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Project # Year
of Award
PI Name(s)
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Abstract Text (Official)
R21 CA092734-01 2001 FARIS, GREGORY W SRI INTERNATIONAL Upconverting Chelates for Cancer Detection and Diagnosis
The long term objectives of this proposed work are to enhance the tools for diagnosing, predicting, and treating cancer through powerful new molecular upconverting labels. These labels have a number of potential uses, including in vitro diagnostics, in vivo detection and demarkation of diseased tissue, and incorporation in microfluidic systems for high throughput screening. The near term objective of this proposed work is to better understand and develop the capabilities of our molecular upconverters through application to in vitro immunodiagnosis of tissue samples. This new and revolutionary type of reporter is based on upconversion in lanthanide (rare earth) chelates. These materials have no autofluorescent background, do not photobleach, use infrared excitation (which is less phototoxic, offers good tissue penetration, and can be provided using diode lasers), and have narrow emission bands (which is advantageous for multiplexed assays). The upconverting chelates are much smaller than the upconverting phosphors (also developed at SRI); the upconverting chelates have molecular weights of a few hundred. We will apply upconverting chelates to immunodiagnosis of tissue samples, where the multiplexing. capabilities and absence of photobleaching will be of great value. In the first phase of work (R21), our specific aims include producing one type of upconverting chelate and measuring its optical properties, assembling an upconverting microscope for its use, conjugating the chelate to antibodies, and testing the chelate-antibody probe in assays on tissue cell lines. In the second phase (R33), we will extend the chelates to multiplexed assays, with specific aims of preparing two additional upconverting chelates, extending the upconverting microscope to detect all three chelates, and simultaneously detecting three antigens in lymphoma tissue samples. This work will combine the efforts of optical physicists, chemists, immunologists, a cancer biologist, and a pathologist.
The importance of cancer research and the inherent complexity of its challenges make it very important to design strategic, innovative approaches. Investigating the accumulation of multiple abnormalities per cell at the molecular level has prognostic value in cancer research. We propose to develop the capability to study this issue with optimal quantitative ability, discrimination and throughput by (a) focusing our proprietary technologies towards the goal of this solicitation; (b) developing the next-generation imaging cytometry instrument, able to bypass previous imitations; (c) raising the much needed optics/hardware/software /biology/clinical application continuum to a new level. We will concentrate on the imaging of 5-10 molecular species simultaneously within the same cancer cell, with a new approach and instrument that we propose to develop, based on our previous advances in acousto-optic technologies and multispectral imaging. The aims are largely technologic, but their implementation will allow us to (a) elucidate critical sequences of genetic evolutionary changes in solid tumors that are responsible for increasing cancer aggressiveness; (b) identify the steps in the sequence that are most closely associated with cellular acquisition of the capacity to metastasize, and (c) develop a practical overall approach for the timely performance of relevant measurements on individual tumors, analysis of the data, and characterization of each tumor with respect to the degree of its advancement along its particular genetic evolutionary pathway, so that this information can be used for prognosis and adjuvant treatment planning. The ultimate clinical challenge is the elimination of false negatives and false positives in diagnostics, leading to individually optimized treatment and very significant savings. The technological task specifically addressed is the design and development of the ability to quantitative simultaneously, in the same cell, a sufficiently large number of molecular species to enable a qualitative leap in diagnosis and treatment. The emphasis is on fluorescence, due to its very high specificity for labeling intracellular features, and the central new technology is acousto-optic tunable filters, a tool whose use in microscopic imaging we perfected and patented. Specific aims include (1) the building of a prototype instrument with the desired new functionality; (2) the implementation of a second, more user-friendly workstation in our cancer research laboratories; (3) the thorough testing of the latter instrument in experiments focusing on molecular-level prognostic factors for tumor progression within single cells of individuals' tumors, and (4) the development of a productized customizable version of the instrument embodying the new technology, multispectral imaging, ready for use on an array of problems by other researchers. A collaborative, multidisciplinary team is proposed for implementation of these goals.
R21 CA091267-01 2001 WATT, PAUL M TELETHON INSTITUTE FOR CHILD HEALTH RES Mechanisms of genomic instability in cancer
A breakdown in the maintenance of genome stability is an invariant feature of tumor cells and one of the defining features of the transformed phenotype. Much of the work on genome stability has focussed on the role of 'mutator' genes, which when themselves mutated, allow high overall mutation rates in cells, precipitating the rapid disruption of oncogenes and tumor suppressor genes in cancer. Examples of such mutator genes are the mismatch repair genes such as MSH2 which are frequently mutated in HNPCC. Less work has focussed on identifying the key players responsible for abnormal recombination and missegregation of chromosomes in cancer cells. Part of the problem has been the lack of systems for rapidly screening defects in chromosome segregation. If such screening tools were available, it would be possible to identify new genes involved in maintaining the fidelity of chromosome segregation. It would be expected that such genes may be mutated in cancer cells, potentially constituting new tumor suppressor genes. Some key guardians of such genome stability have already been identified from a family of DNA helicases, named after the prototypic member RecQ, a bacterial helicase which is required both for initiating the RecF pathway of homologous recombination and for the suppression of illegitimate replication. Members of this RecQ gene family are associated with the intriguing cancer susceptibility syndromes, Bloom's syndrome-BLM, Werner's syndrome-WRN and Rothmund-Thompson syndrome. The overall goal of the proposed application is to establish and validate robust vertebrate models to identify defects in chromosome segregation. The proposed models will allow a sufficiently high throughput that they could be used for genome-wide mutational screens and large-scale screening of candidate peptide blockers of complexes indicated in the maintenance of genome stability The first goal of this project is devoted to the isolation and testing of such peptide blockers isolated using a yeast screen. We suggest that the peptide blockers themselves will prove to be very useful dominant negative reagents to unravel the pathways involved in genome stability, since they allow the uncoupling of different activities of multifunctional proteins. In this way they will simulate the effects of dominant mutations and may even allow 'epistasis' analyses to determine the relationship between different gene products. The goal of the second phase is the establishment and validation of zebrafish and mouse models to detect defects in chromosome segregation during mitosin and meiosis. Since these models employ robust fluorescent screening technology they will be suitable for high throughput industrial application.
R21 CA084704-01A2 2001 YAMAMOTO, FUMIICHIRO SANFORD-BURNHAM MEDICAL RESEARCH INSTIT Technology to Detect Genome wide DNA Methylation Changes
Somatic epigenetic alterations in DNA methylation are tightly linked to development, cell differentiation and neoplastic transformation. For instance, hypermethylation of CpG islands in promoter regions has been increasingly associated with transcriptional inactivation of tumor suppressor genes in carcinogenesis. Although techniques to determine the degree of methylation in specific DNA segments or in total DNA have been available, there are few techniques to efficiently scan and identify changes in methylation in the entire genome. We have developed a method called Methylation Sensitive-Amplified Fragment Length Polymorphism (MS-AFLP). This PCR-based unbiased DNA fingerprinting technique permits the identification of the cleavage sites that exhibit DNA methylation alterations and subsequently allows the isolation of DNA fragments with these sites at their ends. Hyper/hypomethylation can easily be differentiated by the decrease/increase of band intensity, respectively. MS-AFLP requires low amounts of template DNA and electrophoresis of multiple samples in parallel enables easy identification of consistent common differences. Notl-Msel MS-AFLP experiments using matched normal/tumor DNA have shown highly reproducible differences in banding patterns some of which were specifically linked with the tumor phenotype. Sequencing some of these bands has identified multiple numbers of homeotic genes and the genes involved in the regulation of homeotic gene expression. These results demonstrate the potential of MS-AFLP in identifying epigenetic alterations associated with cell differentiation and cancer. We will further develop this powerful MS-AFLP method by transforming the gel electrophoresis-based fingerprinting technique into a DNA microarray-based hybridization technique for general use of methylation alteration analysis of several biological problems. In the R21 phase, we will construct a pilot DNA microarray panel, examine the feasibility and sensitivity of several hybridization-based MS-AFLP and non-PCR methods using the pilot DNA microarray, and determine the best method(s) for further development. In the R33 phase, we will search for the prostate and breast cancer-specific DNA methylation alterations, analyze the gene expression, re-examine some of the identified alterations in DNA methylation and gene expression by the sodium bisulfite modification method and multiplex RT -PCR. We will also construct a cancer-specific DNA microarray for the clinical detection of DNA methylation alterations.
R21/R33 CA091216-02 2001 ALLBRITTON, NANCY L UNIVERSITY OF CALIFORNIA IRVINE Profiling Ras Activated Signal Transduction Pathways
The molecular analysis of the signal transduction pathways driving uncontrolled growth in tumor cells will have a dramatic impact upon cancer biology and patient care. New technologies such as those to identify the genome and proteome of cells hold great promise. However these methods do not provide direct measurements of the activity of molecules involved in signal transduction. Ultimately it is the activation state of molecules such as enzymes that control cell behavior, for example, fueling the growth of tumor cells. A new technology and biochemical assay, the Laser Micropipet System (LMS), has the potential to perform simultaneous biochemical analysis of the activation state of multiple signal transducing enzymes within a single cell. Such data will enable misregulated signaling of tumor cells to be assessed in both linear signaling pathways and in interconnected networks of signaling proteins. The goal of this proposal is to apply the LMS to the Ras signaling cascades which are of immense importance in both the basic and clinical investigation of cancer. An interdisciplinary team with strengths in analytical chemistry, cancer biology, and organic chemistry has been assembled. The research will draw on methods from analytical chemistry to analyze, separate, and detect kinase substrates from single cells. The strengths of combinatorial chemistry and synthetic organic chemistry will be brought to bear on the development of new kinase substrates to be used as specific reporters of Ras-regulated kinase activation. Molecularly engineered tumor cell lines in which individual proteins have been selectively mutated will be used to demonstrate the capabilities of the LMS in measuring the activation of kinases in the Ras-regulated signaling cascades. Finally, a hypothesis of intense interest to cancer research will be addressed with this newly developed and expanded single biochemical assay system. The successful completion of this work will provide a new and powerful tool for basic research, drug discovery and screening, cancer classification, and potentially clinical decision making.
We propose to develop clinically relevant mouse models that resemble human breast tumors in etiology and histology. We propose to use these mouse models to procure pure populations of motile and invasive tumor cells from primary tumors in live intact mice under direct visualization using novel imaging technology. We propose to develop high sensitivity DNA microarrays for use with these cells so as to examine gene expression patterns unique to the invasive and metastatic population of cells in the primary tumor compared to other populations of tumor and normal cells. The coupling of cell behavior to gene expression will allow the rational interpretation of gene expression patterns in metastatic tumors. The specific aims for the R21 phase of the project are: 1. Prepare mouse models that develop metastatic breast tumors as seen in patients and that can be used for imaging of tumor cell behavior in vivo by multi-photon microscopy. 2. Develop methods for multi-photon imaging of normal and tumor bearing mouse mammary glands. 3. Refine methods for collecting chemotactic cells from the primary tumor that have been characterized by direct imaging and that represent the population of cells capable of chemotaxis to vessels and surrounding tissue in response to serum and growth factors. The specific aims for the R33 phase of the project are: 1. Develop sensitive DNA microarray techniques for comparing small numbers of chemotactic tumor cells and white cells collected from the primary tumor with tumor cells and white cells collected from elsewhere. 2. Demonstrate the utility of comparing gene expression patterns of 8 categories of cells collected from various mouse models to identify patterns unique to different subpopulations of cells. 3. Develop methods to correlate the gene expression patterns of cells collected with microneedles from the primary tumor with the histology and behavioral phenotypes of cells at the collection sites. 4. Evaluate a variety of clustering algorithms, with DNA expression patterns obtained in specific aims 2 and 3, to identify genes related to cell behaviors believed necessary for metastasis.
R21/R33 CA089833-02 2001 FRANK-KAMENETSKII, MAXIM D BOSTON UNIVERSITY Studies of Hematologic Malignancies by PNA Technology
The project goal is a robust assay for detecting episomal or genome- integrated oncoviral DNAs in hematologic malignancies. Considering a limited amount of viral DNA, both high specificity and high sensitivity of DNA diagnostics are required. To reach high specificity, a synthetic DNA mimic, peptide nucleic acid (PNA), will be used for sequence- selective formation of PD-loops in double-stranded (ds) DNA targets. High sensitivity will be provided by rolling-circle amplification (RCA), a powerful contamination-immune isothermal method. Large multiplexing capacity intrinsic in RCA enables mutation-insensitive parallel detection of different oncoviruses. EBV and HHV-8 herpesviruses and HTLV-1 retrovirus will be prototype oncoviruses. Their genomes have PD-loop forming sites (PD-sites) that are unique and very constant. These features make PD-sites promising viral markers, which have never been used before in DNA diagnostics. The assay to be developed includes three steps. First, an oligonucleotide probe hybridizes to dsDNA through PD-loops with subsequent probe circularization. This step, assisted by viral-specific PNA openers, is mostly responsible for the overall specificity of the assay. Secondly, the RCA hyperamplification of circular probes at a single temperature yields the dsDNA product in which the original PD-site repeats more than million times. This step will provide requisite sensitivity. Finally, thus multiply copied PD-sites will be selectively exposed by PNA openers and fluorescently detected with molecular beacons. The last step provides a convenient detection and additionally secures the specificity of the assay. Choice of various PD-sites as markers, together with multiplex RCA and multicolor beacons, will ensure reliable diagnosis of several specific oncoviruses. In Phase I, proof-of-principle experiments will be performed on a model system comparable to Phase II samples. The goal is to detect, after initial optimization, the PD-site in a hundred-fold molar excess over 1 mu g of human DNA. These studies will prove that episomal oncoviruses can be characterized in acute blood infections. In Phase II, the assay will be developed for detecting oncoviruses in cell lines and clinical samples with different viral loads. Detection of selected oncoviral marker PD- sites will be optimized using plasmid models to further increase the sensitivity on the excessive human DNA background. The yield and the specificity of the circular probe assembly will be optimized for different configurations of PD-loops. RCA will be optimized using various DNA polymerases and amplification strategies. Optimal conditions and constructions of beacons in terms of their hybridization kinetics with PD-sites will be thoroughly searched. As a result, detection of less than one oncovirus per human genome is projected, which will be tested on DNA samples from oncoviral-infected cell lines and, finally, on clinical specimens. The success in the project will yield a fluorescent assay for a reliable and highly sensitive isothermal diagnosis of oncoviruses in lymphomas/leukemias. It will allow fool roof characterization of malignant samples by detecting viral DNA in a very low number of copies like in case of provirus.
Detection of tumor point mutations is essential to both cancer research and diagnosis. This Phased Innovation Award application is designed to develop a novel mass spectrometric based method for the automated, high-throughput, sensitive, and specific detection of tumor point mutations. This technology integrates Matrix-Assisted-Laser-Desorption-- Ionization Time-Of-Flight (MALDI-TOF) mass spectrometry to Allele-Specific- Amplification (ASA) or Mutant-Enriched PCR Amplification (ME-PCR). Its novelty is that it takes advantage of both methods, namely high-throughput and high amenability to automation of the MALDI-TOF detection and ASA?s or ME-PCR?s highly specific amplification of mutant alleles within a vast background of normal alleles. ASA is the simplest PCR based method requiring only one-stage PCR to detect point mutations and a combination of ASA and MALDI-TOF may be the method of choice for detection of tumor mutations. Thus, the major effort of this project will be devoted to develop the ASA-MALDI-TOF method. In addition, MALDI-TOF, in conjunction with ME-PCR, will also be examined since ME-PCR can be used as an alternative approach to ASA. Our specific goal is to demonstrate the feasibility of this technology, to optimize this technology, and to develop an instrumental system that can deliver a high-throughput DNA analysis. It is our intention that we have not only a well-proven technology, but also a well developed instrumental system that can carry out a highthroughput and sensitive analysis of tumor point mutations by the end of this project. To achieve this goal, we will demonstrate the feasibility of this technology using the synthetic DNA samples resembling the sequences of widetype and mutated K-ras genes in Year 1 (the R21 phase). This demonstration will be carried out using our currently available one-tube instruments and facilities. The issues that we will address in this phase include whether MALDI-TOF has an adequate mass resolution and sensitivity to detect mutant DNA amplified by PCR. If so, can MALDI-TOF detect mutant DNA in the presence of a large background of wide-type DNA? Year 2-4 will be the second phase of this project (the R33 phase). During this period, we will assemble an automated 96- well system that allows an automated and parallel analysis of 96 DNA samples, and develop optimal experimental conditions for this proposed technology and the instrument system. We will also study the application of the microfabricated PCR to this technology since it can further improve the throughput and cost-effectiveness of this technology. Since this technology should work equally well for the detection of other gene alternations including deletion, insertion, and polymorphisms, we will also examine the application of this technology to the detection of these gene alternations.
R21/R33 CA091357-03 2001 LIEBER, CHARLES M HARVARD UNIVERSITY Carbon Nanotube Probes for Direct DNA Sequence Analysis
The Human Genome Project is providing massive amounts of genetic information that should revolutionize the understanding and diagnosis of inherited diseases. In particular, it is hoped that the detection of single nucleotide polymorphism (SNPs) in gene coding and regulatory regions will lead to a greater comprehension of the genetic contribution to risk for cancer, to the elucidation of the genetic factors that affect treatment and prevention, and to the design of powerful new drugs. Achieving these goals will require substantial effort in several areas, including detection or discovery of SNPs and development of high throughput methods for characterizing and comparing nucleotide variations in specific regions of the genome. In addition, recent observations suggest that the haplotype of a subject - the specific alleles associated with each chromosome homologue - is a critical element in SNP mapping, although current methods for determining haplotypes have significant limitations that have prevented their use in large-scale genetic screening. To address this substantial technology gap and provide critical information for molecular level cancer research and medicine, this project will develop a novel technology, which is based upon direct molecular scale imaging of DNA using carbon nanotube atomic force microscopy probes, for the detection and characterization of nucleotide variations, and in particular, direct haplotype determination. In the R21 phase of the project, we will develop the materials science and chemistry required to make nanotube probe tips that provide reproducible detection capability, and to integrate methods from biology, chemistry and engineering for preparation and deposition of DNA samples suitable for reproducible analysis. In the subsequent R33 phase of the project, we will be extend significantly the throughput of our novel technology by developing (i) an integrated system capable of automated deposition of sample arrays, array imaging and image analysis, (ii) system and software to increase significantly sample detection throughput, and (iii) multiprobe arrays for ultrahigh throughput parallel sample imaging.
R21/R33 CA091435-03 2001 PARKOS, CHARLES A EMORY UNIVERSITY Phage Display and Prostate Neoplasia Progression
The goal of this proposal are to utilize a novel technology, random peptide phage display, to identify and characterize peptides that differentiate phases of progression of prostate neoplasia by identifying molecular alterations in tissues. In the developmental phase (R21) of these studies, the technology will be advanced for use in identification of peptides that differentiate benign from malignant tissues, using human prostate cancer as a model system. To do this, new affinity selection procedures will be developed. To detect peptide-bearing phage particles when bound to both formalin-fixed, paraffin-embedded, and fresh tissues, novel histochemical techniques will be defined. The identified phage will be tested on a large series of human prostate cancer cases to confirm binding specificity and reproducibility in histochemical studies. The developed methodologies and first generation phage reagents will then be utilized in a pilot application (R33 phase) in which phage will be identified that bind to and discriminate between the precursor of prostate cancer (prostatic intraepithelial neoplasia) and prostate cancer, on the one hand, and organ-confined prostate cancer and metastatic cancer on the other. The identified phage bearing specific peptides from these experiments will be tested in well-defined clinicopathologic studies on a large series of cases to assess their value as predictors of disease progression (from pre-cancer to cancer, and from cancer to metastatic disease). Important correlations will be made with clinical and pathologic data that will be available on each of the cases used in these studies. The techniques and reagents developed in these studies may have broad applications in basic studies of prostate cancer biology by providing tools to characterize structures that differentiate the phases of cancer progression. Moreover, these studies will yield tools that will be highly useful clinically with direct applications in the field of diagnostic pathology, and may have implications for the management of prostate cancer patients.
R21/R33 CA092725-02 2001 SPEICHER, DAVID W. WISTAR INSTITUTE Novel Comprehensive Proteome Analyses of Metastasis
The overall goal of this proposal is to apply several novel techniques to comprehensive quantitative comparisons of human breast cancer protein profiles focusing on changes associated with invasive/ metastatic potential. Our global proteome strategy should result in reliable quantitative comparisons of up to 10,000 proteins instead of the 1,500 to 2,500 protein spots typically detected on high resolution 2D gels. The most novel and key part of this strategy is a new method that we recently developed for prefractionating whole cell extracts prior to 2D PAGE. This method, termed microscale solution IEF (mu sol-IEF), uses small chambers separated by large pore acrylamide discs with immobilines at specific pH's to separate cell extracts into well resolved pools based upon pI's. A second novel feature of our global proteome strategy is use of slightly overlapping (about +/-0.1 pH) custom-made narrow pH range IPG gels tailored to match mu sol-IEF pools, rather than existing commercial 1 pH unit IPG gels with either 0 or 0.5 pH unit overlaps. A third novel feature is use of high-resolution 1D gels coupled with LC- MS-MS to analyze the insoluble proteins and large soluble proteins (100-500+ kDa). These two protein groups represent >25% of total cell protein mass, but are usually ignored in 2D protein profile comparisons. Finally, we recently optimized femtomole in-gel trypsin digestion to improve sensitivity and reliability of MALDI MS and LC-MS/MS identifications of targeted proteins. Our preliminary results show each of these techniques is very promising, but the feasibility of reliably quantitating 7,500 to 10,000+ proteins in human tumor cells remains to be demonstrated. The one year R21 phase will further optimize and integrate these methods to demonstrate proof-of-principle of our integrated global proteome analysis strategy using human breast cancer cells. The three year R33 phase will then apply the optimized global strategy to comparisons of multiple human breast cancer cell lines and tumors to identify protein changes associated with increased invasion and metastatic potential.
R21/R33 CA092792-02 2001 ZHOU, PENGBO WEILL MEDICAL COLLEGE OF CORNELL UNIVERSITY Protein Knockout Technology/Molecular Analysis of Cancer
The ubiquitin-proteasome pathway is a major cellular proteolysis machinery that selectively targets cellular proteins for degradation. Protein knockout, a new technology we recently developed, harnesses the specificity of the ubiquitin proteolytic system to direct the degradation of otherwise stable cellular proteins. The long-range goal of this research plan is to apply the protein knockout system as a rapid molecular analysis tool to decipher the function of cellular oncoproteins and to validate their potential use as drug targets. The objective of this application is to improve the efficiency and specificity of the protein knockout system and to assess its efficacy in the proteolytic removal of the overexpressed c-myc oncoprotein in established cell culture and animal models for leukemogenesis. The rationale is that recognition of specific cellular proteins by the engineered substrate receptors of the ubiquitination machinery allows for their degradation by the ubiquitin-proteasome pathway. The research plan has been formulated on the basis of strong preliminary data, and on recent studies by many laboratories including our own. We are uniquely prepared to undertake the proposed research, because we have strong preliminary data demonstrating the efficacy of the protein knockout technology in the selective degradation of stable cellular proteins. The objective of the application will be accomplished by pursuing the following specific aims: (1) R21 phase: To improve the efficiency, specificity and delivery of the protein knockout system. (2) R33 phase: To evaluate targeted c-myc degradation in the inhibition of oncogenic transformation in established cell culture systems. (3) R33 phase: To antagonize myc-mediated tumorigenecity by protein knockout in mouse models for leukemogenesis. The proposed work is innovative, because it capitalizes on the recently identified properties of the ubiquitination machinery by our group and by others. It is our expectation that this approach will offer an efficient means to downregulate the c-myc oncoprotein at the protein level and to inhibit c-myc-mediated neoplastic transformation. These results are significant, because they are expected to provide a comprehensive evaluation of the protein knockout technology as a simple and cost effective molecular analysis tool to elucidate the function of cancer-related proteins and genetic pathways, and to validate their potential use as targets for therapeutic intervention.
Ras, the product of the ras protoncogene, relays mitogenic signals from growth factor receptors and plays a prominent role in human carcinogenesis. Rho, a member of the greater Ras family, is necessary for Ras-induced transformation and plays an essential role in the processes of tumor cell invasion and metastasis. Both Ras and Rho are small G proteins which cycle between an active GTP-bound state and an inactive GDP-bound state. We developed a manual method to measure Ras activation in human tissue, i.e., determining the percentage of Ras molecules in the active GTP-bound state, and we recently modified this method to allow assessment of Rho I activation as well. Ras or Rho are isolated from tissue lysates and GTP and GDP bound to the proteins are eluted and quantitatively converted to ATP; ATP is measured by the firefly luciferase system which is sensitive to 1 fmol. We have found that Ras is highly activated in a significant number of breast, lung, and ovarian cancers, in the absence of a genetic ras mutation, with increased Ras activation correlating with increased expression of growth factor receptors. In a limited number of metastatic ovarian cancers, we have found Rho to be more highly activated in the metastatic lesion than in the primary tumor. Under the support of an R2l award, we developed a prototype instrument that allows full automated measurement of Ras and Rho activation (when combined with a commercially-available automated micro plate luminometer). We are now applying for an R33 award to test the instrument's accuracy, reproducibility, sensitivity, and specificity in measuring Ras and Rho activation in a series of breast, lung, and ovarian cancers. Results obtained with the instrument will be compared to results using the well-validated manual methods. Ras is one of the prime targets for cancer chemotherapy and a number of Ras inhibitors, some of which also inhibit Rho, have been developed and are in clinical trials. These drugs are most likely to be effective in tumors with increased Ras and/or Rho activation and, thus, measuring Ras/Rho activation in tumors will be of significant clinical relevance. On completion of the pilot testing phase proposed in these studies, the Automated Ras/Rho Activation Measurement Device should be ready for use in clinical laboratories.
The completion of many genome sequences presents the challenge of understanding how their derived proteome contributes to a living organism. To begin to understand the complex matrix of interactions in a living cell, we propose a pilot project aimed at obtaining a comprehensive map of the protein interactions within the first fully sequenced eukaryote, Saccharomyces. We will combine four newly available technologies to produce a powerful, rapid high-throughput methodology to study protein interactions in the normal cellular context. First, we will develop more versatile variants of our rapid genomic tagging technique, which creates functional protein chimeras expressed at their normal level and carrying a Protein A affinity tag at their C-termini. Second, we will further refine our subcellular fractionation protocols, in order to routinely dissect cells from the tagged strains into fractions enriched in the tagged protein and its specifically interacting partners. Third, we have developed novel and extremely efficient protocols for the immunopurification of the tagged subcomplexes from these different fractions. Fourth, the proteins so isolated will be rapidly identified by mass spectrometry (MS), using both our current and developing techniques. The speed and sensitivity of these MS techniques permits the analysis of large numbers of proteins made from relatively small amounts of cells. For this pilot project, we will select a subset of approximately 200 proteins to analyze, corresponding to approximately 3% of the yeast proteome. The members of this group will be chosen such that they represent the broad spectrum of characteristics found in the proteome, and include proteins with strong interest to the biological community. Each member will be tagged and co-isolated with its specifically interacting partners. We will use these proteins for two purposes. First, we will refine our analytical approach, defining and then eliminating any bottlenecks that we encounter, and extending our methodology to include transient interactions and regulatory modifications of proteins. We will explore the possibilities for automation at every stage, to increase speed and throughput. Second, we will apply the technology to case studies on functionally interrelated subsets of proteins within the group. For a case study in yeast, we will investigate the cell cycle control protein-interaction and modification network occupied by cyclins (UNIVERSITYersal regulators of the eukaryotic cell cycle). Our case study in humans will be the p53-MDM-2 complex, a key player in preventing oncogenesis. We will analyze the protein interactions and modifications relating to the function of this complex. The resulting wealth of information will be used to construct a pilot functional proteomic database, providing a matrix of functional interactions between proteins within a living cell.
R33 CA091342-01 2001 DAVIS, RONALD WAYNE STANFORD UNIVERSITY Target Identification via Haplo Insufficiency Profiling
We have developed a genomic approach to target identification in yeast that may be broadly applicable to human therapeutics. This approach is based on our observation that drug targets can be identified by their ability to confer sensitivity to a cell when present at a reduced copy number; in this a case change in copy number from two copies in a diploid yeast strain to one copy in a heterozygous deletion strain. We have assessed the feasibility of this approach by confirming the molecular targets of several currently available drugs (Giaever et al. 1999). Briefly, in our method, a complete genome set of molecularly bar-coded heterozygous yeast strains are pooled, grown competitively in drug and analyzed for relative growth rates using high-density oligonucleotide arrays. Growth rate is a metric of relative sensitivity of each strain. The strain most sensitive to drug in many cases identifies the gene encoding the drug target. In this way, each strain represents a potential drug target and thus the complete set of potential targets is screened in a single assay. This method has the advantage of generating targets in an unbiased manner, as it is the organism itself that reports its most essential targets in the presence of any given compound. For this reason, we expect that we will uncover novel molecular targets and compounds in a manner that would up-end the current drug discovery paradigm. Instead of the subjective approach of pre-selecting targets, we aim to simultaneously identify and validate novel, essential molecular targets and the compounds that inhibit them in the "genomically" accessible yeast S. cerevisiae. Given that approximately 50% of all known yeast genes have human homologs (Foury 1997), it is our contention that the identification of all gene products essential for yeast growth will be relevant to human cancer. In addition, because each strain can be ranked in order of sensitivity, sensitive strains other than the strain that identifies the drug target will assist in the deconvolution of the involved pathways. These pathways include the drug target pathway as well as drug response pathways. Through the generation of a database containing thousands of profiles of known drugs and unknown compounds, it may be possible to gain insight into mechanisms of drug action and compound structural similarities through the clustering of compound profiles. Furthermore, because our technology defines a novel genome= wide measurement, we believe it will significantly contribute to the elucidation of gene function ultimately arising from the combined analysis of genomic data sets collected using diverse genomic technologies.
With the invention of hybridoma technology by Kohler and Milstein (1975), numerous monoclonal antibodies (MoAbs) against cell surface antigens or receptors have been developed and used clinically as diagnostic agents. In the last two decades there has been enormous effort in both academia and pharmaceutical industry to develop monoclonal antibodies to treat human cancers. The recent clinical success of Rituxan (anti-CD 20 MoAb against B lymphoma) and Herceptin (anti-Her2/neu MoAb against breast cancer) in the treatment of human cancers has validated the cell-surface targeting approach for cancer therapy. Evaluation of biopsy specimens for the presence of CD20 and Her2/neu is now routinely done for non-Hodgkin lymphoma and breast cancer, respectively. Combinatorial chemistry has become one of the most important technologic advances in recent years for basic research and drug discovery. Millions of compounds can be generated and screened for their ability to bind to a specific target macromolecule or to elicit a specific biological response (Lam 1997). In this proposal, we hypothesize that by using the state-of-the-art "one-bead one-compound" combinatorial library method, we can rapidly identify a large number of cell surface binding peptides that are unique to different tumor types. We further hypothesize that with the novel chemical microarray technique that was recently developed in our laboratory, we can rapidly characterize the binding specificities as well as functional effects of the identified peptide ligands on a large number of tumor cell lines. Peptides that are unique to human tumors can then be used to determine the ligand binding profile of human cancer biopsy specimens.
With the rapidly expanding availability of entire genome sequences, the potential for analyzing whole-genome expression patterns is attaining reality. The availability of this vast pool of comparative data will have a major impact on cancer research. Clearly, however, the success of these approaches depends critically on being able to define the relationship between expression patterns and downstream events that define phenotype. For the most part, these downstream events are mediated by the biological activities of protein molecules, which are in turn controlled by protein level and post-translational modification. In this application, we propose to develop a second generation scheme for mRNA expression analysis. In order to develop this technology and at the same time generate biologically important information, we have chosen as our model cell-cycle regulation of gene expression in the yeast Saccharomyces cerevisiae. The core technology in this proposal is what we have termed translation state array analysis (TSSA). TSSA provides, in addition to the absolute levels of individual mRNA molecules, information on the degree to which these mRNAs are engaged in protein synthesis. The results from TSAA will be correlated with datasets generated from proteomic analysis. Combining these three measurements (total mRNA, translated mRNA and protein level) from the same biological system will enable us to make statements about detailed mechanisms of regulation of specific genes and also identify clusters of genes that are regulated through the same mechanisms. This study will provide more finely honed high-throughput tools to provide insight into both mechanisms of regulation of individual genes and the levels and activities of the proteins that ultimately dictate phenotype.
Cancers of unknown primary site are a diagnostic and therapeutic dilemma in oncology. Information from DNA micro array technologies on the gene expression profile of cancers has led to the hypothesis that there are diagnostic sets of genes which can resolve the origin of unknown primary cancers (UPC) with a high degree of confidence. The purpose of this project is to test this hypothesis, by both retrospective and prospective analysis of cases of UPC from Stanford Medical Center and the Sarah Cannon Cancer Center, in the context of a rapidly evolving database of site-specific clusters of gene expression. Specific Aims are: (1) Definition of the gene expression profile of known human cancers. We now have extensive information on the profiles of lymphomas, leukemia, and carcinomas of the breast, prostate, lung, ovary, and liver. Additional tumors to be accrued from our tumor bank and ongoing sample acquisitions include sarcomas, germ cell cancers, melanomas, mesotheliomas, and carcinomas of the colon, stomach, pancreas, bladder, and kidney. (2) Determination of the diagnostic cluster of gene expression for each of the above tumor types. We anticipate that several hundred genes may differentiate one from the others of these known tumors. (3) Acquisition, gene expression profiling, and diagnostic classification of unknown primary cancer specimens. This aim will involve a close collaboration with the world's leading center for the clinical evaluation of unknown primary cancers, the Sarah Cannon Cancer Center in Nashville. (4) Evaluation of a panel of histospecific antisera for diagnostic utility with UPC specimens. This aim will utilize retrospective archived specimens as well as prospectively acquired samples from this project. (5) Identification of specific or clustered gene expression associated with known prognostic factors, response to therapies, and survival of patients with UPC.