Cell Proliferation Assay against the TMD8 Cell Line
In recent years, using advanced gene expression profiling techniques the laboratory of Dr. Louis Staudt at NCI/NIH has revealed that a class of lymphoma referred to as Diffuse Large B-Cell Lymphoma (DLBCL) is composed of at least 3 different sub-groups, each having distinct oncogenic mechanisms that respond differently to therapies. The sub-group classified as activated B cell-like diffuse large more ..
In recent years, using advanced gene expression profiling techniques the laboratory of Dr. Louis Staudt at NCI/NIH has revealed that a class of lymphoma referred to as Diffuse Large B-Cell Lymphoma (DLBCL) is composed of at least 3 different sub-groups, each having distinct oncogenic mechanisms that respond differently to therapies. The sub-group classified as activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) demonstrates activation of B-cell receptors and the NFkappaB signaling pathway. One class of targeted therapeutics showing early clinical success in killing of ABC DLBCL lines are small molecule inhibitors of the Bruton tyrosine kinase (BTK) 11. Thus, we chose to investigate what additional compounds, if any, would synergize with the BTK inhibitor Ibrutinib (PCI-32765).
Prior to any combination assays, using an NCATS developed oncology compound library called the Mechanism of Interrogation Plate (MIPE), two well studied ABC DLBCL lines (TMD8 and HBL1) were screened using a cell viability assay (CellTiterGlo) and a 12-point titration series for each single agent. These initial experiments offered insight into the concentration range that offered the best coverage of activity and non-activity to assure relevant data is generated. In addition to the viability signature associated with this library, we examined the apoptotic response of each agent versus the TMD8 line as judged by caspase 3/7 activation. Activity was assayed at both 8 and 16 hour time-points to gain an appreciation of which agents induced an apoptotic response in a reasonable timeframe from a therapeutic exposure perspective. MIPE was also assayed for single agent viability in a human mesenchymal stem cell (hMSC) line as a surrogate of each library member's potential therapeutic index.
A total of 500 log growing TMD8 cells are seeded per well in 5 uL of a RPMI with glutamine (or Glutamax), without phenol red plus 5% FBS and 1X Pen/Strep into 1536 -solid white high base tissue culture treated Greiner One Bio Plates (789173-F) using a multidrop combi dispenser and a small sterile cassette. Compounds and controls totaling 23nL per well (negative control DMSO and positive control 9.2 uM Bortezomib-final) are immediately added to the plates using a 1536 head pin tool from Kalypsys. The plates are then covered with stainless steel gasket lids from Kalypsys and incubated for 48 hours at 37C under 95% relative humidity with 5% CO2. After 48 hours, the plates are removed and allowed to reach room temperature before 3 uL of Cell Titer Glo reagent (Promega) is added using an Aurora Flying Reagent Dispenser Bioraptor. The plates are then spun at 1000 rpms to remove bubbles and incubated for 15 minutes at room temperature before read on a ViewLux using a luminescent filter with a 10 second exposure. The relative luciferase units are used to calculated percent activity using DMSO as 100% and Bortezomib as 0%.
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)