Ive to cAMP. This widely-known transcription factor is generally implicated in cell proliferation and transformation. However, cFos has been INNO-206 reported to have a role in the end-stage maturation of hematopoietic cells [23,24,25,26], including osteoclasts [27,28,29], as well as in the end-stage maturation of keratinocytes [30] or neuronal cells [31,32]. We therefore investigated whether cFos played a role in reprogramming resistant APL cells towards differentiation in response to cAMP/ATRA cooperation. We first studied its expression in NB4-LR1 cells treated with cAMP, ATRA or a combination of ATRA and cAMP, both at the transcriptional and protein levels. As previously reported, ATRA or cAMP signaling is not self-sufficient to provoke the maturation of retinoid-resistant cells, and a crosstalk between the two drugs is necessary to trigger differentiation [4,5]. As shown in Figure 2A, whereas no effect of ATRA was seen in resistant NB4-LR1 cells, 8CPT-cAMP treatment strongly increased cfos mRNA levels after 15 min. Western blotting analysis (Figure 2B) also showed a transient induction of cFos protein, with a peak at 90 min. In absence of maturation, both mRNA and protein levels rapidly returned to control values within 1h30 and 3 h, respectively. In association with ATRA, 8-CPT-cAMP induced a biphasic response in cfos mRNA and protein expression (Figure 2A, B), i.e. an early induction identical to that observed in NB4-LR1 cells treated with cAMP alone, followed by a second one, more sustained, which was associated with a terminal stage in the progression of promyelocytic cells to mature KB-R7943 (mesylate) neutrophils (Figure 2C). Of note, in retinoid-sensitive NB4 parental cells, which are responsive to ATRA alone, only a late induction of cFos is observed (Figure 3A ), associated with the terminal differentiation of cells, as has been reported for osteoclasts or other terminally maturated cell types. ATRA was found incapable to mediate a fast induction of cFos, suggesting a differential effect of ATRA and cAMP/ATRA association on the transduction of maturation signals in NB4 and NB4-LR1 cells, respectively. These results raise the hypothesis that the early induction of cFos by cAMP may represent an important signal enabling resistant cells to differentiate. A gene-silencing approach has then been considered to determine whether cAMP-induced cFos was implicated in relieving ATRA resistance. A pool of specific short interferingRNA (siRNA) was used, and their efficiency in knocking down cfos mRNAs was confirmed (Figure 4A). In a first set of experiments, we showed that suppressing cAMP-induced cfos by siRNAs greatly inhibited the expression of c-jun (Figure 4B), as well as those of CD44s and its splice v9-10 variant (Figure 4C, D). We further assessed the implication of cFos in the transduction of differentiation signals. We first examined whether its silencing might affect the production of reactive oxygen species (ROS), considered as an important marker of neutrophil maturation. Extinction of cAMP-induced cfos significantly inhibited the generation of intracellular ROS (Figure 5A), as well as the expression of CD11c, a cell surface marker of NB4 maturation (Figure 5B), evaluated by fluorescent analysis. Consistently, cfos silencing also resulted in the inhibition of differentiation of resistant APL cells into neutrophils (Figure 5C). NB4-LR1 cells cultured for 2 K days with the combination of cAMP and ATRA presented typical morphological changes. They decrea.Ive to cAMP. This widely-known transcription factor is generally implicated in cell proliferation and transformation. However, cFos has been reported to have a role in the end-stage maturation of hematopoietic cells [23,24,25,26], including osteoclasts [27,28,29], as well as in the end-stage maturation of keratinocytes [30] or neuronal cells [31,32]. We therefore investigated whether cFos played a role in reprogramming resistant APL cells towards differentiation in response to cAMP/ATRA cooperation. We first studied its expression in NB4-LR1 cells treated with cAMP, ATRA or a combination of ATRA and cAMP, both at the transcriptional and protein levels. As previously reported, ATRA or cAMP signaling is not self-sufficient to provoke the maturation of retinoid-resistant cells, and a crosstalk between the two drugs is necessary to trigger differentiation [4,5]. As shown in Figure 2A, whereas no effect of ATRA was seen in resistant NB4-LR1 cells, 8CPT-cAMP treatment strongly increased cfos mRNA levels after 15 min. Western blotting analysis (Figure 2B) also showed a transient induction of cFos protein, with a peak at 90 min. In absence of maturation, both mRNA and protein levels rapidly returned to control values within 1h30 and 3 h, respectively. In association with ATRA, 8-CPT-cAMP induced a biphasic response in cfos mRNA and protein expression (Figure 2A, B), i.e. an early induction identical to that observed in NB4-LR1 cells treated with cAMP alone, followed by a second one, more sustained, which was associated with a terminal stage in the progression of promyelocytic cells to mature neutrophils (Figure 2C). Of note, in retinoid-sensitive NB4 parental cells, which are responsive to ATRA alone, only a late induction of cFos is observed (Figure 3A ), associated with the terminal differentiation of cells, as has been reported for osteoclasts or other terminally maturated cell types. ATRA was found incapable to mediate a fast induction of cFos, suggesting a differential effect of ATRA and cAMP/ATRA association on the transduction of maturation signals in NB4 and NB4-LR1 cells, respectively. These results raise the hypothesis that the early induction of cFos by cAMP may represent an important signal enabling resistant cells to differentiate. A gene-silencing approach has then been considered to determine whether cAMP-induced cFos was implicated in relieving ATRA resistance. A pool of specific short interferingRNA (siRNA) was used, and their efficiency in knocking down cfos mRNAs was confirmed (Figure 4A). In a first set of experiments, we showed that suppressing cAMP-induced cfos by siRNAs greatly inhibited the expression of c-jun (Figure 4B), as well as those of CD44s and its splice v9-10 variant (Figure 4C, D). We further assessed the implication of cFos in the transduction of differentiation signals. We first examined whether its silencing might affect the production of reactive oxygen species (ROS), considered as an important marker of neutrophil maturation. Extinction of cAMP-induced cfos significantly inhibited the generation of intracellular ROS (Figure 5A), as well as the expression of CD11c, a cell surface marker of NB4 maturation (Figure 5B), evaluated by fluorescent analysis. Consistently, cfos silencing also resulted in the inhibition of differentiation of resistant APL cells into neutrophils (Figure 5C). NB4-LR1 cells cultured for 2 K days with the combination of cAMP and ATRA presented typical morphological changes. They decrea.
