Jim Klostergaard - US grants

We will continue purification and characterization of lymphotoxins (LTS) derived from PMA-stimulated human B-lymphoblastoid cell lines. Purification of alpha-LT from the RPMI 1788 line will be acomplished with one final anticipated step. The electrophoretically homogeneous product will be analyzed by SDS-PAGE. Two models for the structure of the 70-90kd alpha-LT molecule will be examined: a) The alpha-LT is a multimer of a 20kd subunit, as suggested by evidence obtained with currently available cDNA or, b) the alpha-LT is a heterodimer of the 20kd subunit and a 70kd subunit, as suggested by several studies analyzing purified LTs derived from lectin-stimulated human lymphocytes or from a B-lymphoblastoid cell line. If our evidence using the RPMI 1788 alpha-LT further supports b) and the multi-gene nature of the LT system we will i.) develop rabbit antisera and/or rat and mouse monoclonal antibodies to the separate peptides, ii.) analyze by peptide mapping, in SDS gels or by HPLC, the 20kd and 70kd peptides, and iii.) scale up production of the alpha-LT to allow partial amino acid sequencing of the 70kd peptide in collaboration with Biogen. These studies will further characterize the human LT system at the peptide, mRNA, and genomic levels, and will generate elegant probes to assess the expression of LT gene(s) in activated T- and NK-effector cell populations.

The goal of this revised competing application is unchanged; we remain interested in hyperthermia's effect on macrophage/monocyte cytotoxic interactions with tumor cells. However, the focus of the specific aims have evolved toward understanding the interaction of a key cytotoxic molecule of these effector cells, tumor necrosis factor (TNF), with heated tumor cells. We have shown that over-expression of heat shock protein (HSP)27 in TNF-sensitive L929 cells renders these cells highly resistant to TNF. The mechanism(s) for this protection need to be identified and related to our observation of a sequence dependency in heat's sensitization of TNF's cytotoxicity. We will study heat's augmentation of TNF's cytotoxicity and the basis for HSP-mediated protection against TNF-cytotoxicity and the experiments will illuminate the signaling pathways for TNF cytotoxicity and protection and help identify functions of HSP27. The specific aims test the following hypothesis: (1) The protection from TNF cytotoxicity afforded by human HSP27 is not unique to murine L929 cells and is observed when HSP27 is overexpressed in other TNF-sensitive cells. To address this hypothesis, the murine cell line WEHI-164 has already been transfected with sense and anti-sense HSP27 cDNAs, and the human cell lines CaOV-3 and MCF7 will be similarly transfected. These cells will be tested for their response to TNF relative to parental cells and for protection from heat-induced TNF sensitization. (2) Phosphorylation of HSP27 at Ser sites is required for protection against TNF cytotoxicity and against heat-induced TNF sensitization. To examine this hypothesis, L929 and WEHI-164 cells transfected with mutant HSP27 cDNAs encoding Ser to Gly and Ser to Ala substitutions have already been obtained and will be tested for protection against TNF and heat compared to already established sense and anti-sense L929 and WEHI-164 HSP27 stable transfectants. (3) The TNF protection afforded by HSP27 lies in inhibiting the sphingomyelin signaling pathway initiated by TNF treatment. Transfections expressing wild type or mutant HSP27 will be compared to parental cells for the effect of TNF and heat on, first, proximal and, later, downstream components of this pathway. (4) The TNF protection afforded by HSP27 lies in modulating a diacylglycerol (DAG)/protein kinase C (PKC) signaling pathway initiated by TNF treatment. To test these hypotheses, transfectants expressing wild type or mutant HSP27 will be compared to parental cells for the effect of TNF and heat on, first, proximal and, later, downstream components of this pathway. (5) Hyperthermic enhancement/deregulation of one or both of the signaling cascades in Aims Three and Four leads to enhancement of TNF cytotoxicity by apoptosis and the inhibition of this (these) cascades) by HSP27 inhibits the downstream apoptotic endpoints. Under this aim, parental and HSP27-transfected cells, as obtained as above, will be studied for the effect(s) of TNF and heat on the expression of genes associated with apoptosis and for alterations in the apoptotic endpoints of morphological changes and DNA fragmentation following heat and TNF treatments.

DESCRIPTION (provided by applicant): The overall goal of this initiative is to investigate the cellular uptake, trafficking, and sub-cellular localization of different classes and subtypes of nanoparticles (NPs) with well-defined physiochemical properties for the creation of a reference table that relates the sub-cellular distribution of NPs to their intrinsic physiochemical properties across a range of cell lines. The subcellular fate of NPs is relevant both in terms of the therapeutic efficacy and biosafety of the NPs. The effective impact of size, shape, charge, and chemical composition of nanomaterials, in the presence of serum opsonins, on both cellular entry and subsequent subcellular localization will be investigated. The expected outcome of this project is to create a reference table that accelerates the transition of nanomaterials from the bench to the clinic by rapidly expanding our knowledge of the effect of a material's intrinsic characteristics on its intracellular destination. The final product, a comprehensive table of NPs and their subcellular locations, will guide the future development of NP drug delivery systems for rapid expansion of biomedical applications, including cancer therapy, cardiovascular imaging, and gene therapy. PUBLIC HEALTH RELEVANCE: What this project seeks to deliver is a multi-dimensional reference table that relates the subcellular distribution and toxicity of NPs to their intrinsic physiochemical properties across a range of diverse cells and cell lines. It is our hope that the data generated from this project will serve as a resource for future research and encourage model development and new insights into nanotechnologies for imaging and drug delivery.