TNFα modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts.

INTRODUCTION: Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts. METHODS: Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability. RESULTS: RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells. CONCLUSIONS: TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue.

Induction of apoptosis in Eµ-myc lymphoma cells in vitro and in vivo through calpain inhibition.

Calpains are cysteine proteases that have been implicated as both effectors and suppressors of apoptosis. Previously, we showed that c-myc transformation regulated calpain activity and sensitized cells to apoptosis induced by calpain inhibition. The objective of this study was to investigate the role of calpain in the Eµ-myc transgenic model of B-cell lymphoma. Calpain activity assays, apoptosis, cell cycle assays, and expression measurements were used to determine the activity and role of calpain in vitro and in vivo. We found that Eµ-myc transgenic cells have highly elevated calpain activity. Calpastatin, the negative calpain regulator, was expressed at much lower levels in Eµ-myc lymphoma cells compared to normal splenic B cells. The primary isoform in Eµ-myc lymphoma is calpain 1. Treatment of Eµ-myc lymphoma cells with the calpain inhibitors PD150606 or calpain inhibitor III induced caspase-3-dependent apoptosis in vitro. General caspase inhibitors or caspase-3/7 inhibitor protected cells from death induced by calpain inhibitor, whereas caspase-9 inhibitors failed to rescue cells. Human Burkitt's lymphoma (BL2) cells display a pattern of sensitivity and caspase-3 dependence similar to calpain inhibition. Treatment of Eµ-myc lymphoma-bearing mice with PD150606 inhibited calpain activity in vivo and induced cell death in these cells as determined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. Multiple daily treatments resulted in reduced tumor load, particularly in combination with etoposide. In conclusion, calpain is highly elevated in the Eµ-myc lymphoma and calpain inhibition has therapeutic potential.

Anti-tumor immunity in a model of acute myeloid leukemia.

Whole-cell vaccines allow the induction of anti-tumor immune responses without the need to define tumor antigens. We wished to directly compare, for the first time, the capacity of B7-1, B7-2 and 4-1BB ligand (4-1BBL) costimulatory molecules to convert murine and human acute myeloid leukemia (AML) cells into whole vaccines. 32Dc-kit is a murine myeloid cell line, which develops an AML-like disease over a protracted period, emulating human AML disease development. 32Dc-kit cells were modified to express elevated levels of B7-1, B7-2 or 4-1BBL, and each led to tumor rejection, although only mice injected with 32Dc-kit/B7-2 cells were able to reject subsequent parental tumor cell challenge. T-cell deficient nude mice were able to reject the 32Dc-kit variants, but they could not reject parental cell challenge; however, we found no evidence of cytotoxic T lymphocyte or natural killer (NK) activity ex vivo suggesting that tumor cell killing was mediated by an immune response that could not be recapitulated using purified NK or T cells as lone effectors. In human allogeneic mixed lymphocyte reactions (MLRs), we found no single costimulatory molecule was more effective, suggesting that the induction of a universal anti-tumor response will require a combination of costimulatory molecules.

Regulation of calpain activity by c-Myc through calpastatin and promotion of transformation in c-Myc-negative cells by calpastatin suppression.

The c-Myc transcription factor is commonly dysregulated in cancer. c-Myc also sensitizes cells to apoptosis induced by a variety of toxic events. c-Myc turnover is rapid and mediated by the proteasome and intracellular calpains. Therefore, c-Myc accumulation could contribute to cell death associated with protease inhibitors. We investigated the response of c-Myc-positive and c-Myc-negative rat fibroblast cells to proteasome and calpain inhibitors. Apoptosis induced by the proteasome inhibitor, epoxomycin, was c-Myc-independent, whereas apoptosis induced by the calpain inhibitor, PD150606, or by knockdown of calpain small subunit 1 (CPNS1) was strongly dependent on c-Myc. HL60 cells knocked down for c-Myc expression exhibited reduced calpain activity and decreased sensitivity to PD150606 but not epoxomycin. Calpain inhibitor- or CPNS1 knockdown-induced apoptosis in c-Myc-positive fibroblasts was associated with cell detachment and could be prevented by plating cells on fibronectin, suggesting an anoikis phenomenon. c-Myc stimulated calpain activity by suppressing calpastatin expression, the endogenous calpain inhibitor. Knockdown of calpastatin in c-Myc-negative cells led to a restoration of calpain activity, enhanced cell growth, cell cycle redistribution, anchorage independence, and tumorigenicity in immunodeficient mice. Taken together, these results indicate that c-Myc regulates calpain activity through calpastatin; apoptosis induced by calpain inhibition is dependent on c-Myc, and calpastatin knockdown promotes transformation in c-Myc-negative cells.

Mitochondrial regulation by c-Myc and hypoxia-inducible factor-1 alpha controls sensitivity to econazole.

Econazole is an azole antifungal with anticancer activity that blocks Ca(2+) influx and stimulates endoplasmic reticulum (ER) Ca(2+) release through the generation of mitochondrial reactive oxygen species (ROS), resulting in sustained depletion of ER Ca(2+) stores, protein synthesis inhibition, and cell death. c-Myc, a commonly activated oncogene, also promotes apoptosis in response to growth factor withdrawal and a variety of chemotherapeutic agents. We have investigated the role of c-myc in regulating sensitivity to econazole. Here, we show that c-myc-negative cells are profoundly resistant to econazole. c-Myc-negative rat fibroblasts failed to generate mitochondrial ROS in response to econazole and consequently failed to deplete the ER of Ca(2+). HL60 cells knocked down for c-myc expression also displayed decreased ROS generation and decreased econazole sensitivity. Addition of H(2)O(2) restored sensitivity to econazole in both c-myc-negative rat fibroblasts and c-myc knocked-down HL60 cells, supporting a role for ROS in cell death induction. c-Myc-negative cells and HL60 cells knocked down for c-myc have reduced mitochondrial content compared with c-myc-positive cells. The hypoxia sensor, hypoxia-inducible factor-1alpha (HIF-1alpha), interacts antagonistically with c-myc and also regulates mitochondrial biogenesis. Knockdown of HIF-1alpha in c-myc-negative cells increased mitochondrial content restored ROS generation in response to econazole and increased sensitivity to the drug. Taken together, these results show that c-myc and HIF-1alpha regulate sensitivity to econazole by modulating the ability of the drug to generate mitochondrial ROS.

Articular damage is associated with intraosseous inflammation in the subchondral bone marrow of joints affected by experimental inflammatory arthritis and is modified by zoledronate treatment.

OBJECTIVE: We studied histological changes in the cell populations of subchondral bone marrow resulting from inflammation in animal models of arthritis. Inflammation in intraosseous spaces and the effect of treatment with the aminobisphosphonate zoledronate were assessed. METHODS: Peripheral blood, femoral bone marrow, and spleen cells were harvested from carrageenan-induced arthritis Sprague-Dawley rats; fluorescence stained for CD3, CD8, CD4, CD11b/c, anti-mononuclear phagocyte (MNP), CD3/CD8, CD3/CD4, and MNP/CD11b/c; and evaluated by flow cytometry. Arthritis was induced in New Zealand white rabbits in 5 treatment groups: normal, arthritis, zoledronate-treated from arthritis induction, or 2 or 4 weeks after induction. Animals were euthanized after 7 weeks and distal femoral condyles were decalcified, processed, sectioned, and stained. Sections were evaluated for several cell types and histological features relevant to inflammation, which were assigned a categorical grade from 0 (healthy morphology) to 3 (extensive inflammation). Tidemark was evaluated on a continuous scale. RESULTS: Significantly elevated levels of CD3+CD4+ lymphocytes and CD11b/c+MNP+ monocytes were observed in the bone marrow of arthritic rats, consistent with intraosseous inflammation. No corresponding increase was observed in peripheral blood or spleen. In rabbits, the histology of lymphocytes, mononuclear cells, fibrocytes, fibrosis, lipocytes, and plasma cells varied significantly across treatment groups. Controls exhibited normal histology. In arthritis, these cell types and tissues displayed a shift of 1-2 grades higher (toward inflammation). Intact tidemark was reduced. Following zoledronate treatment, mononuclear cells, fibrocytes, fibrosis, and lipocytes were significantly altered toward normal, and tidemark shifted toward normal. CONCLUSION: Intraosseous bone marrow inflammation was observed in carrageenan-induced inflammatory arthritis in rats and rabbits. Zoledronate administration diminished the intraosseous inflammatory response.

Signaling pathways influencing SLF and c-kit-mediated survival and proliferation.

Steel factor (SLF) and c-Kit are a ligand-receptor pair that regulates growth and activation of a variety of hemopoietic and non-hemopoietic cells. This review describes our work investigating downstream signaling pathways activated by SLF, with particular emphasis on signaling differences associated with soluble vs membrane- bound ligand, and our identification of an important role for PLC activation and Ca2+ influx in supporting c-Kit positive cells in vitro and in vivo. This work led to the identification of a unique form of cell death termed activation enhanced cell death (AECD) that involves stimulating a cell with a growth or activation signal while concurrently blocking Ca2+ influx. Approaches that we have taken toward identifying cellular factors associated with sensitivity and resistance to AECD are summarized, as is our experience with a variety of experimental models. The use of econazole as a calcium channel blocker and its mechanism of action are described, as is its potential for development as an anticancer therapeutic.

Inhibition of Ca2+ influx is required for mitochondrial reactive oxygen species-induced endoplasmic reticulum Ca2+ depletion and cell death in leukemia cells.

Disturbances of endoplasmic reticulum (ER) Ca2+ homeostasis or protein processing can lead to ER stress-induced cell death. Increasing evidence suggests that oxidative stress (OS) plays an important role in a variety of cell death mechanisms. To investigate the role of OS in ER stress, we measured OS in response to three ER stress agents: econazole (Ec), which stimulates ER Ca2+ release and blocks Ca2+ influx; thapsigargin (Tg), a sarco(endo)plasmic reticulum Ca2+ ATPase inhibitor that releases ER Ca2+ and stimulates Ca2+ influx; and tunicamycin (Tu), a glycosylation inhibitor that causes protein accumulation in the ER. Ec, but not Tg or Tu, caused a rapid increase in OS. Reactive oxygen species (ROS) generation was observed within mitochondria immediately after exposure to Ec. Furthermore, Ec hyperpolarized the mitochondrial membrane and inhibited adenine nucleotide transport in cell-free mitochondria, suggesting a mitochondrial target. Antimycin A, an inhibitor of complex III in electron transport, reversed mitochondrial hyperpolarization, OS generation, ER Ca2+ depletion, and cell death by Ec, suggesting complex III dependence for these effects. Antioxidants butylated hydroxytoluene and N-Acetyl-L-cysteine prevented ER Ca2+ depletion and cell death by Ec. However, inhibition of Ca2+ influx by Ec was unaffected by either antimycin A or the antioxidants, suggesting that this target is distinct from the mitochondrial target of Ec. Atractyloside, an adenine nucleotide transport inhibitor, generated ROS and stimulated ER Ca2+ release, but it did not block Ca2+ influx, deplete the ER or induce cell death. Taken together, these results demonstrate that combined mitochondrial ROS generation and Ca2+ influx blockade by Ec is required for cell death.

Development of a whole cell vaccine for acute myeloid leukaemia.

We describe the modification of tumour cells to enhance their capacity to act as antigen presenting cells with particular focus on the use of costimulatory molecules to do so. We have been involved in the genetic modification of tumour cells to prepare a whole cell vaccine for nearly a decade and we have a particular interest in acute myeloid leukaemia (AML). AML is an aggressive and difficult to treat disease, especially, for patients for whom haematopoietic stem cell (HSC) transplant is not an option. AML patients who have a suitable donor and meet HSC transplant fitness requirements, have a 5-year survival of 50%; however, for patients with no suitable donor or for who age is a factor, the prognosis is much worse. It is particularly poor prognosis patients, who are not eligible for HSC transplant, who are likely to benefit most from immunotherapy. It would be hoped that immunotherapy would be used to clear residual tumour cells in these patients in the first remission following standard chemotherapy treatments and this will extend the remission and reduce the risk of a second relapse associated with disease progression and poor mortality rates. In this symposia report, we will focus on whole cell vaccines as an immunotherapeutic option with particular reference to their use in the treatment of AML. We will aim to provide a brief overview of the latest data from our group and considerations for the use of this treatment modality in clinical trials for AML.

Increased calcium influx and ribosomal content correlate with resistance to endoplasmic reticulum stress-induced cell death in mutant leukemia cell lines.

Cell clones were derived by treatment of HL-60 cells with stepwise increasing concentrations of econazole (Ec), an imidazole antifungal that blocks Ca2+ influx and induces endoplasmic reticulum (ER) stress-related cell death in multiple mammalian cell types. Clones exhibit 20- to more than 300-fold greater resistance to Ec. Unexpectedly, they also display stable cross-resistance to tunicamycin, thapsigargin, dithiothreitol, and cycloheximide but not doxorubicin, etoposide, or Fas ligand. Phenotypic analysis indicates that the cells display increased store-operated calcium influx and resistance to ER Ca2+ store depletion by Ec. E2R2, the most resistant clone, was observed to maintain protein synthesis levels after treatment with Ec or thapsigargin. Expression of GRP78, an ER-based chaperone, was induced by these ER stress treatments but to equal degrees in HL-60 and E2R2 cells. By using microarray analysis, at least 15 ribosomal protein genes were found to be overexpressed in E2R2 compared with HL-60 cells. We also found that ribosomal protein content was increased by 30% in E2R2 as well as other clones. The resistance phenotype was partially reversed by the ribosome-inactivating protein saporin. Therefore, increased store-operated calcium influx, resistance to ER Ca2+ store depletion, and overexpression of ribosomal proteins define a novel phenotype of ER stress-associated multidrug resistance.

Ketotifen reverses MDR1-mediated multidrug resistance in human breast cancer cells in vitro and alleviates cardiotoxicity induced by doxorubicin in vivo.

PURPOSE: To investigate the effect of the antihistamine ketotifen on multidrug resistance in human breast cancer cells and doxorubicin toxicity in mice. METHODS: Clonogenicity assays were used to test the effect of ketotifen on human multidrug resistant breast cancer cell lines exposed to chemotherapeutic agents. Flow cytometry was used to measure accumulation of doxorubicin in cells. Fluorimetry was used to measure accumulation of doxorubicin in cardiac tissues. Histological analysis and toxicity studies in mice were used to test the effect of ketotifen on doxorubicin-induced toxicity. RESULTS: Ketotifen was found to restore the sensitivity of P-glycoprotein-overexpressing multidrug-resistant MCF-7/adr cells to doxorubicin, mitoxantrone, VP-16 and vinblastine, but not to methotrexate or camptothecin. Ketotifen, however, was unable to restore sensitivity of BCRP-overexpressing MCF-7/mx cells or MRP-overexpressing MCF-7/vp cells to mitoxantrone or VP-16, respectively. In vivo, pretreatment of mice with ketotifen caused an increased accumulation of doxorubicin in cardiac tissue, consistent with a block in drug clearance. However, unlike verapamil, ketotifen pretreatment did not enhance doxorubicin toxicity but in fact provided protection, both at the level of cardiac tissue damage and in terms of survival. CONCLUSIONS: Taken together, these observations show that ketotifen is unique in its ability both to reverse multidrug resistance due to P-glycoprotein overexpression and to provide cardioprotection to doxorubicin.

Sensitivity of myeloid leukemia cells to calcium influx blockade: application to bone marrow purging.

OBJECTIVE: The aim of this study was to assess the potential of store-operated Ca(2+) channel (SOC) antagonists as purging agents for leukemia cells. MATERIALS AND METHODS: Clonogenic, limiting dilution, and nuclear condensation assays were used to evaluate SOC antagonist efficacy. SOC activity and endoplasmic reticulum Ca(2+) content were measured by flow cytometry. Murine bone marrow transplantation was used to determine purging efficacy and effects on hemopoietic reconstitution. RESULTS: Econazole (Ec) and ketotifen (Ke) were variably effective against human and murine leukemia cell lines after 24 hours of incubation. However, a 2-hour serum and bovine serum albumin-free treatment protocol with Ec was found to maximize differential sensitivity between leukemic cells and normal hemopoietic progenitors. Primary acute myelogenous leukemia blast cell viability was reduced 4.2 to 5.1 logs by 2-hour Ec treatment as measured by limiting dilution. An inverse relationship between endoplasmic reticulum Ca(2+) content and Ke sensitivity in leukemia and untransformed cells was observed. Nuclear condensation, an index of apoptosis, which occurred after 24-hour treatments with either Ec or Ke, was not observed after 2-hour serum- and bovine serum albumin-free Ec exposures; however, condensed nuclei were observed after an additional 10-hour incubation in growth medium without drug. Using bone marrow deliberately contaminated with 1% P815 cells, we showed that highly effective in vitro purging can be accomplished using Ec with no adverse effects on bone marrow reconstitution in mice. CONCLUSIONS: These studies suggest that SOC antagonists have potential as purging agents for residual leukemia cells present in bone marrow in the context of high-dose chemotherapy and autologous transplantation for leukemia.