Allan James Yates, MD, University of Alberta, PhD, University of Toronto - US grants

The long range goals of this project are: (a) to elucidate at the molecular level pathogenetic mechanisms involved in the uncontrolled growth of human gliomas; (b) to find means for manipulating these mechanisms in ways that could be applied clinically. One of the important factors responsible for the aggressive behavior of gliomas may be their resistance to natural killer cell (NK) cytolysis. We have demonstrated a correlation between the glycolipid composition of cultured human glioma and fetal brain cells and their resistance to NK lysis. This supports our working hypothesis that the glycoconjugate (ie. glycolipid and glycoprotein) composition of glioma cells is an important determinant of their susceptibility to NK attack. The experiments in this proposal have been designed to define the importance of several glycoconjugates and cellular differentiation in determining the sensitivity or resistance of glioma cells to NK. To do this we will subject cultured glioma and fetal brain cells to environmental alterations to change either their glycoconjugate composition or degree of differentiation, and determine their degree of binding to and lysis by NK. Glycoconjugate contents of target cells will be decreased by glycosidases and cycloserine, and increased by non-enzymatic glycosylation or serum-free medium. The degree of target cell differentiation will be altered by phorbol myristate acetate, and glial maturation factor, and analyzed using flow cytometry (glial fibrillary acidic protein, fibronectin, DNA, low angle forward light scatter). Neutral glycolipid and ganglioside compositions of target cells will be chemically quantiated using HPLC, HPTLC and scanning densitometry. Cell surface glycoconjugates will be analysed using galactose oxidase and NaB(3H)4. Membrane fluidity, cholesterol and phospholipids will also be quantitated. Results of chemical analyses and immunological assays will be statistically analysed to determine if glycoconjugates on target cells correlate with levels of target binding and lysis by NK. This information should identify which target cell glycoconjugates are involved in binding, and/or lysis of human gliomas by NK, and provide insight into the molecular mechanisms through which they operate.

astrocytoma; neoplasm /cancer diagnosis; neoplasm /cancer classification /staging; glycolipids; oligosaccharides; prognosis; cerebrum; biomarker; cerebellum; glycosphingolipids; gangliosides; immunocytochemistry; flow cytometry; human tissue;

There is a group of independent investigators at OSU with interests that span basic, preclinical and clinical aspects of brain tumor research. Funds from this Feasibility Grant plus considerable institutional support (contingent upon funding for this grant) will permit our group to establish three major components essential to develop into a cohesive Center with the theme of "glioma growth control". For the first 3 years (Phase I) the major goals are the following: 1. Support interactive research projects of three new investigators who are bringing necessary breadth and depth to the group's expertise in the areas of molecular biology, flow cytometry, and epidemiology. These projects are all designed to develop the Center's theme of "glioma growth control", and they will be used as a test system for the Neuro-Oncology Translator and Information System. 2. Develop a Neuro-Oncology Translator and Information System that will provide easy access and sharing of data among distributed, heterogeneous neuro-oncology databases. It will be tested with data generated from the three new, interactive research projects and heterogeneous databases distributed throughout the neuro-oncology community as OSU. By the end of Phase-I, it will be functional within our Center and ready to distribute to other institutions, permitting easy sharing of data among collaborating Brain Tumor Research Centers. 3. Establish three core units (Molecular Biology; Neuropathology; Biostatistics) that will provide needed support to all of the new and several established research projects studying glioma growth control. The addition of these components to the existing excellent clinical and research facilities in a rich academic milieu will provide the essential ingredients to transform a group of independent investigators and clinicians with a common interest in glioma growth control into a coordinated, integrated Brain Tumor Research Center.

There is a group of independent investigators at OSU with interests that span basic, preclinical and clinical aspects of brain tumor research. Funds from this Feasibility Grant plus considerable institutional support (contingent upon funding for this grant) will permit our group to establish three major components essential to develop into a cohesive Center with the theme of "glioma growth control". For the first 3 years (Phase I) the major goals are the following: 1. Support interactive research projects of three new investigators who are bringing necessary breadth and depth to the group's expertise in the areas of molecular biology, flow cytometry, and epidemiology. These projects are all designed to develop the Center's theme of "glioma growth control", and they will be used as a test system for the Neuro-Oncology Translator and Information System. 2. Develop a Neuro-Oncology Translator and Information System that will provide easy access and sharing of data among distributed, heterogeneous neuro-oncology databases. It will be tested with data generated from the three new, interactive research projects and heterogeneous databases distributed throughout the neuro-oncology community as OSU. By the end of Phase-I, it will be functional within our Center and ready to distribute to other institutions, permitting easy sharing of data among collaborating Brain Tumor Research Centers. 3. Establish three core units (Molecular Biology; Neuropathology; Biostatistics) that will provide needed support to all of the new and several established research projects studying glioma growth control. The addition of these components to the existing excellent clinical and research facilities in a rich academic milieu will provide the essential ingredients to transform a group of independent investigators and clinicians with a common interest in glioma growth control into a coordinated, integrated Brain Tumor Research Center.

DESCRIPTION (Adapted from Applicant's Abstract):Gangliosides are associated with the abnormal growth and differential of human gliomas, and it has become increasingly clear that one of the major ways that they act is by modulating systems that control protein phosphorylation. Using U-1242MG human glioma cell lines as a model, the investigators have shown that specific gangliosides inhibit platelet-derived growth factor (PDGF) stimulated growth, and this is associated with characteristic changes in both the phosphorylation states of several proteins, and intracellular free calcium concentrations ([Ca2+]i). They are now focusing on the relation of gangliosides to protein phosphorylation and [Ca2+]i because of their universal importance in regulating critical cellular events.During the next funding period they will conduct three groups of experiments to elucidate the molecular mechanisms through which gangliosides cause these biochemical and biological effects. The first group will identify specific proteins whose phosphorylation states are altered by mitogenic stimuli, and determine the effects that exogenous gangliosides have on phosphorylation of these protein. Identification and cellular localization of substrates for the PDGF receptor tyrosine kinase, and determinations of the effects of gangliosides on the activity of this enzyme will also be made. The second group will examine effects of PDGF and gangliosides on [Ca2+]i, which is known to play a major role in signal transduction and protein phosphorylation systems. The third group will gain information on the uptake, metabolism and distribution of exogenous gangliosides in U-1242MG cells. This is essential to understand the specific molecular mechanisms through which gangliosides act. During the first funding period the investigators have established several important phenomena relating gangliosides, growth, protein phosphorylation, and [Ca2+]i. The experiments designed for the next funding period build on these findings, and will provide some of the information essential to develop a more specific and detailed model of how these factors interact.Such knowledge could lead to the development of new diagnostic and therapeutic strategies based on understanding the biological and molecular mechanisms responsible for aberrant growth of human gliomas.

Our original hypothesis was that glycolipids can be of value in improving the pathological classification of human gliomas. During the current funding period we obtained considerable supporting information for this hypothesis. Most significantly, we found that a decrease in the proportion of gangliosides of the 1b pathway and the presence of 6'-LM1 correlate with shorter survival times. The group of tumors on which these observations were made is the training set. Here we propose; (1) To test these findings independently in a validation set that we are establishing, and then (2) To evaluate their practical usefulness in a large group-wide study that will focus on creating a panel of markers to improve upon the classification of human gliomas in a clinically beneficial way. In addition, we will examine several other selected glycolipids and their correlation with diagnosis and/or survival using the training set for which glycolipids and consensus diagnoses of the neuropathology panel are available. Those found to correlate with diagnosis and/or survival in the training set will then be studied further using both the validation set and the group-wide set of gliomas. The goal of these studies is to find a pattern of glycolipids that can provide prognostic information beyond that of currently available methods. Such a panel of diagnostic glycolipid markers will provide the basis for improvements in the pathological classification of human gliomas. We will also continue to cooperate with other members of the Glioma Marker network to advance the goals of the RFA by participating in the Coordinating Committee (Yates, Pearl, Mam-ak), neuropathological review sessions (Yates), statistical collaboration with other institutions (Pearl), and maintenance of a group database (Pearl and Mamrak).

DESCRIPTION: During the initial two funding periods, the investigators have determined that specific gangliosides (GM-1) inhibit ligand induced dimerization and tyrosine phosphorylation of PDGFR. Results of these studies clearly demonstrated that gangliosides play a regulatory role in several platelet derived growth factor (PDGF)-mediated events involved in cell growth. The investigators believe that they have uncovered an important previously unrecognized regulatory mechanism through which glycoconjugates can modulate and coordinate entire signal transduction pathways by regulating the receptor dimerization (activation of dimerizable receptors). The investigators further proposed to continue the study to define critical gangliosides-PDGF receptor interactions. The investigators hypothesized that there is a specific ganglioside binding domain on the PDGFR through which gangliosides regulate PDGFR dimerization and function. They will test this hypothesis for GM-1, a ganglioside found to have pronounced inhibitory effects on the PDGFR. The investigators propose to continue their studies for further understanding which will be answered by the results obtained from three general specific aims. Aim 1 is to transfect PDGFR in cell lines and characterize several receptor and post receptor functions. Furthermore, they will use GM-1 to inhibit such functions. In Specific Aim 2, they propose to locate the GM-1 binding domain in PDGFR by gene deletion experiments. Subsequently, they propose to mutate this specific domain and identify specific amino acids necessary for GM-1 binding. In Specific Aim 3, they propose to characterize several receptor functions and preceptor events in U251MG and Sf9 cells transfected with GM-1 binding negative mutants identified in Specific Aim 2. The investigator believes that the results of these experiments will test their hypothesis and allow them to define the polypeptide sequence of GM-1 binding domains in the PDGFR through which GM-1 can regulate PDGFR function.

[unreadable] DESCRIPTION (provided by applicant): This is an extensively revised grant application for a training program to support 3 students in each of the first 2 years of an Integrated Biomedical Science Graduate Program (IBGP) having the theme "The Biology of Human Disease." Students for this program will be selected from the pool of students in the first year of the IBGP on the basis of academic achievements and an interest in interdisciplinary studies on mechanisms of human disease. During these 2 years students take a rigorous, broadly based core curriculum consisting of lectures, seminars, group discussions and laboratory rotations. At the end of year 1 all students choose a dissertation advisor and advisory committee who guide them in an interdisciplinary research project, and advise them on the selection of advanced courses and seminars. These will help them to integrate material related to their cross-disciplinary dissertation research on complex interactions underlying the mechanisms responsible for human disease(s). After the first year, students in the program will take an advanced seminar in integrated biomedical science for at least one quarter each year. To assist students in the choice of courses and seminars after completion of the core courses, we have designed suggested curricula in 9 traditional research disciplines, which if followed will result in their final transcripts designating this area as a graduate specialization. Many students in this program will probably want to obtain graduate specialization in more than one area (referred to as areas of research emphasis). This training program has excellent graduate faculty in each of these 9 areas of research emphasis who can serve as dissertation advisors and advisory committee members, and one of these faculty members serves as a faculty liaison for an area. This faculty member is available to discuss with students different aspects of the area of research emphasis he represents. The candidacy exam will be taken early in the third year. It will consist of: (1) A grant application written by the student on the proposed interdisciplinary dissertation research project; (2) An oral examination to test the student's integration of information and concepts related to the proposed cross-disciplinary research project. Subsequent to this most of the students' efforts will be spent on research that will culminate in a dissertation that will be defended orally; the time to graduation should be no more than 5 years. We believe that the curriculum, faculty and resources supporting this training program will attract outstanding students who will become distinguished, productive scientists in integrative biomedical research. [unreadable] [unreadable]