Therapeutic enhancement of ER stress by insulin-like growth factor I sensitizes myeloma cells to proteasomal inhibitors.

PURPOSE: Multiple myeloma is a clonal plasma cell disorder in which growth and proliferation are linked to a variety of growth factors, including insulin-like growth factor type I (IGF-I). Bortezomib, the first-in-class proteasome inhibitor, has displayed significant antitumor activity in multiple myeloma. EXPERIMENTAL DESIGN: We analyzed the impact of IGF-I combined with proteasome inhibitors on multiple myeloma cell lines in vivo and in vitro as well as on fresh human myeloma cells. RESULTS: Our study shows that IGF-I enhances the cytotoxic effect of proteasome inhibitors against myeloma cells. The effect of bortezomib on the content of proapoptotic proteins such as Bax, Bad, Bak, and BimS and antiapoptotic proteins such as Bcl-2, Bcl-XL, XIAP, Bfl-1, and survivin was enhanced by IGF-I. The addition of IGF-I to bortezomib had a minor effect on NF-κB signaling in MM.1S cells while strongly enhancing reticulum stress. This resulted in an unfolded protein response (UPR), which was required for the potentiating effect of IGF-I on bortezomib cytotoxicity as shown by siRNA-mediated inhibition of GADD153 expression. CONCLUSIONS: These results suggest that the high baseline level of protein synthesis in myeloma can be exploited therapeutically by combining proteasome inhibitors with IGF-I, which possesses a "priming" effect on myeloma cells for this family of compounds.

Silencing of tubulin binding cofactor C modifies microtubule dynamics and cell cycle distribution and enhances sensitivity to gemcitabine in breast cancer cells.

Tubulin binding cofactor C (TBCC) is essential for the proper folding of α- and ß-tubulins into microtubule polymerizable heterodimers. Because microtubules are considered major targets in the treatment of breast cancer, we investigated the influence of TBCC silencing on tubulin pools, microtubule dynamics, and cell cycle distribution of breast cancer cells by developing a variant MCF7 cells with reduced content of TBCC (MC-). MC- cells displayed decreased content in nonpolymerizable tubulins and increased content of polymerizable/microtubule tubulins when compared with control MP6 cells. Microtubules in MC- cells showed stronger dynamics than those of MP6 cells. MC- cells proliferated faster than MP6 cells and showed an altered cell cycle distribution, with a higher percentage in S-phase of the cell cycle. Consequently, MC- cells presented higher sensitivity to the S-phase-targeting agent gemcitabine than MP6 cells in vitro. Although the complete duration of mitosis was shorter in MC- cells and their microtubule dynamics was enhanced, the percentage of cells in G(2)-M phase was not altered nor was there any difference in sensitivity to antimicrotubule-targeting agents when compared with MP6 cells. Xenografts derived from TBCC variants displayed significantly enhanced tumor growth in vivo and increased sensitivity to gemcitabine in comparison to controls. These results are the first to suggest that proteins involved in the proper folding of cytoskeletal components may have an important influence on the cell cycle distribution, proliferation, and chemosensitivity of tumor cells.

Preclinical studies on the mechanism of action and the anti-lymphoma activity of the novel anti-CD20 antibody GA101.

GA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies.

Sensitivity and gene expression profile of fresh human acute myeloid leukemia cells exposed ex vivo to AS602868.

PURPOSE: The need for new treatment options for acute myeloid leukemia (AML) is increasing. AS602868 is a novel investigational drug with reported activity on AML cells. METHODS: We studied gene expression profiles in AML blasts exposed to AS602868 in order to better understand its mechanism of action. We analyzed the in vitro cytotoxicity of AS602868 alone or in combination with daunorubicin, etoposide or cytarabine. To document AS602868-induced IKK2 inhibition in fresh AML cells, a flow cytometry analysis of IκB was performed. Finally, the effect of AS602868 on gene expression in fresh AML cells was analyzed. RESULTS: The results show that AML cells are globally as sensitive to AS602868 as they are to cytarabine, with large interindividual variations. Combinations with conventional antileukemic agents showed enhanced cytotoxic activity in subsets of patients. IKK2 appeared to be effectively inhibited by 100 µM AS602868 in fresh leukemic cells. Gene expression profiling and gene ontology analyses identified several groups of genes induced/inhibited by exposure to AS602868 and/or exhibiting a correlation with sensitivity to this agent in vitro. Of note, the expression of several genes related to immune function was found to be significantly altered after exposure to AS602868. CONCLUSION: These data suggest that AS602868 is cytotoxic against fresh human AML blasts and provide insights regarding the mechanisms of cytotoxicity.

Tubulin binding cofactor C (TBCC) suppresses tumor growth and enhances chemosensitivity in human breast cancer cells.

BACKGROUND: Microtubules are considered major therapeutic targets in patients with breast cancer. In spite of their essential role in biological functions including cell motility, cell division and intracellular transport, microtubules have not yet been considered as critical actors influencing tumor cell aggressivity. To evaluate the impact of microtubule mass and dynamics on the phenotype and sensitivity of breast cancer cells, we have targeted tubulin binding cofactor C (TBCC), a crucial protein for the proper folding of alpha and beta tubulins into polymerization-competent tubulin heterodimers. METHODS: We developed variants of human breast cancer cells with increased content of TBCC. Analysis of proliferation, cell cycle distribution and mitotic durations were assayed to investigate the influence of TBCC on the cell phenotype. In vivo growth of tumors was monitored in mice xenografted with breast cancer cells. The microtubule dynamics and the different fractions of tubulins were studied by time-lapse microscopy and lysate fractionation, respectively. In vitro sensitivity to antimicrotubule agents was studied by flow cytometry. In vivo chemosensitivity was assayed by treatment of mice implanted with tumor cells. RESULTS: TBCC overexpression influenced tubulin fraction distribution, with higher content of nonpolymerizable tubulins and lower content of polymerizable dimers and microtubules. Microtubule dynamicity was reduced in cells overexpressing TBCC. Cell cycle distribution was altered in cells containing larger amounts of TBCC with higher percentage of cells in G2-M phase and lower percentage in S-phase, along with slower passage into mitosis. While increased content of TBCC had little effect on cell proliferation in vitro, we observed a significant delay in tumor growth with respect to controls when TBCC overexpressing cells were implanted as xenografts in vivo. TBCC overexpressing variants displayed enhanced sensitivity to antimicrotubule agents both in vitro and in xenografts. CONCLUSION: These results underline the essential role of fine tuned regulation of tubulin content in tumor cells and the major impact of dysregulation of tubulin dimer content on tumor cell phenotype and response to chemotherapy. A better understanding of how the microtubule cytoskeleton is dysregulated in cancer cells would greatly contribute to a better understanding of tumor cell biology and characterisation of resistant phenotypes.

Transfection of cells in suspension by ultrasound cavitation.

Sonoporation holds many promises in developing an efficient, reproducible and permanent gene delivery vector. In this study, we evaluated sonoporation as a method to transfect nucleic acids in suspension cells, including the human follicular lymphoma cell line RL and fresh human Chronic Lymphocytic Leukemia (CLL) cells. RL and CLL cells were exposed to continuous ultrasound waves (445 kHz) in the presence of either plasmid DNA coding for green fluorescent protein (GFP) or fluorescent siRNA directed against BCL2L1. Transfection efficiency and cell viability were assessed using fluorescent microscopy and flow cytometry analysis, respectively. Knock-down of target protein by siRNA was assessed by immunoblotting. Moreover, sonoporation was used to stably transfect RL cells with a plasmid coding for luciferase (pGL3). These cells were then used for the non-invasive monitoring of tumorigenesis in immunodeficient SCID mice. Sonoporation allows a highly efficient transfection of nucleic acid in suspension cells with a low rate of mortality, both in a tumor cell line and in fresh human leukemic cells. It also allowed efficient transfection of BCL2L1 siRNA with efficient reduction of the target protein level. In conclusion, ultrasound cavitation represents an efficient method for the transfection of cells in suspension, including fresh human leukemic cells.

Vinorelbine induces beta3-tubulin gene expression through an AP-1 Site.

Although the correlation between beta3-tubulin expression level in tumors has been well correlated with clinical outcome in patients receiving microtubule-targeted agents, the regulation of this protein remains poorly understood. Recently, new insight of regulatory processes was offered with the cloning of the gene promoter. In this study beta3-tubulin gene expression was induced in response to exposure to various antimicrotubule agents such as vinorelbine and paclitaxel. The exploration of the beta3-tubulin gene promoter by successive deletions followed by site-directed mutagenesis led to the localization of a vinorelbine-responsive element containing an activator-protein 1 (AP-1) site. Among the various antimicrotubule agents tested, it appeared that the implicated AP-1 site was activated only after exposure to vinorelbine. This study confirms the inducible nature of the beta3-tubulin gene promoter.