The Expression of Fas Antigen (CD 95) on Clonogenic Acute Myeloid Leukemia Cells Correlates with Their In Vitro Sensitivity to Farnesyltransferase Inhibitor (FTI; Zarnestra TM).

Abstract

The farnesyl transferase inhibitors (FTIs) have demonstrated significant antitumor activities in vitro and in vivo due to their ability to inhibit of Ras- oncogen farnesylation. Besides Ras, there are many other farnesylated proteins in cancer pathways which are potential targets for FTI. In this study, the in vitro effect of the FTI (Zarnestra, Z: provided kindly by Johnson & Johnson Pharmaceutical Research and Development) on clonogenic leukemia cells, expressing CD 34 and Fas were examined. All evaluated patients fullfilled the diagnostic criteria for high-risk AML and demonstrated the CFU-L in vitro growth, or/and expression of CD34 antigen on the myeloblast cells. Bone marrow mononuclear cells from 30 AML patients (M0=3, M1=15, M2=5, M3=1, M4=5, M5=1) were isolated and were plated in serum-free methylcellulose medium with or without growth factors (Methocult, Stem Cell Technologies, Vancouver, Canada) for the 14 days in vitro cultures. The expression of Fas (CD 95) on AML cells were also studied (anti-Fas APO-1, FITC- CD 95, Becton Dickinson, USA) by flow cytometric analysis using Cell Quest Software (Becton Dickinson, USA). MNC incubated with increasing concentrations of Z for 24, 48 and 72 days were analysed for apoptosis (staining for annexin-possitive cells, Annexin V Kit, Pharmingen, USA).The percentage of Fas positivity was variable (range 20–94%) and was related to the morphologic FAB classification with the highest expression in AML-M1 patients (range 28–94, mean 72%, p<0.01).. No relationship was found between Fas and the CD 34 antigen expression. The in vitro CFU-L growth in the presence of cytokines was observed in 19/30 patients (63%) and 37% patients showed the spontaneous CFU-L or no colony growth of blast cells in the in vitro culture. In the majority of evaluated AML cases, a time and dose dependent proapoptotic in vitro effect of Z was found. The above effect was the most frequent in AML-M1 patients (11/23). Within Z concentration between 20–300 nM, the significant increase of apoptosis cell number (mean 60% AN+/PI− cells) was observed. After 24, 48 and 72 h, incubation with Z the number of patients whose cells underwent apoptosis were 49%, 13% and 8% respectively. The Z induced apoptosis showed significant correlation with Fas expression on AML cells.(P<0.01). The myeloblast apoptotic was not detected in the patients (40 %) demonstrated spontaneous CFU-L growth and the lack of Fas expression on blast cells (even after 120 h Z. exposure).Our observations suggest that Z induced apoptosis of AML cells might be mediated at least in part by Fas, expressed on primitive, clonogenic leukemic progenitor cells. Induction of apoptosis throught the Fas pathway might be one of the new approach for effective leukemia therapy.