Abstract:
Background: Almost all mammalian cells contain an endogenous clock which ensures appropriate timing of a plethora of cellular processes including proliferation and inflammatory responses. Aberrant proliferation and inflammation are hallmarks of leukaemia. Abnormal expression of Brain and Muscle Arnt-Like protein 1 (BMAL1), a core mammalian clock protein, has recently been reported in leukaemic cells, but its contribution to the leukaemic phenotype is unclear. Aim: To determine if aberrant BMAL1 expression in leukaemic cell lines affect cell proliferation and inflammation. Methods: CMY, Jurkat, and Meg-01 cell lines were each transfected with BMAL1-targeting siRNA (siBMAL1) or a non-targeting control siRNA (siCntrl) for transient knockdown or a plasmid encoding BMAL1 or GFP (control) for ectopic BMAL1 expression. Transfections were validated using RT-PCR and Western blot analysis. The clock was “re-set” by serum starving. Cell proliferation was measured by BrdU incorporation assay and cell death by lactate dehydrogenase release. Affected cell cycle regulators and pro-inflammatory cytokines were identified using RT-qPCR. Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), was measured using Western blot analysis. Results: Ectopic BMAL1 expression increased the rate of proliferation in all cell lines, heightened the expression of CCND1 and reduced the expression of CDKN1A to support cell cycle progression. Knockdown of BMAL1 reduced the expression of the pro-inflammatory cytokine, TNF, in Jurkat and Meg-01 cell lines, whereas ectopic BMAL1 expression resulted in higher TNF expression in Meg-01 cells. BMAL1 was involved in regulating NF-κB activation in both Jurkat and Meg-01 cells. In Jurkat cells BMAL1 knockdown resulted in a significant reduction in level of activated NF-κB and in Meg-01 cells ectopic BMAL1 expression led to a significant increase in the level of activated NF-κB. Discussion: These results imply that increased BMAL1 expression supports the leukaemic phenotype, with elevated proliferation rate and expression of inflammatory cytokines observed after ectopic BMAL expression. Part of BMAL1’s action may be influenced by NF-κB activity. Maintenance of the circadian clock may therefore be critical in suppressing leukaemia, suggesting the molecular clock may provide a novel target for therapy.