A region within the third extracellular loop of rat Aquaporin 6 precludes trafficking to plasma membrane in a heterologous cell line.

The inability to over-express Aquaporin 6 (AQP6) in the plasma membrane of heterologous cells has hampered efforts to further characterize the function of this aquaglyceroporin membrane protein at atomic detail using crystallographic approaches. Using an Aquaporin 3-tGFP Reporter (AGR) system we have identified a region within loop C of AQP6 that is responsible for severely hampering plasma membrane expression. Serine substitution corroborated that amino acids present within AQP6194-213 of AQP6 loop C contribute to intracellular endoplasmic reticulum (ER) retention. This intracellular retention signal may preclude proper plasma membrane trafficking and severely curtail expression of AQP6 in heterologous expression systems.

An uncharacterized region within the N-terminus of mouse TMC1 precludes trafficking to plasma membrane in a heterologous cell line.

Mechanotransduction by hair cell stereocilia lies at the heart of sound detection in vertebrates. Considerable effort has been put forth to identify proteins that comprise the hair cell mechanotransduction apparatus. TMC1, a member of the transmembrane channel-like (TMC) family, was identified as a core protein of the mechanotransduction complex in hair cells. However, the inability of TMC1 to traffic through the endoplasmic reticulum in heterologous cellular systems has hindered efforts to characterize its function and fully identify its role in mechanotransduction. We developed a novel approach that allowed for the detection of uncharacterized protein regions, which preclude trafficking to the plasma membrane (PM) in heterologous cells. Tagging N-terminal fragments of TMC1 with Aquaporin 3 (AQP3) and GFP fusion reporter, which intrinsically label PM in HEK293 cells, indicated that residues at the edges of amino acid sequence 138-168 invoke intracellular localization and/or degradation. This signal is able to preclude surface localization of PM protein AQP3 in HEK293 cells. Substitutions of the residues by alanine or serine corroborated that the information determining the intracellular retention is present within amino acid sequence 138-168 of TMC1 N-terminus. This novel signal may preclude the proper trafficking of TMC1 to the PM in heterologous cells.

Hypoxia-induced mixed-lineage leukemia 1 regulates glioma stem cell tumorigenic potential.

Normal stem cells reside in functional niches critical for self-renewal and maintenance. Neural and hematopoietic stem cell niches, in particular, are characterized by restricted availability of oxygen and the resulting regulation by hypoxia-inducible factors (HIFs). Glioblastoma multiforme (GBM) is the most common malignant brain tumor and also contains high degrees of hypoxia. Heterogeneity within the neoplastic compartment has been well characterized in GBM and may be derived from genetic and epigenetic sources that co-evolve during malignant progression. Recent experimental evidence has supported the importance of hypoxia in glioma stem cell (GSC) niches. We hypothesized that HIFs require epigenetic-modifying proteins to promote tumor malignancy in GBM. Here we demonstrate that in GBM the histone methyltransferase mixed-lineage leukemia 1 (MLL1) is induced by hypoxia and enhances hypoxic responses. Loss of MLL1 reduces the expression of HIF transcripts and HIF2α protein. Targeting MLL1 by RNA interference inhibited the expression of HIF2α and target genes, including vascular endothelial growth factor (VEGF). GSCs expressed higher levels of MLL1 than matched non-stem tumor cells and depletion of MLL1 reduced GSC self-renewal, growth, and tumorigenicity. These studies have uncovered a novel mechanism mediating tumor hypoxic responses linking microenvironmental regulation of epigenetic-modifying proteins to cellular heterogeneity and provide rationale for the design of more sophisticated clinical approaches targeting epigenetic regulation.

Adoptive immunotherapy in patients with recurrent malignant glioma: preliminary results of using autologous whole-tumor vaccine plus granulocyte-macrophage colony-stimulating factor and adoptive transfer of anti-CD3-activated lymphocytes.

OBJECT: This trial was designed to determine the ability of autologous whole-tumor cell vaccines to induce cell mediated immune responses in patients with recurrent malignant glioma, as well as to determine whether combining such vaccination with adoptive transfer of in vitro activated T lymphocytes prolongs patient survival. METHODS: Nineteen patients with recurrent malignant glioma, in whom previous external beam radiotherapy and at least one course of chemotherapy had failed were vaccinated twice with irradiated autologous whole tumor cells by using granulocyte-marcrophage colony-stimulating factor as an adjuvant. Patients then underwent leukapheresis followed by adoptive transfer of peripheral blood lymphocytes activated in vitro with anti-CD3 and interleukin-2. In vivo immune response, radiological response, clinical outcome, and survival were monitored. Seventeen patients developed a delayed-type hypersensitivity (DTH) response to vaccination that appeared to be directed against the autologous tumor. In eight patients there was radiological evidence of a response and in five there was evidence of clinical improvement. Median survival was 12 months (range 6-28 months), and both the presence of a DTH response and the radiological response correlated with survival (p < 0.02 and p < 0.04, respectively). CONCLUSIONS: These preliminary results suggest that autologous whole-tumor cell vaccines induce a cell-mediated immune response, which appears to be tumor specific in most patients. Furthermore, vaccination combined with adoptive immunotherapy with in vitro activated cells may induce a radiologically demonstrated tumor response and improved survival despite a condition of advanced disease and immunosuppression resulting from previous treatment or tumor burden. Further studies of immunotherapy are warranted.

Enhanced T cell migration to sites of microscopic CNS disease: complementary treatments evaluated by 2- and 3-D image analysis.

Novel therapies are being developed to attack tumour or other abnormal cells within the brain. A general problem is the need for delivery to sites of microscopic disease. Leukocytes offer an attractive solution; they are able to both move through tissue and recognize abnormal targets. Leukocytes may act as effectors, or as vehicles for drugs, retroviral vectors or other agents. Here, we illustrate complementary ways of enhancing leukocyte migration to sites of microscopic central nervous system (CNS) disease. Enhanced T cell migration to sites of disseminated tumour is used as the example. Computer-assisted image analysis is used to evaluate migration patterns in 2 and 3 dimensions. Shared regulatory features in the migration of tumour and responding cells, and the opportunities and questions they imply, are discussed.