James W. Jacobberger - US grants

The goal of this project is to produce airway epithelial cell lines with well characterized phenotypes and the Cystic Fibrosis (CF) genotype. An immortalization protocol designed to introduce minimal oncogenic transformation using defective retroviral vectors will be employed. Immortalizing human cells with little alteration of differentiated function is possible because 1) mouse and human cell lines with a reduced transformation phenotype have been made this way, 2) several immortalizing genes have been cloned into expression vectors, 3) transformation is quantitatively related to phenotype, 4) these genes and vectors can be genetically manipulated, and 5) phenotypic analysis can be quantified. Significant progress in CF research has been made at the cellular and molecular level by identification of abnormal ion transport regulation in epithelial cells of the airways, and the finding that these properties are retained when airway cells are cultured in the laboratory. Complete understanding of the CF defect at the molecular and cellular level progresses slowly due to scarcity of tissue and the short life time of cultured cells. Well characterized cell lines will relieve this bottle-neck. Advances in organic virology and tumor biology have identified several viral and cellular genes that control cell growth and differentiation that, when abnormally expressed, will transform normal cells in a step-wise manner to a tumorigenic state. One independent step in this process is cell immortalization defined as the ability to escape normal senescence. Several studies have indicated that introduction and expression of a single gene (from a list of several) will immortalize tissue specific cell types that retain most if not all of the differentiated phenotype expressed at the time of immortalization. It also seems clear, especially from analysis of leukemias and glial cell lines, that immortal cells are arrested at a differentiation stage along a pathway leading to a terminal phenotype. Therefore, cell lines can be made that express tissue-specific, differentiation-specific and transformation-specific phenotypes. The details of an individual cell line's composite phenotype, are likely governed by which immortalizing gene is used, its expression level, the differentiation state of the cell at the time of immortalization, and genotype. This proposal describes a program to 1) immortalize airway epithelial cells from non-CF and CF tissue using a defective retroviral vector system, 2) assess the level of CF, epithelial, and differentiated phenotype retained as well as the level of transformation phenotype induced, and 3) use phenotype information to genetically modify the immortalizing vectors to reduce the transformed phenotype to a minimum. Modifications of vectors include switching oncogenes, using mutant alleles, and/or modulation of oncogene expression by switching promoters or reinfecting with antisense oncogene RNA vectors.

flow cytometry; biomedical equipment purchase; cell sorting;

DESCRIPTION: (Adapted from the Applicant's Abstract) Flow cytometry of cellular DNA content is used routinely to evaluate cell proliferation. It is common to couple this parameter assay with immunofluorescence to measure important molecules in a cell cycle specific manner. These molecules include proliferation and phase- specific markers as well as regulatory molecules with cell cycle rate limiting function, e.g., cyclins, c-myc, Rb. Most interesting molecules are intracellular. This cytometric approach to molecular quantification is useful in basic research and in the clinical evaluation of hemotological and solid tumors. In clinical analysis, DNA content provides prognostic information and many two parameter analyses provide more accurate DNA content measurements. Expression levels of regulatory proteins may have prognostic significance, however, it is unlikely that a measurements of a single molecule will be useful, except in rare instances, because of the highly complex and apparently redundant nature of cell regulatory mechanisms. The literature on single markers attests to this. A cytometric approach provides the unique ability to quantify on a single cell basis two or more proteins and DNA content, thus providing correlated gene expression analysis resolved at the cell cycle level, and thus, by inference in a dynamic (time resolved) manner. Current problems with this approach are technological rather than rational. These are (1) multiple markers are required but the multiparametric methodology has not been developed, (2) lack of standardization does not permit direct comparison of data generated across time or between laboratories, (3) the reported levels are relative, and (4) the sensitivity level is known to need improvement but the required lower level has not been established. The ability to solve each of these problems exists but technological development has been slow, largely, because no single unified effort has been made by any one laboratory. The research proposed herein seeks to rectify this by developing (1) multiparameter immunofluorescence cell cycle analysis employing up to three antigens coupled with DNA content, (2) development of methodology to determine the absolute number of molecules rather than relative fluorescence, (3) standardization by creating cell line and/or microbead staining standards, and (4) increasing assay sensitivity through development of improved chemistry, hardware and software.

Flow cytometry (FC) is an essential resource in modern biomedical research. Cytometry resources at Case Western Reserve University (CWRU) are limited to a Coulter Elite 6 parameter cytometer that will electronically sort to 15,000 events per second, but is limited by coincidence to approximately 6000 cells/s. This instrument 's in the Flow Cytometry Core of the NCI supported CWRU Comprehensive Cancer Center and is the only cell sorter shared resource at CWRU. The core currently supports the research efforts of more than 70 laboratories with -85% funded by NIH, DOE, or NSF grants. The supported research is diverse and the facility operates as a flexible core that strives to meet the needs of all users who access the core on a formal first-come-first serve basis. The Medical School has expanded its research base significantly and steadily since the early 1980s and currently ranks 13th in NIH funding for US Medical Schools with $142,335,793 funding in 1999, and is the major research institution in Northeast Ohio. The FC core plays a significant role in the research infrastructure. The Medical School and University Hospitals faculty increased significantly over the last 3 years and plans indicate continued growth over the next 3 years. This proposal seeks funds to purchase a multiparametric high speed sorter to enhance the research efforts of 30 major users and 37 intermediate or minor users. If funded, his instrument will be added to the core facility and provide the following: (1) upgraded flow cytometric /cell sorting technology that will support funded programs; (2) increased instrument capacity that will allow the Core to respond to increased numbers of faculty and NIH funded projects; (3) insurance against significant disruption of research efforts during breakdown or scheduled maintenance - especially important for experiments that take weeks of planning; (4) permit creation of BL3 level sorting capacity to respond to NIH funded AIDS projects.

DESCRIPTION (provided by applicant): Flow cytometry (FC) is an essential resource in modern biomedical research. The Cancer Center Flow Cytometry Core at Case Western Reserve University (CWRU) and University Hospitals of Cleveland (UHC) has continuously functioned since 1985 with an average annual percentage growth of 12% per year. The facility maintains flow cytometers and cell sorters and provides cytometry expertise to support research and clinical trials at CWRU/UHC. Current instrumentation is up-to-date, well maintained, and heavily used. The Flow Cytometry Core has been continuously funded through an NIH funded P30 Comprehensive Cancer Center grant (since 1987) and user support. The current user base is approximately 134 principal investigators (principal investigators accessing resources within last 5 years) with an average use by 70 laboratories per year. We have been fortunate to receive funding through the Shared Instrumentation Program in 1990 and 2000 for cell sorters. Currently, a group of six NIH funded investigators, most of whom are major users of flow cytometry, need to make similar quantitative measurements of gene expression on cells and cells within tissues that are correlated with parameterized morphology and sub-cellular localization. The Compucyte iCyte Imaging Cytometer is a commercially available cytometer that offers this capability. This is an application to fund acquisition of this instrument for installation within and administration by the Cancer Center Flow Cytometry Core. Although the instrument would currently enhance the research of 6 investigators, the administration of the instrument within this core facility would ensure that additional investigators could utilize the instrument by a formal, non-restrictive access mechanism.

DESCRIPTION (provided by applicant): The long-range objective is to create a robust, precise, and accurate model of the mammalian cell cycle based on N dimensional clustering of cytometric measurements of the activity of rate limiting molecules for cell cycle traverse. At present, this is encapsulated by an intermediate Transition State Model of Mitosis that is based on measurements of DNA content, two morphological parameters (DNA pulse shape and cell size), the expression of cyclin A, cyclin B1, and phospho-serine 10 histone H3. In mitosis, quantification of cyclin A is a measurement of the activity of the Anaphase Promoting Complex (APC) in conjunction with Cdc20; Cyclin B1 reads out the activity of the APC in conjunction with Cdhl. Phospho-S10-histone H3 immunoreactivity reports the activity of Aurora B kinase. For human cells lines, the conjunction of these measurements results in the ability to quantify the frequency of multi-dimensional clusters with sharply defined boundaries that represent transition states that closely approximate morphological mitosis i.e., post- G2 -> prophase --> prometaphase -> metaphase --) anaphase/telophase --> cytokinesis --) new born GI. Because these clusters are frequencies of cells residing in each transition state, the size of the cluster is proportional to the time the analyte population existed in each state at sampling time. Within this analysis, but dependent on fewer parameters, the remainder of the cell cycle (G1, S, G2) can be quantified. The specific aims of this proposal are: (1) to enhance the current analysis by adding 2 parameters aimed at simultaneous quantification of GO and apoptotic cells, and add a third variable parameter; (2) develop a primary set of cytometry-grade cell cycle probes with a uniform and low Kd with the yeast display system. These include phospho-epitopes of substrates of Nek2, PIk-1, cyclin Bl/Cdkl, and Aurora B kinase. (3a) determine the relationship between classical mitosis and cytometrically-defined cell state clusters. (3b) test the hypothesis that the conjunction of phospho-cyclin B1 measurements and other mitotic enzyme activity measurements define unique, novel cell states. (3c) test the hypothesis that the Transition State model can be generalized through GI.

The Cytometry Core provides flow and image cytometry and cell sorting instrumentation, expertise, training, consultation, and other services to Case Comprehensive Cancer Center (Case CCC) investigators. There are two sites, one at Case and one at the Lerner Research Institute, that work together under a Federation Model. These two sites, under one Case CCC administration, provide efficient maximization of resources and providing uniform quality of services to all cancer investigators. The resource serves a base of approximately 170 laboratories in any one year and a broader base over time. Services include consulting, training/teaching, low, intermediate, and high-end (multi-laser, multi-color) cytometry, low and high speed cell sorting and single cell deposition (cloning), multilaser/multicolor laser scanning cytometry, absolute cell counting, data analysis, and specialized staining. Services are delivered in styles to fit individual need, ranging from independent use of instruments to fully dependent, sample drop-off mode. Instruments are upto- date and the Core has been the beneficiary of multiple Shared Instrumentation Grants. Core usage has increased at both sites, growing at 13% per year over the last 5 years (data from Case). The core is used by 8 of the 9 programs of the Case CCC with the heaviest use in programs 2, 3, 6, and 7 (Cell Proliferation and Cell Death, Radiation and Cellular Stress Response, Stem Cells and Hematologic Malignancies, and Developmental Therapeutics, respectively), and the developing Vascular Biology initiative. Peer-reviewed cancer member use is high. Case CCC members receive discounted service.

DESCRIPTION (provided by applicant): The long term objectives of this proposal are to develop a system to monitor patients with pre-leukemic conditions like myelodysplastic syndrome (MDS) and myeloid proliferative neoplasms (MPN) for early detection of leukemic transition. This general system is stimulation of unfractionated bone marrow aspirates with cytokines and measuring the fine kinetics of modified epitopes on signaling proteins by immunofluorescence flow cytometry. Leukemia is an evolutionary process that works by clonal selection. Cytometry is a measurement sys- tem that works at the level of single cells, therefore, the specificity of detection and signal to noise ratio can be very high. In addition, cytometry is an established clinical tool used for diagnosis of hematopoietic malignancies based on measuring the levels of differentiation antigens. One model for the genesis of acute myeloid leukemia (AML) is that at least two mutations are leukemogenic: 1) in a transcription factor that alters differentiation, and 2) in a signaling pathway protein that affects cell proliferation and/or apoptosis. A significant component of cell signaling involves phosphorylation/de-phosphorylation cycles on pathway proteins, essentially from membrane surfaces to the genome, and between pathways (networks). We have developed the ability to measure cell signaling in committed myeloid precursor cells in unfractionated bone marrow by flow cytometry. Published results on normal, healthy volunteers demonstrates remarkable uniformity of response across age and gender, and published results on 4 (Jacobberger lab) and 14 (Goolsby lab) leukemias demonstrates clear, robust detection of abnormal signaling in eight pathways (Kit?Erk; ?Akt; ?S6; ?Stat5, and Flt3?Erk; ?Akt; ?S6; ?Stat5) that demonstrate classifiable complexity. Thus, we propose that cell signaling can be used as a biomarker to detect leukemic cells with high fidelity. Additionally, since the enzymes of these pathways are focus of targeted therapy, this approach provides information with therapeutic implications. Because a small fraction of abnormal cells can be detected by this approach, this should work well for early detection. Current diagnosis, therapeutic choices, and selection for clinical trials depend to some degree on genetic analysis, and in the future, this dependence will be greater. The signaling patterns, identified by our approach, will associate with specific mutations in signaling protein genes. Thus, cell signaling may provide rapid genetic inferences. To test this idea, we propose to correlate phenotype (signaling) and genotype, determined by high throughput sequencing. Our specific aims are 1) to measure signaling for 2 ligands (Stem cell factor and Flt3-ligand) and 5 endpoints (Erk, Akt, S6, Stat3, and Stat5) on committed myeloid precursor cells in 60 healthy human donors to define the normal ranges for each component of the signal analysis and determine any age or gender effects; 2) to measure signaling in 75 MDS and 75 AML patient blast cells to test the hypothesis that signaling can be classified by patterns and determine any age or gender effects; 3) to sequence 182 gene exons that may should impact on the signaling patterns for 10 MDS and 10 AML patients to demonstrate the feasibility of this approach for associating phenotype to genotype.

? DESCRIPTION (provided by applicant): The Case Comprehensive Cancer Center (Case CCC) Cytometry & Microscopy Core provides flow cytometry; cell sorting; imaging flow cytometry; laser scanning cytometry; laser scanning / high resolution / two-photon confocal microscopy; TURF; digital video microscopy; and imaging fluorescence microscopy to investigators at Case Western Reserve University (CWRU) and other institutions in the wider Cleveland area. The cytometry core was created in 1985 and has been continuously funded by NCI the P30 mechanism since 1987. The Core has cooperative agreements with the Case Center for Aids Research (CFAR) and the Lerner Institute, leveraging expertise, providing wider access to equipment, and enabling joint planning and decision-making. More than 150 principal investigators access Core services per ~5 year period. The average number of laboratories accessing in a given year is >100. The core houses three flow cytometers - BD Biosciences LSR I and LSR II and a Beckman Coulter XL. We acquired the XL in 1999 with institutional funds; the LSR I in 2001 with funds from the Fannie Ripple Foundation, and the LSR II in 2003 with funds from NIH S10 grant RR015967. The XL and the LSR I are no longer supported by the manufacturer, dysfunctional, and very difficult to maintain. Parts are hard to find and breakdowns are frequent. The LSR I has been disabled since 2012 and the XL has not been repaired after laser failure in April, 2015. Therefore, we have only one modern, multi-laser flow cytometer (LSR II). This creates scheduling conflicts and over-capacity run-time difficulties. During periods of overflow, we shift investigators to the CFAR Immune Function Core Fortessa or send investigators to the Lerner Institute's Flow Cytometry and Imaging Core, but for most investigators both our LSR II and the CFAR Fortessa are not the appropriate instrument, and both instruments are heavily subscribed. The Lerner Institute Core is also heavily used and unable to absorb the over-flow. Therefore, we are requesting funds to purchase an intermediate Attune Nxt flow cytometer with acoustic focusing, which will relieve work-flow stress on the LSR II, provide a low cost LSR II alternative, provide high quality no-wash assays that will reduce sample preparation labor, and enable higher flow rates for DNA content, providing cost saving benefits to all users.