have suggested that Bcl-B is important for the survival of some
plasma cell malignancies.
Several types of solid tumors show
pathological elevation of Bcl-B protein, sometimes correlating with
poor patient prognosis.
Selective inhibitors of Bcl-B, therefore,
might find applications for autoimmune diseases, perhaps either by
impairing the survival of long-lived autoantibody-producing cells or
as a complement to chemical inhibitors of Bcl-2/Bcl-X
clinical development for cancer.
In summary, this study illustrates a novel approach leading to the
identification of chemical modulators of antiapoptotic Bcl-2 family
proteins, in which flow cytometry was employed as a platform for
simultaneous HTS of six antiapoptotic proteins Bcl-2, Bcl-B, Bcl-W,
, Bfl-1, and Mcl-1 against a large collection of compounds.
Several future applications of multiplex flow cytometry can be
envisioned for targets in the field of apoptosis, including assays
using the BIR domains of IAP family proteins (there are eight
IAP family members in humans), many of which are known to
bind a tetrapeptide ligand derived from endogenous antagonists
(second mitochondria-derived activator of caspase, SMAC, and
high temperature requirement A 2, HtrA2, also named OMI) that
suppresses their antiapoptotic activity. This multiplex assay method
offers the advantage of assessing upfront the differential binding
activities of chemicals for members of these multigene families,
thus efficiently creating opportunities for identifying starting
points for optimization of chemical inhibitors with unique spec-
trums of activity that may translate into distinct efficacy and tox-
icity profiles f or different clinical indications.
This research was supported by an NIH grant (No. U54MH074425).
AUTHOR DISCLOSURE STATEMENT
L.A.S. and B.S.E. declare competing financial interests as co-
inventors of HyperCyt and cofounders of Intellicyt.
1. Carson RT, Vignali DA: Simultaneous quantitation of 15 cytokines using a
multiplexed flow cytometric assay. J Immunol Methods 1999;227:41–52.
2. Tessema M, Simons PC, Cimino DF, Sanchez L, Waller A, Posner RG, Wandinger-
Ness A, Prossnitz ER, Sklar LA: Glutathione-S-transferase-green fluorescent
protein fusion protein reveals slow dissociation from high site density beads
and measures free GSH. Cytometry A 2006;69:326–334.
3. Zuck P, Lao Z, Skwish S, Glickman JF, Yang K, Burbaum J, Inglese J: Ligand-
receptor binding measured by laser-scanning imaging. Proc Natl Acad Sci U S A
4. Adams JM, Cory S: The Bcl-2 apoptotic switch in cancer development and
therapy. Oncogene 2007;26:1324–1337.
5. Oltersdorf T, Elmore SW, Shoemaker AR, Armstrong RC, Augeri DJ, Belli BA,
Bruncko M, Deckwerth TL, Dinges J, Hajduk PJ, Joseph MK, Kitada S, Korsmeyer
SJ, Kunzer AR, Letai A, Li C, Mitten MJ, Nettesheim DG, Ng S, Nimmer PM,
O’Connor JM, Oleksijew A, Petros AM, Reed JC, Shen W, Tahir SK, Thompson CB,
Tomaselli KJ, Wang B, Wendt MD, Zhang H, Fesik SW, Rosenberg SH: An
inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature
6. Reed JC, Pellecchia M: Apoptosis-based therapies for hematologic
malignancies. Blood 2005;106:408–418.
7. Chen L, Willis SN, Wei A, Smith BJ, Fletcher JI, Hinds MG, Colman PM, Day CL,
Adams JM, Huang DC: Differential targeting of prosurvival Bcl-2 proteins by
their BH3-only ligands allows complementary apoptotic function. Mol Cell
8. van Delft MF, Wei AH, Mason KD, Vandenberg CJ, Chen L, Czabota r PE,
Willis SN, Scott CL, Day CL, Cory S, Adams JM, Roberts AW, Huang DC:
The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently
induces apoptosis via Bak/Bax If Mcl-1 is neutralized. Cancer Cell
9. Luciano F, Krajewska M, Ortiz-Rubio P, Krajewski S, Zhai D, Faustin B, Bruey JM,
Bailly-Maitre B, Lichtenstein A, Kolluri SK, Satterthwait AC, Zhang XK, Reed JC:
Nur77 converts phenotype of Bcl-B, an antiapoptotic protein expressed in
plasma cells and myeloma. Blood 2007;109:3849–3855.
10. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ: Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings. Adv Drug Deliv Rev 2001;46:3–26.
11. Zhai D, Jin C, Satterthwait AC, Reed JC: Comparison of chemical
inhibitors of antiapoptotic Bcl-2-family proteins. Cell Death Differ 2006;
12. Edwards BS, Young SM, Oprea TI, Bologa CG, Prossnitz ER, Sklar LA: Biomolecular
screening of formylpeptide receptor ligands with a sensitive, quantitative, high-
throughput flow cytometry platform. Nat Protoc 2006;1:59–66.
13. Black CB, Duensing TD, Trinkle LS, Dunlay RT: Cell-based screening using high-
throughput flow cytometry. Assay Drug Dev Technol 2010;9:13–20.
14. Simons PC, Young SM, Carter MB, Waller A, Zhai D, Reed JC, Edwards BS,
Sklar LA: Simultaneous in vitro molecular screening of protein–peptide
interactions by flow cytometry using six Bcl-2 family proteins as examples.
Nature Protocols, in press.
15. Labi V, Grespi F, Baumgartner F, Villunger A: Targeting the Bcl-2-regulated
apoptosis pathway by BH3 mimetics: a breakthrough in anticancer therapy?
Cell Death Differ 2008;15:977–987.
16. Zhang H, Nimmer P, Rosenberg SH, Ng SC, Joseph M: Development of a high-
throughput fluorescence polarization assay for Bcl-x(L). Anal Biochem
17. Zhang JH, Chung TD, Oldenburg KR: A simple statistical parameter for use in
evaluation and validation of high throughput screening assays. J Biomol Screen
18. Zhai D, Ke N, Zhang H, Ladror U, Joseph M, Eichinger A, Godzik A, Ng SC, Reed
JC: Characterization of the anti-apoptotic mechanism of Bcl-B. Biochem J
19. Zhai D, Jin C, Huang Z, Satterthwait AC, Reed JC: Differential regulation of Bax
and Bak by anti-apoptotic Bcl-2 family proteins Bcl-B and Mcl-1. J Biol Chem
20. Krajewska M, Kitada S, Winter JN, Variakojis D, Lichtenstein A, Zhai D, Cuddy M,
Huang X, Luciano F, Baker CH, Kim H, Shin E, Kennedy S, Olson AH, Badzio A,
Jassem J, Meinhold-Heerlein I, Duffy MJ, Schimmer AD, Tsao M, Brown E,
Sawyers A, Andreeff M, Mercola D, Krajewski S, Reed JC: Bcl-B expression in
human epithelial and nonepithelial malignancies. Clin Cancer Res
Address correspondence to:
Larry A. Sklar, PhD
Department of Pathology
UNM Center for Molecular Discovery
University of New Mexico Health Sciences Center
Albuquerque, NM 87131
CURPAN ET AL.
474 ASSAY and Drug Development Technologies OCTOBER 2011