Fluorescent HTS Cytotoxicity/Cell viability assay (HPDE-C7 cells)
One of the most deadly forms of cancer in humans is pancreatic cancer. Typically few individuals survive beyond 12 months after diagnosis. The oncogene KRAS has been suggested to play a role in this disease. Mutations which activate KRAS are almost always found in pancreatic adenocarcinoma. ..more
BioActive Compounds: 1123
Sanford-Burnham Center for Chemical Genomics (SBCCG)
Sanford-Burnham Medical Research Institute (San Diego, CA)
NIH Molecular Libraries Screening Centers Network (MLSCN)
One of the most deadly forms of cancer in humans is pancreatic cancer. Typically few individuals survive beyond 12 months after diagnosis. The oncogene KRAS has been suggested to play a role in this disease. Mutations which activate KRAS are almost always found in pancreatic adenocarcinoma.
This assay was developed to determine the cytotoxic effects of small molecule compounds on HPDE-C7 cells in a 384 well format. HPDE-C7 cells are primary human pancreatic duct epithelial cells that have been immortalized via the insertion of E6/E7 genes from the human papilloma virus (HPV-16). HPDE-C7 cells are not tumorigenic. These are the parental cell line for the cells used in AID 431. Thus this assay can be used as a selectivity assay for AID 431.
In this assay, cell viability/proliferation is measured using Alamar Blue. The internal environment of viable cells is more reduced than that of nonproliferating or dead cells. This reduced state can be measured by the reduction of Alamar Blue which in turn generates a change in both absorbance and fluorescence. We have opted for the fluorescent readout for this assay.
This RAS-based screening was performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). XO1 submission, MH076387-01, Chemical genetic screening of oncogene RAS-based inhibitors for pancreatic cancer, Assay Provider Dr. Xiaodong Cheng, University of Texas, Galveston.
1) HPDE-C7 cells are cultured in Keratinocyte-SFM (Invitrogen 17005-042, with rhEGF and pituitary extracts) supplemented with penicillin/streptomycin.
2) 22.5ul of cells (1000 cells/well) are added to each well of a 384 well microtiter plate (Greiner 781086).
3) Cells are left to adhere overnight at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
4) After the overnight incubation, 2.5ul of diluted compound (final compound concentration is 10uM, final DMSO concentration is 0.5%) are added to appropriate wells.
5) 2.5ul of 5% DMSO are added to negative control wells.
6) 2.5ul of 0.15uM Doxorubicin in 5% DMSO are added to positive control wells.
7) The cells are incubated with compound for 48hr at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
8) After incubation 5ul of 10 fold diluted Alamar Blue (Biosource International DAL1100) are added to each
9) The plates are incubated for 4 hrs at 37 Celsius degrees in a humid environment in the presence of 5% CO2.
10) After incubation, the plates are read in a Molecular Devices Analyst HT, multimode plate reader. The filters used were 530/25nm excitation, 580/10nm emission, and a 561nm dichroic mirror. Due to high background of the Alamar Blue the plates are read at a medium attenuation setting for this plate reader.
Data calculation: Raw data are sent to our biological database (CBIS) whereby % activity of the compounds are calculated based on the intraplate controls of 15uM Doxorubicin (positive control) and 0.5% DMSO (negative control). Compounds that are not cytotoxic will have a low % activity, while cytotoxic compounds will have a high %.
For information regarding these cells please contact:
Xiaodong Cheng, Ph.D.
Department of Pharmacology & Toxicology
University of Texas Medical Branch
Galveston, Texas 77555-1031
Potential artifacts of this assay are compounds that quench at the excitation, or emission wavelengths used in the assay. Compounds that precipitate would also lend to artifacts due to their not being tested at the assumed concentration. Due to the high overall signal of the assay, there were no naturally occurring fluorescent compounds that interfered with the assay at the concentration of compounds used.
Activity scoring rules:
1. 0-40 scoring range is reserved for primary screen data
a. If primary % Activity is greater than 100%, then the assigned score is 40
b. If primary % Activity is less than 0%, then the assigned score is 0
c. If primary % Activity is between 0% and 100%, then the calculated score is (% Activity)*0.4
2. 41-80 scoring range is reserved for dose response confirmation dat
a. False positives that failed the reconfirmation are assigned a score of 41.
b. True positives that have EC50 are assigned a score in the range 42-80
c. The score is linearly correlated with a compound's potency and, in addition, provides a measure of the likelihood that the compound is not an artifact based on the available information.
d. The Hill coefficient is taken as a measure of compound behavior in the assay via an additional scaling factor QC:
QC = 2.6*[exp(-0.5*nH^2) - exp(-1.5*nH^2)]
This empirical factor prorates the likelihood of target-specific compound effect vs. its non-specific behavior in the assay. This factor is based on expectation that a compound with a single mode of action that achieved equilibrium in the assay demonstrates the Hill coefficient value of 1. Compounds deviating from that behavior are penalized proportionally to the degree of their deviation.
e. Summary equation that takes into account the items discussed above is
Score = 44 + 6*(pIC50 - 3)*QC,
where pIC50 is a negative log(10) of the EC50 value expressed in mole/L concentration units. This equation results in the Score values above 50 for compounds that demonstrate high potency and predictable behavior. Compounds that are inactive in the assay or whose concentration-dependent behavior are likely to be an artifact of that assay will generally have lower Score values.
f. Note that compounds that did not generate EC50s were assigned >20 uM in the EC50 column.
g. The screen was performed in 2 campaigns. The criteria for choosing which compounds were tested in serial dilutions was dependent on which primary screening campaign the compounds were tested.
Criteria Set 1 (for the first screening campaign): The compound exhibited >50% activity in AID 431, but <30% activity in AID430, or >90% activity in AID 431 and AID 430.
Criteria Set 2 (for the second screening campaign): The compound exhibited >50% activity in AID 431, but <30% activity in AID 430.
3. 81-100 scoring range is reserved for resynthesized true positives and their analogues
Categorized Comment - additional comments and annotations
Assay Type: Toxicity
Assay Cell Type: HPDE-C7
Assay Format: Cell-based
Assay Type: Functional
* Activity Concentration. ** Test Concentration.
Data Table (Concise)