Susceptibility to cytotoxic T lymphocyte-induced apoptosis is a function of the proliferative status of the target

The enforced expression ofc-myc did not alter the membrane lysis between these targets ( Fig. 3 A) but markedly enhanced their susceptibility to CTL-induced DNA fragmentation ( Fig. 3 (3) and used as targets ~ on day 0. Just before use, purified I.CMV-CTL were resuspended in medium containing the desired concentration of FBS. Cytotoxicity assays were performed as described (5) with Con A (Sigma Chemical Co.) at 5/~g/ml. L929 cells were cultured identically for both the slCr-and 125I-IUDRrelease portion of the assay. Assay time for A and B, performed in parallel, was 6 h. Standard error of mean (SEM) for slCr release was <5%. (B) Infuence of serum on target cell's susceptibility to CTMnduced DNA fragmentation. L929 target cells were cultured as above and labeled with [125I]iododeoxyuridine (12sI-IUDR.) at 1 #Ci/ml on day -3 for 18 h as described (3) . SEM: 12SI-DNA release <10%. quiescent targets with 10% FBS added during the assay. 3T3-A31 target cells were cultured in DMEM+10% calf serum (Hyclone Labs., Logan, UT). 5 d before use (day -5) 3T3-A31 cells 6:1 12:1 25:1 50:1 were subcultured into T150 cm 2 Effector to Target Ratio flasks to obtain ,'o75% confluency, On day -4, they were labeled with 125I-IUDR at 1/~Ci/ml in 5 ml for 18 h. After labeling, the medium was removed, and fresh RPMI 1640+5% CPSR-2 (Sigma Chemical Co.) was added, and the cells were allowed to quiesce for a minimum of 72 h. CPSR.-2 is a nonmitogenic serum substitute. For cells in log phase growth, 3T3-A31 cells were subcultured to ,'050% confluency 2 d before use. The cells were labeled as above on day -1. Cytotoxicity assays were performed with I.CMV-CTL in lectin-dependent assays in AIM-V media without or with 10% FBS. Assay time for parts A and B performed in parallel, was 10 h. SEM, 12SI-DNA release <15%. growth rate under these conditions, had an intermediate level of DNA fragmentation even though they showed a lower levd of membrane lysis than the 3T3-A31 cells (Fig. 3, A and B ). To further dissect the relationship between the proliferative status of the target cell and CTL-induced DNA fragmentation, we analyzed CTL-induced apoptosis in the prototypic targets for apoptosis, P815 (ta = 11 _+ 2 h) and A20 (td = 14 _+ 1 h) cells. These suspension-grown cells were extremely sensitive to apoptosis and consequently were analyzed in 4-6-h cytotoxidty assays. Although the levd of membrane lysis was slightly greater in A20 cells than in P815 ceils ( Fig. 3 C) , the level of DNA fragmentation was greater in the faster growing P815 cells (Fig. 3/9 ). Thus, P815 and A20 cells underwent apoptosis more rapidly than 3T3-A31, A31m F , or L929 cells, but in each case the faster growing cell underwent apoptosis more readily (P815>A20; A31-myc>L-929>3T3-A31). Demonstration that each cell line underwent apoptosis associated with distinct oligonucleosomal ladders is presented in Fig. 4 , which shows that the level of DNA fragmentation in cells not exposed to CTL (C), and in the cell pellets (P) and culture medium (M) from cells exposed to CTL in 6-(P815, A20) or 10-(L929, 3T3-A31, A31-mF) h assays. Note both the presence of the ladders and the disappearance of high molecular weight DNA, the latter being most noticeable with the P815 cells. Pig. 4 also shows that the DNA fragmentation in A31-myc ceils is far more extensive than in the 3T3-A31 cells.