These
results were consistent with our previous data [19]. The BCR signaling, which is mimicked by PMA/ionomycin, is regulated through novel PKCs (mainly PKC-δ) and atypical PKC-ζ, but not conventional PKCs in DT40 [19] and [34]. As shown in Fig. 2, gene expressions of four PKCs (PKC-δ, PKC-ε, PKC-η and PKC-ζ) were remarkably increased in Helios−/−. Therefore, we examined effects of the PMA/ionomycin treatment on viability of Helios−/− and DT40 ( Fig. 3A). As expected, the viability of Helios−/−, as well as DT40, remained unchanged in the absence of the two drugs. On the other hand, in the presence of PMA/ionomycin, the viability of Helios−/− (to ∼75% by 24 h) was remarkably higher PI3K inhibitor than that of DT40, which was dramatically reduced (to ∼45% by 24 h). Next, we examined influences GSK-3 activity of the PMA/ionomycin treatment on DNA fragmentation, one of typical apoptosis indices ( Fig. 3B). DNA fragmentation was less severe for Helios−/− than for DT40 in the presence of the two drugs at 24 h. These results revealed that the Helios-deficiency suppressed apoptosis of the DT40 cell line induced by PMA/ionomycin. It
is known that Go6976 preferentially inhibits conventional PKCs, whereas Rottlerin selectively inhibits novel PKCs (especially PKC-δ) and atypical PKC-ζ. To know participation of Helios for the BCR-mediated apoptosis signaling, we treated Helios−/− with each of these two inhibitors in the presence of PMA/ionomycin for 24 h. Cell death and DNA fragmentation of PMA/ionomycin-treated Helios−/− were significantly accerelated by Rottlerin as compared to Go6976, while these two inhibitors showed insignificant effects on both the viability and DNA fragmentation in the absence of PMA/ionomycin ( Fig. 4). Methocarbamol We showed that DT40 generates O2− upon stimulation in a similar manner to mammalian B lymphocytes [36]. It is well known that several PKCs
are involved in activation of the O2−-generating system in leukocytes [39]. Especially, PKC-δ is required for both full assembly of the O2−-generating system and activation of the respiratory burst [39], [40], [41] and [42]. Therefore, we examined effects of the Helios-deficiency on the O2−-generating activity. As expected, the O2−-generating activity in Helios−/− increased more than 4-fold compared to DT40 ( Fig. 5A). Next, to know the participation of PKC-δ on the increased O2−-generating activity, we treated Helios−/− and DT40 with PMA in the absence or presence of Rottlerin. The drastic inhibition by Rottlerin of the O2−-generating activity in DT40 revealed that the O2−-generating activity was preferentially regulated by PKC-δ ( Fig. 5B). Similarly, the increased O2−-generating activity in Helios−/− was significantly reduced by Rottlerin.