Clonogenic assay of leukemic cells in serum-free culture

Dai, Yucheng; Lu, Xiao-Quang; Zheng, Fang

doi:10.1007/bf02631048

Cited by 1 publication

(1 citation statement)

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“…Clonogenic Assay-Colony formation following UV-C or ␣-Fas CH-11 treatment was evaluated using a soft agar cloning assay as described previously (45). Briefly, cells were preincubated with nystatin and ␣-ceramide monoclonal antibody, or vehicle and control IgM in RPMI plus 0.5% lipid-free fetal bovine serum, and stimulated with increasing doses of UV-C or ␣-Fas.…”

Section: Methodsmentioning

confidence: 99%

Caspase-dependent and -independent Activation of Acid Sphingomyelinase Signaling

Rotolo

Zhang²,

Donepudi³

et al. 2005

Journal of Biological Chemistry

153

139

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Recent evidence suggests clustering of plasma membrane rafts into ceramide-enriched platforms serves as a transmembrane signaling mechanism for a subset of cell surface receptors and environmental stresses (Grassme, H., Jekle, A., Riehle, A., Schwarz, H., Berger, J., Sandhoff, K., Kolesnick, R., and Gulbins, E. Translocation of the secretory form of acid sphingomyelinase (ASMase) into microscopic rafts generates therein the ceramide that drives raft coalescence. This process serves to feed forward Fas activation, with ϳ2% of full caspase 8 activation sufficient for maximal ASMase translocation, leading to death-inducing signaling complex formation within ceramide-rich platforms, and apoptosis. Here we report that treatment of Jurkat T cells with UV-C also induces ASMase translocation into rafts within 1 min, catalyzing sphingomyelin hydrolysis to ceramide and raft clustering. In contrast to Fas, UV-induced ASMase translocation and activation were caspase-independent. Nonetheless, ceramide-rich platforms promoted UV-C-induced death signaling, because ASMase inhibition or raft disruption inhibited apoptosis, improving clonogenic cell survival. These studies thus define two distinct mechanisms for biologically relevant ASMase activation within rafts; a Fasmediated mechanism dependent upon caspase 8 and FADD, and a UV-induced mechanism independent of caspase activation. Consistent with this notion, genetic depletion or pharmacologic inhibition of caspase 8 or FADD, which render Jurkat cells incapable of sphingolipid signaling and apoptosis upon Fas ligation, did not impair these events upon UV-C stimulation.

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Section: Methodsmentioning

confidence: 99%