Cell death of bioenergetically compromised and transcriptionally challenged CLL lymphocytes by chlorinated ATP

Abstract

Myeloid cell leukemia-1 (MCL-1) acts as a key survival factor for chronic lymphocytic leukemia (CLL) cells. In addition, dissipation of cellular bioenergy may impose a lethal effect on these quiescent cells. Previously, in multiple myeloma cell lines we demonstrated that halogenated adenosine (8-Cl-Ado) was phosphorylated to triphosphate (8-Cl-adenosine triphosphate [ATP]), which preferentially incorporated into mRNA and inhibited RNA synthesis by premature transcription termination. Furthermore, 8-Cl-ATP accumulation was associated with a decline in cellular bioenergy. Based on these actions, we hypothesized that 8-Cl-Ado would be ideal to target CLL lymphocytes. In the present study we demonstrate that leukemic lymphocytes incubated with 8-Cl-Ado display time- and dose-dependent increase in the accumulation of 8-Cl-ATP, with a parallel depletion of the endogenous ATP pool. Inhibition of global RNA synthesis resulted in a significant decline in the expression of transcripts with a short half-life such as MCL1. Consistent to this, protein expression of MCL-1 but not B-cell lymphoma-2 (BCL-2) was decreased. Furthermore, 8-Cl-ATP induced programmed cell death, as suggested by caspases activation, cleavage of caspase 3, and PARP (poly-adenosine diphosphate [ADP]-ribose polymerase), and increased DNA fragmentation. In conclusion, 8-Cl-Ado induces apoptosis in CLL lymphocytes by targeting cellular bioenergy as well as RNA transcription and translation of key survival genes such as MCL1.

Figure 1.

Structure of 8-Cl-adenosine.

Figure 2.

Accumulation of 8-Cl-ATP and depletion of ATP pool with the treatment of 8-Cl-Ado in primary CLL cells from 7 patients (1, 3-8). The CLL lymphocytes were incubated with 10 μM 8-Cl-Ado for indicated times and PCA-extracted nucleotides were analyzed by HPLC. The accumulation of 8-Cl-ATP (▴) and the depletion of ATP (•) are plotted together for 7 hours (A) and up to 24 hours (B). Symbols indicate mean ± standard deviation.

Figure 3.

Dose-dependent accumulation of 8-Cl-ATP and its relation to the ATP pool. CLL lymphocytes from 12 patients (8, 23-33) were incubated with indicated concentration of 8-Cl-Ado for 4 hours and PCA-extracted nucleotides were analyzed by HPLC. Dose-dependent increase of 8-Cl-ATP was plotted (A) and accumulation of 8-Cl-ATP at 30 μM exogenous 8-Cl-Ado was compared with endogenous ATP pool from each patient (B); each symbol represents one patient.

Figure 4.

Effect of 8-Cl-Ado on expression of antiapoptotic proteins MCL-1 and BCL-2 in CLL lymphocytes. Immunoblot analysis was carried out for the expression of MCL-1 and BCL-2 proteins after incubating CLL lymphocytes with 10 μM 8-Cl-Ado with varying times. Actin was used as a loading control.

Figure 5.

Induction of CLL cell death by 8-Cl-Ado. Several experimental approaches were used to determine cell death of primary leukemic lymphocytes by 8-Cl-Ado. Immunoblot analysis of cleavage of procaspase 3 to active caspase 3 (A), caspase activation detection by fluorescent assay (B), time-dependent cleavage of PARP (C), and apoptotic death measured by sub G_O-G₁ DNA content (D).