Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G 2 phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G 2 checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G 2 checkpoint inhibitors. It inhibited the G 2 checkpoint with an IC 50 of 8 M and showed moderate cytotoxicity (IC 50 ‫؍‬ 25 M) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC 50 ‫؍‬ 3 M) and Chk2 (IC 50 ‫؍‬ 3.5 M) but not ataxiatelangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.

Acute lymphoblastic leukaemia (ALL) is the most common paediatric malignancy and, although current therapy is widely effective, relapse remains a significant clinical problem for which new treatment strategies are required. The ligation of Toll‐like receptors (TLR) on antigen‐presenting cells stimulates the generation of strong T‐cell helper type 1 (Th1) adaptive immune responses. Although TLR9 ligation has been shown to enhance immunogenicity of a number of leukaemia cell types, there have been few reports of the effects mediated through other TLR. In this study we analysed both the expression of TLR by B‐cell precursor ALL cell lines and the effects of individual TLR ligation on the ability of ALL cells to stimulate allogeneic T cells. While ligation of TLR2, TLR 7 and TLR9 led to detectable changes in ALL costimulatory molecule expression, only TLR2 and TLR9 stimulation influenced T‐cell responses. The TLR2 ligand Pam3CysSerLys4 provoked the most significant changes in T‐cell response, dramatically augmenting interferon‐γ production. These results suggest that TLR ligands, in addition to TLR9 agonists, may provide a strategy to enhance the generation of anti‐ALL immune activity by skewing responding T cells towards a Th1 response.

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