PURPOSE: Pathogenesis of primary pulmonary mucosa-associated lymphoid tumors (MALT) and lymphomas remains unknown, but it is thought to involve antigen stimulation leading to immortalization, and then subsequent transformation into a more aggressive phenotype. We have previously shown that HOXB4 can induce apoptosis in malignant cell lines through a caspase-mediated pathway involving FLASH and caspase-8, with caspase-3 as the effector caspase. We now demonstrate that HOXB4 induces apoptosis through an alternate novel pathway.
METHODS: HOXB4 protein was purified using a Ni-NTA purification system and confirmed with a Western blot. HOXB4 protein was added, at varying concentrations up to 1 mcg/ml, to cell cultures of several malignant cell lines including WSU-DLCL2, WSU-FSCCL and REH. Initial growth curve were obtained demonstrating cell death with the addition of HOXB4. Western blots demonstrated increased expression of FLASH, caspase-3, and caspase-8. Cell lines treated with HOXB4 were then individually treated with inhibitors to each of the caspases, growth curves were plotted, and Western blots obtained. Cells treated with the caspase-3 inhibitor exhibited cell growth. However, cells treated with caspase-8 inhibitor still underwent apoptosis.
RESULTS: HOXB4 induced apoptosis has previously been shown to increase the expression of FLASH, caspase-3, and caspase-8, implicating a role for caspase-8 in apoptosis. Our data shows that cells treated with caspase-8 inhibitor fail to demonstrate an arrest of apoptosis.
CONCLUSION: HOXB4 induced apoptosis has been implicated through a pathway consisting of FLASH, caspase-8 and caspase-3. The failure of apoptosis to be abolished by a caspase-8 inhibitor indicates a role for a second pathway. We hypothesize that FLASH may activate a second pathway via activation of caspase-10, with subsequent activation of caspase-3. Alternatively in Drosophila, the homolog of HOXB4, called Deformed, activates the homolog of SMAC, called Reaper. SMAC, a second mitochondria-derived activator, or caspase, has been implicated to function through a caspase-9 pathway in humans. This is presently under investigation.
CLINICAL IMPLICATIONS: HOXB4 induced apoptosis may be derived from several distinct pathways. This provides a potential therapeutic advatange against rapidly dividing cancer cells.
DISCLOSURE: Russell Blair, None.