Flavopiridol (2 μM) robustly induces a distinct pattern of ER stress in CLL cells that contributes to cell death through IRE1-mediated activation of ASK1 and possibly downstream caspases[1]. Flavopiridol results in potent upregulation of a number of PRGs in treatments lasting 4-24 h. Flavopiridol has and immediate and long-term effect on the expression of several PRGs. In serum starved cells re-stimulated with serum, flavopiridol also inhibits the expression of these genes, but subsequently, JUNB, GADD45B and EGR1 are upregulated in the presence of flavopiridol[2].
Kinase Assay
Briefly, lysates containing approximately 3×106 cells are incubated with 50 μM LEVD-AFC (caspase 4 substrate) or LETD-AFC (caspase 8 substrate) containing 10 mM dithiothretiol (DTT). caspase 4 activity is measured one hour after addition of substrate and caspase 8 activity is measured 30 minutes after addition of substrate. Release of free AFC is measured with a Beckman-Coulter DTX 880 multimode detector.
Cell Assay
The cells treated with flavopiridol are washed after 4 hours with PBS and resuspended in regular growth medium (RPMI 1640) supplemented with 10% human serum and antibiotics for the remainder of the incubation time. In the case of flavopiridol/chloroquine samples, chloroquine is re-added in the fresh media after flavopiridol is washed at 4 hours. For all the other conditions, cells are incubated with the respective drugs for 24 hours continuously.
Animal Administration
References
[1]. Mahoney E, et al. ER stress and autophagy: new discoveries in the mechanism of action and drug resistance of the cyclin-dependent kinase inhibitor flavopiridol.Blood. 2012 Aug 9;120(6):1262-1273.
[2]. Keskin H, et al. Complex effects of flavopiridol on the expression of primary response genes. Cell Div. 2012 Mar 29;7:11.
[3]. Kim KS, et al.Thio- and oxoflavopiridols, cyclin-dependent kinase 1-selective inhibitors: synthesis and biological effects. J Med Chem. 2000 Nov 2;43(22):4126-34.
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