High Throughput Screen to Identify Compounds that Suppress the Growth of Cells with a Deletion of the PTEN Tumor Suppressor - Dose Response
Numerous genes have been identified which participate, either through activation (oncogenes) or inactivation (tumor suppressors), in the multifactorial process of carcinogenesis (Vogelstein B and Kinzler KW, 2004). PTEN is one of the most important cancer-related genes currently known. Inactivating mutations of PTEN are found in approximately 30-40% of all malignant human tumors (Sulis ML and more ..
BioActive Compounds: 1382
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Submitted by Dr. Todd Waldman (Lombardi Cancer Center, Georgetown University School of Medicine)
Numerous genes have been identified which participate, either through activation (oncogenes) or inactivation (tumor suppressors), in the multifactorial process of carcinogenesis (Vogelstein B and Kinzler KW, 2004). PTEN is one of the most important cancer-related genes currently known. Inactivating mutations of PTEN are found in approximately 30-40% of all malignant human tumors (Sulis ML and Parsons R, 2003). Importantly, this gene is in a pathway that is critically important to human cancer pathogenesis. PTEN modulates signaling by the PI3K signaling pathway, which is well known to be activated by numerous mechanisms during human cancer pathogenesis. Therefore, by screening against this target there is the potential to identify pathway modulators that could have efficacy against a wide range of common human tumors.
PTEN is a PIP3 lipid phosphatase, and as such works in opposition to PI3K. Inhibitors of PI3K are commercially available, and isoform-specific inhibitors of PI3K are currently under development (Kim JS et. al., 2007). However, most cells with inactivating mutations of PTEN (including HCT116 PTEN deleted cells used in the present study) do not depend on their high levels of PI3K activity for growth or survival (Samuels Y, et. al. 2005). Therefore, we did not expect to identify PI3K inhibitors in the present assay. Instead, we would expect to identify other types of compounds that specifically target PTEN-deficient cells through novel pathways. At the time of this screen, no such compounds have yet been reported in the literature.
The goal of this dose response study was to confirm the activity of compounds identified in a screen to identify pharmacological probes of the PTEN pathway (AID824, AID827, AID999). The present study and the primary single dose screen used the HCT116 clonal cell line which has a targeted integration of a plasmid into the PTEN loci (knockout) (Lee et al, 2004). In a companion screen (AID818, AID823, AID1004), a different HCT116 clonal cell line has a targeted integration of a plasmid into the beta-catenin locus, while retaining the PTEN tumor suppressor (Kim et. al. 2002). As such, each cell line serves as the clonal control for the other.
In the present assay, we treated HCT116 PTEN "knockout" colon tumor cells with library compounds for 60 h over a 10 point 2-fold dilution series, ranging from 0.059 uM to 30 uM. Following 60 hours of treatment, relative viable cell number was determined using a commercially available assay (Cell Titer Glo, Promega). Each plate contained 32 replicates of vehicle treated cells which served as negative controls, and 32 replicates treated with Hyamine 1622 (100 micromolar), which served as positive controls.
Cell Culture: HCT116 PTEN "knockout" colon tumor cells were subcultured every 3-4 days in McCoy's 5A medium with 10% fetal bovine serum, 100 U/mL penicillin, 100 ug/mL streptomycin and 2 mM glutamine (complete growth medium), incubated at 37 degrees C in 5% carbon dioxide, and maintained at sub-confluent density for no more than 20 passages.
Compound Dosing/Plating: Carrier or Hyamine 1622 (Benzethonium chloride, Sigma-Aldrich) were diluted in complete growth medium to prepare a 5X concentrated dosing solution (100 uM Hyamine/1.5% DMSO) which was dispensed into 384-well black clear-bottom tissue culture treated plates (5 uL volume). This resulted in a final culture volume in assay plates of 25 uL, with final concentrations of 20 uM Hyamine and 0.3% DMSO for the library compounds and controls.
Cell Plating: Twenty uL of complete growth medium containing 1250 cells were dispensed per well, while stirring. Plates were incubated at room temperature for 30 min prior to incubation at 37 degrees C in 5% carbon dioxide for 60 h.
Endpoint/Detection: At the end of the treatment period, assay plates were removed from the incubator and equilibrated to room temperature 10 min. Twenty five uL of Cell Titer Glo reagent was added and plates were incubated for an additional 10 min in the dark. At the end of the incubation, assay plates were analyzed using a PerkinElmer Envision microplate reader with an integration time of 0.1 s in luminescence mode.
Data Analysis: Thirty-two control wells containing cells treated with DMSO vehicle and 32 wells containing Hyamine 1622 were included on each assay plate to calculate Z value for each plate and to normalize the data on a per plate basis. Z values ranged from 0.76 to 0.88 with a mean of 0.82. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100*(Cmpd Lum-Med Ctrl Drug)/(Med Cell Ctrl - Med Ctrl Drug). The normalized % viability was plotted against the tested concentrations of 0.059 to 30 uM. The EC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm allowing extrapolation to identify weakly active compounds.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Compounds that returned EC50 values were considered active within the context of this assay, although compounds with EC50 values greater than 100 were considered to be weak actives.
Score: In this confirmatory dose response screen, active compounds were scored on a scale of 41-80 using an inverse linear correlation to EC50 values between 0 and 100 uM. Compounds that were not confirmed as active in the dose response screen were given the score 0. In later stage probe development screening, active resynthesized confirmatory screen compounds and active analogues thereof will score in a range of 81-100.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)