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
). 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 (). 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 (). 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 (more ...)
Green luciferase expression reflects active cellular proliferation and is significantly lower when cellular proliferation is slowed in the absence of serum (). 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 (). 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
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 () and the LC50
value for U0126 was determined to be 96 μM using dual-luciferase assays (), 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) (), which is consistent with previous reports where nocodazole was used at this dose to induce complete growth arrest (22
). 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 (). 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
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 (more ...)
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 (). 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.
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 (). 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) () 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 (). 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 (more ...)
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 (). 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
). 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 (). 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 (, ), and the cytotoxic compound, deoxybouvardin (, ). 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 (). 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 (more ...)
At least 5 of the cytostatic compounds identified in the HTS screen were found to alter the cellular morphology of the astrocytoma cells (). 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 (). 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 (). NSC#676693 also reaches Emax at 55 % inhibition of the E2F1 promoter over a 2-fold log concentration from 1 μM to 10 μM () and washout experiments confirm that cells recover after treatment with NSC#676693 similarly to nocodazole () 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 (more ...)
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 (). 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. (), 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.