Anti-apoptotic Function Of Bcl-2 Research Articles

Role of oxidative/nitrosative stress‐mediated Bcl‐2 regulation in apoptosis and malignant transformation

Bcl-2 is a key apoptosis regulatory protein of the mitochondrial death pathway. The oncogenic potential of Bcl-2 is well established, with its overexpression reported in various cancers. The antiapoptotic function of Bcl-2 is closely associated with its expression levels. Reactive oxygen and nitrogen species (ROS/RNS) are important intracellular signaling molecules that play a key role in various physiological processes including apoptosis. We have recently reported that ROS and RNS can regulate Bcl-2 expression levels, thereby impacting its function. Superoxide anion (*O(2)(-)) plays a proapoptotic role by causing downregulation and degradation of Bcl-2 protein through the ubiquitin-proteasomal pathway. In contrast, nitric oxide (NO)-mediated S-nitrosylation of Bcl-2 prevents its ubiquitination and subsequent proteasomal degradation, leading to inhibition of apoptosis. Interestingly, NO-mediated S-nitrosylation and stabilization of Bcl-2 protein was the primary mechanism involved in the malignant transformation of nontumorigenic lung epithelial cells in response to long-term carcinogen exposure. We describe a novel mechanism of Bcl-2 regulation by *O(2)(-) and NO, providing a new dimension to reactive species-mediated Bcl-2 stability, apoptotic cell death, and cancer development.

BCL2 Ala43Thr Is a Functional Variant Associated with Protection Against Azoospermia in a Han-Chinese Population1

Apoptosis is very common during various stages of mammalian germ cell development and differentiation, and the BCL2 gene is one of the most important apoptotic regulators. Although its genetic variants are reported to be involved in cancers and autoimmune diseases, little information is available regarding BCL2 polymorphisms in male spermatogenesis. In the present study, single nucleotide polymorphisms (SNPs) in coding regions of the BCL2 gene were examined in a hospital-based, case-control study including 198 infertile patients with idiopathic azoospermia and 183 fertile controls. Subsequently, a functional study was conducted for comparison of paclitaxel-induced cytotoxicity and apoptosis between the BCL2 variant and the wild type in vitro. Three SNPs were found in exon 2--A21G (rs1801018), G127A (rs1800477), and C300T (rs61733416)--with the latter first reported in the Han-Chinese population. The frequency of G127A (GA+AA) genotype was significantly lower in azoospermic, infertile men compared to the age-matched controls (P = 0.01). This genotype may confer a lower risk of azoospermia (adjusted odd ratio [OR] = 0.448, 95% confidence interval = 0.226-0.889). In addition, HeLa cells expressing the BCL2 Ala43Thr (G127A), similar to the control cells, were more sensitive to paclitaxel-induced cytotoxicity and apoptosis than those expressing wild-type BCL2. Consistently, the cleaved PARP and p-BCL2 proteins were subsequently increased after paclitaxel treatment, as also predicted by the bioinformatics analysis. Considering the decreased antiapoptotic function of BCL2, these results suggest that the Ala43Thr variant is associated with protection against azoospermia in the Han-Chinese population.

Anti-apoptotic Actions of PPAR-γ Against Ischemic Stroke

Stroke is a leading cause of adult disability and mortality. Diabetes is a major risk factor for stroke. Patients with diabetes have a higher incidence of stroke and a poorer prognosis after stroke. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a ligand-modulated transcriptional factor and a therapeutic target for treating type II diabetes. It is well-documented that activation of PPAR-gamma can also attenuate postischemic inflammation and damage. In this review, we focus on the newly revealed anti-apoptotic actions of PPAR-gamma against cerebral ischemia. PPAR-gamma, by increasing superoxide dismutase/catalase and decreasing nicotinamide adenine dinucleotide phosphate oxidase levels, attenuated ischemia-induced reactive oxygen species and subsequently alleviated the postischemic degradation of Bcl-2, Bcl-xl, and Akt. The preserved Akt phosphorylated Bad. Meanwhile, PPAR-gamma also promotes the transcription of 14-3-3epsilon. Elevated 14-3-3epsilon binds and sequesters p-Bad and prevents Bad translocation to neutralize the anti-apoptotic function of Bcl-2. This review further supports the notion that PPAR-gamma may serve as a potential therapeutic target for treating ischemic stroke.

Synergistic Activity of Co-Treatment with PIM1 Kinase Inhibitor SGI-1776 and Histone Deacetylase Inhibitor Panobinostat or Heat Shock Protein 90 Inhibitor AUY922 against Human CML and Myeloproliferative Neoplasm (MPN) Cells.

Abstract 2651Poster Board II-627The oncoprotein PIM1 is a cytoplasmic serine/threonine kinase, which is overexpressed and implicated in the pathogenesis of hematologic malignancies. PIM1 activity promotes cell survival and proliferation, and is a downstream mediator of cytokine receptor signaling involving the JAK/STATs. PIM1 phosphorylates and inactivates Bad, which enhances the anti-apoptotic functions of Bcl-2. PIM1 also mediates the phosphorylation and inactivation of cell cycle inhibitors p21 and p27, thereby promoting progression through G1 and G2/M phases of the cell cycle. PIM1 phosphorylates serine 10 on histone H3, and has been shown to phosphorylate and stabilize Myc, thereby collaborating in Myc-induced transformation. Further, PIM1 is known to phosphorylate 4EBP1 on the same residues (threonine 37 and 46) as mTOR kinase. It can also phosphorylate eIF4B, thereby maintaining high levels of protein translation. PIM1 is known to be chaperoned as a heat shock protein 90 client protein. In the present studies, we demonstrate that treatment with the PIM kinase inhibitor SGI-1776 (SuperGen Inc.) dose-dependently (500 nM to 2.0 μM) depleted p-BAD (Ser112), p-p70S6K, p-4EBP1, p-Ser10 Histone H3 and c-Myc levels, while inducing p27 and p21 expression, in the cultured human CML-BC K562 and erythroleukemia HEL92.1.7 (with homozygous JAK2-V617F mutation). Treatment with SGI-1776 induced apoptosis of K562 and HEL92.1.7 (HEL) cells as well as Ba/F3-hEpoR-JAK2-V617F but not Ba/F3-hEpoR cells. Similar effects of SGI-1776 were observed in primary CML-BC and MPN (myeloproliferative neoplasm) cells. Treatment with the non-geldanamycin analogue hsp90 inhibitor AUY922 (Novartis Pharma) or with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharma) disrupted the chaperone association of PIM1 with hsp90 and increased binding to hsp70, thereby promoting the proteasomal degradation of PIM1 in K562 and HEL cells. Treatment with PS (20–100 nM) also depleted PIM1 mRNA as measured by qPCR. Co-treatment with SGI-1776 (0.5 to 2.0 μM) and clinically achievable concentrations of AUY922 (e.g., 25 nM) caused greater depletion of pBAD (Ser112), pp70S6K, p4EBP1, cyclin B1, c-Raf, c-Myc, and AKT and synergistically induced apoptosis of K562 and HEL92.1.7 cells (combination indices < 1.0). As compared to treatment with either agent alone, co-treatment with SGI-1776 and AUY922 or PS also exerted greater cytotoxicity against primary CML-BC and MF-MPN cells. Importantly, co-treatment with PS significantly enhanced SGI-1776-induced apoptosis of purified primary CD34+CD38-Lin- hematopoietic stem cells expressing JAK2-V617F derived from a patient with primary myelofibrosis. Taken together, these pre-clinical findings indicate that simultaneously depleting PIM kinase levels by AUY922 or PS and inhibiting PIM1 kinase activity with SGI-1776 induces synergistic apoptosis against cultured and primary CML-BC and MPN cells. Additionally, our findings suggest that co-treatment with PS and SGI-1776 may be an effective treatment strategy against hematopoietic stem cells expressing JAK2-V617F in patients with MF-MPN. Disclosures:Atajada:Novartis: Employment. Quadt:Novartis: Employment. Bhalla:Merck: Honoraria; Novartis: Honoraria, Research Funding.

Vacuolar H+‐ATPase inhibitors overcome Bcl‐xL‐mediated chemoresistance through restoration of a caspase‐independent apoptotic pathway

The anti-apoptotic oncoproteins Bcl-2 and Bcl-xL play crucial roles in tumorigenesis and chemoresistance, and are thus therapeutic cancer targets. We searched for small molecules that disturbed the anti-apoptotic function of Bcl-2 or Bcl-xL, and found vacuolar H(+)-ATPase (V-ATPase) inhibitors, such as bafilomycin A1 (BMA), that showed such activity. Bcl-xL-overexpressing Ms-1 cells displayed resistance to anticancer drugs, but underwent apoptosis following treatment with a combination of V-ATPase inhibitors at doses similar to those that caused inhibitory activities of V-ATPase. We investigated the apoptosis mechanism induced by cotreatment of Bcl-xL-overexpressing Ms-1 cells with BMA as a V-ATPase inhibitor and taxol (TXL) as an anticancer drug. With BMA, TXL triggered mitochondrial membrane potential loss and cytochrome c release, whereas downstream caspase activation was not observed. In contrast, pronounced nuclear translocation of mitochondrial apoptosis-inducing factor and endonuclease G, known as effectors of caspase-independent apoptosis, was observed with BMA and TXL cotreatment. Moreover, depletion of apoptosis-inducing factor and endonuclease G using each siRNA significantly rescued cells from BMA- and TXL-induced apoptosis. Hence, the apoptosis-inducing factor- and endonuclease G-dependent pathway was critical for apoptosis induction by BMA and TXL cotreatment. Our data suggest that V-ATPase inhibitors could not only suppress anti-apoptotic Bcl-2 nor Bcl-xL but could also facilitate the caspase-independent apoptotic pathway. V-ATPase inhibition will be a promising therapeutic approach for Bcl-2- or Bcl-xL-overexpressing malignancies.

An Open-Label, Multicenter, Phase I/II Study of Single-Agent AT-101 in Men with Castrate-Resistant Prostate Cancer

AT-101 binds and inhibits the antiapoptotic function of Bcl-2, Bcl-xL, Mcl-1, and Bcl-w and is a potent stimulator of proapoptotic proteins. In this multi-institution phase I/II trial, we evaluated the safety and efficacy of single-agent AT-101, in men with chemotherapy naïve, castrate-resistant prostate cancer (CRPC). Patients with progressive CRPC were to be treated with escalating doses of AT-101 on a continuous daily basis until the maximally tolerated dose was achieved. At the recommended phase 2 dose, an additional 21 patients were planned to assess for preliminary evidence of efficacy. Twenty-three patients were enrolled. The phase I starting dose was 30 mg/day on a continuous basis; however, ongoing trials with AT-101 showed increased gastrointestinal toxicity with this daily schedule when given for repetitive cycles. As a result, the phase II starting dose was chosen to be 30 mg/day for 21 of 28 days. The most frequent observed adverse events (any grade) were diarrhea (43.5%), fatigue (34.8%), nausea (21.7%), anorexia (21.7%), and small intestinal obstruction (21.7%). Due to the high incidence of grade 3 small intestinal obstruction (n = 5; 21.7%), a reduction in dose to 20 mg/day for 21 of 28 days was mandated for all patients. Two patients had a confirmed > or =50% posttherapy prostate-specific antigen decline. No objective responses (Response Evaluation Criteria in Solid Tumors) were observed. AT-101 administered at 20 mg/day for 21 of 28 days was well-tolerated. Evidence of single-agent clinical activity was observed with prostate-specific antigen declines in some patients. Further investigation of AT-101 in prostate cancer is warranted and trials combining AT-101 with androgen deprivation, as well as with docetaxel chemotherapy are ongoing.

Bcl-2 complexed with Beclin-1 maintains full anti-apoptotic function

The binding of Bcl-2 to Beclin-1 reduces Beclin-1's capacity to induce autophagy. Here, we have tested whether the interaction is reciprocated by loss of Bcl-2's anti-apoptotic function. We targeted Bcl-2 to mitochondria or endoplasmic reticulum (ER) and induced apoptosis using several apoptotic stimuli that initiate ER and/or mitochondrial signaling pathways (UV radiation, TNF and cycloheximide, staurosporine, thapsigargin and tunicamycin). When Beclin-1 and Bcl-2 were expressed together in HeLa cells, Beclin-1 (but not Beclin-1 lacking the Bcl-2-binding domain) followed Bcl-2 to the appropriate organelle with complete or near-complete overlap (comprising 60 and 30% of cells, respectively). The interaction between Beclin-1 and Bcl-2 was verified by immunoprecipitation, and a membrane-proximate localization of Beclin-1 was shown by immunoelectron microscopy. Apoptosis was followed by measuring changes in nuclear morphology, caspase-3 activity, poly-ADP-ribose polymerase cleavage or punctation of mRFP-Bax on mitochondria. Binding of Beclin-1 to Bcl-2 did not modify apoptosis irrespective of Bcl-2 concentration, location or apoptotic stimulus. A similar result was obtained in Atg5-/- cells that are autophagy-deficient, arguing against compensation for the loss of protection by Bcl-2 by autophagy-mediated survival induced by Beclin-1. Hence, although Beclin-1 contains a BH3-only motif typical of pro-apoptotic proteins, it is a negligible modulator of Bcl-2's anti-apoptotic function.

Apoptosis of t(14;18)-positive lymphoma cells by a Bcl-2 interacting small molecule

Overexpression of Bcl-2 protein occurs via both t(14;18)-dependent and independent mechanisms and contributes to the survival and chemoresistance of non-Hodgkin lymphomas. HA14–1 is a nonpeptidic organic small molecule, which has been shown to inhibit the interaction of Bcl-2 with Bax, thereby interfering with the antiapoptotic function of Bcl-2. In this study, we sought to determine the in vitro efficacy of HA14–1 as a therapeutic agent for non-Hodgkin lymphomas expressing Bcl-2. Assessment of cell viability demonstrated that HA14–1 induced a dose- (IC50 = 10 μM) and time-dependent growth inhibition of a cell line (SudHL-4) derived from a t(14;18)-positive, Bcl-2-positive, non-Hodgkin lymphoma. HA14–1 effectively induced apoptosis via a caspase 3-mediated pathway but did not affect either the p38 MAPK or p44/42 MAPK pathways. Western blot analyses of Bcl-2 family proteins and other cell cycle-associated proteins were performed to determine the molecular sequelae of HA14–1-induced apoptosis. The results show down-regulation of Mcl-1 but up-regulation of p27kip1, Bad, Bcl-xL, and Bcl-2 proteins, without change in Bax levels during HA14–1-mediated apoptosis. Our findings further elucidate the cellular mechanisms accompanying Bcl-2 inhibition and demonstrate the potential of Bcl-2 inhibitors as therapeutic agents for the treatment of non-Hodgkin lymphomas.

Response of Bladder Carcinoma Cells to TRAIL and Antisense Oligonucleotide, Bcl-2 or Clusterin Treatments

Bladder transitional cell carcinoma is the second most common urological malignancy, of which 80% are superficial disease limited to the bladder. Superficial bladder transitional cell carcinoma has a high propensity for recurrence and progression after initial resection, necessitating adjuvant intravesical therapy. TRAIL (tumor necrosis factor-related apoptosis inducing ligand) can selectively induce apoptosis in most tumor cells while sparing normal cells. TRAIL drives not only the death receptor pathway, but also the mitochondrial pathway through Bid. Due to the anti-apoptotic functions of Bcl-2 and clusterin on the mitochondrial apoptotic pathway the effects of down-regulating these proteins were examined in partially TRAIL resistant bladder transitional cell carcinoma cell lines. Antisense oligonucleotides targeting Bcl-2 and clusterin were used alone or combined with TRAIL and cytotoxicity was examined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolim bromide) proliferation assay. Apoptotic pathway signals were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting after the various combination treatments. All statistical tests were 2 sided. Although no direct correlation between TRAIL sensitivity and the relative expression levels of Bcl-2 and clusterin was found in the bladder transitional cell carcinoma cell lines examined, antisense oligonucleotide mediated the down-regulation of Bcl-2 and clusterin, increasing the sensitivity of the partially resistant cells to TRAIL. This was mediated through increased apoptotic signaling of the mitochondrial pathway, as evident by the increased activation of caspase-9 and 3, and cleaved DFF45. There was no benefit of combined antisense oligonucleotide therapy. This study provides proof of principle that TRAIL combined with antisense oligonucleotide-Bcl-2 may have potential as a novel future treatment strategy for bladder transitional cell carcinoma.

Protein phosphatase 2A inactivates Bcl2's antiapoptotic function by dephosphorylation and up-regulation of Bcl2-p53 binding

AbstractBcl2 is associated with chemoresistance and poor prognosis in patients with various hematologic malignancies. DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membrane results in a Bcl2 conformational change with loss of its antiapoptotic activity in interleukin-3–dependent myeloid H7 cells. Here we find that specific disruption of protein phosphatase 2A (PP2A) activity by either expression of small t antigen or depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation and suppresses cisplatin-stimulated p53/Bcl2 binding in association with prolonged cell survival. By contrast, treatment of cells with C2-ceramide (a potent PP2A activator) or expression of the PP2A catalytic subunit (PP2A/C) inhibits Bcl2 phosphorylation, leading to increased p53/Bcl2 binding and apoptotic cell death. Mechanistically, PP2A-mediated dephosphorylation of Bcl2 in vitro promotes its direct interaction with p53 as well as a conformational change in Bcl2. PP2A directly interacts with the BH4 domain of Bcl2 as a docking site to potentially “bridge” PP2A to Bcl2's flexible loop domain containing the target serine 70 phosphorylation site. Thus, PP2A may provide a dual inhibitory effect on Bcl2's survival function by both dephosphorylating Bcl2 and enhancing p53-Bcl2 binding. Activating PP2A to dephosphorylate Bcl2 and/or increase Bcl2/p53 binding may represent an efficient and novel approach for treatment of hematologic malignancies.

Aranorosin and a novel derivative inhibit the anti-apoptotic functions regulated by Bcl-2

Bcl-2 is an intracellular membrane protein that prevents cells from undergoing apoptosis in response to various cell-death signals. It negatively regulates mitochondrial outer membrane permeabilization, which is responsible for the release of apoptogenic factors and the subsequent activation of caspases. A microbial metabolite, aranorosin, was identified as an inhibitor of the anti-apoptotic function of Bcl-2. Based on its structure, a more potent derivative, K050, was synthesized. Apoptosis could be induced in a cell line that overexpressed Bcl-2 when cells were treated with an anti-Fas antibody in addition to K050, at sub-micromolar concentrations. Furthermore, K050 inhibited anti-apoptotic functions regulated by Bcl-2, resulting in a Fas-triggered mitochondrial transmembrane potential loss, the activation of caspase-9, and a morphological change to apoptosis. Inhibition of cell-based function of Bcl-2 and its anti-apoptotic effects could serve as useful pharmacological effects. Thus, a novel aranorosin derivative, K050, could be a potent therapeutic agent against Bcl-2-overexpressing human malignancies.

P3-303: PP2A activates the antiapoptotic function of BCL-2, while the dephosphorylation of tau by PP2A facilitates apoptosis

P3-303: PP2A activates the antiapoptotic function of BCL-2, while the dephosphorylation of tau by PP2A facilitates apoptosis

Biosynthetic pathway of 24-membered macrolactam glycoside incednine

Incednine was isolated from Streptomyces sp. ML694-90F3 as an inhibitor of anti-apoptotic function of Bcl-2/Bcl-xL oncoproteins. The structure of incednine is quite unique with a characteristic 24-membered macrocyclic lactam aglycone and two unusual aminosugars. To understand its biosynthetic pathway, the incorporation studies were carried out with [1- 13C]acetate, [1,2- 13C 2]acetate, [1- 13C]propionate, l-[ 13C 5, 15N]glutamate, [1,2,3- 13C 3]glycerol, d-[6,6- 2H 2]glucose, and l-[CH 3– 13C]methionine. As a result, acetate, propionate, and glycerol were well incorporated into the elongation units of the macrolactam moiety, which indicates that its basic skeleton could be constructed by standard polyketide synthase, whereas all atoms of the starter unit were labeled by [ 13C 5, 15N]glutamate suggesting that glutamate is somehow decarboxylated and rearranged into 3-aminobutyrate as the unique starter unit. The origins of the sugar moieties and methyl groups were also clarified. Based on the incorporation pattern, a plausible biosynthetic pathway for incednine is proposed.

The Bax Inhibitor-1 (BI-1) Family in Apoptosis and Tumorigenesis

The signaling pathways that determine the fate of a cell regarding death or survival depend on a large number of regulatory proteins. The Bax Inhibitor-1 (BI-1) family is a highly preserved family of small transmembrane proteins located mostly in the endoplasmic reticulum (ER). Although most members of this family are still not characterized an antiapoptotic effect has been described for BI-1, Lifeguard (LFG), and the Golgi anti-apoptotic protein (GAAP). The cytoprotective activity has been associated to the control of ion homeostasis and ER stress but includes other cell death stimuli as well. Recent data describes multiple interactions between the proteins of the BI-1 family and the Bcl-2 family either stimulating the antiapoptotic function of Bcl-2 or inhibiting the proapoptotic effect of Bax. The potent cell death suppression makes this protein family an interesting target for the development of new drugs and gene therapeutic approaches for diseases caused by apoptotic dysregulation, such as cancer.

A Novel Calmodulin-Ca2+ Target Recognition Activates the Bcl-2 Regulator FKBP38

The FK506-binding protein 38 (FKBP38) affects neuronal apoptosis control by suppressing the anti-apoptotic function of Bcl-2. The direct interaction between FKBP38 and Bcl-2, however, requires a prior activation of FKBP38 by the Ca2+ sensor calmodulin (CaM). Here we demonstrate for the first time that the formation of a complex between FKBP38 and CaM-Ca2+ involves two separate interaction sites, thus revealing a novel scenario of target protein regulation by CaM-Ca2+. The C-terminal FKBP38 residues Ser290-Asn313 bind to the target protein-binding cleft of the Ca2+-coordinated C-terminal CaM domain, thereby enabling the N-terminal CaM domain to interact with the catalytic domain of FKBP38 in a Ca2+-independent manner. Only the latter interaction between the catalytic FKBP38 domain and the N-terminal CaM domain activates FKBP38 and, as a consequence, also regulates Bcl-2.

Bcl2 enhances induced hematopoietic differentiation of murine embryonic stem cells

Bcl2 is a potent antiapoptotic gene that can increase resistance of adult bone marrow hematopoietic progenitor cells to lethal irradiation, and thereby preserve their ability to differentiate. However, the effect of Bcl2 on murine embryonic stem (ES) cells induced to undergo hematopoietic differentiation in the absence of a toxic stress is not known. To test this, murine CCE-ES cells that can be induced to undergo hematopoietic differentiation in a two-step process that results in upregulation of Bcl2 were used. Upregulation of Bcl2 precedes formation of hematopoietic embryoid bodies (EB) and their further differentiation into hematopoietic colony-forming units, when plated as single cells in methylcellulose. ES cells stably expressing a Bcl2 siRNA plasmid to "knock-down" endogenous expression or cells expressing wild-type (WT) Bcl2 or phosphomimetic Bcl2 mutants were examined. ES cells expressing the Bcl2 siRNA or those expressing a dominant-negative, nonphosphorylatable Bcl2 display a strikingly reduced capacity to form hematopoietic EBs and colony-forming units compared to cells expressing WT or phosphomimetic Bcl2 that demonstrate an increased capacity. Bcl2's effect on induced-hematopoietic differentiation of ES cells does not result from either decreased apoptosis or a reduced number of cells. Rather, Bcl2-enhances hematopoietic differentiation of ES cells by upregulating p27, which results in retardation of the cell cycle at G1/G 0. Thus siRNA silencing of p27 reverts Bcl2's enhancement phenotype in a manner similar to that of Bcl2 "silencing" or expression of a nonphosphorylable Bcl2. In addition to Bcl2's well-described antiapoptotic and cell-cycle retardant effect on somatic cells, Bcl2 may also function to enhance induced hematopoietic cell differentiation of murine ES cells. These findings may have potential relevance for expanding hematopoietic stem/progenitor cell numbers from an ES cell source for stem cell transplantation applications.

Oligomerization of BH4-truncated Bcl-x L in solution

BH4 domain is critical for the anti-apoptotic functions of Bcl-2 and Bcl-x L and their binding abilities with other members of the Bcl-2 family. The cleavage of the BH4 domain in Bcl-x L and Bcl-2 by caspase 1 or 3 converts the anti-apoptotic Bcl-x L and Bcl-2 into pro-apoptotic proteins that potently induce apoptosis. Herein, we report that recombinant Bcl-x L proteins without N-terminal 61 residues, His 6-NΔ61-Bcl-x L-CΔ21 and NΔ61-Bcl-x L-CΔ21, form oligomers in solution, whereas Bcl-x L-CΔ21 exists as a monomer. The oligomerization of the truncated proteins is independent of protein–lipid interaction, protein concentration or the ion strength of the solution. Circular dichroism spectrum shows a significant decrease in the content of α-helices upon deletion of N-terminal residues. NΔ61-Bcl-x L-CΔ21 also loses its heterodimerization capability with the BH3 peptide derived from Bak. This newly acquired property might be linked to its ability to induce apoptosis in cells.

Protein Kinase Cζ Abrogates the Proapoptotic Function of Bax through Phosphorylation

Protein kinase Czeta (PKCzeta) is an atypical PKC isoform that plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Because Bax is extensively co-expressed with PKCzeta in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKCzeta in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKCzeta activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKCzeta specific inhibitor (a myristoylated PKCzeta pseudosubstrate peptide). Purified, active PKCzeta directly phosphorylates Bax in vitro. Overexpression of wild type or the constitutively active A119D but not the dominant negative K281W PKCzeta mutant results in Bax phosphorylation at serine 184. PKCzeta co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKCzeta by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of wild type or A119D but not K281W PKCzeta mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKCzeta can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKCzeta may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of the proapoptotic function of Bax.

Protein Phosphatase 2A Inactivates Bcl2's Antiapoptotic Function by Dephosphorylation and Up-Regulation of Bcl2-p53 Binding.

DNA damage-induced p53/Bcl2 interaction at the outer mitochondrial membranes results in a Bcl2 conformational change and loss of its antiapoptotic function. Our data now indicate that either treatment of cells with the protein phosphatase 2A (PP2A) inhibitor, okadaic acid (10 nM), or specific disruption of PP2A activity by the expression of SV40 small tumor antigen enhances Bcl2 phosphorylation and suppresses the cisplatin-stimulated Bcl2-p53 interaction in association with prolonged cell survival. By contrast, C2-ceramide, a potent PP2A activator, reduces Bcl2 phosphorylation and increases Bcl2-p53 binding and promotes apoptotic cell death, suggesting that PP2A may function as a physiological regulator of Bcl2 by, at least in part, affecting its association with p53. Overexpression of the PP2A catalytic subunit (PP2A/C) suppresses Bcl2 phosphorylation in association with increased p53-Bcl2 binding and apoptotic cell death. By contrast, specific depletion of PP2A/C by RNA interference enhances Bcl2 phosphorylation, suppresses p53-Bcl2 interaction and prolongs cell survival. Purified PP2A can directly enhance the formation of the p53-Bcl2 complex in vitro in an okadaic acid-sensitive manner, supporting a direct mechanism. Importantly, PP2A directly interacts with Bcl2 at its BH4 domain which may function as the PP2A ‘docking site' to potentially ‘bridge' PP2A to the flexible loop domain which contains the physiological serine 70 phosphorylation site. Thus, PP2A may provide a double whammy to Bcl2's survival function by both dephosphorylating and enhancing p53-Bcl2 binding. Therapeutically stimulating Bcl2 dephosphorylation and/or increasing Bcl2/p53 binding by activating PP2A may represent an efficient and novel antineoplastic approach.

Protein Mapping and Characterization of Post-Translational Modifications of Ku86 Protein Variants in Multiple Myeloma Cell Lines.

A single biological event, immunoglobulin heavy chain (IgH) isotype class switch recombination (CSR) is thought to mark the onset of multiple myeloma (MM). The DNA repair enzyme, DNA-dependent protein kinase (DNA-PK), which consists of a heterodimeric regulatory subunit (Ku70/Ku86) and its catalytic subunit (DNA-PKcs), is the principal mediator of IgH isotype CSR. Studies in Ku70 and Ku86 knockout mice have suggested that Ku proteins could have a role to play in carcinogenesis. Since we have previously detected truncated variants of Ku86 (Ku86v) in 86% to 100% of freshly isolated patient MM cells, we therefore hypothesize that Ku86v could be a putative oncoprotein. Two variants of Ku86 are predominantly found in MM cells: a 69 kDa variant truncated at the C-terminus (Ku86v-N), which has lost its DNA repair function; and a 56 kDa N-terminus truncated variant (Ku86v-C), which is not translocated into the nucleus and aberrantly expressed on the cell membrane. Our prior studies using Northern blotting, whole cell polyacrylamide gel electrophoresis (PAGE) and electrophoretic mobility shift assays (EMSA) have demonstrated that Ku86vs in MM cells are not the result of alternative RNA splicing; or in vitro degradation, as has been described in human lymphocytes. Moreover, since many proteins in MM cells are known to be heavily glycosylated, and deglycosylation could lead to shorter forms of the protein, we now show using Endo H digestion that Ku86vs are also not the result of N-linked deglycosylation. Rather, Ku86vs are formed by in vivo proteolytic cleavage via a trypsin-like serine protease. Since the proteasome degradation pathway is highly active in MM cells; and protease digestion of DNA-PK holoenzyme is enhanced by proteasome inhibition (i.e. bortezomib treatment), these data support the notion that in vivo activation of putative Ku86v oncoproteins within MM cells could arise via a constitutively active post-translational proteolytic process. To define a functional role for Ku86v, we first demonstrate that Ku86v is physically associated with the most abundant anti-apoptotic protein in MM cells, BCL2. We then used cyanogen bromide immunoprecipitation to purify BCL2-bound Ku86vs followed by 2-dimensional gel electrophoresis (2DGE) to resolve these purified proteins, and demonstrate that only the 56 kDa Ku86v-C (pI 5.0) bound to BCL2. Both full length Ku86 and Ku86v-N were not detected. We then isolated Ku86v-C from the 2D gel and preliminary mass spectrometric analyses suggest that the N-terminus truncation retains the Ku autoantigen related protein 1 (KARP1) motif of Ku86. The significance of this is still under investigation. In conclusion, we have found that Ku86v-C (56 kDa pI 5.0) is formed by post-translational proteolytic cleavage. Since, Ku86v-C is significantly associated with BCL2 oncoprotein, we therefore speculate that Ku86v-C could possibly potentiate BCL2's anti-apoptotic function. Accordingly, Ku86v-C could be considered as a potential target for therapeutic intervention in MM.