Protein kinase Cbeta modulates ligand-induced cell surface death receptor accumulation: a mechanistic basis for enzastaurin-death ligand synergy.

Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-alpha-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2-5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCalpha, PKCbeta, PKCepsilon, and PKC, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCbeta shRNA uniquely reversed PMA-induced protection against cell death. The PKCbeta-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCbeta shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCbeta, to tumor necrosis factor-alpha related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death.

Pooled analysis of fluorouracil-based adjuvant therapy for stage II and III colon cancer: who benefits and by how much?

PURPOSE: Although it is well-established that fluorouracil- (FU-) based adjuvant therapy improves survival for patients with resected high-risk colon cancer, the magnitude of adjuvant therapy benefit across specific subgroups and for individual patients has been uncertain. PATIENTS AND METHODS: Using a pooled data set of 3,302 patients with stage II and III colon cancer from seven randomized trials comparing FU + leucovorin or FU + levamisole to surgery alone, we performed an analysis based on a Cox proportional hazards regression model. Treatment, age, sex, tumor location, T stage, nodal status, and grade were tested for both prognostic and predictive significance. Model derived estimates of 5-year disease-free survival and overall survival (OS) for surgery alone and surgery plus FU-based therapy were calculated for a range of patient subsets. RESULTS: Nodal status, T stage, and grade were the only prognostic factors independently significant for both disease-free survival and OS. Age was significant only for OS. In a multivariate analysis, adjuvant therapy showed a beneficial treatment effect across all subsets. Treatment benefits were consistent across sex, location, age, T-stage, and grade. A significant stage by treatment interaction was present, with treatment benefiting stage III patients to a greater degree than stage II patients. CONCLUSION: Patients with high-risk resected colon cancer obtain benefit from FU-based therapy across subsets of age, sex, location, T stage, nodal status, and grade. Model estimates of survival stratified by T stage, nodal status, grade, and age are available at http://www.mayoclinic.com/calcs. This information may improve patients' and physicians' understanding of the potential benefits of adjuvant therapy.

Determination of potential adjuvant systemic therapy benefits for patients with resected cutaneous melanomas.

OBJECTIVE: To facilitate both better physician understanding of prognostic information (baseline and with adjuvant interferon) for individual patients who present with resectable melanomas and more informed patient decisions about whether they should receive adjuvant high-dose interferon therapy after resection of primary melanomas. PATIENTS AND METHODS: Baseline survival estimates were derived from a surgical database composed of 17,600 patients with complete clinical, pathologic, and follow-up data. Potential survival benefits ascribed to adjuvant interferon were obtained from results of a meta-analysis of Eastern Cooperative Oncology Group studies, which provided evidence for a uniform relative benefit of high-dose interferon across different baseline risk groups. A mathematical formula was then applied to these data to allow for individual prognostic information. RESULTS: The 5-year survival benefits in patients who received high-dose interferon after surgery, using the assumptions of the provided prognoses and interferon survival improvements, ranged up to 13%. CONCLUSIONS: These data should allow for a better understanding of baseline prognosis in individual patients and a better understanding of the potential benefits of adjuvant interferon. They should also help patients make more informed decisions regarding their treatment options.

Dynamin 2 regulates T cell activation by controlling actin polymerization at the immunological synapse.

Actin reorganization at the immunological synapse is required for the amplification and generation of a functional immune response. Using small interfering RNA, we show here that dynamin 2 (Dyn2), a large GTPase involved in receptor-mediated internalization, did not alter antibody-mediated T cell receptor internalization but considerably affected T cell receptor-stimulated T cell activation by regulating multiple biochemical signaling pathways and the accumulation of F-actin at the immunological synapse. Moreover, Dyn2 interacted directly with the Rho family guanine nucleotide exchange factor Vav1, and this interaction was required for T cell activation. These data identify a functionally important interaction between Dyn2 and Vav1 that regulates actin reorganization and multiple signaling pathways in T lymphocytes.

Heat shock protein 90 inhibition sensitizes acute myelogenous leukemia cells to cytarabine.

Previous studies demonstrated that ataxia telangiectasia mutated- and Rad3-related (ATR) kinase and its downstream target checkpoint kinase 1 (Chk1) facilitate survival of cells treated with nucleoside analogs and other replication inhibitors. Recent results also demonstrated that Chk1 is depleted when cells are treated with heat shock protein 90 (Hsp90) inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The present study examined the effects of 17-AAG and its major metabolite, 17-aminogeldanamycin (17-AG), on Chk1 levels and cellular responses to cytarabine in human acute myelogenous leukemia (AML) cell lines and clinical isolates. Cytarabine, at concentrations as low as 30 nM, caused activating phosphorylation of Chk1, loss of the phosphatase Cdc25A, and S-phase slowing. Conversely, treatment with 100 to 300 nM 17-AAG for 24 hours caused Chk1 depletion that was accompanied by diminished cytarabine-induced S-phase accumulation, decreased Cdc25A degradation, and enhanced cytotoxicity as measured by inhibition of colony formation and induction of apoptosis. Additional studies demonstrated that small inhibitory RNA (siRNA) depletion of Chk1 also sensitized cells to cytarabine, whereas disruption of the phosphatidylinositol 3-kinase (PI3k) signaling pathway, which is also blocked by Hsp90 inhibition, did not. Collectively, these results suggest that treatment with 17-AAG might represent a means of reversing checkpoint-mediated cytarabine resistance in AML.

Phorbol 12-myristate 13-acetate inhibits death receptor-mediated apoptosis in Jurkat cells by disrupting recruitment of Fas-associated polypeptide with death domain.

Regulation of death receptor-mediated apoptosis is incompletely understood. Previous studies have demonstrated that phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, inhibits Fas (CD95)-mediated apoptosis in Jurkat (type II) cells but not SKW6.4 (type I) cells. In this study, we demonstrated that PMA also protects Jurkat cells from apoptosis induced by tumor necrosis factor-alpha and the tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL). Interestingly, PMA failed to protect Jurkat cells from apoptosis induced by other agents, including etoposide, camptothecin, and gamma-irradiation. Analysis of the initial events induced by agonistic anti-Fas antibodies revealed that PMA inhibited Fas binding to Fas-associated polypeptide with death domain (FADD) in Jurkat cells but not in SKW6.4 cells. Although the protein kinase inhibitor bisindoylmaleimide VIII increased apoptosis induced by agonistic anti-Fas antibody, tumor necrosis factor-alpha, and TRAIL, these effects were not observed with the protein kinase C inhibitor H7 and were not associated with increased FADD recruitment to Fas. These results indicate that PMA inhibits death signaling induced by a number of discrete receptors and suggest that the effects are mediated at the level of receptor-mediated adaptor molecule recruitment.

Synthetic Smac/DIABLO peptides enhance the effects of chemotherapeutic agents by binding XIAP and cIAP1 in situ.

Inhibitor of apoptosis proteins (IAPs) interact with and inhibit caspases-3, -7, and -9. This interaction can be inhibited by Smac/DIABLO, a polypeptide released from mitochondria upon initiation of the apoptotic signaling process. Here we demonstrate that the first 4-8 N-terminal amino acids of Smac/DIABLO fused to the Drosophila antennapaedia penetratin sequence, a carrier peptide, enhance the induction of apoptosis and long term antiproliferative effects of diverse antineoplastic agents including paclitaxel, etoposide, 7-ethyl-10-hydroxycamptothecin (SN-38), and doxorubicin in MCF-7 breast cancer cells. Similar effects were observed in additional breast cancer and immortalized cholangiocyte cell lines. Further analysis demonstrated that the Smac-penetratin fusion peptide crossed the cellular membrane, bound XIAP and cIAP1, displaced caspase-3 from cytoplasmic aggregates, and enhanced drug-induced caspase action in situ. These studies demonstrate that inhibition of IAP proteins can modulate the efficacy of antineoplastic agents.

Microtubule destabilization and nuclear entry are sequential steps leading to toxicity in Huntington's disease.

There has been a longstanding debate regarding the role of proteolysis in Huntington's disease. The toxic peptide theory posits that N-terminal cleavage fragments of mutant Huntington's disease protein [mutant huntingtin (mhtt)] enter the nucleus to cause transcriptional dysfunction. However, recent data suggest a second model in which proteolysis of full-length mhtt is inhibited. Importantly, the two competing theories differ with respect to subcellular distribution of mhtt at initiation of toxicity: nuclear if cleaved and cytoplasmic in the absence of cleavage. Using quantitative single-cell analysis and time-lapse imaging, we show here that transcriptional dysfunction is "downstream" of cytoplasmic dysfunction. Primary and reversible toxic events involve destabilization of microtubules mediated by full-length mhtt before cleavage. Restoration of microtubule structure by taxol inhibits nuclear entry and increases cell survival.

Heterogeneous role of caspase-8 in fenretinide-induced apoptosis in epithelial ovarian carcinoma cell lines.

The mechanism of action of fenretinide, a synthetic retinoid currently undergoing testing as a chemopreventive and chemotherapeutic agent, is incompletely understood. In the present study, fenretinide caused apoptotic changes, including DNA fragmentation and cleavage of caspase substrates, in six low-passage ovarian cancer cell lines. However, the caspase activation pathway used by this agent varied. Transient transfection of cDNA-encoding cytokine response modifier A (CrmA), a caspase-8 inhibitor, diminished fenretinide-induced death in OV177 cells. Likewise, IETD(OMe)-fluoromethylketone (fmk) inhibited fenretinide-induced apoptosis by >80% in OV177 or OV266 cells and by approximately 50% in OV17, OV167, or OV207 cells. Further analysis demonstrated that inhibition of Fas ligand, tumor necrosis factor-alpha, or TRAIL signaling with blocking reagents did not affect fenretinide-induced apoptosis, raising the possibility that fenretinide activates caspase-8 in a death receptor-independent manner. In contrast, CrmA transfection or IETD(OMe)-fmk treatment did not inhibit fenretinide-induced apoptosis in OV202 cells. These divergent behaviors did not correlate with increased levels of procaspase-10, which is relatively resistant to CrmA and IETD(OMe)-fmk, nor with the expression of procaspase-8 and -9, apoptotic protease activating factor-1, or cellular FLICE-like inhibitory protein. Similarly, fenretinide treatment increased ceramide levels equally in cells that do (OV177) and do not (OV202) rely on caspase-8 to initiate apoptosis. These results indicate that synthetic retinoids can use caspase-8 as an initiating caspase, but they also indicate unexpected heterogeneity in caspase activation pathways among closely related cell lines.