John F. McDonald - US grants

The Drosophila gypsy retrovirus will be used as a model system for the study of the processes underlying retrovirus regulatory evolution. Regional duplications within the cis-regulatory regions located in the long terminal repeat (LTR) and/or translated leader region (ULR) is a characteristic feature of retrovirus regulatory evolution. Gypsy elements within Drosphila melanogaster popluations are in the process of evolving new regulatory sequences which provides a unique opportunity to study retroviral regulatory evolution in progress. Such cis-regulatory changes seem to be an adaptive response to host (chromatin dependent) silencing mechanisms. We proposed to utilize the gypsy model to investigate the molecular and population genetic principles underlying retrovirus regulatory evolution.

Integrins initiate signaling pathways that impact tissue organization, inflammation, blood clotting, oncogenesis and angiogenesis upon binding of extracellular matrix proteins, in the regulation of biological processes. In this proposal, the role of integrin-associated protein (IAP, CD47), a co-receptor in mediating integrin signaling, will be studied by manipulation of both the receptor apparatus and the ligand structure. The C- terminal cell-binding domain (CBD) of thrombospondin (TS) is a ligand of IAP, and peptides derived from the CBD stimulate integrin-dependent processes via heterotrimeric G proteins. To define the mechanism of G protein coupling to the integrin-IAP complex, 1) a model expression system will be established for integrins, IAP and G protein subunits to evaluate receptor function in cellular assays upon treatment with IAP agonists; 2) direct measurements of G protein coupling will be carried out in the transfected cells and correlated with functional assays to identify the role of each component in the signaling complex; and 3) expression and mutagenesis of TS and CBD will be carried out to identify the determinants for IAP activation. By correlating changes in the interactions of the receptor and G protein components with downstream or "read-out" cellular functions, these studies should begin to define the molecular basis for the role of IAP in integrin-regulated biological processes such as angiogenesis, tumorigenesis and wound healing.

The Drosophila gypsy retrovirus will be used as a model system for the study of the processes underlying retrovirus regulatory evolution. Regional duplications within the cis-regulatory regions located in the long terminal repeat (LTR) and/or translated leader region (ULR) is a characteristic feature of retrovirus regulatory evolution. Gypsy elements within Drosphila melanogaster popluations are in the process of evolving new regulatory sequences which provides a unique opportunity to study retroviral regulatory evolution in progress. Such cis-regulatory changes seem to be an adaptive response to host (chromatin dependent) silencing mechanisms. We proposed to utilize the gypsy model to investigate the molecular and population genetic principles underlying retrovirus regulatory evolution.

DESCRIPTION (provided by applicant): Ovarian cancer is the leading cause of death from gynecologic cancer. Despite high initial tumor response rates of 80% to surgical debulking and chemotherapy, most women with advanced ovarian cancer will eventually develop drug-resistant disease. Because second-line chemotherapeutic agents provide a response rate of only 15-25%, there is clearly a need to develop better therapeutic strategies. Small interfering RNAs (siRNAs) are a class of RNA molecules that previously have been demonstrated to be highly effective in inactivating cancer-causing genes (oncogenes) in cancer cells grown in culture. However, the development of siRNAs as clinically significant therapeutic agents has been hampered by the fact that siRNAs are extremely unstable at physiological conditions. In addition, there has been no effective way to target these potentially therapeutic molecules specifically to cancer cells. We have recently demonstrated that gelatinous-like nanoparticles called nanogels can be easily loaded with siRNAs and targeted to deliver their potentially therapeutic payload to ovarian cancer cells grown in culture. Nanogels protect the siRNA molecules from degradation until released into the cancer cells. The proposed studies seek to demonstrate that these non-toxic nanogels can be used to effectively target siRNAs to cancer cells in living organisms (experimental mice) as well. Such studies are pre-requisite to the potential clinical application of the technology for the treatment of cancer in humans. Specifically, siRNAs directed against the EGFR (epidermal growth factor receptor) gene will be loaded into nanogels engineered to specifically target proteins expressed on the surface of ovarian cancer cells. The EGFR gene is highly expressed in ovarian and most other types of cancer cells and is known to induce cells to rapidly divide and become resistant to most commonly used chemotherapy agents. Thus, the inactivation of EGFR in cancer cells has great therapeutic value. Demonstration that nanogels can be an effective vehicle for the delivery of therapeutic siRNAs to cancer cells in mice will open the door to the development of this exciting new technology of clinical application in humans. PUBLIC HEALTH RELEVANCE: The ability to deliver siRNA against oncogenes specifically targeted to cancer cells will be a major contribution to the treatment of cancer. Development of a mouse model in which to demonstrate and refine this targeted delivery nanotechnology is a key step in moving the technology into clinical practice.

? DESCRIPTION (provided by applicant): The examination of cancer cells collected from effusions and ascites is an important step to identify or determine the progress of cancer by pathologists or in preparation of performing analytical techniques by cancer scientists. However this examination is complicated due to the presence of many noncancerous cells, including mesothelial, macrophage, and lymphocyte cells. Standard preparation techniques such as centrifugation can be utilized to increase the density of total cells in the sample, but further purification of the collected sample to highly enrich the cancer cells is troublesome and expensive, typically requiring laborious processing and expensive labeling reagents such as antibodies. As such, enriching cancer cells from ascites is rarely performed in the clinic and infrequently in the research laboratory. The objective of this research proposal is to test a label free microfluidic cell sorting technology that utilizes differences in cell stiffness and cell sizeto enrich cancer cells from noncancerous cells present in ascites fluid. The technology utilizes the observed fact that the cancer cells frequently undergo biophysical and morphological changes to be distinct from other noncancerous cell types. The technology consists of microchannels with periodic constrictions that are designed to direct soft cells along a different path than stif cells. We will examine how variations in channel geometry and other system parameters can be harnessed to provoke cancer cell segregation through inherent biophysical differences. A high throughput and label-free method to purify cancer cells from noncancerous cells will greatly benefit the accuracy and efficiency of both cancer science and clinical diagnoses. Successful completion of this project will create a new enrichment method to facilitate the detection of very low levels of diseased cells in complex fluids or to enhance the purity of cancer cell subpopulations within heterogeneous mixtures, including effusions and ascites fluid.