Evaluation of Division-Arrested Cells for Cell-Based High-Throughput Screening and Profiling

Abstract

Just-in-time cell supply for cell-based high-throughput screening (HTS) is frequently problematic. In addition to scheduling and logistical issues, quality issues and variability due to passage effect, cell cycle, or confluency contribute to day-to-day signal variability in the course of cell-based HTS campaigns. Cell division-arrest and cryopreservation technologies permit the use of cells as assay-ready reagents for HTS and other cell-based profiling and structure-activity studies. In this report, the authors compare division-arrested and dividing cells in 2 assay types that are dependent on movement of proteins within or through cell membranes: a receptor tyrosine kinase assay involving A431 cells responsive to epidermal growth factor, and a secretion reporter assay, which measures secretion of a reporter gene, secreted alkaline phosphatase. In both assays, dividing and division-arrested cells yielded similar basal and maximal signals at a given cell density. Similar IC₅₀s were obtained for reference inhibitors in each assay, type in both dividing and division-arrested cells. In addition, for the secretion reporter assay, when comparing IC₅₀s obtained from 44 compounds randomly chosen from a primary screening hit list, the rank order of potency obtained from dividing cells and division-arrested cells was essentially identical. Furthermore, the results show that, under certain assay conditions, data generated using division-arrested cells are less variable than those generated using dividing cells. In summary, the results suggest that, in many cases, division-arrested cells can substitute for dividing cells and offer certain advantages for cell-based assays.

Keywords

high-throughput screening cell-based assay receptor tyrosine kinase division-arrest cryopreservation secretion assay assay comparison

References

Cong M , Fursov N , Federici M , Tacke R , Livelli T , Zhong Z : Improving consistency of cell-based assays by using division-arrest cell reagents [poster P04005]. Paper presented at the 10th Anniversary Conference and Exhibition of the Society for Biomolecular Screening, Orlando, FL, September 2004.

Fursov N , Cong M , Federici M , Platchek M , Haytko P , Tacke R , et al : Improving consistency of cell based assays by using division-arrested cells. Assay and Drug Dev Tech 2005;3:7-15.

Kanapuli P , Zheng W , Weber M , Solly K , Mull R , Platchek M , et al : Application of division arrest technology to cell-based HTS: Comparison with frozen and fresh cells. Assay and Drug Dev Tech 2005;3:17-26.

Traxler P , Allegrini PR , Brandt R , Brueggen J , Cozens R , Fabbro D , et al : AEE788: a dual family epidermal growth factor receptor/ErbB2 and vascular endothelial growth factor receptor tyrosine kinase inhibitor with antitumor and antiangiogenic activity. Cancer Res 2004;64:4931-4941.

Rietz B , Guilbault GG : Fluorometric assay of serum acid or alkaline phosphatase, either in solution or on a semisolid surface. Clin Chem 1975;21:1791-1794.

Parker PJ , Young S , Gullick WJ , Mayes EL , Bennett P , Waterfield MD : Monoclonal antibodies against the human epidermal growth factor receptor from A431 cells: isolation, characterization, and use in the purification of active epidermal growth factor receptor. J Biol Chem 1984;259:9906-9912.

Zhang J-H , Chung TC , Oldenburg KR : A simple statistical parameter for use in evaluation and validation of high throughput screening assays. JBiomol Screen 1999;4:67-73.

Harant H , Eldershaw SA , Lindley IJ : Human macrophage inflammatory protein-3alpha/CCL20/LARC/Exodus/SCYA20 is transcriptionally upregulated by tumor necrosis factor-alpha via a non-standard NF-kappaB site. FEBS Lett 2001;509:439-445.

Vasudevan C , Maunder P , Haskins J , Fursov N , Zhong Z : The use of division arrested cells improves the robustness of high content screening assays [poster P11008]. Paper presented at the 10th Anniversary Conference and Exhibition of the Society for Biomolecular Screening, Orlando, FL, September 2004.

10.

Chow TY , Alaoui-Jamali MA , Yeh C , Yuen L , Griller D : The DNA double-stranded break repair protein endo-exonuclease as a therapeutic target for cancer. Mol Cancer Ther 2004;3:911-919.

11.

Chen D , Carter TH , Auborn KJ : Apoptosis in cervical cancer cells: implications for adjunct anti-estrogen therapy for cervical cancer. Anticancer Res 2004;24:2649-2656.

12.

Weetall M , Hugo R , Freidman C , Maida S , West S , Wattanasin S , et al : A homogeneous fluorometric assay for measuring cell adhesion to immobilized ligand using V-well plates. Anal Biochem 2001;293:277-287.

13.

DeBiasio R , Ghosh RN : The use of cryopreserved primary neurons in high content screening assays [poster P10028]. Paper presented at the 10th Anniversary Conference and Exhibition of the Society for Biomolecular Screening, Orlando, FL, September 2004.