Abstract
Cell proliferation is dependent upon iron, and numerous studies have shown that iron limitation arrests cells in the G1 phase of the cell cycle. A recent study of the molecular basis of these observations (Richardson, et al. Blood2007;109:4045) examined the ability of iron chelators to inhibit cell proliferation and to induce apoptosis, focusing on the role of iron chelation on cyclin D1. Cyclin D1 assembles with cdk-4 or cdk-6, generating an active holo-enzyme that catalyzes a rate limiting step in G1/S progression. This complex phosphorylates substrates, including the retinoblastoma protein, which regulate S phase entrance. Richardson’s group demonstrated that the G1/S arrest after Fe depletion is mediated, in part, by a decrease in cyclin D1 via ubiquitin-independent proteasomal degradation. Studies looking specifically at mantle cell lymphoma cell lines, however, have not yet been reported. Mantle Cell lymphoma is an interesting target for potential iron chelation as it is associated with a balanced translocation (t11;14) which leads to upregulation of BCL1 and to the constitutive overproduction of cyclin D1. We studied five different cell lines - JeKo (Mantle Cell Lymphoma), BL-41 (Burkitt Cell Lymphoma), DG-75 (Burkitt Cell Lymphoma), SUDHL-6 (Diffuse Large B cell Lymphoma) and EBV-immortalized lymphocytes from normal controls - and incubated them with four different iron chelators - deferoxamine (DFO), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), Pyridoxal Isonicotinoyl Hydrazone (PIH), and Salicylaldehyde Isocotinoyl Hydrazone (SIH). We then measured and compared cell cycle proliferation (using the Cellometer Auto T4, an instrument that measures cell count, cell viability, and cell size) and the rate of apoptosis (via propidium iodide FACS analysis). At 24 hours incubation, the mantle cell lymphoma lines showed significantly increased rates of apoptosis compared with non-chelated mantle cell controls (5% vs. 48%, p=0.04). The diffuse large B cell lymphoma line showed a lesser increase in apoptosis that did not reach statistical significant (6.5% vs. 14%, p=0.07), while the Burkitt’s lymphoma lines and the EBV immortalized lymphocytes showed no significant difference (BL-41, 3.4% vs. 4.1%, p=0.50; DG-75, 6% vs. 5.9%, p=0.99; EBV lymphocytes, 12.5% vs. 12.7%, p=0.96). At 72 hours of incubation with chelators, the EBV lymphocytes showed increased apoptosis compared to untreated controls (2.5% vs. 44.5%, p=0.002), while the apoptotic rate increased in the diffuse large B cell lymphoma line (3.8% vs. 48%, p=0.001) and even more dramatically in the mantle cell lymphoma line (1.5% vs. 64%, p=0.0006). The two Burkitt’s lymphoma lines were affected to a lesser degree at 72 hours by the presence of iron chelators (BL-41, 0.9% vs, 3.9%, p=0.02; DG-75, 5.5% vs. 8.9%, p=0.11). Although iron chelation, especially at longer incubation times, did affect all cell lines to various degrees, the chelator-mediated effects do appear to be specific for cell type, with mantle cell lymphoma cells displaying higher rates of apoptosis compared with other lymphomas and normal lymphocytes. These initial results will now be followed by examination of cyclin D1 expression after iron chelation. If overexpression of cyclin D1 in mantle cell lymphoma releases cells from their normal controls and acts as an oncogene, then a decrease in cyclin D1 levels via iron chelation could be added to the therapeutic armamentarium of mantle cell lymphoma.
The anaphase-promoting complex/cyclosome: a new promising target in diffuse large B-cell lymphoma and mantle cell lymphoma