LT SV 40 Large T Antigen

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LI E T A L .
largely unknown (Urban and Schreiber, 1992). Their charac- was confirmed by sequence analysis. Reti-ovirases were proterization requires tumor-specific cytotoxic T cells whose cul- duced as described (Cayeux et al, 1995). The amphotropic tivation and specificity depends on the availabUity of the tamor packaging cell lines PA317 and GP-envA12 were infected witii line. A notable exception is malignant melanoma that is easier ecotropic viras of psi2 or G P ^ E-86 that wereti-ansfectedwith to culture and for which a number oftamor-associatedor spe- plasmid D N A of Lox P-HyTk-large T and Cre-puro by use of cific antigens have been cloned (Boon et al, 1994); (c) In gen- a mammalian transfected kU (Stratagene). PA317 cells that proeral, for many tumors, the availability of early passage lines duced virastitersof 1 X 10= (LoxP-HyTk-large T) and 5 X IO'' would allow to phenotypically characterize them in a more care- cfu/ml (Cre-puro) were used. GP+envA12/LT, which produced ^ E-86 ful way. The large T antigen of SV40 has been shown to pow- viras titer of 7 X 10*, were obtained by cloning G P erfuUy immortalize primary cells of both mouse and human ori- viras-infected GP-l-envA12 and identifying by immunofluoresgin (Fanning and Knippers, 1992; Manfredi and Prives, 1994). cent staining of large T. In some experiments, vUus was conHowever, the continuous presence of large T may change the centrated to 5 X 10^ cfu/ml by centrifugation. For mock-infecantigenicity of the cells, alter gene expression, or cause muta- tion tgLS(4-)HyTk or Babe-Puro viras was used. tions not specific for the tumor. Transient expression may be achieved by use of temperature-sensitive large T mutants (Jat Cell culture and virus infection and Sharp, 1989). However, this poses theriskof 'leakiness' Tumor specimens of breast or colon carcinomas were obor a not so total absence of the protein. The bacteriophage re- tained from surgery with patients' informed consent. Tumor tiscombinase Cre catalyses recombination in a sequence (LoxP) sues were washed with R P M I 1640 medium supplemented with specific way and, if two LoxP sites are oriented as direct re- antibiotic cocktail. Stroma and necrotic areas were removed, peats, the intervening sequence can be deleted and is lost by the tumor samples minced to about 1 m m ^ pieces, and digested the ceUs (Gu et al, 1993; Bergemann et al, 1995). Here, w e for 1-3 h (colon carcinoma) or 3-16 h for breast carcinoma at show that by efficient gene transfer through retrovimses and 37°C in 10-20 ml of medium plus 1 mg/ml collagenase type use of the Cre/LoxP system large T can be expressed in a time- IV (Sigma), 0.01% hyaluronidase type V (Sigma), and 0.001% dependent fashion. Furthermore, retroviral large T expression DNAse I (Boehringer, Mannheim). After organoids had formed, seems to delay the crisis primarytamorsusually undergo in cul- they were placed in 25-cm^ flasks coated for 2-4 h with a mixture. This system facilitates the ability to rapidly obtain larger ture of 30 /xg/ml of Collagen I and 10 /ig/ml BSA. The cells amounts of primary cells and thus minimize theriskof cell culwere cultured in low-calcium and low-semm D M E M / F 1 2 ture artifacts. medium supplemented with 0.1 m M calcium, 5 ng/ml EGF, 1 /xg/ml hydrocortisone, 1 /tg/ml insulin, 1.7 /ig/ml transferrin, 5 ng/ml sodium selenite, 35 /ig/ml pituitary extract, 0.01 m M deoxycholic acid, 2 % fetal brovine seram (FBS), 100 units/ml penicillin, and 100 /ig/ml streptomycin for colon carcinoma, and with 12.5 /ig/ml EGF, 1 /tg/ml hydrocortisone, 35 /ig/ml pitaitary extract, 10 n M triiodothyronine, 2 n M Estradiol, 1 ng/ml cholera toxin, 1 /ig/ml insulin, 10 /ig/ml transferrin, 15 n M sodium selenite, 2 m M L-glutamine, 5 % F B S and antibiotics as just described for breast cancer specimens. Cancer cells as well as primary human fibroblasts, mouse primary kidney cells, and NIH3T3 cells were infected by exposure to the viras containing supematants in the presence of polybrene (8 /ig/ml) for 24 h as described (Cayeux et al, 1995). Selection was begun between 24 h (NIH3T3) and 72 h for primary cells after infection. Selection occurred in medium containing hygromycin B (500 /ig/ml), puromycin (2 //.g/ml), and gancyclovir (GCV) (1 /ig/ml) for 10-14 days.
'Max-Delbriick-Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13122 Berlin, FRG. 2Robert-Rossle-Hospital, Humboldt University, Lindenberger Weg 80, Berlin 13125. 1695