ABSTRACT
BACKGROUND: Multiple myeloma is an incurable disease requiring the development of effective therapies which can be used clinically. We have elucidated the potential for manipulating the cAMP signaling pathway as a target for inhibiting the growth of multiple myeloma cells. METHODS: As a model system, we primarily used the murine multiple myeloma cell line MOPC315 which can be grown both in vivo and in vitro. Human multiple myeloma cell lines U266, INA-6 and the B-cell precursor acute lymphoblastic leukemia cell line Reh were used only for in vitro studies. Cell death was assessed by flow cytometry and western blot analysis after treatment with cAMP elevating agents (forskolin, prostaglandin E2 and rolipram) and cAMP analogs. We followed tumor growth in vivo after forskolin treatment by imaging DsRed-labelled MOPC315 cells transplanted subcutaneously in BALB/c nude mice. RESULTS: In contrast to the effect on Reh cells, 50 µM forskolin more than tripled the death of MOPC315 cells after 24 h in vitro. Forskolin induced cell death to a similar extent in the human myeloma cell lines U266 and INA-6. cAMP-mediated cell death had all the typical hallmarks of apoptosis, including changes in the mitochondrial membrane potential and cleavage of caspase 3, caspase 9 and PARP. Forskolin also inhibited the growth of multiple myeloma cells in a mouse model in vivo. CONCLUSIONS: Elevation of intracellular levels of cAMP kills multiple myeloma cells in vitro and inhibits development of multiple myeloma in vivo. This strongly suggests that compounds activating the cAMP signaling pathway may be useful in the field of multiple myeloma.
Subject(s)
Apoptosis/physiology , Cyclic AMP/metabolism , Disease Models, Animal , Multiple Myeloma/metabolism , Animals , Apoptosis/drug effects , Blotting, Western , Caspase 3/metabolism , Caspase 9/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Colforsin/pharmacology , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Dinoprostone/pharmacology , Dose-Response Relationship, Drug , Humans , Intracellular Space/drug effects , Intracellular Space/metabolism , Membrane Potential, Mitochondrial/drug effects , Mice , Mice, Inbred BALB C , Mice, Nude , Multiple Myeloma/drug therapy , Multiple Myeloma/pathology , Poly(ADP-ribose) Polymerases/metabolism , Thionucleotides/pharmacology , Time Factors , Tumor Burden/drug effectsABSTRACT
It is desirable to have an early and sensitive detection marker of autoimmune disease in intact animals. Nuclear factor (NF)-kappaB is a transcription factor that is associated with inflammatory responses and immune disorders. Previously, we demonstrated that so-called idiotypic-driven T-B cell collaboration in mice doubly transgenic for paired immunoglobulin and T cell receptor transgenes resulted in a systemic autoimmune disease with systemic lupus erythematosus-like features. Here, we investigated NF-kappaB activation by including an NF-kappaB-responsive luciferase reporter transgene in this animal model. Triply transgenic mice developed bioluminescence signals from diseased organs before onset of clinical symptoms and autoantibody production, and light emissions correlated with disease progression. Signals were obtained from secondary lymphoid organs, inflamed intestines, skin lesions, and arthritic joints. Moreover, bioluminescence imaging and immunohistochemistry demonstrated that a minority of mice suffered from an autoimmune disease of the small intestine, in which light emissions correlated with antibodies against tissue transglutaminase and gliadin. Detection of luciferase by immunohistochemistry revealed NF-kappaB activation in collaborating B and T cells, as well as in macrophages. These results demonstrate that bioluminescent in vivo imaging of NF-kappaB activation can be used for early and sensitive detection of autoimmune disease in an experimental mouse model, offering new possibilities for the evaluation of anti-inflammatory drugs.
