Pathogenesis of primary pulmonary mucosa-associated lymphoid tumors (MALT) and lymphomas remains unknown, but is thought to involve antigen stimulation leading to immortalization, and then subsequent transformation into a more aggressive phenotype. HOXB4 is a homeobox transcription factor that is known to clonally expand hematopoietic stem cells. We have previously shown that HOXB4 is also an apoptotic agent in the pre-B acute lymphoblastic leukemia cell line REH via the extrinsic pathway that activates caspase-8 and caspase-3 leading to DNA disintegration. We now demonstrate that despite the fact that HOXB4 is homologous to the Drosophila gene Deformed, HOXB4-induced apoptosis in REH cells is not mediated via the intrinsic pathway cascade that involves the second mitochondria-derived activator of caspase (SMAC) and caspase-9.
The HOXB4 cDNA was ligated into the mammalian expression vector pLNCX2 and REH cells were transfected with the plasmid construct using lipofection. Constitutive expression was confirmed by immunocytochemistry.
The REH cells transfected with control plasmid began to grow exponentially 4 weeks after transfection. Cells transfected with pLNCX2-HoxB4 declined in number and died. Immunocytochemical staining with anti-SMAC antibody was negative at 2, 3, and 4 weeks after transfection. Similarly, no caspase-9 activity was detected at 2, 3, or 4 weeks. Parallel experiments re-demonstrated that FLASH (FLICE-associated huge protein) expression was expressed faintly at 2 weeks, and more strongly at subsequent time points. Caspase-8 and caspase-3 were faintly expressed at 3 weeks and more strongly thereafter.
Despite the sequence homology between the human protein HOXB4 and the Drosophila protein Deformed, the pathway of apoptosis induced by these proteins is different. Deformed activates Reaper, but HOXB4 does not activate SMAC (a homolog of Reaper). Apoptotic pathways have diverged during the course of evolution.
Our results provide further mechanistic insight into the apoptosis induced by HOXB4 in cell lines derived from hematological malignancies. Since HOXB4 causes clonal proliferation of hematopoietic stem cells and apoptosis of hematological malignant cell lines, targeting this pathway may be of therapeutic benefit.
Kamal Mubarak, None.