We identified here that pentamidine is a novel inhibitor of hMRE11 nuclease (Fig. ). Pentamidine also repressed ATM kinase activity in vitro (Fig. ). Low concentrations of pentamidine enhanced radio-sensitivity of HeLa cells (Fig. ), suggesting that pentamidine might influence other factors in DNA damage response. Pentamidine reduced IR-induced focus formation of DDR proteins such as γ-H2AX and NBS1, although a remarkable effect on ATM-dependent phosphorylations was not observed (Fig. and ). Pentamidine also repressed HR activity but not NHEJ (Fig ). Furthemore, pentamidine reduced acetylation of histone H2A (Fig. ), which is suggested to influence IR-induced focus formation and HR repair. H2A mutated at acetylation sites decreased both IR-induced focus formation of NBS1 and HR activity (Fig. ). Knockdown of Tip60, which is known as a histone acetyl transferase, resulted in effects on H2A acetylation and HR activity similarly to that seen upon pentamidine treatment (Fig. ). Moreover, pentamidine reduced HAT activity of Tip60 in vitro and Tip60-dependent acetylation of p53 in vivo (Fig. ), suggesting that the inhibitory effects of pentamidine on DDR may be mediated, at least in part, by inhibitin of Tip60. However, the effects of Tip60-knockdown were less pronounced than those by pentamidine treatment (Fig. ). Taken together, pentamidine might influence IR-induced DNA damage response through the inhibitory effect on not only Tip60, but also hMRE11 and ATM.
Pentamidine is known to be as one of the most effective agents against Pneumocystis carinii. Pentamidin is capable of binding to the minor groove of double-strand DNA and inhibits protein synthesis, DNA synthesis and the activity of endo-exonuclease in Pneumocystis carinii [24
]. Pentamidine also repressed Saccharomyces cerevisiae RNC1/TRM2 endo-exonuclease, which displayed 5'->3' exonuclease activity on double-strand DNA and endonuclease activity on single-strand DNA in vitro [35
]. Recently, it was reported that pentamidine also inhibited human endo-exonuclease activity in vitro [26
]. These observations suggest that pentamidine might show an inhibitory effect on other nuclease such as hMRE11 in mammalian cells. Indeed, our results demonstrate that addition of 2 mM pentamidine abolished hMRE11 nucelase activity in vitro (Fig. ), suggesting that pentamidine could be used as an effective inhibitor in human cells. We also showed that pentamidine reduced ATM kinase acitivity. As ATM requires MRN complex for optimal activation [27
], the effect of pentamidine on ATM kinase activity might occur via repression of hMRE11 nuclease by pentamidine. Future experiments will be aimed at clarifying whether pentamidine reduces ATM kinase activity directly.
Recently, several reports suggest a tight relationship between histone acetylation and DNA damage response. Tamburini and Tyler showed that acetylation of histone H3 and H4 increased at HO endonuclease-restricted DSB sites in yeasts by ChIP assay and also showed that the histone acetyltransferases Gcn5 and Esa1 were recruited at these DSB sites [36
]. Downs and his colleagues also reported that Arp4, a subunit of NuA4 HAT complex interacts with phosphorylated H2A directly and Arp4 was recruited to HO-related DSB sites [37
]. Further, human homologous of NuA4 HAT, Tip60 interact with hitstone H2AX and could acetylated it, and also showed that acetylation of histone H2AX increased in response to DSB damage [33
]. Furthermore, the strains of S. pombe or S. cerevisieae expressing mutated H3 or H4 at acetylation sites or HAT-deficient yeasts increased the sensitivity to DNA damaging agents in yeast and dominant-negative Tip60-expressing HeLa cells showed the delay of DSB repair following IR by comet assay [32
]. These reports suggest that acetylation of histon is important for DNA damage response and, perhaps, DNA repair. This is consistent with our results showing that the H2A-K5/9R (acetylation site mutant)-expressing cells showed a decrease in HR repair activity (Fig. ) and Tip60-knockdown cells also reduced the HR activity (Fig. ). We also indicated that pentamidine treatment and the H2A (K5/9R)-expressing cells repressed both focus formation and HR (Figs , and ), suggesting that focus formation of DNA damage-related factors such as NBS1 and γ-H2AX contributes to HR repair pathway through histone acetylation. In fact, a partner protein of Tip60, TRAPP-knockout mouse cells showed a decrease in accumulation of histone acetylation at DSB sites, IR-induced focus formation such as BRCA1 or Rad51 and HR activity [31
]. Moreover, we reported that the defect of MRE11 focus formation in mutated NBS1-expressing cells leads to reduction of HR activity [20
]. Thus, focus formation of DNA damage response factors could be closely related with homologous recombination repair. Moreover, pentamidine might influence HR repair through repression of histone acetylation, related with IR-induced focus formation.
So far, it was reported that the bisbenzamidine derivative, pentamidine showed the growth repression against several tumor cells such as MCF7 and HeLa cells via repression of endo-exonuclease activity [26
]. The paper also showed that normal human diploid fibroblats are not sensitive to pentamidine and pentamidine displayed an inhibitory effect on tumor growth in mouse model. These facts suggest that pentamidine might be an effective anti-tumor reagent. Moreover, pentamidine has a synergistic inhibitory effect to tumor growth with mitomycin C or other anti-growth reagent, suggesting that pentamidine may show a synergistic action with ionizing radiation [38
]. In fact, Fig. shows the expected effect with irradiation of γ-ray, and hence lower concentration (0.05 mM) of pentamidine might be an effective sensitizer for radiotherapy. However, much higher concentration of pentamidine was required for inhibitory effects on Mre11 nuclease and ATM kinase activity in vitro (Fig. ), while middle range concentration of pentamidine reduced Tip60 activity in vitro and in vivo (Fig. ). These results suggest that the effect of pentamidine (low concentration) as a radiation sensitizer could be due to an inhibitory effect on Tip60. In order to use pentamidine or other deamidine analogues with radiotherapy or chemotherapy, we need to clarify all the targets of these reagents in DNA damage response and the underlying mechanisms in detail.