Abstracts

Department of Pathology, Biochemistry, and Molecular Biology, Jake Gittlen Cancer Research Center, Pennsylvania State University

1 R33 CA118591-01

This proposal seeks to develop an RNA Sensor to be employed for detection of circulating tumor cells. RNA detection is based upon a hybridization “sandwich.” Two target RNAs have been chosen for clinically important cancers (prostate, breast, and melanoma), and library selection protocols will be utilized to identify/optimize accessible sites for antisense oligonucleotide (ASO) binding. Silicon nanowires will then be covalently derivatized with ASO to a library-selected site (ASO-) in the target RNA. The ASOi nanowires will then be deposited by fluidic deposition onto chips, and integrated into the underlying CMOS circuitry. Target RNA will be purified from cellular preparations, and will then be hybridized to the ASd-nanowires. An ASO2, targeted to a second library-selected site, will be covalently attached to 12 nm gold particles (ASO2-nanoprobe). Binding of the ASO2-nanoprobe to the target RNA-ASOi-nanowire complexes will induce a resonance frequency shift in the nanowires, which is greatly amplified by the mass of the gold particle. This resonance frequency shift (R) will be detected by direct electrical read-out, with voltage (quantitatively) related to binding events (R) will initially be detected optically. We have successfully measured R of 300 nm silicon nanowires (with high Quality-Factors) under ambient conditions. Theoretical calculations predict very good Quality-Factors for silicon nanowires in H20, and detection of single binding events should be achievable.

Preliminary data related to all aspects of RNA Sensor development have been obtained. These include library selection of target sites in prostatic DD3 RNA, sandwich hybridization specificity “off-chip” synthesis and derivatization of nanowires, R measurements with nanowires, and fluidic deposition of nanowires on chips. After basic developmental steps are completed, experiments will include quantitative determination of target RNAs using the detection device compared to QPCR amplification. The Specific Aims for this funding period are designed to develop an RNA Sensor appropriate for subsequent use in clinical validation studies for circulating tumor cells. Successful development of this RNA Sensor would provide a major advantage over PCR-based assays, and could form the basis for high-throughput screening tests for simultaneous detection of many different circulating tumor cell types.

Department of Cell Biology, The Scripps Research Institute

1 R33 CA118696-01

Proteases are suspected to play major roles in cancer, including the activation/inactivation of growth factors and the degradation of extracellular matrix components to promote cancer cell migration and invasion. Consistent with this premise, transcript and protein levels for many proteases are upregulated in cancer cell lines and primary tumors. However, whether these changes in protease expression correlate with changes in protease activity remains a critical, but largely unanswered, question. Indeed, proteases are regulated by a complex series of posttranslational events, meaning that their expression levels, as measured by conventional genomic and proteomic methods, may fail to accurately report on the activity of these enzymes.

To address this important problem, we have introduced a chemical proteomics technology referred to as activity-based protein profiling (ABPP) that utilizes active site-directed probes to determine the functional state of large numbers of proteases directly in whole cell, tissue, and fluid samples. We have applied ABPP to identify several protease activities upregulated in human cancer cells and primary tumors. Recently, we have created an advanced antibody-based microarray platform for ABPP that enables profiling of protease activities with an unprecedented combination of sensitivity, resolution, and throughput, while requiring only minute quantities of proteome. The goal of this R33 application is to extend these studies to create the first ABPP microarray for the parallel analysis of key cancer-associated protease activities in any proteomic sample. These studies will deliver valuable new reagents and methods for the functional characterization of proteases that will be made freely available to the cancer research community. We envision that the general implementation of these innovative technologies will greatly accelerate the discovery of proteases with altered activity in human cancer. These proteases may in turn represent valuable new markers and targets for the diagnosis and treatment of cancer.

SRI International

1 R21 CA118526-01A1

Understanding the basic genetic and molecular markers of cancer at the cellular level is vital for preventing, diagnosing, and treating cancer. Recent work in our lab has led to new technologies for manipulating small aqueous drops containing biological molecules. We use laser light to induce surface tension gradients, which allows us to selectively move very small individual droplets (nl_-pL), and we have performed simple enzymatic assays with this approach. Because our technique uses laser heating as a basis for droplet control, we believe that it is well suited to genomic analysis methods, such as polymerase chain reaction (PCR), which rely on thermal cycling. We propose to combine our laser-based droplet control techniques with genomic analysis tools to develop a device for screening large numbers of individual cells. To test the capabilities of our apparatus, we propose an R21 project with the following specific aims: Aim 1: Optimize and automate the liquid handling system for genomic analysis. We will choose the optimal materials and reagents for use in our apparatus. We will automate both the droplet delivery and the droplet handling capabilities of our device. Aim 2: Perform a real-time PCR in our droplet-based system. We will test the overall sensitivity and quantify nonspecific amplification in our binary liquid system. Aim 3: Examine single cells within small droplets. This examination will include testing for short- and long-term cell viability and PCR amplification of single-cell genomic material. This research is fully consistent with the goals outlined in RFA-CA-06-002. If this program is successful, we believe that the resulting technology will prove invaluable in helping physicians and scientists better understand the molecular basis of cancer while also providing tools to help diagnose specific variants of disease, plan and assess therapy, and monitor disease recurrence.

Department of Chemistry, University of Arizona

1 R21 CA122630-01

The long-term objective of the proposed research project is to provide a robust, sensitive, and rapid method for the direct detection of CpG island methylation in the promoter region of specific genes implicated in cancer. Cytosine methylation occurs at CpG dinucleotides in 70%-80% of the human genome, most often in repetitive genomic regions. On the other hand CpG islands, defined as short sequences with statistically high CpG content, present in the promoter region of many genes (60%) are primarily protected from methylation in normal tissues. These CpG islands have been found to be methylated in cancer leading to transcriptional repression. Recent experiments provide strong correlation between CpG hypermethylation at promoter sites of numerous genes and the incidence of cancer, thus making specific promoter hypermethylation a valuable marker for early detection. Current methods for detection of specific CpG island methylation rely on extensive bisulfite treatment of methylated DNA followed by PCR-based amplification, sequencing, or microarray techniques. These current methods, though powerful, are also laborious, time-intensive, and expensive for characterizing known sites of hypermethylation. Towards the goal of rapidly determining promoter CpG hypermethylation, we will apply our newly developed technology called Sequence Enabled Reassembly (SEER) of proteins. The SEER system allows for the recognition of specific sequences of double-stranded DNA that result in the concomitant assembly of functional protein reporters (green fluorescent protein and p-Lactamase). In the proposed detection of specific CpG hypermethylation, we will target CpG islands utilizing the methyl-CpG binding domain (MBD) of the MBD2 protein, while targeting the correct promoter sequence utilizing a designed zinc-finger. Our approach has the potential to provide a sensitive turn-on sensor for directly reporting upon CpG methylation at known promoter sites. This approach if successful will rapidly distinguish between normal and cancerous tissues in a clinical setting without the requirement for bisulfite treatment, PCR amplification, and sequencing. We will provide proof of concept by (1) designing and optimizing turn-on biosensors for detecting specific methylation events in model DNA constructs and (2) designing and testing biosensors that target promoter regions of genes (BRCA1, CDH1, p15, p16, MGMT, GSTpl) implicated in cancer.

Livermore Laboratories, University of California, Berkeley

1 R33 CA1184791-01A1

Though great progress has been made in the area of DNA analysis for cancer, understanding the proteins encoded by DNA can provide more answers, but is also more challenging. As the study of cancer proteomics advances, it is clear that new analytical tools and technology are needed for the comprehensive profiling of the proteins in a cell so that our understanding of carcinogenesis and the differences between healthy and cancerous cells can progress. Further understanding of cancer proteomics will drive the discovery of new drug targets as molecular changes in the cell are observed without preconceived ideas about what changes would be the most valuable to monitor. Due to the very large number of proteins in a cell, comprehensive analyses require the use of separation methods that have high peak capacities. Capillary isoelectric focusing (cIEF) has shown great promise in this area with a peak capacity in excess of 1400. This greatly exceeds traditional separation methods, such as liquid chromatography (LC), capillary electrophoresis (CE), or mass spectrometry (MS), which often have peak capacities of less than 200.

An increase in the total peak capacity of a system can be achieved when multiple separation techniques are combined, leading to the popularity and performance of tandem methods such as LC/LC or LC/MS. Though the superior performance of cIEF over CE and LC would seem to make it a preferred choice in a tandem system, it is not able to be efficiently interfaced with other methods. This is the primary reason it is not widely used. The proposed research will continue the development of dynamic isoelectric focusing, which is a new technology developed by the PI that will be able to provide the high peak capacity of cIEF while also efficiently coupling with other techniques. The combined systems made possible will easily outperform other tandem methods and will have a high impact on the molecular analysis of cancer because they will permit the acquisition of a more comprehensive profile of the proteins in cancerous cells than is currently possible. The capabilities of dynamic IEF will be demonstrated by interfacing it to MALDI-MS and using the system to analyze and observe differences in extracts from treated and untreated PC-3 prostate cancer cells. The cell treatment will be based on compounds currently researched by the Co-PI, such as bisdehydrodoisynolic acid, which is an estrogenic carboxylic acid shown to be effective at reducing the proliferation of prostate cancer.

Academic Health Center, University of Minnesota Cancer Center

1 R21 CA118600-01

Prostate cancer is the second leading cause of male cancer death in the United States and often results in a reduced quality of life for those living with or treated for this disease. Prostate cancer is commonly treated by androgen ablation therapy and although many tumors initially respond to this treatment, many eventually progress to hormone refractory prostate cancer (HRPC). The genetic basis for the transition to hormone insensitivity is poorly understood. We propose to use a mouse model for invasive prostate cancer that results from prostate-specific loss of the tumor suppressor gene Pten. This mouse model is relevant to human disease as PTEN expression is lost in many human prostate tumors and the tumors that form in the mice remain partially sensitive to hormone withdrawal. We will use a novel method for cancer gene discovery in mice, the Sleeping Beauty (SB) transposon system, to promote aggressive tumor formation in this model. The SB transposon is a DMA element that is capable of mobilizing and inserting in a different location in the genome. If a mobilized transposon reinserts near a cancer gene, it can promote changes in expression of that gene that promote the transition from a normal cell to a transformed cancer cell. We have previously generated mice engineered with all the components necessary for mobilizing SB transposons in various tissues in the adult mouse. In unpublished experiments, we have successfully used the SB system to identify genes involved in sarcoma and lymphoma formation in mice, and we believe that SB will prove to be equally as successful in prostate tumor models. By using SB to promote HRPC formation we can both identify the genetic changes that cause a tumor to become insensitive to hormone withdrawal and also generate a useful mouse model of HRPC that will be useful for discovery and testing of novel chemotherapeutic agents for advanced prostate cancer. Finally, this approach represents a novel method for the unbiased molecular/genetic analysis of cancer development and could be used widely in the study of important clinical cancer problems.

Hematology-Oncology Division, Department of Pediatrics, University of Texas, Dallas

1 R21 CA120681-01

The goal of this research is to develop methods for the precise modification of specific target genes in two important genetic model organisms, the nematode Caenorhabditis elegans and the zebrafish Danio rerio. Both nematodes and fish are powerful experimental systems that combine elegant developmental biology with large-scale genetics. Both systems have contributed to our understanding of fundamental problems in cancer biology, including programmed cell death, the control of organogenesis, the interaction of cancer susceptibility genes with the environment, and the genetics of melanoma. An important limitation of these model systems is that techniques for site-specific manipulation of the genome are not currently available in either nematodes or fish. Thus, in contrast to murine embryonic stem cells and the yeast S. cerevisiae, it is not possible to knock out specific genes or to precisely control the time and place of gene expression. In the last 2 years, a powerful new approach to gene-targeting has been developed and successfully used in flies and in mammalian somatic cells. This technique uses chimeric zinc finger nucleases to stimulate precise targeting of specific genes in their native genomic context. The aim of this proposal is to induce targeted, heritable genetic changes via zinc finger nuclease-mediated homologous recombination in C. elegans and D. rerio. Initially we will employ a well-characterized zinc finger nuclease that recognizes the green fluorescent protein (GFP) gene. We will introduce the nuclease into transgenic nematodes and zebrafish that express GFP. We expect the resulting double-strand DNA breaks to stimulate mutagenic non-homologous end joining (NHEJ), leading to the loss of GFP signal. In the second phase, we will simultaneously introduce the nuclease and a repair template that will allow us to create precise mutations in the target locus by homologous recombination. Based on the success of this work we will then target native genes in the worm and the fish by designing novel nucleases and testing them in vitro and in vivo for activity against the targeted gene. We expect that, if successful, this novel approach would be a practical, flexible, and powerful technique that would find wide application, significantly increasing the power of these systems to illuminate human cancer biology.

The Molecular Science Institute

1 R33 CA114306-01A1

Biologists have long known that cancer cells sometimes announce their presence by shedding certain molecules into the blood. More recently, many have come to believe that some cancers might be detectable by “signatures,” patterns of sets of molecules, perhaps normally present in the blood, but, for certain cancers, present in higher or lower amounts than normal. Recently, we learned to make new kinds of molecules, which we call “tadpoles.” We have demonstrated that we can use them to detect and count small numbers of molecules. These assays are relatively simple and relatively inexpensive, and they should be applicable to both kinds of cancer detection. During the next 3 years, we seek funding to develop and “harden” these assays to the point that they can be tested in clinical cancer diagnosis.

Department of Chemistry and Biochemistry, Southern Illinois University

1 R21 CA120691-01

Though great progress has been made in the area of DNA analysis for cancer, understanding the proteins encoded by DNA can provide more answers, but is also more challenging. As the study of cancer proteomics advances, it is clear that new analytical tools and technology are needed for the comprehensive profiling of the proteins in a cell so that our understanding of carcinogenesis and the differences between healthy and cancerous cells can progress. Further understanding of cancer proteomics will drive the discovery of new drug targets as molecular changes in the cell are observed without preconceived ideas about what changes would be the most valuable to monitor. Due to the very large number of proteins in a cell, comprehensive analyses require the use of separation methods that have high peak capacities. Capillary isoelectric focusing (cIEF) has shown great promise in this area with a peak capacity in excess of 1400. This greatly exceeds traditional separation methods, such as liquid chromatography (LC), capillary electrophoresis (CE), or mass spectrometry (MS), which often have peak capacities of less than 200.

An increase in the total peak capacity of a system can be achieved when multiple separation techniques are combined, leading to the popularity and performance of tandem methods such as LC/LC or LC/MS. Though the superior performance of cIEF over CE and LC would seem to make it a preferred choice in a tandem system, it is not able to be efficiently interfaced with other methods. This is the primary reason it is not widely used. The proposed research will continue the development of dynamic isoelectric focusing, which is a new technology developed by the PI that will be able to provide the high peak capacity of cIEF while also efficiently coupling with other techniques. The combined systems made possible will easily outperform other tandem methods and will have a high impact on the molecular analysis of cancer because they will permit the acquisition of a more comprehensive profile of the proteins in cancerous cells than is currently possible. The capabilities of dynamic IEF will be demonstrated by interfacing it to MALDI-MS and using the system to analyze and observe differences in extracts from treated and untreated PC-3 prostate cancer cells. The cell treatment will be based on compounds currently researched by the Co-PI, such as bisdehydrodoisynolic acid, which is an estrogenic carboxylic acid shown to be effective at reducing the proliferation of prostate cancer.

Department of Bioengineering, University of Pennsylvania

1 R21 CA116102-01A1

We propose to develop a molecular imaging probe that will provide quantitative information on the expression level of mRNA with spatial and temporal resolution. Specifically, an oligonucleotide-based probe will be designed to form a stem-loop structure and will be labeled with a “reporter” fluorophore at one end and a quencher at the other, analogous to a molecular beacon; however, the oligonucleotide will also be labeled with a second optically distinct “reference” dye/nanoparticle, which will be selected such that it is unquenched regardless of the conformation of the probe. Fluorescently labeled neutravidin and quantum dots will be tested for their suitability in serving as the reference dye. We hypothesize that beneficial features of this novel probe compared with conventional molecular beacons will include (1) the ability to monitor transfection efficiency due to the presence of the unquenched reference dye. This will reduce false-negatives by allowing for the differentiation between untransfected cells and cells with low levels of gene expression. (2) The ability to remove via ratiometric imaging (i.e., reporter fluorescence/reference fluorescence) the impact of instrumental and experimental variability.

(3) The ability to quantitatively compare variations in gene expression levels between samples, between cells within individual samples, and even between sub-cellular compartments by using the reference dye as a point of reference. (4) The ability to quantify gene expression with spatial and temporal resolution since the covalent linkage between the reporter and reference dye ensures they exhibit an equivalent intracellular lifetime and co-localization pattern. (5) The ability to use the quantum dot/neutravidin as a platform to attach targeting agents, opening up the possibility for in vivo imaging. (6) The possibility of an improved signal-to-background due to quenching of the “reporter” dye by both the quencher molecule and the “reference” dye. To evaluate these features we will pursue two major aims during the proposed research: (1) We will design, synthesize, and characterize the “quantitative” molecular beacon (QMB) in terms of its signal-to-background and lower detection limit ( in vitro and in vivo ) and (2) We will evaluate the ability of the QMBs to quantify endogenous mRNA expression in breast cancer cells in real time. It is envisioned that the approach proposed here will allow significant advancements in our understanding of human health and disease and could potentially prove to be a powerful diagnostic tool.

Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins

1 R21 CA116214-01A1

One of the experimental challenges in cancer molecular biology is assessing the validity and generality of biomarkers. This has become a critical bottleneck in the development of biomarkers from differential gene expression revealed by microarray studies. In this proposal, we develop the concept of using vertical arrays for exploration of differential gene expression in cancer. Vertical arrays explore the expression of a gene in many biological samples simultaneously, whereas standard microarrays explore the expression of many genes in response to one biological variable at a time. Vertical arrays are like dot blots in this regard, but vertical arrays are printed on glass slides, giving them better signal-to-noise behavior, and, rather than spotting the entire complexity of the RNA population in each spot, the RNA population is divided up among multiple spots. These low-complexity representations have superb signal-to-noise performance. The work in this proposal will focus on establishing the feasibility of making a vertical array for studying gene regulation in many cancer samples simultaneously. Potential throughput is very high, such that multiple regions from each tumor can be studied simultaneously. This approach will be useful in confirming that a gene is indeed differentially regulated, in determining the distribution of expression of the gene in the transformed and surrounding normal tissue, and in determining whether the gene behaves in a similar manner in different cases of the same type of cancer and in different kinds of cancer. The goals require extensive and efficient microdissection, and we have built a novel instrument, the “tissue mill,” to achieve these ends. Relevance: Biomarkers are useful for diagnosis, prognosis, and as potential therapeutic targets for cancer. There are hundreds of potential biomarkers, but further validation is needed before they can be exploited.

Department of Medicine, University of California, San Diego

1 R21 CA118595-01

Many cancer-implicated proteins are integral membrane proteins (IMPs). There is a pressing need for improved methods for the production of IMP constructs for use in high-resolution structure determination efforts. Three years ago, we completed a fifteen-year effort to develop methods for the performance of peptide amide hydrogen/deuterium exchange-mass spectrometry (DXMS). In collaboration with the Joint Center for Structural Genomics (JCSG), we recently demonstrated that DXMS can provide precisely the information needed to guide the design of well-crystallizing constructs of otherwise poorly-crystallizing soluble proteins. The NCI IMAT Program is now funding our efforts to optimize DXMS-guided construct design for soluble cancer-implicated proteins (R33 CA099835). Until recently, we thought it unlikely that successful DXMS analysis of membrane proteins would be possible, and this funded grant contains no reference to membrane proteins (IMPs), nor does it support work on them. However, insights and preliminary studies described in the present application now make it likely that, with intensive development work, we can devise highly modified methods that will allow the facile DXMS analysis of IMPs. Development of membrane protein DXMS will greatly impact the structural biology of cancer implicated IMPs, which are particularly difficult to prepare in crystallizable form. Initial year 1 development efforts will focus on the integrin allbbS, with which I have had considerable experience. Integrins are widely implicated in cancer cell and cancer vasculature biology, and findings with the prototypic allbbS integrin have proven applicable to the understanding of all integrins. The resulting IMPDXMS methods will be further refined and validated in year 2 through study of additional cancer-relevant IMPs and daughter constructs provided by Dr. Raymond Stevens, P.I. of the newly NIH-funded JCSG Center for Innovative Membrane Protein Technologies (JCIMPT). Once IMP-DXMS has been fully developed and validated, it will be made available to investigators studying cancer-implicated IMPs, by integrating the methods with our soluble-protein DXMS resource now supported by the NCI IMAT Program. Thus the NCI’s investment in presently funded DXMS work will be greatly leveraged by the relatively modest support requested for the development of IMP-DXMS.

Pathology Department, Weill Medical College of Cornell University

1 R21 CA122673-01

Activation of phosphatidylinositol 3-kinase (PI3K) and the downstream serine/threonine kinase Akt (also known as protein kinase B) triggers a cascade of responses that are critical for tumorigenesis, from cell growth and proliferation to survival and mobility. Aberrations of components in the PI3K/Akt pathway have been shown to be present in a majority of tumors. We hypothesize that aberrant PISK/Akt activation could be characterized by combined activity profiles and used as a diagnostic marker in cellular activity-profiling. To test this hypothesis, we propose the following specific aims: (1) To analyze the activities of PI3K and Akt in breast cancer cell lines and to further develop fluorescent activity sensors for various components in the PISK/Akt pathway and (2) To develop cellular assay platforms for high-throughput activity profiling of oncogenic PISK/Akt signaling. These studies will take advantage of a series of Fluorescence Resonance Energy Transfer (FRET)-based reporters we have recently developed for measuring the activities of Akt and PI3K in living mammalian cells. Fluorescent activity sensors and cellular assay platforms developed in this study can be used in systematic analysis of the critical components in PISK/Akt pathway in various cancers to generate activity profiles. Correlation of genetic alterations with activity profiles and phenotypes should provide new insights into the molecular mechanisms of cancer development. On the other hand, molecular diagnostics based on such activity profiling could identify the molecular defects and the malfunctioned key nodes in the signaling network for a given cancer and guide appropriate molecular therapeutics as well as facilitate their development and evaluation.

The Michigan Nanotechnology Institute for Medicine and Biological Sciences, University of Michigan, Ann Arbor

1 R33 CA112141-01A1

Methods using fluorescent probes to identify cancer signatures and biological activities of cancer cells hold great promise. Probes based on fluorescence resonance energy transfer (FRET) and techniques such as fluorescence correlation spectroscopy (FCS) and other similar technologies offer the ability to identify specific RNA or protein molecules that can identify a cancer and provide information on oncogenic pathways used by the tumor cells. Other probes can give insight into drug response by measuring apoptosis induction by chemotherapies and radiation. However, fluorescent analysis has several limitations. Most ex vivo analyses use a flow cytometer or complex, confocal microscope to perform analyses, and this requires that tissue be removed from the body and often disrupted into cells, then fixed and analyzed in a static manner. The problems with in vivo fluorescent analysis are even greater since background fluorescence and tissue scattering, even in the near-infrared range, limit signal acquisition to the skin. Two-photon excitation has been a critical advance in optics, facilitating FRET, FCS and CARS techniques in vitro . However, these applications are limited by the complex technology (confocal microscopy) necessary to employ these techniques. We have demonstrated the use of two-photon fluorescence analysis through optical fibers for analysis of cancer cells in vitro and human tumors in vivo in SCID mice. This prior work constitutes the equivalent of an R21 proposal, as we achieved our major objectives: to develop sensing system optics and electronics and to document the ability of this system to obtain and analyze fluorescence signals in vitro and in vivo . The primary goal of this R33 application is to develop a more sensitive prototype device based on a novel dual-clad photonic crystal fiber (DCPCF) that we hypothesize will provide the sensitivity and redundancy necessary for the clinical evaluation of fluorescence signals in vivo using several fluorescence techniques. We plan to carry out our studies in three Specific Aims: (1) Develop DCPCF for use in a two-photon optical fiber fluorescence probe (D-TPOFF). (2) Utilize the D-TPOFF to quantify cancer signatures in vitro and monitor drug effects in tumor cells using targeted nanoparticles ex vivo and in vivo . (3) Utilize D-TPOFF to adapt other fluorescent techniques to examine events in tumors in vivo . At the end of these studies, this technology will be at a point where it is ready for commercialization.

Radiology Department, University of Massachusetts Medical School

1 R21 CA116144-01

Molecular dissection of gene expression pathways in cancer has revealed many new targets for cancer therapy. Those targets include the components of abnormal transcription machinery. Proteins involved in regulation of transcription attained high priority due to the convergence of many signal transduction pathways at the transcriptional level. New molecular therapies directed to transcriptional targets have significant advantages over traditional therapies due to a precision of their interference with target gene expression. Consequently, small molecule inhibitors, DNA binding polyamides, protein-binding oligonucleotide decoys, as well as small interfering RNAs and their combinations are being developed for cancer therapy. While rapid progress in molecular genetics and medicinal chemistry delivers new “attenuators” of gene expression, the technologies of early and non-invasive assessment of cancers that would be amenable to these therapies are currently lacking. In particular, imaging technologies that report directly on gene transcription in cancer cells are critically important for both cancer phenotyping and staging, as well as for evaluating new therapies. The goal of the proposed research is to optimize and characterize far-red fluorochrome labeled oligodeoxyribonucleotide molecular reporter (ODMR) probes followed by the investigation of their transcription-factor reporting properties. Using NF-kB as a model cancer-relevant transcription factor, the following specific aims will be pursued: (1) Optimization of design, synthesis, and in vitro testing of transcriptional factor reporter probes (ODMR) and (2) Investigation of the potential of ODMR probes in detecting active transcription factor in live cells.

Department of Neurological Surgery, Ohio State University

1 R21 CA114487-01

Differences in the expression of several genes between normal brain and its tumors (such as glioma) can provide information useful for malignant glioma diagnosis and therapy. Gliomas arise in the brain and are characterized by heterogeneous regions of necrosis, apoptosis, proliferation, invasion and angiogenesis. Drugs are being developed to target such phenotypes but it is unclear what imaging or molecular correlate will such drugs use to assay responses. Recently, serial analysis of gene expression (SAGE) data for malignant glioma has become available through efforts of the Cancer Genome Anatomy Project (CGAP) and several genes have been identified that are overexpressed in glioma and not in normal brain. Some of these genes have been postulated to correlate with a particular glioma phenotype, such as invasion or angiogenesis. We propose a set of technologies useful to translate CGAP knowledge into imaging the transcriptional activation of these genes and correlate such activation with the observed phenotypic heterogeneity in in vitro and in vivo models. We plan to combine the ability of our infectious bacterial artificial chromosome (iBAC) technology to rapidly isolate and deliver into cells large genomic fragments (up to 150 kb) with the ability of MRI and bioluminescence imaging to image gene expression. Specifically, the R21 phase this project proposes to (1) Verify that large 5’ flanking regions of one glioma expressed gene (for SPARC) transcriptionally activates the MRI-imaging gene ETR and luciferase, (2) Image the transcriptional activation of this region in in vitro models of glioma, (3) Image the transcriptional activation of this region in in vivo models of glioma. Transgenic mouse models have shown that large regions of 5’ flanking area are best at providing complex tissue-specific and developmentally correct gene expression. A large 5’ flanking region of SPARC will thus be cloned upstream of luciferase and/or ETR in the iBAC system. Imaging will be performed in in vitro and in vivo glioma models. The information obtained in this R21 will justify further grants (R33, R01) where the transcriptional activation of other genes can be imaged, thus providing a quantitative anatomic map of transcriptional activation in different heterogeneous regions of gliomas (invading, angiogenic, hypoxic, proliferating areas). This provides a baseline for assessing the effects of therapies (drugs, radiation) on these tumor phenotypes. The conceptual scheme presented herein will also be applicable to a variety of diseases for which gene profiling analyses are available.

Department of Cell Biology, University of Connecticut School of Medicine

1 R21 CA114489-01

Immune-dependent responses are selective in defining non-self or aberrant antigen presentation. Unfortunately, long-term antibody production to human cancer antigens is limited. In an innovative and novel approach, we have developed a method to utilize primary immune reactions in tumor draining lymph nodes to provide the means to define biologically active tumor antigens originating from breast cancers. This novel approach combines a series of steps to coordinate the construction of low complexity antibody cDNA libraries and protein production that are used to identify tumor antigens using sensitive antibody microscale “antigen-trap” assays followed by LC-MS/MS antigen identification. Tumor antigens identified can then be verified as potential tumor antigens using biochemical, immunological, and molecular methodologies. The methodologies applied are medium throughput platform based designed to rapidly evaluate matched lymph node and tumor samples from the same patient. This project applies innovative technologies that demonstrate that (a) Tumor draining lymph nodes are immuno-reactive to aberrant breast cancer antigens and produce antigen-dependent somatic hypermutation in proliferative B-cell germinal centers; (b) Antigen binding domains of somatic hypermutated antibodies synthesized as recombinant VH and/or VHVl/Vk ScFv proteins can specifically recognize and identify breast cancer antigens; and (c) Antigens identified can be verified as diagnostic for breast cancer sub-phenotypes. This R21 application proposes to expand and refine our methodologies to (1) Determine the diversity and effectiveness of immune-selection of tumor antigens in a larger patient population; (2) Expand our ability to produce antibody molecules with appropriate structure and antigen binding; and (3) Develop methodologies to incorporate antibody proteins synthesized into highly sensitive assays that can screen primary cancer, histological material, and/or biological fluids necessary to evaluate the potential of antigens as diagnostic biomarkers.

Department of Chemistry and Biochemistry, University of Texas at Austin

1 R21 CA107887-01A1

There are numerous methods for identifying and probing genomic markers in tumor cells, most of which are based on nucleic acid amplification technologies. However, in many instances protein markers are going to be of even greater utility in identifying and classifying tumor cells than genetic markers. To this end, it would be extremely useful to have methods by which protein (rather than nucleic acid) markers could be amplified. In this proposal we outline a series of novel methods for transducing tumor cell antigens to amplicons, which can in turn be sensitively detected using methods common to nucleic acid diagnostics. In particular, it has previously been shown that nucleic acid binding species (aptamers) selected from random sequence pools can specifically interact with a wide range of protein targets, including those relevant to tumor biology. Aptamers typically bind their cognate targets with dissociation constants in the nanomolar range and can readily discriminate between even closely related proteins. For these reasons, aptamers should prove useful for recognizing a wide range of protein markers associated with cellular transformation. We have previously developed automated methods for the selection of aptamers. We now propose to use such automated methods to target cell surface antigens of tumor cells. Selected anti-tumor aptamers will be adapted to a number of important diagnostic methods, including methods to label tumor cells and methods to transduce tumor protein antigens into nucleic acid amplification assays, via two novel methods, proximity ligation assays and rolling circle amplification.

Department of Biomedical Engineering, Boston University
1 R21 CA112418-01

A radically new approach for the fluorescence in situ detection of DNA is proposed, which makes it possible to detect short (about 20-bp-long) single-copy DNA sequences in metaphase chromosome spreads and in interphase nuclei under non-denaturing conditions. The method of fluorescence in situ detection of short sequences (FISDOSS) to be developed will be exceedingly specific because a circular probe will be assembled via ligation of synthetic oligonucleotides on short DNA sequences opened up by specially designed peptide nucleic acids (PNAs). A high sensitivity will be provided by an efficient contamination-immune isothermal method of signal amplification: rolling-circle amplification (RCA) of the assembled circular probes with incorporation of numerous fluorescently labeled nucleotides. All procedures will be performed directly on slides and the final detection of interphase nuclei and metaphase chromosomes will be done by standard techniques using a fluorescent microscope. In Phase I, proof-of-principle experiments will be performed on arbitrarily chosen sites unique for the human genome. The goal of Phase I is to demonstrate, after initial optimization, that the short specific sequences can be effectively and specifically detected within non-denatured metaphase human chromosomes. The method will be extended to parallel multiple detections of various unique sites in the human genome. To demonstrate that FISDOSS is applicable to detect genetic markers of cancer, 12 appropriate sites associated with chronic lymphocytic leukemia (CLL) will be tested. The implementation of the project will yield a convenient fluorescent in situ technique with a great potential for reliable and highly sensitive diagnosis of cancer on the DNA level.

Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic
1 R21 CA112586-01

Identification of genes and pathways that contribute to tumorigenesis should lead to the defining of novel targets for therapeutic intervention and provide biomarkers for better diagnosis, staging, and risk assessment for individual cancer patients. Further progress in molecular genetics of cancer would greatly benefit from a reliable methodology of assigning gene functions based on phenotypic changes resulting from modulations in gene expression. Existing techniques of this kind are based on screening of genetically modified cells for genetic elements favoring cell growth under restrictive conditions (positive selection). Recently, a novel gene discovery methodology, named selection subtraction approach (SSA), was developed that allowed for a direct negative selection. Although proven useful for isolation of killing or growth suppressive genetic elements, SSA capabilities are limited by the necessity to construct expression libraries. This proposal is focused on developing a new Insertional Selection Subtraction Approach (ISSA) that combines the advantages of SSA with the power of retroviral insertional mutagenesis and is based on a completely new vector system. The insertional mutagenesis arm is enhanced by the addition of a regulatable promoter, splice donor sequences, and the ability to trap polyadenylation signals. The second arm of ISSA involves “tagging” or “bar-coding” each mutant (SSA), thereby allowing monitoring of the relative abundance of mutants within the population during selection by using “retrophage arrays,” the key component of SSA. ISSA technique promises to become a universal functional screening tool, free from major drawbacks of its precursors. After “technical” testing of ISSA, its power will be determined by identification of genes involved in regulation of cell sensitivity to TNF, a well-characterized system that has been already well studied by numerous approaches, including functional selection.

Van Andel Research Institute

1 R21 CA112153-01

Serum markers hold great promise for improving the care and treatment of cancer patients. Although many proteins have serum levels associated with various cancers, each has limited clinical usefulness when measured individually at single time points. The lack of sensitivity and specificity of current serum markers stems from heterogeneity in the baseline levels of the marker proteins and heterogeneity in the tumors and patients. A biomarker discovery strategy that accounts for the heterogeneity in people and tumors is to use individualized thresholds, based on longitudinal measurements, to precisely define abnormal levels for each individual. Previous research has shown that biomarkers defined by longitudinal measurements could have greatly improved specificity and sensitivity over current markers. No systematic study of this topic has been performed, largely because of the lack of a convenient technology for that purpose. A well-developed and validated antibody microarray technology in the laboratory of Dr. Haab now makes this exploration possible. The multiplex detection capability of the antibody microarray will allow us to test the performance improvement upon using longitudinal measurements for many different proteins and to establish the general principles that define the use of longitudinal markers. In addition, the multiplex detection capability ultimately will allow the use of combined longitudinal measurements for even further biomarker performance improvement. To test this strategy, we will evaluate the sensitivity, specificity, and time of the detection of prostate cancer recurrence using both longitudinal and single-time-point measurements of many different prostate cancer-related proteins in serum. This new approach addresses fundamental issues in biomarker research and should result in valuable information for a wide variety of research areas. The successful demonstration of the approach to prostate cancer diagnostics will signal its potential usefulness for all types of biomarker studies.

Chemistry Department, Boston College

1 R21 CA114135-01

This proposal focuses on a novel system for the electrochemical detection of cancer-related protein targets using a nanoscale electrode platform. The assay proposed relies on an electro catalytic process involving two transition-metal ions that reports on biomolecular complexation events. Because the reporter system responds to changes in the electrostatics of an electrode surface, it will enable the analysis of nucleic acids/protein and peptide/protein complexes. The proposed project has 3 specific aims: (1) Electro catalytic detection of DNA repair proteins implicated in cancer using DNA-modified electrodes; (2) Optimization of electro catalytic detection and multiplexed analysis of cellular DNA repair activities; and (3) Development of a generalized electro catalytic protein detection method using prostate cancer biomarkers. The electro catalytic protein detection system described is advantageous because it will allow the analysis and discrimination of multiprotein complexes in addition to uncomplexed analytes and will have high selectivity and specificity. Additionally, the protein detection assays will be conducted using nanoelectrodes that will allow multiplexed detection of panels of different biomolecular targets.

Cancer Research Institute, University of South Alabama

1 R21 CA116070-01

The quote that “aberrant glycosylation is the hallmark of cancer cells” is reflected in numerous reports in the literature documenting changes in glycosylation on specific membrane proteins in cancer cells relative to normal cells. These changes have been shown to be involved in the release of cancer cells into the extracellular matrix and in the formation of metastasis. Glycosylated proteins represent a huge, almost untapped source of biomarkers, considering the wealth of evidence documenting their significance in cancer. Unique glycoforms could be used for diagnostic purposes, to target drugs at cancer cells, and for the development of immunotherapy. Despite the evolution of new mass spectrometry-based methods for protein analysis, few of these involve the determination of post-translational modifications, especially glycosylation. As routine methods (e.g., MS/MS based sequencing methods) yield little light on glycosylated peptides, this proteomics research facility has established a new approach to automatically identify glycan structures on pure proteins from commercial or recombinant sources. It involves the acquisition of molecular weight only spectra and the detection of the glycosylation patterns using accurately determined mass gaps between the various glycoforms. The presence of multiple glycoforms is used to enhance the analysis rather than to confuse it. The approach has been shown to be reliable and extremely fast (taking less than 1 second) at identifying and characterizing such sites, including in proteins with highly complex glycosylation patterns. The aim of this proposal is to prove its utility in cancer where changes in glycosylation are interlaced with the progression of the disease. It will concentrate on both the cell surface proteins and those secreted from cells. Data will be compared to previously published reports where available. The glycans on previously uncharacterized proteins will be established and validated against the best hand-interpreted results. The long-term aim is to make glycoanalyses routine to all cancer investigators, and the software integral to the approach will be made publicly available on a www site. This will represent the first step, with the glycoanalysis of full proteomes being an ultimate objective.

Biochemistry Department, Wake Forest University School of Medicine

1 R21 CA112145-01

Oxidative damage and redox signaling are important components of oncogenic cell transformation, yet the molecular details of how these phenomena impact cellular proteins remain largely uncharacterized. The goal of this proposal is to develop experimental and predictive methods for the identification and molecular analysis of proteins undergoing oxidative modifications at cysteine residues, impacting cancer-related redox signaling pathways in cells. With these efforts, we will be able to apply our new proteomics-based technology to the in situ identification of redox-sensitive signaling proteins in a multi-protein, multi-pathway, whole cell format for the first time. This will allow for investigations to proceed in both discovery- and hypothesis-driven modes to unravel the molecular details of cell signaling pathways responsive to the production of reactive oxygen species (ROS). Multiple studies have highlighted the importance of activated (low pKa) cysteinyl residues in proteins as the primary targets of oxidative modifications, and have shown that hydrogen peroxide is the most important ROS involved in receptor-stimulated cell signaling. The immediate product of peroxide-linked cysteine oxidation is cysteine sulfenic acid (Cys-SOH). Therefore, trapping of Cys-SOH upon its formation in cellular proteins using a detectable reagent will yield a sensitive and comprehensive way of locating redox-responsive cysteinyl residues in cell signaling pathways. To date, no methods exist to trap this species in a manner that is amenable to proteomics type approaches; thus, identification of Cys-SOH containing proteins must be done on an individual, targeted basis. Therefore, we propose four specific aims to (i) Create and validate fluorophore- and biotin-linked reagents reactive toward Cys-SOH based on the known sulfenic acid reagent dimedone; (ii) Develop “redox-profiling” protocols, using dimedone and reagents created in Aim 1, to trap Cys-SOH in proteins as formed within cells and detect the extent and location of Cys-SOH formation in proteins involved in a particular signaling pathway; (iii) Develop “active site profiling” methods using bioinformatics approaches to identify particular cysteinyl residues likely to be targets of redox modifications; and (iv) Validate and apply the experimental and predictive methodologies of Specific Aims 2 and 3 as tools in a total cell protein/proteomics format to analyze protein modification during the course of cancer-relevant cell signaling events. These tools will usher in a new era of redox proteomics and enable proteome-scale studies of effects of oxidative stress and antioxidant therapies on cell pathways and signaling networks.

Bioengineering Department, University of Washington

1 R21 CA114143-01

New technologies for molecular analysis of cancer identify patterns of genetic and protein expression changes that have occurred in tumorigenic cells. Application of these tools for in vivo analysis is critical for a complete understanding of metastatic cancer; sadly, such studies have been limited by the lack of effective methods for delivery to metastases. Nanoparticle formulations of these agents offer in vivo protection and concentrated tumor delivery and are therefore promising delivery entities. However, a major limitation of nanoparticles for tumor delivery is restricted interstitial transport. Here, we propose to harness forces generated by actin polymerization to propel nanoparticles within the interstitial space by energy-mediated, cell-to-cell transfer, thus resulting in more efficient nanoparticle penetration. This goal can be achieved by realizing the following aims: (i) Modifying nanoparticles with ActA, a bacterial protein that initiates actin polymerization resulting in propulsive forces, and optimizing formulations for motility in cytoplasmic extract; (ii) Achieving actin-mediated, cell-to-cell transfer of nanoparticles in cultured monolayer cells; and (iii) Demonstrating improved nanoparticle penetration in three-dimensional spheroid cultures. Efficient delivery systems are crucial for both research and clinical applications; thus, successful completion of this project would result in a major step toward realizing the full potential of molecular analysis, detection, and treatment of cancer.

University of Wisconsin, Madison

1 R33 CA111933-01A1

The proposed aims of this project center on constructing whole genome maps from 30 different oligodendroglioma tumor samples - a solid tumor that has confounded conventional genome analysis approaches to associate loss of heterozygosity (LOH) with a distinct set of gene(s). The research proposed reflects a multidisciplinary collaborative effort to use a robust single molecule platform (Optical Mapping) to construct high-resolution restriction maps from a selected group of characterized tumors bearing a heterogeneous genome population. Chromosomal aberrations will be scored and classified on a whole genome basis in the absence of any hypothesis, outside of the established link between 1p/19q LOH and diagnostic purposes. Genomic aberrations in the tumor samples - deletions, insertions, translocations, tandem amplifications, and gross rearrangements - will be precisely located and characterized. Map coverage of 20-50x will ensure discernment of separate populations of chromosomal aberrations within each sample at 50-500 kb genome intervals. New algorithms will be developed, based on Optical Mapping data, to identify breakpoints within a heterogeneous population of aberrant genomes based on the local alignment of single molecule barcodes, or Optical Maps, with the latest build of the human genome sequence. Aberrations will be statistically assessed to discern the percentage of the tumor cell population bearing a given genomic lesion. To synergize this, a new generation of microfluidic device to incorporate cell lysis and DNA loading within the same disposable silicone fabrication will be perfected. These first-ever whole genome maps of oligodendroglioma tumor genomes and comprehensive determinations of aberrations will be entered into a customized Santa Cruz Genome Browser as additional annotation tracks. This technology provides a unique platform to decipher the complex molecular anatomy of cancer cells, on a whole genome basis, at high resolution.

Department of Biochemistry, Cellular, and Molecular Biology, Johns Hopkins University

1 R21 CA116210-01

Change in genome structure can occur in mitotic and meiotic cell lineages, and this contributes to individual variation, evolution, and disease. It has been argued that stochastic somatic genome instability makes an early and important contribution to the development of human cancer, largely through loss of wild-type tumor suppressor genes. Many tumor suppressor genes themselves are guardians of genome structure and proper cell cycle control, and their loss may cause additional somatic instability. Thus, high levels of genomic instability may be viewed as possible cause and/or effect of steps in tumorigenesis. To distinguish these, a method is needed for continuous monitoring of genome stability in cell lineages that give rise to cancer. Genome stability in vertebrates is currently followed using karyotype analysis, fluorescence in situ hybridization, or measurement of endogenous marker loss using cell selection procedures. At this time, an in vivo system that can be used to determine genome stability in situ (i.e., without tissue disruption) is lacking. We propose the zebrafish Danio rerio as an ideal model system in which to develop this view of vertebrate biology, and we outline a novel method for following marker stability in fish. The method is based on a transcriptional repression design in which repressor loss leads to expression of the fluorescent protein EGFP. Using transgenic zebrafish, we will perform proof-of-concept tests for detection of repressor loss and characterization of repressor loss mechanisms. The zebrafish is ideally suited for development of this strategy due to the ease of organ visualization and its well-developed genetics and genomics tools. Furthermore, the rapid generation time and small size of the zebrafish supports cost-effective observation of many individuals, providing statistical power. In future work, this measurement of genome stability in situ will be important for understanding the relationship between gene function and genome instability in different tissues, and between genome instability and tumor development.

Chemistry Department, Georgia State University

1 R21 CA113917-01

Early detection helps to increase the survival rate in cancer patients. One way to achieve this is the detection and analysis of molecular signatures or biomarkers that have been correlated to cancer development and prognosis. Along this line, there is a need for the development of new technologies for the molecular analysis of various cancer markers. Such spirit is reflected in an RFA (RFA-CA-05-002) requesting applications on developing new “detection technologies and sensors of cancer and the structures and molecules important in its development and diagnosis,” among other things. In response to this RFA, we propose this feasibility study of a new platform technology that can be used for the rapid construction of fluorescent sensors for glycoproteins. We focus on glycoproteins because numerous such proteins have been implicated in cancer development. This method is based upon (1) The power of systematic evolution of ligands by exponential enrichment method (SELEX) in search of optimal oligonucleotide aptamers that can afford high affinity and specificity recognition of the target analytes; (2) The unique ability of boronic acids to recognize diol structures present on the saccharide part of glycoproteins; and (3) Our own development of several fluorescent boronic acid compounds that show very significant fluorescence intensity changes (17- to 200-fold) upon saccharide or glycoprotein binding. We hope to build synergy between the SELEX approach and the unique recognition of glycoprotein by boronic acids in making DNA aptamer-based fluorescent sensors that (1) Have high affinity and specificity for the target glycoprotein and (2) Exhibit very significant fluorescence intensity changes upon binding. Specifically, the project intends to develop a method to prepare DNA aptamers modified with our fluorescent boronic acid reporter compounds. The specific aims of the projects include (1) The synthesis of fluorescent boronic acid compounds that show great fluorescence changes upon binding to saccharides; (2) Incorporation of the fluorescent boronic moieties into nucleotides; (3) Using the SELEX approach for the selection of sensors with optimal specificity and affinity; and (4) Validation of the sensor binding with glycoproteins in solution. For this feasibility study (R21), we have selected prostate-specific antigen (PSA) as our model glycoprotein because of its importance in cancer diagnosis and the fact that glycosylation variations distinguish between physiological and pathological PSA isoforms. Such fluorescent sensors, if developed, offer the advantage of rapid and sensitive detection, the potential for high-throughput screening, and low cost. Furthermore, the same technology, once developed, can also be used for the construction of fluorescent sensors for other cancer-related glycoproteins.

Department of Medicine, Boston University Medical Center

1 R21 CA112228-01A1

A detailed understanding of the genetic basis of neoplastic diseases has emerged during recent decades which has encouraged efforts to develop genetically targeted reagents both as experimental tools and as novel anti-cancer drugs. Peptide nucleic acid (PNA), a DNA mimic in which the phosphate deoxyribose backbone of DNA has been replaced by a pseudopeptide polymer, first described in 1991, has attracted particular interest as a gene-targeting reagent, since it is highly stable and binds to complementary RNA and DNA with high affinity and specificity. However, because PNA resists cellular uptake, its potential usefulness as a tool for modifying gene expression in whole animal studies or as a potential therapeutic agent has been limited. In preliminary studies, we have found that Anthrax toxin “protective antigen” (PA), the component of this microbial toxin that mediates cellular delivery, is able to transport antisense PNA oligomers into cells. To explore the feasibility and potential of using native and modified forms of Anthrax PA as vehicles for delivering PNA into cells for the purpose of altering cancer-related gene expression, we propose studies with two specific aims. First, we will define the kinetics and dose limits of RA-mediated cellular delivery of antisense PNA using PNA oligomers linked to varying polypeptide sequences derived from selected toxin protein functional domains. Stably transfected cell lines engineered to express a luciferase gene interrupted by a mutant p-globin intron-2 (SIVS2-654) with an aberrant splice site that can be blocked by antisense PNA, thereby allowing luciferase expression, will be used to detect antisense activity and effective cellular delivery of PNA. Second, we will determine whether Anthrax PA permits antisense PNA-peptide constructs to alter Bcl-xL gene expression and induce apoptosis in human cancer cell lines (e.g., PCS cells) in vitro . The potential impact of this research is substantial, not only with regard to the development of experimental tools for modulating cancer-related gene expression selectively and combinatorially in cancer cells in vitro and in vivo , but also with regard to the ultimate goal of developing novel agents for cancer treatment that are both genetically targeted and cell selective.

Department of Molecular, Cellular, and Developmental Biology, Yale University

1 R21 CA111993-01

There is strong evidence that gap junctional communication (GJC) is a regulator of cell proliferation and that interruption of this is one of the steps in the malignant transformations of cancer. Gap junctions occur in all animal species and in most tissues from extremely early in development: the eight cell stage in mice, gastrulation in Drosophila and the two-cell stage in nematodes. Yet gap junctions are the cell structures about which the least is known; their role in cell biology and development is still barely explored. Gap junctions are difficult to detect. The standard way to determine whether cells are GJ coupled is to inject dye into one cell and see if it spreads to neighboring cells. in vivo this requires microinjection, which limits the technique to large and unusually accessible cells. We propose to develop a molecular biological method for the in vivo detection of both enduring and transient GJC without the need for intracellular injection. In the simplest version of the technology, transgenic animals will be made with tissue-specific expression of b-galactosidase (b-gal). The intact animal will be injected with a b-gal substrate (e.g., X-gal), which is taken up by cells and is hydrolyzed to a small colored reporter molecule. b-Gal is too large to pass through gap junctions, but the reporter molecule can. Cells expressing b-gal can be detected with antibodies; any cell not expressing b-gal, but filled with the reporter color must have received its color via GJC. The technology will be validated for uniform cells of a single tissue type, different cell types in a complex tissue, in gap junctions made from a variety of GJ proteins, and in heterotypic junctions made from two different GJ proteins. Quantitative measures will be taken of in-animal and across-animal statistical reliability, extent of spread, and spatial and temporal resolution of the method. Aside from b-gal and X-gal as an enzyme-substrate pair, the method will be validated using other b-gal substrates. Another similar method will be tested using tissue-specific expression of transporters to load the presynaptic cells and a detection method for trans-junctional passage. The method will be applied to tumors to assess GJ coupling between tumor cells, between tumor cells and normal cells before and after tumor induction. GJ proteins will be expressed in tumors and the method used to assess GJ coupling after expression and to determine whether tumor growth has been suppressed.

Institute for Systems Biology

1 R21 CA114852-01

Cancers develop over a period of several years and they are characterized by molecular changes prior to invasion and metastasis. Development of a technology that enables screening of cancer from body fluids could permit cancer detection at early and treatable stages. It is expected that the composition of the serum proteome contains valuable information about the state of the human body in health and disease, and that this information can be extracted via quantitative proteomic measurements. Suitable proteomic techniques need to be sensitive, reproducible and robust, to detect potential biomarkers below the level of highly expressed proteins, to generate data sets that are comparable between experiments and laboratories, and have high throughput to support studies with sufficient statistical power. In this proposal, we will develop a method for high-throughput quantitative analysis of serum proteins. It consists of the selective isolation of the peptides that are N-linked glycosylated in the intact protein using solid-phase extraction of glycopeptides (SPEG) on a robotic workstation, the analysis of these now de-glycosylated peptides by liquid chromatography mass spectrometry (LC-MS), and the comparative analysis of the resulting patterns. By focusing selectively on a few formerly N-linked glycopeptides per serum protein, the complexity of the analyte sample is significantly reduced, and the sensitivity, reproducibility, and throughput of serum proteome analysis are increased compared with the analysis of total tryptic peptides from unfractionated samples. We will explore the feasibility to identify cancer-specific serum proteins in the background of normal variation using a carcinogen-induced skin cancer mouse model. The specific aims are (1) To develop chemistries and protocols for an automatic robotic system to isolate N-linked glycopeptides from serum in a high throughput and highly reproducible fashion; (2) To develop efficient and reproducible procedures for LC-MS analyses, and sequence identification of discriminatory peptides by tandem mass spectrometry; and (3) To explore the feasibility of this method for the identification of distinctive serum peptides specific to cancer-bearing mice in the background of normal variations. If successful, the proposed research could subsequently be used for profiling human serum samples from cancer patients and normal individuals to identify the cancer-associated proteins in serum. The identified biomarkers will open a new paradigm for performing screening and detection of human cancer at an early stage and for clinical therapeutic management.