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

We next sought to determine the susceptibility of serumstarved quiescent calls to CTL-induced DNA fragmentation. These experiments required that the target cells be able to reach a state of quiescence upon serum deprivation or growth factor removal. Initial experiments with prototypic apoptotic CTL targets, i.e., the P815 mastocytoma, the A20 B cell lymphoma, and the T cell lymphomas YAC-1 and EL-4 (9) proved unsuitable, as these cells underwent serum deprivation-induced apoptosis during our attempts to render them quiescent. L929 cells have been reported to enter a G0-1ike state after prolonged serum deprivation (10) , but in our hands the L929 cells displayed significant levels of DNA synthesis even after a 3-d serum starvation protocol. We, therefore, selected for these studies the NIH BALB/c 3T3 cell, clone A31 (3T3-A31), which would undergo quiescence upon serum deprivation (11) . 3T3 cells are reported not to undergo high levels of DNA fragmentation when lethally hit by CTLs (9) . We found, however, that, in 9-12-h assays, lethally hit 3T3-A31 cells did undergo apoptosis, which allowed for tenable DNA fragmentation comparisons. When quiescent 3T3-A31 cells were exposed to LCMV-CTLs, the level of membrane lysis was similar to 3T3-A31 cells in log phase growth ( Fig. 2 A) , but the level of DNA fragmentation was dramatically lower (Fig. 2 B) . When quiescent 3T3-A31 cells were assayed in the presence of 10% FBS, added at the beginning of the cytotoxicity assay, the level of lysis (Fig. 2 A) and the level of DNA fragmentation increased only slightly (Fig. 2 B) . These results support the concept that the DNA fragmentation of the target cell may be mediated by a mechanism(s) regulated by cell cycle controls.