In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-γ1 (PLC-γ1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-γ1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-γ1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-γ1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-γ1 and Bcr-Abl inhibition. The pivotal role of PLC-γ1 was further confirmed in a mouse leukemogenesis model.
Chronic myeloid leukemia (CML) is one of the best characterized diseases with respect to the initiating molecular event, activated signaling cascades, interacting protein networks and biological outcome (Konopka et al., 1985; Van Etten, 2002; Saglio and Cilloni, 2004; Marley and Gordon, 2005; Ren, 2005). Defining Bcr-Abl as the molecule driving the disease led to development of the tyrosine kinase inhibitor imatinib as the first targeted therapy for leukemia (Druker et al., 2001). Nevertheless, identification of new pathways and molecules activated by Bcr-Abl offers new insights into the pathophysiology of CML and may also be useful to further develop molecular therapy. For example, it has been shown that inhibition of a relevant signaling intermediate in combination with Bcr-Abl may result in synergism at the level of suppression of proliferation and induction of apoptosis potentially including leukemic stem cells (Walz and Sattler, 2006; Copland et al., 2007).
In imatinib resistance, in addition to BCR-ABL resistance mutations, activated phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) pathway has recently been implicated in the survival and expansion of leukemic cells (Burchert et al., 2005). Activation of PI3K has emerged as one of the essential signaling mechanisms contributing to Bcr-Abl leukemogenesis (Kharas and Fruman, 2005; Ren et al., 2005) by stimulating cellular proliferation, cell cycling and survival (Skorski et al., 1995, 1997). mTOR has a critical function in transducing proliferative signals from the PI3K/Akt signaling cascade. mTOR is both a nutrient sensor and an important target in response to growth factors and oncoproteins (Wullschleger et al., 2006). This protein is a member of the PI3K-related kinase family and its catalytic domain is highly homologous to the lipid kinase domain of PI3K. mTOR activates downstream protein kinases that are required for both ribosomal biosynthesis and translation of key mRNAs of proteins required for G1 to S-phase transition, including the 40S ribosomal protein p70S6-kinase and the eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) (Hay and Sonenberg, 2004; Sarbassov et al., 2005). mTOR signaling is deregulated in many diseases including cancer and metabolic disorders and is considered an attractive therapeutic target in these diseases (Giles and Albitar, 2005; Easton and Houghton, 2006; Shaw and Cantley, 2006).
So far, the mechanism by which Bcr-Abl activates mTOR/p70S6-K pathway in hematopoietic cells has not been precisely defined, but recent studies show that the mTOR pathway is controlled by constitutive PI3K activity in BCR-ABL-positive cells (Ly et al., 2003). Therefore, it is commonly accepted that activation of mTOR follows the classical pathway, including PI3K/Akt-dependent phosphorylation of TSC2 and disruption of the TSC1/TSC2 complex (Ly et al., 2003; Richardson et al., 2004; Kharas and Fruman, 2005).
mTOR is currently being evaluated as a therapeutic target in CML, as its inhibition using rapamycin or similar compounds suppresses growth of primary CML cells and, in particular, of imatinib-resistant leukemic cells. In addition, the combination of rapamycin and imatinib was shown to synergistically suppress proliferation of leukemic cells in culture and in mouse models and therefore may be useful for treatment of imatinib-resistant CML (Ly et al., 2003; Mohi et al., 2004).
While studying the involvement of PI3K/Akt/mTOR pathway in the development of imatinib resistance we uncovered the existence of an additional, Akt-independent mechanism of activation of mTOR/p70S6-kinase pathway, which we describe here.
Short-term treatment with imatinib leads to downregulation of mTOR/p70S6-kinase pathway, but has no effect on Akt phosphorylation/activity
Initially, we sought to investigate the functional role of the Bcr-Abl-dependent downstream activation of mTOR/p70S6-kinase pathway in CML cells. Ba/F3 cells expressing the p185 isoform of Bcr-Abl, LAMA84 and KCL22 cells were treated for 4 h with 1 μM imatinib, with 5 nM of the mTOR inhibitor RAD001 or with a combination of both. This short-term treatment, as expected, inhibited the mTOR/p706-kinase pathway as judged by the phosphorylation status of S6 ribosomal protein (serine 240/244), which is the endpoint of this signaling cascade (Figure 1; please see also the scheme). Surprisingly, upon imatinib treatment, S6 dephosphorylation was not coupled to inactivation of Akt, as measured by phosphorylation of serine 473. Identical results were obtained in Ba/F3 cells expressing the p210 isoform of Bcr-Abl and in the AR230 and K562 cell lines derived from CML blast crisis (Supplementary Figure S1A and B). Effectiveness of imatinib-induced Bcr-Abl kinase inhibition was controlled at the level of two downstream targets, namely by the phosphorylation status of CrkL and STAT5 proteins (Supplementary Figure S1B). We also assessed the Akt phosphorylation at threonin 308, another site that is involved in Akt activation and here, as for Ser473, we did not detect change upon imatinib treatment (Supplementary Figure S1B). The absence of Akt inactivation upon short-term imatinib treatment was confirmed by lack of alteration in the phosphorylation of one known downstream Akt target, namely glycogen synthase kinase-3β (GSK-3β) at serine 9 (Supplementary Figure 1C). In contrast, when cells were incubated with PI3K inhibitor wortmannin, we detected, as expected, decrease in Akt and GSK-3β phosphorylation
Our findings were unexpected, because Akt is known to be upstream of mTOR/p70S6-kinase pathway and has been shown to be the major player in its activation (Ly et al., 2003; Richardson et al., 2004; Kharas and Fruman, 2005).
Inhibition of Akt alone does not lead to downregulation p70S6-K/S6 pathway
Our observations led us to consider the existence of an alternative mechanism for mTOR/p70S6-K stimulation, which may work independently and in addition to the Akt-driven activation. If this hypothesis is correct, then suppression only of Akt will not be sufficient to inhibit the pathway and consequently to inhibit the S6 phosphorylation. Indeed, when KCL22 and Ba/F3-p185 cells were treated for 1 h with 200, 500 or 1000 nM of the specific Akt-inhibitor-VIII we observed efficient downregulation of Akt, but still, the phosphorylation of S6 ribosomal protein was apparently not affected in both examined cell lines (Figure 2a). Furthermore, 24 h treatment of cells with Akt-inhibitor-VIII or siRNA-mediated suppression of isoforms 1/2 of Akt did not influence significantly the phosphorylation of S6. These experiments supported our hypothesis for the existence of a new, so far undiscovered mechanism involved in activation of p70S6-K/S6 pathway in BCR-ABL-positive cells.
We speculated that suppression of constitutive Akt activation by imatinib requires prolonged application. To test for this, we subjected K562 cells to 24 h treatment with imatinib and indeed, Akt activity was significantly suppressed as measured by its phosphorylation at serine 473, as well as by the phosphorylation of its downstream target GSK-3β (Supplementary Figure 1D).
Phospholipase C-γ1-dependent activation of p70S6-K/S6 pathway
In 1994, Gotoh et al. have shown that p185-Bcr-Abl is physically associated with phospholipase C-γ 1 (PLC-γ1) and the latter is tyrosine-phosphorylated in cells expressing p185-Bcr-Abl (Gotoh et al., 1994). These data suggested that PLC-γ1 may participate in Bcr-Abl downstream signaling. PLC-γ1 is known to be activated by phosphorylation at tyrosines 783 and 1253 by receptor- and non-receptor tyrosine kinases such as PDGF-R, FGF-Rs, EGF-R, Kit and Src.
To determine whether PLC-γ1 is involved in Bcr-Abl-induced p70S6-kinase/S6 activation, we first examined its Tyr783 phosphorylation. In the BCR-ABL-positive cell lines (KCL22, Ba/F3-p185, 32D-p210), we could detect strong PLC-γ1 activation. Figure 3 depicts a representative experiment, in which Ba/F3-p185 cells were treated with imatinib, RAD001, the PLC-specific inhibitor U73122, or the inactive analog U73343, respectively. In PLC-γ1 immunoprecipitates we observed strong Tyr783 phosphorylation, which was effectively suppressed by imatinib treatment (Figure 3a). Incubation of cells with U73122, in contrast to the inactive analog U73343, significantly blocked this phosphorylation. More importantly, U73122 treatment inhibited p70S6-K and S6 phosphorylation in aliquots from the same cell lysates (Figure 3b). Likewise the phosphorylation of another target of mTOR, namely 4E-BP1 was also suppressed by pharmacological or siRNA-mediated suppression of PLC-γ1 (Figure 3c). Thus, our results presented here suggest that PLC-γ1 has a critical role in the activation of mTOR/p70S6-kinase pathway. In contrast to BCR-ABL-positive cells, parental Ba/F3 cells do not exhibit PLC-γ1 activation, even when cultured in the presence of interleukin-3 (IL-3) and fetal calf serum (FCS) (Figure 3d). Therefore, activation of PLC-γ1 seems to be specific for Bcr-Abl.
Recruitment of PLC-γ1 to the membrane by PI3,4,5P3 (PIP3), produced by the action of PI3Ks, has been shown to have a function in PLC-γ activation (Falasca et al., 1998; Rameh et al., 1998; Maffucci and Falasca, 2007). In agreement with the literature, we detected PI3K activation in BCR-ABL-positive K562 cells (Supplementary Figure 2, right). Furthermore, suppression of PI3Ks by wortmannin led to inhibition of PLC-γ1 phosphorylation (left panel) suggesting involvement of PI3Ks in its stimulation. The downregulation of S6 phosphorylation detected here is most likely result of inactivation of multiple upstream molecules—PI3Ks, PLC-γ, Akt, but also consequence of direct suppression of mTOR by wortmannin because its catalytic domain shares high homology to the lipid kinase domain of PI3Ks (Brunn et al., 1996).
Downregulation of p70S6-kinase/S6 by inhibition of PLC-γ1 signaling
Activation of PLC-γ results in hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to diacylglycerol and inositol 1,4,5-triphosphate (IP3), which in turn leads to activation of different PKC isoforms and increase in Ca-dependent signaling, including Ca/Calmodulin-dependent-kinase (CaMK) (Rhee et al., 2000; Rhee, 2001) (please see the scheme in Figure 4a). We used several inhibitors of these pathways and RNAi technology to examine their involvement in the activation of p70S6-kinase. Treatment of Ba/F3-p185 cells with the Ca2+ chelators EDTA (10 μM) or the cell permeable BAPTA-AM (10 μM) was followed by profound suppression of p70S6-kinase and S6 phosphorylation (Figure 4a). Again, activation of Akt was found to be unchanged, as well as no unspecific inhibition of PLC-γ1 was observed (Figure 4a). Moreover, KN93, a CaMK inhibitor attenuated S6 phosphorylation in two different Bcr-Abl cell lines (Figure 4b, left and middle panels), without exerting unspecific effects on the phosphorylation of Akt and PLC-γ1. We further sought to confirm our findings by siRNA knockdown of CaMK as an independent approach. For our experiments, we choose CaMKIIγ as it has been shown that this is the isoform predominantly expressed in myeloid cells and that it has a critical role in regulation of proliferation in variety of myeloid leukemia cells, including BCR-ABL-positive cells (Si and Collins, 2008). Suppression of CaMKIIγ in K562 cells brought down the phosphorylation of S6 ribosomal protein, but did not cause unspecific inactivation of Akt and PLC-γ1 (Figure 4b, right panel). This indicates that CaMK has a functional role in p70S6-kinase activation.
Another group of molecules that can be activated by PLC-γ are the various isoforms of protein kinase C. We used PKC 412, a potent inhibitor of several of these isoforms to treat Ba/F3-p185 cells to examine whether they are involved in this additional mTOR/p70-S6-K activation cascade (Figure 4c). Treatment with 100 nM PKC 412, which is sufficient to inhibit PKC isoforms α, β and γ (Marte et al., 1994), as well as 2 μM PKC 412 (necessary for inhibition of PKC isoforms δ, η and ɛ) led to inhibition of S6 phosphorylation. These results suggest that Ca-signaling and PKCs are major players in the novel pathway.
To gain further inside as to the relevance of the described pathway in CML we incubated mononuclear cells (MNCs) from newly diagnosed CML patient with imatinib or U73122 (Figure 4d). In accordance with experiments performed in cell lines, pharmacological PLC-γ1 inhibition here also resulted in downregulation of S6 phosphorylation, providing additional support for our model.
In BCR-ABL-positive cells, PLC-γ is involved in the control of apoptosis
Our next aim was to determine whether blockage of PLC-γ activity or of its downstream signaling influences the biological functions of BCR-ABL-positive cells such as proliferation and inhibition of apoptosis. PLC-γ1 regulated activation of p70S6-kinase appears to be important for the proliferation of BCR-ABL-positive cells as U73122 treatment led to 40–70% suppression of the proliferation, depending on the cellular background (Supplementary Figure S3). Combination of Bcr-Abl and PLC-γ inhibition resulted in further suppression of proliferation. Interestingly, in Ba/F3 cells expressing Bcr-Abl WT, F317V, E255K and T315I mutation, respectively, combined inhibition of mTOR and PLC-γ was most efficacious in suppression of cellular growth even without targeting Bcr-Abl itself (Supplementary Figure S3A and B).
Next, we examined the consequences of PLC-γ inhibition on the control of apoptosis. Ba/F3-p185 and 32D-p210 cells when treated with U73122 or BAPTA-AM alone showed weak to moderate increase in apoptosis (Figures 5a and b). Strikingly, a combination of these compounds with imatinib resulted in almost complete eradication of cells, in particular in the case of the combination of imatinib with U73122. Of note, this strong enhancement of apoptosis appeared to be strictly Bcr-Abl dependent and not a result of general toxicity, as the presence of IL-3 in the cell culture medium was able to almost completely compensate for the action of the inhibitors (Figures 5a and b) and same inhibitors did not induce substantial apoptosis in the parental Ba/F3 and 32D cells (Figure 5c).
The functional role of PLC-γ1 signaling in the control of apoptosis was also analyzed in primary MNC derived from newly diagnosed, untreated CML patients. Remarkably, here PLC-γ inhibition alone was sufficient to cause a considerable increase in apoptotic cells, which was further enhanced by imatinib (Figure 5d). In contrast, the same treatment failed to induce apoptosis rates above 5–10% in MNC isolated from healthy donors indicating that treatment of normal MNC with 1 μM U73122 is nearly non-toxic. This suggests that PLC-γ1-induced downstream signaling indeed has a critical role in primary BCR-ABL-positive cells. The results were also confirmed in CD34-positive hematopoietic progenitor cells isolated from CML patients in chronic phase. As in the case of BCR-ABL-positive MNC, PLC-γ1 inhibition was sufficient to dramatically induce apoptosis in CD34-positive CML cells within a short period of incubation, which was additionally enhanced by imatinib (Figure 5e). The same treatment of CD34-positive cells derived from a healthy donor did not lead to induction of apoptosis.
Downregulation of PLC-γ1–p70S6-kinase/S6 pathway by siRNA
To further confirm the role of PLC-γ1 for activation of p70S6-K/S6 pathway in BCR-ABL-positive cells, we used siRNAs to knock down PLC-γ1 expression. Mouse or human cells were transiently transfected with negative-control or PLC-γ1-targeting siRNAs. Within 24 h efficient PLC-γ1 downregulation was observed in all cell lines studied and this resulted in strong suppression of S6 ribosomal protein phosphorylation. It seemed that efficacy of PLC-γ1 knockdown paralleled the level of pS6 downregulation. An example of these experiments is presented in Figure 6a. In addition, cells in which PLC-γ1 was knocked down by siRNA were also treated with imatinib. Here, we observed substantial increase in apoptosis levels as compared with the cells transfected with negative-control siRNA (Figure 6b), thus confirming our results obtained by pharmacological inhibition of PLC-γ. siRNA-mediated suppression of PLC-γ1 in the imatinib-resistant KCL22-r cells converted them to an imatinib-sensitive phenotype, indistinguishable from the imatinib-sensitive KCL22-s cells (Figure 6b; upper panel). Again, these results point out to an essential involvement of PLC-γ1 in the control of apoptosis in BCR-ABL-positive cells.
Bcr-Abl-induced leukemogenesis is impaired by PLC-γ1 knockdown
To specifically address the question of PLC-γ1 involvement in Bcr-Abl-induced leukemogenesis we used a mouse model. Ba/F3-p185 cells, additionally transduced with retroviral vectors for expression of either non-targeting shRNA (control) or PLC-γ1-targeting shRNAs, were used to inject Balb/C mice. This approach allows investigation of PLC-γ1 inactivation in vivo while avoiding off-target effects of currently available PLC-γ inhibitors. First of all, we tested two different shRNAs for their ability to suppress PLC-γ1 in comparison to the control, non-targeting sequence (Figure 7a). Both selected shRNAs successfully inhibited PLC-γ1 without exhibiting unspecific effects on the expression of PLC-γ2. Next, the Ba/F3-p185-shRNA cells were investigated in the mouse leukemogenesis model. Injection of mice with cells carrying Bcr-Abl together with the control shRNA led to fast disease development and death of the animals within less than 20 days. In contrast, injection with PLC-γ1-deficient cells (shPLC-γ1A) showed delay in the disease appearance and prolonged survival of the mice (Figure 7b). Cells recovered from spleens of diseased mice were still Bcr-Abl positive and were expressing the shRNAs (Figures 7c and d). This indicates that in this experimental model other pathways are able to compensate for PLC γ1 downregulation. Together, these results strongly support our previous notion on the functional role of PLC-γ1 in controlling cell survival and proliferation in BCR-ABL-positive leukemia. In addition, they suggest that inhibiting PLC-γ1 in vivo may have a therapeutic potential.
We here describe a novel Akt-independent, PLC-γ1-driven mechanism of activation of mTOR/p70S6-K in BCR-ABL-positive cells. This signaling pathway operates together and in parallel to the classical PI3K/Akt mode of activation.
Although a link between Bcr-Abl and PLC-γ1 has been reported earlier (Gotoh et al., 1994), the mechanistic consequences of such association remained unclear. On the other hand, PLC-γ1 has been extensively investigated in the context of signaling initiated by other receptor- and non-receptor tyrosine kinases, including PDGF-R, EGF-R, FGF-R, NGF-R, VEGF-R, Src and Fyn (Rhee, 2001; Wilde and Watson, 2001). The PLC-γ1 isoform is widely distributed and activated by receptor- and non-receptor tyrosine kinases, whereas the second isoform PLC-γ2 is expressed primarily in cells of hematopoietic lineage (Wilde and Watson, 2001; Liao et al., 2002). PLC-γ1 activation is induced upon its binding to specific phosphotyrosine residues in activated RTKs or to adaptor proteins (Rhee et al., 2000). As a consequence, PLC-γ1 itself becomes phosphorylated on tyrosine residues 771, 738 and 1254, with the last two sites correlating with stimulation of its enzymatic activity (Kim et al., 1990, 1991).
Our results show that PLC-γ1 is activated in BCR-ABL-positive cells by a yet unknown molecular mechanism. Downstream signaling was shown to include calcium/calmodulin-dependent protein kinase and the PKC family. The observation that downregulation of PLC-γ1 by siRNA led to dephosphorylation of mTOR and of p70S6-K and S6 was intriguing as activation of mTOR has been reported to mediate coordinated changes in cellular protein translation, which are required for cell growth, cell-cycle progression and regulation of apoptosis (Kharas and Fruman, 2005). In addition, mTOR contributes in a non-translational manner to the control of these cellular programs (Edinger and Thompson, 2002). Our findings suggested that PLC-γ1 might also have a critical role in these functions. We have investigated this hypothesis by both pharmacologic and siRNA-mediated inhibition of PLC-γ1, which led to repression of cell proliferation and, more importantly, enhanced apoptosis of the BCR-ABL-positive cells. Combined suppression of Bcr-Abl and PLC-γ1 dramatically increased apoptosis. In primary CML MNCs and in CML CD34-positive hematopoietic progenitor cells, pharmacologic inhibition of PLC-γ was also shown to have strong pro-apoptotic activity, which was further enhanced by imatinib. Additional support for our in vitro results was provided by the in vivo experiments showing that Bcr-Abl-induced leukemogenesis is impaired by PLC-γ1 knockdown. Thus, our findings point out that inhibition of the PLC-γ1 pathway not only attenuates cell proliferation, but also serves to eliminate BCR-ABL-positive cells by inducing cell death.
Our results that demonstrate the absence of Akt inhibition after imatinib exposure, but at the same time show efficient suppression of p70S6-kinase and S6 phosphorylation seem to contradict the accepted pathway which include PI3K/Akt-dependent phosphorylation of TSC2, disruption of the TSC1/TSC2 complex and thus activation of mTOR (Ly et al., 2003; Richardson et al., 2004; Kharas and Fruman, 2005). Most likely, this apparent discrepancy is explained by the short-term imatinib incubation used in our experiments (up to 4 h), which has not been investigated in previously published experiments where cells were incubated with the inhibitor for 18 or more hours. Short-term imatinib treatment is obviously not sufficient to suppress constitutive Akt activation, but is enough to downregulate PLC-γ1. Thus, the differences in the inhibition kinetics allowed us to discriminate between the Akt-dependent and Akt-independent mechanisms of activation of mTOR/p70S6-kinase pathway.
Our finding of the co-existence (co-operation) of Akt-dependent and Akt-independent mechanisms of mTOR/p70S6-kinase activation is in accordance with several reports in the literature. For instance, FGF-9 could induce a PLC-γ1-controlled mTOR and p70S6-kinase activation, although PI3K and Akt were not at all activated in the analyzed human endometrial stroma cells (Wing et al., 2005). Two other examples of Akt-independent mTOR/p70S6-kinase activation are provided from transformed B lymphocytes (Wlodarski et al., 2005) and from prostaglandin-F2α-treated bovine luteal cells (Arvisais et al., 2006), even though the mechanisms involved here might be different.
Additional support for our hypothesis of an Akt-independent mode of mTOR/p70S6-K activation comes from analyses of the phosphorylation/activation status of Akt and p70S6-K in primary acute myeloid leukemia (AML) and CML samples. Grandage et al. (2005) failed to detect any relationship between PI3K/Akt signaling upregulation and p70S6-K phosphorylation in primary AML cells. Analyses of Akt-signaling activation in primary CML samples revealed a very heterogeneous picture with several cases of p70S6-K phosphorylation, which did not correlate with Akt activation (Burchert et al., 2005). As these studies were carried out in distinct cellular backgrounds, we hypothesize that this PLC-γ1-driven, Akt-independent pathway may be a more general mechanism, not restricted to a particular oncogene as Bcr-Abl. This idea is supported by our own experiments in cells expressing activated mutant (ITD) of the receptor tyrosine kinase FLT3, in which we could show that downregulation of PLC-γ1 expression by siRNA leads to substantial inhibition of p70S6-kinase/S6 signaling (unpublished data). FLT3 belongs to the same class of RTK as PDGF-R and Kit, and shares similar mechanisms of action with other RTKs, some of which are also involved in the tumorigenicity. Thus, PLC-γ1 may have an important function in oncogenesis not only in CML but also in other malignancies, driven by activated tyrosine kinases.
Materials and methods
Murine Ba/F3 cells expressing the ‘wild type’ p185 isoform of Bcr-Abl or the kinase domain E255K, T315I, F317V mutants were described by Dengler et al. (2005). LAMA84, AR230 and the imatinib-sensitive and -resistant KCL22-s and KCL22-r lines are human cells derived from CML patients and were described earlier (Mahon et al., 2000). Murine 32D-p210 cells were created by retroviral transduction of the Bcr-Abl-p210 isoform, cloned in the pMX vector (kindly provided by Dr T Skorski, Philadelphia, PA, USA). All cells were maintained in RPMI1640 medium, supplemented with 10% heat inactivated FCS. Parental Ba/F3 cells were cultured in RPMI1640 containing 10% FCS and 10% WEHI-conditioned medium as a source of IL-3 as described (Heidel et al., 2006). Ba/F3-p185 cells retrovirally infected with pLMP vector encoding for non-silencing (control) or PLC-γ1-targeting shRNA were maintained in RPMI1640 containing 10% FCS and 2 ng/ml recombinant IL-3 (R&D Systems, Minneapolis, MN, USA). Retroviral packaging cell line ΦNX-Eco, kindly provided by Gary P Nolan (Stanford, CA, USA), was cultured in DMEM containing 10% FCS.
Plasmids and retroviral transduction
MIY-p185-EYFP was generated by subcloning of Bcr-Abl-p185 from pBluescript into the retroviral MIY vector. pLMP-EGFP construct encoding non-silencing shRNA was purchased from OpenBiosystems (Huntsville, AL, USA). PLC-γ1 shRNAs were generated according to the manufacturer's instructions. The sequences of oligonucleotides required for cloning of the PLC-γ1 shRNAs into pLMP vector are available on request. Preparation of retroviruses and transduction of Ba/F3 and Ba/F3-p185 cells were performed as described earlier (Miething et al., 2006).
Antibodies against phospho-Akt (Ser473 or Thr308), phospho-S6 ribosomal protein (Ser240/244 or Ser235/236), phospho-p70S6-kinase (Thr389), phospho-PLC-γ1 (Tyr783), phospho-CrkL (Tyr207), phospho-4E-BP1 (Ser65), phospho-GSK-3β (Ser9), total-PLC-γ1, total-Akt, total-S6 and total-4E-BP1 were purchased from Cell Signaling Technology (Frankfurt, Germany). Anti-phosphotyrosine (4G10), anti-p85 and phospho-STAT5 (Tyr694/699) antibodies were from Upstate (Lake Placid, NY, USA), anti-actin from MP Biomedicals (Aurora, OH, USA), anti-tubulin from Sigma-Aldrich (Munich, Germany) and anti-GAPDH from Biodesign International (Saco, ME, USA). Antibodies against CaMKIIγ (C18) and PLC-γ2 (Q20) were obtained from Santa Cruz (Heidelberg, Germany). Anti-Abl (8E9) antibody was from BD Biosciences (San Jose, CA, USA).
KN-93, wortmannin, U73122, a specific PLC inhibitor and the inactive analog U73343 were obtained from Alexis Biochemicals (Switzerland). Akt-inhibitor-VIII and BAPTA-AM were from Calbiochem (Schwalbach, Germany), imatinib mesilate (Im), RAD001 and PKC 412 were kindly provided by Novartis (Basel, Switzerland).
After indicated treatments, cells were collected, washed with ice-cold PBS and lysed in buffer containing 50 mM HEPES (pH 7.4), 150 mM NaCl, 0.5% Nonidet P40, 1 mM EDTA, 2 mM EGTA, 25 × complete protease inhibitor cocktail (Roche, Mannheim, Germany), 1 mM sodium orthovanadate and 50 mM NaF. For PLC-γ1 immunoprecipitation, cell lysates were incubated for 4 h at 4 °C with the specific antibody and A/G-Sepharose beads (Santa Cruz). The immunoprecipitates were washed three times. Cell lysates or immunoprecipitates were resolved by SDS–polyacrylamide gel electrophoresis, transferred onto PVDF membranes (Immobilon-P, Millipore Corporation, Billerica, MA, USA) and further analyzed by immunoblotting with appropriate antibodies.
The percentage of apoptotic cells was determined by measurement of the fraction with sub-G1 DNA content upon propidium iodide incorporation as described earlier (Kindler et al., 2003; Heidel et al., 2006). Briefly, 4 × 104 cells were treated with various inhibitors for 24 or 48 h, collected and washed with PBS. DNA was propidium iodide stained by incubation for 30 min at 4 °C in buffer containing 0.005% propidium iodide, 3.4 mM sodium citrate, 0.1% Triton-X 100. Cell-cycle analyses were performed using flow cytometry. As a control, cellular apoptosis was further measured using the Annexin V-FITC apoptosis detection kit I (BD Biosciences, Heidelberg, Germany) according to the manufacturer's protocol.
AllStar-Alexa-Fluor-488 negative-control siRNA was ordered from Qiagen (Hilden, Germany). siRNAs targeting human or mouse PLC-γ1 were designed using BLOCK-iT RNAi Designer tool of Invitrogen (Groningen, the Netherlands). siRNAs targeting human CaMKIIγ and Akt1/2 were obtained from Santa Cruz. siRNAs were dissolved to a final concentration of 20 μM and for each transfection 10 μl (∼3 μg) from the siRNA stock solutions were electroporated (350 V, 975 μF) into 2.5 × 106 cells. Protein expression and phosphorylation were analyzed 24 h after the transfection by immunoblotting. For assessment of apoptosis, cells were left to recover from the transfection for 6 h and were then treated with imatinib. The percentage of apoptotic cells was measured after 24 and 48 h incubation as described above.
Isolation of primary MNC and CD34+ cells
A measure of 20–40 ml heparin-treated peripheral blood samples were collected from CML patients or normal donors after informed consent was obtained. MNCs were isolated by Ficoll density-gradient centrifugation as described earlier (Kindler et al., 2003). For purification of CD34+ cells, fresh leukapheresis, peripheral blood or bone marrow samples from CML patients or healthy donor were collected. The study was approved by the ethics review boards of the medical faculties of the Universities of Hamburg and Mainz, Germany. CD34+ cells were selected using a Midi-MACS CD34 Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and were cultured as described (Bartolovic et al., 2004).
In vivo leukemogenesis studies
Suspension of 1 × 106 Ba/F3 cells expressing Bcr-Abl-p185 together with either PLC-γ1-suppressing shRNA or non-silencing shRNA was injected into 6–8-week-old female Balb/C mice through tail vein. The mice were followed up for disease development judged by symptoms such as abnormal gait and labored breathing as described earlier (Miething et al., 2006). Moribund animals were killed by cervical dislocation. The Ba/F3-p185-derived leukemic cells were rescued from spleens of the diseased mice immediately after killing and cultured in RPMI1640 medium containing 10% FCS for 7–10 days.
Conflict of interest
The authors declare no conflict of interest.
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We thank Dr E Buchdunger and J Roesel (Novartis Pharma, Basel, Switzerland) for the provision with imatinib, RAD001 and PKC 412, and Dr T Skorski (Temple University, Philadelphia, PA, USA) for Bcr-Abl expression constructs. We thank Dr M Schuler for his support during the preparation of this paper.
Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
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Markova, B., Albers, C., Breitenbuecher, F. et al. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-γ1-driven activation of mTOR/p70S6-kinase pathway. Oncogene 29, 739–751 (2010). https://doi.org/10.1038/onc.2009.374
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