Evidence that the apoptotic actions of etoposide are independent of c-Jun/activating protein-1-mediated transregulation

We recently demonstrated that physiological induction of apoptosis by cytot oxic sphingolipid messengers proceeds via activating protein-1 (AP1)-depend ent and AP1-independent mechanisms in U937 human monoblastic leukemia cells . Here we examine involvement of the stress-activated protein kinase (SAPK) cascade and API in the initiation of apoptosis in U937 cells by podaphyllo toxin-derived inhibitors of topoisomerase II. Induction of apoptotic cell d eath and DNA damage by treatment of U937 cells with etoposide (100 mu M) wa s associated with phosphorylation and activation of the c-Jun NH2-terminal kinase (JNK1) SAPK enzymes p46 and p54-JNK2 and transient increases in expr ession of the transcription factor c-Jun, a primary JNK substrate. These re sponses were accompanied by a modest, but sustained, recruitment of the mit ogen-activated protein kinases p42-extracellular signal receptor-activated kinase (ERK)1 and p44-extracellular signal receptor-activated kinase 2. The capacity of etoposide to promote double-stranded DNA degradation and cell death was unaffected by manipulations that interfere with SAPK signaling ou tflow through c-Jun/AP1, including: 1) pharmacological inhibition of AP1 ac tivity by diferuloylmethane and 2) molecular ablation of normal c-Jun funct ion by the Jun dominant-negative mutant TAM-67. Cytotoxicity of the structu rally related compound teniposide was similarly unaffected. In parallel tri als, the lethal actions of ceramide (but not of sphingosine) were markedly diminished by pretreatment with diferuloylmethane or expression of TAM-67, confirming the effectiveness of these interventions in suppression of SAPK/ AP1-dependent apoptosis. The involvement of API in the proapoptotic actions of other inhibitors of topoisomerase II activity was also evaluated. Induc tion of cell death by the anthracyclines daunorubicin, daunorubicin, and id arubicin was found to be insensitive to pretreatment with diferuloylmethane or expression of TAM-67. Collectively, the present data indicate that indu ction of apoptosis by etoposide and related inhibitors of topoisomerase II is mediated through a cell death pathway that does not require SAPK-depende nt recruitment of AP1. These findings additionally suggest that activation of the SAPK represents a consequence, rather than an underlying cause, of e toposide-induced apoptosis in myeloid leukemia cells.