6 ± 5.0 to 66.8 ± 2.0). Considering the fact that in erythroid-induced K562 cells the growth efficiency is lower (see Tables 1 and 2), these evidences support

the concept that benzidine-negative cells at day 6 still can differentiate even in the absence of irradiated compounds in the medium (this “commitment-like” effect is present in several inducers of K562 cell differentiation). In any case, the data suggest that the induced differentiation observed at day 6 is irreversible. Since 5′-methylpsoralen (5′-MP), 4′,5′-DMP and 5,5′-dimethylpsoralen (5,5′-DMP) for psoralens and 4,6,4′-TMA for angelicins were the most active compounds, further experimental activity was carried out with these molecules. Moreover, the lower UV-A (1 J/cm2) dose was this website chosen to minimize the phototoxic effect. The mechanism by which erythroid differentiation Metabolism inhibitor induced by furocoumarin takes place is still

unknown. However, the DNA photobinding is considered the main effect for the photoantiproliferative activity of the PUVA therapy. Thus, some preliminary experiments were carried out to verify whether furocoumarin DNA photodamage could be involved also in the erythroid differentiation process. K562 cells were irradiated in the presence of the tested compounds and of the inhibitors of some phosphoinositide kinase-related kinases, such as DNA-dependent protein kinase (DNA-PK), ataxia telangiectasia mutated (ATM) and the ataxia- and Rad3-related protein (ATR), which can be activated after different kinds of DNA damage not [27]. In particular, wortmannin was used as inhibitor of the catalytic subunit of the PI3-kinase family of enzymes [28], and caffeine as inhibitor of ATM and ATR but not of DNA-PK [29]. Cell viability was not affected

by the presence of these two inhibitors (data not shown). As it can be observed in Fig. 3, the amount of benzidine positive cells was significantly reduced, even if not completely abolished, for all tested compounds, when the irradiation was carried out in the presence of those inhibitors. Thus, the processes activated by DNA damage could be involved, at least in part, in the erythroid differentiation process. The effects of furocoumarins on the expression of human globin genes were determined by RT-qPCR analysis using probes amplifying the α-like α-globin and ζ-globin and the β-like ε-globin and γ-globin mRNA sequences. Effects on production of β-globin mRNA were not analyzed, since it is well known that K562 cells do not efficiently transcribe the β-globin genes [10] and [30]. In Fig. 4, globin mRNA expression for 4′,5′-DMP and 4,6,6′-TMA is presented; these two molecules were selected as an example for linear (4′,5′-DMP) and angular (4,6,4′-TMA) most active furocoumarins in inducing erythroid differentiation (Table 1).