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Theodore Lewis
Theodore Lewis

Activation BIM 360 Plan 2008


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activation BIM 360 Plan 2008



An attractive mechanism for the antitumor activity exerted by IFNα is the induction of apoptosis. We and others have shown that IFNα can induce apoptosis in several transformed cell lines from many different tissues and in primary tumor cells (Manabe et al., 1993 ; Thyrell et al., 2002 ). We have also demonstrated that IFNα-induced apoptosis proceeds via the mitochondrial pathway with activation of the Bcl-2 family members Bak and Bax, subsequent release of cytochrome c (cyt c), and activation of caspases (Panaretakis et al., 2003 ). Many different proapoptotic stimuli activate Bak and Bax, causing the release of cyt c from mitochondria; however, the upstream signaling leading to this common pathway is most probably more agent specific (Wang et al., 2001 ; Panaretakis et al., 2002 ). Several signaling pathways are activated by IFNα, including the canonical Janus tyrosine kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, largely responsible for the antiviral activities of IFNα, and a cohort of other pathways whose role for the IFN-induced biological outcomes remains controversial, such as the phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR), the p38 mitogen-activated protein kinase (MAPK), protein kinase C (PKC)δ, and JNK/stress-activated protein kinase (SAPK) pathways (Parmar and Platanias, 2003 ; Katsoulidis et al., 2005 ). In a recent publication, we showed that IFNα-induced Bak activation and apoptosis in several tumor cell lines required activation of PI3K and mTOR (Thyrell et al., 2004 ).


PKCδ, ERK, and JNK are involved in IFNα-induced apoptosis. U266-1984 cells were treated with or without 300 U/ml IFNα for 48 h in the presence or absence of rottlerin, U0126, SP600125, or a combination of inhibitors. To assess apoptosis, cells were stained for annexin V (a and c) and for Bak activation (b) and analyzed by flow cytometry. The percentages of annexin V- and active Bak-positive cells were analyzed by flow cytometry. The bars represent the mean value of three independent experiments.


PKCδ, ERK, and JNK are sequentially activated downstream of PI3K and mTOR. (a) U266-1984 cells were treated for the indicated time points with 5000 U/ml IFNα in the presence or absence of Ly294002 (10 μM) or rapamycin (1 μM), respectively. The cleavage of PKCδ was detected with immunoblotting. PKCδ-CL: PKCδ cleaved. Tubulin was used as loading control. (b) Immunoblot analysis of IFNα-stimulated phosphorylation of ERK1/2 and JNK1/2 after treatment of U266-1984 cells with Ly294002 (10 μM), rapamycin (1 μM), rottlerin (2 μM), U0126 (10 μM), or SP600125 (10 μM) in the presence or absence of IFNα 5000 U/ml for 16 h. β-Actin was used as a loading control. (c) To further confirm JNK activation as being most proximal to the apoptotic machinery, cells were cultured in the absence or presence of IFNα, with or without pre-incubation with the indicated kinase inhibitors and stained for the phosphorylated form of JNK (Thr183/Tyr185) and analyzed by flow cytometry. The percentages of pJNK-positive cells were quantified using CellQuest software (BD Biosciences) and expressed as relative induction compared with the control cells. (d) Immunoblot analysis of cJun phosphorylation on the serine (S63) residue after treatment of U266-1984 cells with SP600125 (10 μM) in the presence or absence of 5000 U/ml IFNα for 16 h. Tubulin was used as loading control. All results shown are representative of two independent experiments.


JNK inhibition was also achieved by transient transfection of RHEK-1 cells with a dominant-negative form of JNK1 (dnJNK). Expression of the dnJNK in RHEK-1 cells abolished the IFNα-induced caspase activity as assayed by FLICA positivity in individual cells, further demonstrating that JNK activation is involved in apoptosis induced by IFNα and that JNK activity is required upstream of the mitochondrial events (Figure 2b).


IFNα induces cell death in enucleated cells. Live cell imaging by time-lapse microscopy. RHEK-1 cytoplasts and nucleated cells were either treated with 5000 U/ml IFN for 45 h (a) or left untreated (b). Images were taken every 15 min, and the indicated time points were selected to demonstrate changes in the apoptotic markers. RHEK-1 cytoplasts and nucleated cells were stained for nuclear morphology (Hoechst, blue) in combination with TMRE (red) and annexin V (green). Cytoplasts are indicated by arrows. (c) Immunocytochemical analysis of caspase-3 activation (green) and cyt c (red). Release of cyt c manifests as absence of mitochondrial staining. Cytoplasts are indicated by arrows.


IRS-1 is an adaptor protein commonly involved in growth factor receptor signaling. IRS-1 is also activated through tyrosine phosphorylation by JAKs in response to IFNα (Burfoot et al., 1997 ), and it contains a Scr homology 2-domain that recruits p85, the regulatory subunit of PI3K, to the receptor, thus leading to the activation of PI3K (Shoelson et al., 1992 ). To verify that the link between the IFN receptor and PI3K was unaffected by the cytoplast preparation, we also examined whether IRS-1 translocates from the cytoplasm to the plasma membrane in response to IFNα treatment. Immunostaining shows that a clear IFNα-induced IRS-1 translocation from the cytoplasm to the plasma membrane occurs both in cytoplast and nucleated cells, (Figure 5b). In addition, we confirmed that the kinase cascade activated in nucleated cells was also activated in cytoplasts. We chose to study JNK activation because we found this kinase to be activated most proximal to the mitochondrial apoptotic machinery and dependent on PI3K, mTOR, PKCδ, and ERK activity. Cells and cytoplasts were stained with Hoechst to allow separation of enucleated cells from nucleated cells by electronic gating and an antibody recognizing the phosphorylated form of JNK (Thr183/Tyr185). The flow cytometric analysis showed a clear activation of JNK in cytoplasts (Figure 5c) and in nucleated cells (data not shown). JNK activation was also confirmed in FACS-sorted cytoplasts by Western blotting (data not shown).


A key event in the induction of the proapoptotic activity of PKCδ is its caspase-mediated cleavage to a 40-kDa fragment (Koriyama et al., 1999 ), and overexpression of this catalytic fragment rapidly leads to apoptosis (Ghayur et al., 1996 ). We found that IFNα induced PKCδ cleavage in U266-1984 myeloma cells. Furthermore, inhibition of both PI3K and mTOR completely blocked this cleavage, indicating that this activation occurs downstream of PI3K and mTOR. It has been previously shown that PKCδ is a direct/indirect downstream target of PI3K and mTOR in other systems (Kumar et al., 2000 ; Baek et al., 2001 ). PKCδ activation has been shown to occur as the result of cleavage, and by phosphorylation (Kikkawa et al., 2002 ), although the exact relationship between these events has not been clearly established. A PI3K/mTOR-dependent phosphorylation of PKCδ may make it amenable for the efficient cleavage to the active proapoptotic 40-kDa fragment. Alternatively, it is possible that PI3K and/or mTOR directly or indirectly phosphorylate/activate a caspase/protease responsible for the cleavage of PKCδ in U266-1984 cells, thus leading to its conversion to its active proapoptotic form.


Although ERK activation has mainly been associated with survival and proliferation, the ERK module is also involved in apoptosis in some systems, such as apoptosis induced by cisplatin and asbestos (Hayakawa et al., 1999 ; Persons et al., 1999 ). Inhibition of PKCδ by rottlerin led to a significant inhibition of ERK phosphorylation and reduced apoptosis in response to IFNα. Indeed, PKCδ has been shown to activate MEK/ERK signaling and through this pathway to promote 12-O-tetradecanoylphorbol-13-acetate-induced apoptosis (Ueda et al., 1996 ).


It is generally believed that the cellular effects of IFNα are exerted by the expression of IFN-stimulated genes, regulated through the JAK/STAT pathway (Ihle and Kerr, 1995 ; Darnell, 1997 ). We and others have previously shown that the IFNα-induced activation of the PI3K/mTOR module, that in the present study was found to act upstream of PKCδ, ERK, and JNK, acts independently of STAT activation (Thyrell et al., 2004 ; Kaur et al., 2005 ). Conversely, it has previously been noted that both type I and type II IFN-induced PKCδ activation can be responsible for the phosphorylation of STAT1 on Ser727 and for STAT-dependent gene expression in some systems (Uddin et al., 2002 ; Kaur et al., 2005 ). However, our data do not support a role for PKCδ in STAT activation in the present system, because pretreatment with rottlerin did not alter STAT1 serine or tyrosine phosphorylation or the induction of several known IFN-stimulated genes. Similarly, inhibition of JNK, which resulted in a clear block of apoptosis, did not affect IFNα-induced STAT phosphorylation or induction of known ISGs. This implies that STAT activation is not sufficient for executing IFN-induced apoptosis. This fact, together with strong evidence for the cytoplasmic phosphorylation cascades being of a major importance, prompted us to explore the possibility that IFNα-induced apoptosis might occur in the absence of nuclear events.


LOD was first introduced by the American Institute of Architects (AIA) in 2008 when it defined five different levels of development to define the detailing levels in a BIM model. But the concept of LOD was present much before that.


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