Adenine deoxynucleotides fludarabine and cladribine induce apoptosis in a CD95/Fas receptor, FADD and caspase-8-independent manner by activation of the mitochondrial cell death pathway

Abstract

The adenine deoxynucleosides cladribine (2CdA) and fludarabine (FAraA) are DNA-damaging agents that interfere with DNA repair and induce apoptosis in nonproliferating lymphoid cells. Although both drugs are clinically used for the treatment of indolent lymphoproliferative diseases, the pathways of apoptosis induction remain largely unknown. In the present work, we demonstrate that both drugs induce apoptosis independently of death receptor signaling but activate the mitochondrial cell death pathway. To dissect the signaling pathways, we employed Jurkat cells either deficient for FADD or caspase-8 or overexpressing Bcl-2. In Bcl-2 overexpressing cells, apoptosis and cytochrome c release were blocked whereas processing of caspase-9, -3 and -8 was partially inhibited. In contrast, neither the deficiency of FADD or caspase-8 nor the interference with death receptor signaling by neutralizing anti-CD95/Fas antibodies affected cell death. Inhibitor experiments revealed that caspase-8 is processed by caspase-3-like caspases. Moreover, cytochrome c release and processing of caspase-9 and -3 occurred to an equal extent in wild-type FADD -/-and caspase-8 -/-Jurkat cells. Likewise, apoptosis induction by cladribine or fludarabine was not hampered upon inhibition of caspase-8 in MOLT-3 and MOLT-4 cells or overexpression of a dominantnegative FADD mutant in BJAB cells. Thus, we conclude that apoptosis induced by nucleoside analogues is independent from death receptor signaling as well as from a proposed direct effect on APAF-1, but rather follows the mitochondrial signaling pathway of cytochrome c release and subsequent processing of caspase-9 and -3.

Arsenic trioxide induces regulated, death receptor-independent cell death through a Bcl-2-controlled pathway

“…Furthermore, As 2 O 3 -mediated cell death was largely undisturbed in FADD-deficient cells and cells trans- Figure 9 and 10, might be related to a putative role of FADD in the death-receptor-independent cleavage of initiator caspases such as caspase-8 (Chandra et al, 2004). In line with this, we recently observed that FADD deficiency slightly reduces the amount of taxol-or fludarabine-induced cell death in a death-receptor-independent fashion von Haefen et al, 2003;Klo¨pfer et al, 2004). Our data, therefore, demonstrate that neither CD95 ligation nor a functional FADD or caspase-8 is necessary for As 2 O 3 -induced cell death.…”

“…To address this and the To test this, Jurkat A3 cells were cultured with As 2 O 3 (6 mM) in the presence or absence of a soluble, anti-CD95 antibody (IgG 2b , clone SM1/23, 5 mg/ml), which was reported to block the induction of apoptosis mediated by anti-CD95 ligation (Klo¨pfer et al, 2004). To validate the effect of the blocking anti-CD95 antibody (clone SM1/23) in our system, we cultured Jurkat A3 cells with plastic-bound, agonistic anti-CD95 antibody (clone 2R2, IgG 3 , 5 mg/ml) in the absence or presence of the soluble blocking antibody (clone SM1/23) for 48 h. Blocking the CD95/Fas receptor reduced total cell death induced by the agonistic anti-CD95 antibody from 37.971.1 to 8.470.9% (Figure 11).…”

Flavopiridol causes early mitochondrial damage in chronic lymphocytic leukemia cells with impaired oxygen consumption and mobilization of intracellular calcium

“…52 Our findings are in contrast to several reports indicating the Bcl-2 and/or Bcl-X L independence of flavopiridol, 3,53 and also suggest there is not a direct effect of flavopiridol on APAF-1/ apoptosome assembly, as Bcl-2 family proteins likely do not affect this process. 28 It is possible that the discrepancy in the involvement of Bcl-2 in CLL versus lung carcinoma cells 3 may be because in CLL cells caspase-8 appears to play a minor role, with release of cytochrome c in the cytosolic fraction. In contrast, flavopiridol activity in lung carcinoma cells was found to be caspase-8 dependent without cytochrome c release.…”

“…27,28 To further understand the pathway of flavopiridol-mediated cell death, we used the 697 lymphoblastic cell line overexpressing Bcl-2. Flow cytometry was conducted on flavopiridol-treated 697 cells to assess both annexin binding and mitochondrial effects.…”

TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway

“…Anticancer drugs like nucleoside analogs (Klo¨pfer et al, 2004), anthracyclines , or taxanes (von Haefen et al, 2003) trigger apoptotic cell death through the death receptor-independent induction of the mitochondrial apoptosis pathway. This occurs through a Bcl-2-dependent sensitive signaling event that involves multidomain proapoptotic Bcl-2 homologs, that is, Bax and Bak.…”