Human Fibroblast Cell Proliferation Assay - Dose Response
Angiogenesis is the process of new blood vessel formation, which is believed to be involved in many human diseases including cancer, diabetic retinopathy, and rheumatoid arthritis (Carmeliet, 2005). Endothelial cell proliferation is known to occur in early stages of angiogenesis (Coultas et al, 2005). As such, the identification of compounds that selectively inhibit endothelial cell proliferation more ..
BioActive Compounds: 314
Southern Research Molecular Libraries Screening Center (SRMLSC)
Southern Research Institute (Birmingham, Alabama)
NIH Molecular Libraries Screening Centers Network (MLSCN)
Assay Provider: Dr. Zhican Qu, Southern Research Institute
Angiogenesis is the process of new blood vessel formation, which is believed to be involved in many human diseases including cancer, diabetic retinopathy, and rheumatoid arthritis (Carmeliet, 2005). Endothelial cell proliferation is known to occur in early stages of angiogenesis (Coultas et al, 2005). As such, the identification of compounds that selectively inhibit endothelial cell proliferation represents an attractive strategy for the development of novel drugs, which act by inhibiting angiogenesis. We had previously reported the results of a high throughput screening of the MLSCN compound library against two primary human cell lines, endothelia (HUVEC) and fibroblasts (LL 47) (AID 648 & 719, respectively) to identify compounds with selective inhibition against human endothelia versus fibroblast.. Compounds that are inactive against LL 47, but active against HUVEC are potential probes for angiogenesis research. Conversely, compounds that selectively inhibit the proliferation of fibroblasts may have utility for other areas of research such as the study of mechanisms involved in tumor desmoplasia or diseases involving fibrosis.
The primary HTS hit compounds with differential inhibitory activity against endothelial cell activation had been selected based on the single dose screening results. The potency of these compounds has been evaluated by a dose response study against endothelial cells (AID 822). To assess selectivity of the identified primary hit compounds, a study of dose response with ten doses ranging from 0.039 to 20 uM in HTS setting with LL 47 fibroblast cell line has been completed and EC50 values on inhibition of LL 47 proliferation have been determined based on the dose response curve of each hit compound.
Cell growth conditions:
Human lung fibroblasts (LL 47:ATCC, cat. no. CCL-135) were grown under standard cell culture conditions involving incubation at 37C with 5% CO2 and 95% humidity. Cells were grown in IMEM media (Mediatech Inc, cat. no. 10-24-CV) supplemented with 15% fetal bovine serum (Hyclone, cat. no. SH30071.03) and 50 ug/mL gentamicin (Mediatech Inc, cat. no. 30-005-CR) and referred to as Complete Growth Media. Cells were harvested 1:5 in 100 mm Petri plates twice a week, after washing with PBS, followed by 1 mL of trypsin (Invitrogen, cat. no. 4671). Cells used for this assay were maintained in culture for no more than 12 passages.
Fibroblasts were dispensed using a Matrix WellMate bulk dispenser into 384 well plates (Corning Flat Clear Bottom Black Polystyrene TC-Treated, cat. no. 3712) at a density of 1,150 cells per well (23 uL). Cell suspensions were maintained at room temperature with stirring during the plating procedure. Following plating, cells were incubated at 37C overnight prior to the addition of library compounds.
Dilution media, carrier and positive control drug were dispensed to dilution plate. Compounds were diluted in complete growth medium to prepare a 10x concentrated dosing solution (0.39-200 uM). A volume of 2.5 uL of the diluted compounds was transferred to the cell plate using a Biomek FX and the plates were returned to the incubator. This resulted in final culture volume in assay plates of 25.5 uL, with final concentrations of 1 ug/mL 6-methyl purine riboside (MePR) and 0.039 to 20 uM for library compounds. DMSO concentrations were maintained at 0.2% in all wells.
Cell viability measurement:
After incubating 72 h at 37C, assay plates were removed from the incubator and equilibrated to room temperature. As per the manufacturer's protocol, an equal volume of CellTiter-Glo reagent (Promega Inc.) was added to each well and plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using an Envision multi-label reader (Perkin Elmer) with an integration time of 0.1 s.
Thirty-two control wells containing cells only and 32 wells containing MePR were included on each assay plate and used to calculate Z value for each plate and to normalize the data on a per plate basis. Z values ranged from 0.66 to 0.84 with a mean of 0.77. Compound data was normalized and reported as % viability which was calculated using the following formula: % viability = 100 x luminescence compound well / median luminescence cell control. The normalized % viability was plotted against the tested concentrations of 0.039 to 20 uM . The EC50 values were calculated using XLfit formula 205, a 4 parameter Levenburg-Marquardt algorithm allowing extrapolation up to 100 uM.
Possible artifacts in this assay include, but are not limited to, compounds that interfere with the luciferase reaction, absorb luminescence, or precipitate.
Outcome: Compounds with EC50 values <= 100 uM were considered Active within the context of this assay. Compounds with LL 47 specific activity were determined by comparing the activity score derived in this assay with the activity score derived in the HUVEC based assay (AID 822).
Score: In this confirmatory dose response screen active compounds were scored on a scale of 41-80 using an inverse linear correlation to EC50s between 0 and 100 uM. Compounds that did not confirm as actives 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.
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Data Table (Concise)