Disease: acute T cell leukemia Cell Type: T lymphocyte;
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The I 2.1 cell line is a FADD mutant of the wild-type Jurkat cell line A3 (see ATCC -CRL-2570).
Wild-type A3 cells were made neomycin resistant and treated with three cycles of exposure to the frameshifting mutagen ICR-191 to isolate clones harboring recessive mutations that were resistant to killing by Fas antibody. [51527]
ICR-191 treated clones were serially diluted in 96-well plates in the presence of Fas Antibody for 3 to 5 weeks. [51529]
Two of these ICR-191 treated clones have been deposited at the ATCC . They are I 9.2 (ATCC CRL-2571), a clone with a mutation in the cysteine protease caspase-8/FLICE and I 2.1 (ATCC CRL-2572), a clone with a mutation in the adaptor FADD.
Unlike the parental line A3 that does express FADD, the I 2.1 cell line does not express FADD protein by immunoblot analysis and is completely resistant to Fas-induced death. [51529]
Complementation of the I 2.1 cell lines with wild-type FADD restores Fas-mediated apoptosis. Fas activation of caspase-2, caspase-3, caspase-7, and caspase-8 is completely defective in the FADD mutant cell line I 2.1. [51529]
This cell line can be used to study the role of FADD in apoptotic signaling pathways in the absence of overxpression.
Propagation:
ATCC complete growth medium: The base medium for this cell line is ATCC-formulated RPMI-1640 Medium, Catalog No. 30-2001. To make the complete growth medium, add the following components to the base medium: fetal bovine serum to a final concentration of 15%. Atmosphere: air, 95%; carbon dioxide (CO2), 5% Temperature: 37.0°C
Subculturing:
Protocol: Cultures can be maintained by addition of fresh medium or replacement of medium. Alternatively, cultures can be established by centrifugation with subsequent resuspension in fresh medium at 3 to 5 X 10(5) viable cells/ml. Maintain cell density between 2 X 10(5) and 2 X 10(6) viable cells/ml. Medium Renewal: Add fresh medium every 2 to 3 days (depending on cell density)
Preservation:
Freeze medium: Complete growth medium supplemented with 5% (v/v) DMSO Storage temperature: liquid nitrogen vapor phase
Related Products:
Recommended medium (without the additional supplements or serum described under ATCC Medium):ATCC 30-2001
recommended serum:ATCC 30-2020
parental cell line:ATCC CRL-2570
References:
51527: Juo P, et al. Essential requirement for caspase-8/FLICE in the initiation of the Fas-induced apoptotic cascade. Curr. Biol. 8: 1001-1008, 1998. PubMed: 9740801 51529: Juo P, et al. FADD is required for multiple signaling events downstream of the receptor Fas. Cell Growth Differ. 10: 797-804, 1999. PubMed: 10616904 51530: Juo P, et al. Fas activation of the p38 mitogen-activated protein kinase signalling pathway requires ICE/CED-3 family proteases. Mol. Cell. Biol. 17: 24-35, 1997. PubMed: 8972182
