole and NSC#676693, but not in those previously treated with deoxybouvardin (Fig. 4). Therefore, G/R-luc dual-reporter astrocytoma cells were able to recover from cytostatic treatment with nocodazole and NSC#676693, but not from the cytotoxicity of deoxybouvardin. These data further suggest that green luminescence in the G/R-luc dual-reporter system can be used to distinguish between cytostatic and cytotoxic inhibition.

Figure 4
The G/R-luc dual-reporter system distinguishes between cytostatic and cytotoxic activities in washout experiments. Cytostatic compounds nocodazole and NSC#676693 and the cytotoxic compound deoxybouvardin (white bars) inhibit green luminescence. When compounds ...

At least 5 of the cytostatic compounds identified in the HTS screen were found to alter the cellular morphology of the astrocytoma cells (Table 1B). Treatment with NSC#676693, NSC#128687, NSC#158383 or NSC#131734 results in an apparent increase in cellular cytoplasm and change in cell shape. On the other hand, treatment with NSC#131053 results in a decrease in total cell size. Furthermore, treatment of anaplastic astrocytoma cells with NSC#676693 results in a clear morphological change that lacks the astrocytic projections and cell-spreading characteristics of KR158 cells and is consistent with inhibition of cytoskeletal regulation (Fig. 5B). Because these compounds are cytostatic and Emax does not approach 100% growth inhibition, the concentration resulting in 50% growth inhibition (GI50) is not equal to the concentration resulting in half of the maximal inhibitory effect (IC50 values). Therefore, IC50 values are more reflective of the activity of cytostatic compounds than GI50 values. The IC50 values for all but one of the morphology-altering compounds are in the low micromolar to nanomolar range. Although these compounds are cytostatic rather than cytotoxic up to 10 μM, all but one of these compounds (NSC#131053) reaches 50% growth inhibition. This is consistent with nocodazole treatment that arrests the cells in mitosis and reaches Emax near 55% growth inhibition (Fig. 2C). NSC#676693 also reaches Emax at 55 % inhibition of the E2F1 promoter over a 2-fold log concentration from 1 μM to 10 μM (Fig. 5A) and washout experiments confirm that cells recover after treatment with NSC#676693 similarly to nocodazole (Fig. 4) thereby suggesting a specific cytostatic rather than cytotoxic function for NSC#676693.

Figure 5
Representative drug response curves for the cytostatic compound NSC#676693 compared to the cytotoxic compound camptothecin (A). NSC#676693 reaches Emax at 50% growth inhibition over two log concentrations. Texas Red-conjugated phalloidin was used to visualize ...

Because the dual-reporter assay has been established in a mouse grade III astrocytoma line, it is important to validate the results of this assay in human tumor lines and in tumors of different grades. GI50 or IC50 values for potent compounds were assessed in other astrocytoma cell lines to determine if the inhibitory effects were common to different astrocytoma cells from both mouse and human (Table 1C). Alamar blue assays, which assess innate metabolic activity and cell viability, were used to generate therapeutic response curves and calculate GI50 or IC50 values for cell lines not containing the red and green luciferase reporters. The alamar blue assay in grade III KR158 mouse astrocytoma cells and the dual-reporter assay in grade III G/R-luc mouse astrocytoma cells yielded similar results. Thus, GI50 and IC50 values obtained from the dual-reporter assay compare with the conventional alamar blue assay. To assess whether cytotoxic and cytostatic effects were specific to tumor cells, GI50 or IC50 values were determined for each compound in primary astrocyte cultures. Only deoxybouvardin exhibited non-specific inhibition. (Table 1C), which suggests that deoxybouvardin cytotoxicity does not discriminate between cancerous and non-cancerous cells.

With conventional HTS assays, it is necessary to run a secondary assay to eliminate false positives that are cytotoxic or non-specific to the target of interest, such as the E2F1 promoter. Because the dual-luciferase assay design discriminates between green and red luminescence, it is possible to distinguish luciferase expression under the control of different promoters in a single assay. This system distinguishes between specific inhibition of the E2F1 promoter (green luminescence) and non-specific transcription factor inhibition and general cytotoxicity (red luminescence) in the initial screen. Thus, the dual-luciferase reporter assay system can significantly cut costs and time by identifying potential therapeutic candidates from a single HTS assay. This system can further be used to determine GI50, LC50 and IC50 values as well as studying the additive or synergistic effects of combinatorial treatments in a HTS fashion.

The G/R-luc astrocytoma dual-reporter assay was validated as a stable and reproducible high-throughput screening tool to identify novel compounds with anti-proliferative activity in astrocytoma cells. Out of 1982 chemically diverse compounds in the NCI Diversity Set compound library, 14 compounds were identified that also have significant activity in other CNS tumor cell lines. Several compounds with known anti-proliferative activity in CNS tumors were also identified, such as camptothecin and several of its derivatives, and served as positive controls for successful identification of therapeutic agents using the G/R-luc dual-reporter assay. Three novel compounds, NSC#207895, NSC#268665, and NSC#606985, were also identified as having potent cytotoxic activity in mouse and human astrocytoma cells with specificity for tumor cells over primary astrocytes. Future experiments will be required to determine the in vivo bioavailability and use of these compounds as anti-astrocytoma therapeutic agents.

The G/R-luc dual-reportor HTS also identified six compounds in the NCI Diversity set that are cytostatic and specifically inhibit cellular proliferation accompanied by alteration of astrocytoma cell morphology. For example, NSC#676693 is highly potent cytostatic inhibitor, specific for astrocytoma cells compared to primary astrocytes, and induces morphological changes in astrocytoma cells. These compounds may be useful tools to understanding astrocytoma tumorgenicity. Future in vivo experiments will determine the utility of these cytostatic compounds as anti-astrocytoma therapeutics.

We describe here a novel dual-reporter assay that uses filtered luminescence to simultaneously assess and distinguish between activity of the E2F1 promoter and non-specific transcription factor inhibition and cytotoxicity. The G/R-luc dual-reporter system is an efficient and promising tool for the identification and study of anti-astrocytoma therapeutic agents in vitro.

Acknowledgments

Funding: This research was supported by the Intramural Research Program of the NIH, NCI. J.J.H. is supported by a National Research Council Research Associateship Award.

This research was supported by the Intramural Research Program of the NIH, NCI. This Research was performed while J.J.H. held a National Research Council Research Associateship Award at the National Cancer Institute. All experiments were conducted in compliance with the current laws of the United States.

References

1. Gilbert MR, Loghin M. The Treatment of Malignant Gliomas. Curr Treat Options Neurol. 2005;7(4):293–303. [PubMed]
2. CBTRUS CBTRotUS. Primary Brain Turmors in the United States-Statistical Report: Central Brain Tumor Registry of the United States. Chicago: 2002.
3. Ohgaki H. Genetic pathways to glioblastomas. Neuropathology. 2005;25(1):1–7. [PubMed]
4. McLendon RE, Halperin EC. Is the long-term survival of patients with intracranial glioblastoma multiforme overstated? Cancer. 2003;98(8):1745–8. [PubMed]
5. Ichimura K, Bolin MB, Goike HM, Schmidt EE, Moshref A, Collins VP. Deregulation of the p14ARF/MDM2/p53 pathway is a prerequisite for human astrocytic gliomas with G1-S transition control gene abnormalities. Cancer Res. 2000;60(2):417–24. [PubMed]
6. Rasheed BK, McLendon RE, Herndon JE, et al. Alterations of the TP53 gene in human gliomas. Cancer Res. 1994;54(5):1324–30. [PubMed]
7. Watanabe K, Sato K, Biernat W, et al. Incidence and timing of p53 mutations during astrocytoma progression in patients with multiple biopsies. Clin Cancer Res. 1997;3(4):523–30. [PubMed]
8. von Deimling A, Eibl RH, Ohgaki H, et al. p53 mutations are associated with 17p allelic loss in grade II and grade III astrocytoma. Cancer Res. 1992;52(10):2987–90. [PubMed]
9. Harris CC. p53 tumor suppressor gene: from the basic research laboratory to the clinic--an abridged historical perspective. Carcinogenesis. 1996;17(6):1187–98. [PubMed]
10. Weinstein JN, Myers TG, O’Connor PM, et al. An information-intensive approach to the molecular pharmacology of cancer. Science. 1997;275(5298):343–9. [PubMed]
11. Huson S, Hughes R. The Neurofibromatoses: A pathogenetic and clinical overview. London: Chapman & Hall Medical; 1994.
12. Martin GA, Viskochil D, Bollag G, et al. The GAP-related domain of the neurofibromatosis type 1 gene product interacts with ras p21. Cell. 1990;63(4):843–9. [PubMed]
13. Cichowski K, Shih TS, Schmitt E, et al. Mouse models of tumor development in neurofibromatosis type 1. Science. 1999;286(5447):2172–6. [PubMed]
14. Vogel KS, Klesse LJ, Velasco-Miguel S, Meyers K, Rushing EJ, Parada LF. Mouse tumor model for neurofibromatosis type 1. Science. 1999;286(5447):2176–9. [PMC free article] [PubMed]
15. Reilly KM, Loisel DA, Bronson RT, McLaughlin ME, Jacks T. Nf1;Trp53 mutant mice develop glioblastoma with evidence of strain-specific effects. Nat Genet. 2000;26(1):109–13. [PubMed]
16. Uhrbom L, Nerio E, Holland EC. Dissecting tumor maintenance requirements using bioluminescence imaging of cell proliferation in a mouse glioma model. Nat Med. 2004;10(11):1257–60. [PubMed]
17. Sanchez JF, Sniderhan LF, Williamson AL, Fan S, Chakraborty-Sett S, Maggirwar SB. Glycogen synthase kinase 3beta-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kappaB signaling. Mol Cell Biol. 2003;23(13):4649–62. [PMC free article] [PubMed]
18. Brian Almond PD, Erika Hawkins MS, Pete Stecha BS, Denise Garvin MS, Aileen Paguio MS, Braeden Butler BS, Michael Beck MS, Monika Wood MS, Keith Wood PD. A New Luminescence: Not Your Average Click Beetle. 2003. [cited; Available from: www.promega.com.
19. Gammon ST, Leevy WM, Gross S, Gokel GW, Piwnica-Worms D. Spectral unmixing of multicolored bioluminescence emitted from heterogeneous biological sources. Anal Chem. 2006;78(5):1520–7. [PMC free article] [PubMed]
20. Uht RM, Amos S, Martin PM, Riggan AE, Hussaini IM. The protein kinase C-eta isoform induces proliferation in glioblastoma cell lines through an ERK/Elk-1 pathway. Oncogene. 2007;26(20):2885–93. [PubMed]
21. Ahn Natalie G, PD, Tolwinski Nicholas S, BA, Hsiao Kevin, MS, Goueli Said A., PhD U0126: An Inhibitor of MKK/ERK Signal Transduction in Mammalian Cells. Promega Corporation. 1999;(71):04.
22. Barnouin K, Dubuisson ML, Child ES, et al. H2O2 induces a transient multiphase cell cycle arrest in mouse fibroblasts through modulating cyclin D and p21Cip1 expression. J Biol Chem. 2002;277(16):13761–70. [PubMed]
23. Yamada HY, Gorbsky GJ. Cell-based expression cloning for identification of polypeptides that hypersensitize mammalian cells to mitotic arrest. Biol Proced Online. 2006;8:36–43. [PMC free article] [PubMed]
24. Zhang JH, Chung TD, Oldenburg KR. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays. J Biomol Screen. 1999;4(2):67–73. [PubMed]
25. Assay Guidance Manual Version 4.1. 2005 [cited Jan. 3, 2008]; Available from: http://www.ncgc.nih.gov/guidance/manual_toc.html
26. Klautke G, Schutze M, Bombor I, Benecke R, Piek J, Fietkau R. Concurrent chemoradiotherapy and adjuvant chemotherapy with Topotecan for patients with glioblastoma multiforme. J Neurooncol. 2006;77(2):199–205. [PubMed]
27. Adwankar MK, Khandalekar DD, Chitnis MP. Combination chemotherapy of early and advanced murine P388 leukaemia with bouvardin, cis-diamminedichloroplatinum and vincristine. Oncology. 1984;41(5):370–3. [PubMed]
28. Jolad SD, Hoffmann JJ, Torrance SJ, et al. Bouvardin and deoxybouvardin, antitumor cyclic hexapeptides from Bouvardia ternifolia (Rubiaceae) J Am Chem Soc. 1977;99(24):8040–4. [PubMed]