Role of protein kinase C ζ isoform in Fas resistance of immature myeloid KG1a leukemic cells

Abstract

Leukemic CD34+ immature acute myeloid leukemia (AML) cells express Fas receptor but are frequently resistant to Fas agonistic reagents. Fas plays an important role in T-cell–mediated cytotoxicity, and recently it has been suggested that altered Fas signaling may contribute to drug resistance. Therefore, Fas resistance could be one of the mechanisms by which AML progenitors escape chemotherapy or T-cell–based immune intervention. However, the molecular mechanism of Fas resistance in AML cells has not been identified. Fas signaling can be interrupted at 3 mains levels: Fas clustering, alteration of death-inducing-signaling-complex (DISC) formation, and effector caspase inhibition of downstream caspase-8. This study shows that in the Fas-resistant CD34+CD38− KG1a cells, Fas agonists resulted in Fas aggregation but not in caspase-8 activation, related to a defect in DISC formation. However, pretreatment with chelerythrin, but not with calphostin C, resulted in the restoration of Fas-induced caspase-8 activation and cytotoxicity, suggesting that some atypical protein kinase C (PKC) isoforms contributed to the lack of DISC formation. Indeed, treatment with antisense oligonucleotides directed against PKCζ and enforced expression of Par-4, a negative regulator of PKCζ activity, restored Fas-induced caspase-8 activity and apoptosis. Moreover, it was found that PKCζ interacts with FADD and that PKCζ immunoextracts prepared from KG1a cells are able to phosphorylate FADD in vitro, whereas this phosphorylation is dramatically reduced in Par-4 transfectant cells. In conclusion, it is suggested that in AML cells, PKCζ plays an important role in Fas resistance by inhibiting DISC formation, possibly by phosphorylating FADD.

Functional analysis of apoptosis induction in acute myeloid leukaemia‐relevance of karyotype and clinical treatment response

“…These data are in agreement with an increasing number of publications that cast doubt on the functional relevance of the FAS/FAS-ligand system in primary AML (Wuchter et al, 1999;Lewis et al, 2000;Rozenfeld-Granot et al, 2001). Interestingly, while FAS expression in primary AML is common (Munker et al, 1995;Landowski et al, 2001) -and therefore its potential absence is an unlikely explanation for resistance in the very first step of the signalling cascade (early phase) -recent data in a leukaemic cell line have cast light on the mechanisms of FAS resistance that are in line with our findings in primary AML: de Thonel et al (2001) reported that, in immature myeloid KG1 cells, resistance to FAS-ligand induced cytotoxicity was explained by a protein kinase C zeta-mediated pathological phosphorylation pattern of FADD (FAS-associated death domain protein) -a crucial member of DISC (death inducing signalling complex) that is normally recruited to the intracellular domains of the ligated FAS receptors. However, at this stage DISC is incomplete and is unable to recruit and activate the 'regulator' caspase 8, which in turn results in the termination of the apoptosis-inducing signalling cascade.…”

ExoS of Pseudomonas aeruginosa induces apoptosis through a Fas receptor/caspase 8-independent pathway in HeLa cells

“…Dephosphorylated FADD interacts with caspase 8 and increases the sensitivity of cells to FasR-mediated apoptosis (Curtin and Cotter, 2004). Overexpression of another FADD kinase, PKCξ can inhibit the interaction between FADD and caspase 8 (DeThonel et al, 2001). Inhibition of Erk activity has also been demonstrated to reduce FADD phosphorylation and increase sensitivity to FasR-mediated apoptosis (Meng et al, 2003).…”

FADD and its Phosphorylation

“…As similar finding has been reported by Tschopp's group, they identified a 130-kD kinase that induces FADD phosphorylation and inhibits Fasmediated Jun NH2-terminal kinase activation, which is designated Fas-interacting serine/threonine kinase/homeodomain-interacting protein kinase (FIST/HIPK3) as a novel Fasinteracting protein (16). Moreover, it was also found that protein kinase C (PKC)z interacts with FADD in vivo and that PKCz immunoextracts prepared from KG1a cells are able to directly phosphorylate FADD in vitro (17). PKC exerts a protective function against Fas death pathway and contribute to the lack of DISC formation.…”