L and Methods). It really is noteworthy that, except for tiny populations, mature monocytes, DCs

L and Methods). It really is noteworthy that, except for tiny populations, mature monocytes, DCs and macrophages do not proliferate in vivo [10]. In truth, as revealed by flow cytometry (Fig. 1B), all 3 cell forms have been arrested in G1 and maintained in vitro in a nonproliferative state. Namodenoson Technical Information remedy with all the methylating anticancer drug TMZ resulted within a substantial induction of apoptosis in monocytes, even though DCs and macrophages had been resistant to this agent (Fig. 1C). DNA methylation following TMZ exposure yields distinctive lesions with N7-methylguanine to become by far the most frequent one particular to appear [2]. Despite the fact that O6-methylguanine was shown to be responsible for the toxicity of methylating agents in proliferating cells [4], this lesion is unlikely to become vital in our cellular system because the cells usually do not proliferate. Also, monocytes express a high degree of MGMT when compared with macrophages and DCs and are therefore capable to get rid of O6-methylguanine from DNA [11]. N7MeG, N3-MeG and the toxic lesion N3-MeA are repaired by BER. This prompted us to decide the expression degree of BER enzymes and we located, equivalent to our GS-626510 site preceding observation [6], that monocytes express XRCC1 and ligase IIIa at an extremely low (nondetectable) level (Fig. 2A,B). Expression of those BER elements was evoked, on the other hand, following cytokine remedy and was absolutely restored on day 6 (Fig. 2A) and day 3 (Fig. 2B) in the course of maturation of monocytes into DCs and macrophages, respectively. We also observed that monocytes lack PARP-1, an important aspect of BER and SSB repair, and that expression of PARP-1 was restored concomitant with XRCC1 and ligase IIIa throughout the maturation of monocytes to DCs and macrophages (Fig. 2A,B). Next we wished to decide no matter if treatment with TMZ has an impact on the expression in the repair proteins. TMZ remedy resulted in decreased PARP-1 levels in DCs and macrophages. Surprisingly, in addition, it resulted in an increase within the level of XRCC1 and ligase IIIa in monocytes (Fig. 2C). Therefore it seems that TMZ remedy provokes upregulation of XRCC1 and ligase IIIa, butPLoS 1 | plosone.orgFigure 1. Differentiation of monocytes into DCs and macrophages and their killing response. (A) Images of monocytes andMonocyte Response to TemozolomideDCs and macrophages derived from them by cytokine stimulated maturation. Blue, nuclear staining with ToPro3; red staining on monocytes and macrophages, CD14 surface marker, which is not expressed on macrophages. (B) Representative histograms of monocytes, DCs and macrophages stained with propidium iodide and measured by flow cytometry. C, non-treated; TMZ, treated with 0.6 mM TMZ and measured 72 h following therapy. (C) Evaluation of apoptosis 72 h following 0.six mM TMZ treatment by quantification on the subG1 fraction of cells by flow cytometry. (p,0.01, p,0.001, t-test comparing monocytes with DCs and macrophages). doi:ten.1371/journal.pone.0039956.gnot PARP1 in monocytes. This prompted us to study the BER activity in monocytes following genotoxic strain by TMZ. With out TMZ treatment, monocytes were unable to restitute the cleaved oligonucleotide (Fig. 2D). Despite the enhance in XRCC1 and ligase IIIa level following TMZ remedy, the BER activity in monocytes was not restored (Fig. 2D). As a result, the initial incision in the lesion (yielding the 19mer fragment) and the following processing (yielding the 19+1mer fragment) was highly effective in monocytes, while the XRCC1/ligase IIIa-dependent re-ligation step (resulting in the restoration.