The Signaling Mechanisms That Control Cell Survival Are Differentially-Regulated during the Progression of B-CLL.

Delineating the molecular mechanisms that trigger the development of leukemic cells is of fundamental importance for the creation of therapeutic interventions to specifically target these rogue cells. B cell-chronic lymphocyte leukemia (B-CLL) is characterized by an accumulation of long-lived B-lineage cells with the distinctive phenotype: CD19hi, CD5+, CD23+, IgMlo, which are refractory to apoptosis and have undergone cell cycle arrest in the G0/G1 phase. The signaling events that control the development of B-CLL are not well defined. To address this, we generated a novel cellular system in which the spontaneous generation of B-CLL-like cells is achieved by stably expressing a plasmid-encoding dominant negative PKCα (PKCα-KR) in mouse FL-derived hematopoietic progenitor cells (HPCs) and then culturing these cells in a B cell generation system. The resultant cells bear hallmark characteristics of human B-CLL cells at the level of: (i) phenotype - CD19hi CD5+ CD23+ IgMlo; (ii) cell cycle phase - halted in G0/G1, and; (iii) resistance to apoptosis. Therefore, this system provides us with a unique opportunity to investigate the signaling events that control the initiation of B-CLL. In addition, we isolated a mouse B-CLL (mB-CLL) cell line during long-term culture of PKCα-KR-HPCs, which proliferates in the absence of growth factors or stroma, thus allowing us to investigate the signaling mechanisms that may be important for the expansion of B-CLL. In an effort to identify the key signaling pathways that are responsible for maintaining survival of B-CLL cells, we treated MIEV (vector control)-, PKCα-KR-HPCs and mB-CLL cells with pharmacological inhibitors against ERK-MAPK- and PI3K-mediated pathways, as these pathways have been implicated in mediating the survival of human B-CLL cells. We assessed the proportion of cells that were induced to undergo apoptosis (Annexin V binding) relative to control (DMSO-treated) cells. We found that treatment of MIEV- and PKCα-KR-HPCs with PI3K inhibitors (LY294002/ wortmannin: 1–50 μM) induced a significant increase in apoptosis compared with the control cells (MIEV: 4.7±0.14; PKCα-KR: 6.8±1.18 at 30 μM LY294002), while the level of apoptosis in these cells was unaffected upon treatment with MEK inhibitors (PD98059/U0126: 1–20 μM) compared with controls (MIEV: 0.9±0.1; PKCα-KR: 0.7±0.01 at 10 μM U0126). Conversely, treatment of mB-CLL cells with MEK1 inhibitors induced an increase in apoptosis compared with the control cells (7.7±4.21 at 10 μM U0126), while the level of apoptosis in these cells was unaffected upon treatment with PI3K inhibitors (1.6±0.84 at 30 μM LY294002). Our results suggest that the signaling pathways responsible for maintaining cell survival are differentially-regulated during the initiation vs. expansion phases of B-CLL. Additional studies of the phosphorylation/ activation status of ERK1/ERK2 in MIEV-, PKCα-KR-HPCs and mB-CLL cells revealed that while ERK1/ERK2 are unphosphorylated/ inactive in MIEV-HPCs, ERK1 only is constitutively phosphorylated in PKCα-KR-HPCs, while mB-CLL cells contain constitutively phosphorylated ERK1 and ERK2. The differential phosphorylation of ERK1/ERK2 may, at least in part, explain the differential regulation of survival in quiescent and proliferating B-CLL cells. Indeed, the activation of ERK-MAPK can occur in a PI3K-dependent or -independent fashion, depending on the cell type. Collectively, these studies suggest that PI3K- and ERK-MAPK signaling pathways are differentially-regulated to induce survival during the progression of B-CLL cells.