p53 MEDIATED APOPTOSIS IN HeLa CELLS: TRANSCRIPTION DEPENDENT AND INDEPENDENT MECHANISMS Ygal HAUPT, Sheldon ROWAN, Eitan SHAULIAN, Anat KAZAZ, Karen VOUSDEN' and Moshe OREN The most frequent target for genetic alterations in human cancers is the p53 tumor suppressor gene. Mutations in p53 abrogate its ability to inhibit cell growth and to suppress tumor progression. The anti-proÜferative activity of p53 can be mediated by the induction of growth arrest and/or programmed cell death (apoptosis). Recent in vivo studies support the involvement of apoptosis in tumor suppression by p53. To gain further insight into the mechanisms by which p53 induces apoptosis. the activity of p53 was studied in HeLa ceils using a transient transfection assay. To define the functional domains of p53 required for apoptosis a C- terminal deletion mutant of p53 was used. This mutant, p53dl2l4, lacks the ohgomerization domain, the nuclear localization signal and a large part of the core DNA binding domain. This mutant was shown to be deficient in sequence specific transactivation activity. Overexpression of wt p53 induced an efficient apoptosis in transiently transfected HeLa cells. Surprisingly p53dl214, containing only the first 214 N-terminal residues induced extensive apoptosis. The induction of apoptosis by p53dl214 is slower than that induced by wt p53. Furthermore, p53dl2l4 suppressed the transformation of rat embryo fibroblasts by several oncogene combinations, such asmycplus ras. In view of the fact that p53dl214 lacks measurable transactivation potential, our findings suggest the existence of two p53 dependent-apoptotic pathways - one involves activation of specific target genes, and the other is independent of it. Transactivation independent apoptosis may play a centra! role in tumor suppression by p53. INTRODUCTION Mutations in the p53 tumor suppressor gene are found in a significant proportion of many types of human cancers. These mutations abrogate the ability of p53 to suppress tumor growth by eliminating one or more ofthe anti-proliferative activities of wt p53. Activation of p53 in response to DNA damage induces a growth arrest, usually at the Gl phase, but also at the G2 phase of the cell cycle. This arrest permits sufficient time for damaged DNA to be repaired, and hence, the genomic integrity of the cell is maintained (Lane, 1992). When the damage is excessive the cell may be triggered to undergo programmed cell death (apo- ptosis). The apoptotic activity of p53 can also be induced by withdrawal of essential growth-survival factors. To date the apoptotic activity of p53 has been demonstrated in a variety of cell types, using different model systems (reviewed in Oren, 1994). Significantly, recent evidence suggests that the apoptotic activity of p53 is essential for its tumor suppression activity (Morgenbesser et al., 1994; Pan and Griep, 1994; Symonds et al., 1994; Howes et ai., 1994). The Weizmann Instituteof Science, Rehovot 76100, Israel, and 'NCI-Frederick Research and Development Center. Frederick. MD, 21702-1201, USA. The best studied activity of p53 is the sequence specific transcriptional activation (SST) of specific genes. An increasing number of p53-target genes have been identified (Vogelstein and Kinzler, 1992). The identification of p2r'''''"''', an inhibitor of cyclin de- pendent kinases (Cdks), as a target for p53 was seminal to linking p53 to the control ofthe cell cycle, and for unravelling the pathway by which p53 induces a Gl cell growth arrest. Thus, a correlation has been es- tablished between SST by p53 and the ability to induce cell cycle arrest. On the other hand, the mechanisms by which p53 induces apoptosis, in particular with regard to the contribution of SST to apoptosis, have not been defined clearly. The study reported here sheds new light on this important issue. RESULTS AND DISCUSSION To study the mechanisms by which p53 can induce apoptosis and to define the functional domains of p53 that are essential for apoptosis, a transient transfection assay in HeLa cells was utilized. HeLa cells contain very low levels of p53 due to the expression of the human papilloma virus (HPV) E6 protein that targets p53 for degradation. Overexpression of wt p53 in HeLa cells induces an efficient apoptotic response 337