Requirement for the CD95 receptor-ligand pathway in c-myc-induced apoptosis

Science

Volumn 278, Issue 5341, 1997, Pages 1305-1309

  Hueber, Anne Odile ^a   Zörnig, Martin ^a   Lyon, Debbie ^a   Suda, Takashi ^a   Nagata, Shigekazu ^a   Evan, Gerard I ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

FAS ANTIGEN;

APOPTOSIS; ARTICLE; CELL INTERACTION; HUMAN; HUMAN CELL; HUMAN TISSUE; ONCOGENE C MYC; PRIORITY JOURNAL; RECEPTOR BINDING; RECEPTOR UPREGULATION; SIGNAL TRANSDUCTION;

3T3 CELLS; ADAPTOR PROTEINS, SIGNAL TRANSDUCING; ANIMALS; ANTIGENS, CD95; APOPTOSIS; AUTOCRINE COMMUNICATION; CARRIER PROTEINS; CELL LINE; CELL MEMBRANE; CELLS, CULTURED; FAS LIGAND PROTEIN; FAS-ASSOCIATED DEATH DOMAIN PROTEIN; GENE EXPRESSION REGULATION; GENES, MYC; INSULIN-LIKE GROWTH FACTOR I; MEMBRANE GLYCOPROTEINS; MICE; PROTO-ONCOGENE PROTEINS C-BCL-2; PROTO-ONCOGENE PROTEINS C-MYC; RATS;

EID: 0030667376     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.278.5341.1305     Document Type: Article

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36. 32P-labeled internal oligonucteotide CD95L5. A single round of PCR amplification was carried out with 2 μl of cDNA and the primer pair Act1 and Act2 (1 μM each). Reaction conditions were identical to those used in the first-round PCR. Primer sequences: CD95L1, 5′-GTATTTTTCATGGTTCTGGTGG-3′; CD95L2, 5′-ATGAATTCCTGGTGCCCATG-3′; CD95L3, 5′-AAGCTTCAGCTCTTCCACCTG-3′; CD95L4, 5′-TAAAGAATAGTAGATCATTT-3′; CD95L5. 5′-AAGTATACTTCCGGGGTGAGT-3′; Act1, 5′-GTGGCCATCTCCTGCTCGAAGTC-3′; and Act2, 5′-GTTTGAGACCTTCAACACCCC-3′.
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38. The Fadd dominant-negative molecule [FADD(80-208)] (11) was cloned into the retroviral expression vector pBabe Hygro and transfected into the retroviral packaging cell line GP+E. Recombinant retrovirus was harvested 48 hours later and used to infect S3T3 c-MycER cells in the presence of polybrene (8 μg/ml). Control retrovirus was prepared by transfecting the GP+E packaging line with pBabe Hygro vector alone. Cells were selected with Hygromycin (Sigma; 200 μg/ml), and resistant cells were pooled.
39. The pBabe puro c-MycER construct was transfected into the retroviral packaging cell line BOSC
40. Recombinant retrovirus was harvested 48 hours later and used to infect MEFs prepared from WT, lpr, or gld embryos in the presence of polybrene (8 μg/ml). Twenty-four hours after infection, the medium was replaced and the cells were cultivated in 0.5% FCS in the presence or absence of OHT.
41. We thank A. M. Bernard and M. Chautan for providing lpr MEFs; V. Dixit for providing the mutant DN Fadd construct; P. Golstein for the L12.10 CD95 cells; T. Möröy, T. Schmidt, and H. Karsunky for help and support with the MEF experiments; and our colleagues at ICRF for advice and criticism. A.-O.H. is supported by a fellowship from the European Molecular Biology Laboratory and M.Z. by a Marie Curie TMR Fellowship (ERBFMBICT 950498) from the European Community (EC). We are grateful for support from MRC-LINK G9321238.EC and from EC grants BioTech BIO4-CT96-0052 and BioMed2 BMH4-CT96-0010.