This KO8 application is to support a 4 year PhD training program of Dr. Keiko Watanabe in molecular genetics at the University of Illinois at Chicago. During this training, Dr. Watanabe will retain her appointment in the College of Dentistry and will continue to be involved in teaching/patient care for 10% time. The program consists of didactic (10 courses) and research components. The didactic portion will be completed by the end of year 2, at which time an oral qualifying examination is taken. Given that the applicant has already chosen a laboratory and research project, she will initiate the laboratory phase in the beginning of year one. Completion of the research component will culminate in a formal thesis and its defense. LPS is believed to be an important virulence factor in periodontopathogenic bacteria. One of its biological properties is the ability to act as a cytotoxin which directly kills human cells, including periodontal fibroblasts, possibly by activating apoptotic pathways. The goal of this proposal is to identify genetic suppressor elements (GSEs) that can inhibit the sensitivity of human fibroblasts to LPS derived from the putative periodontopathogen, Actinobacillus actinomycetemcomitans. This experimental approach, pioneered in the laboratory of Dr. Roninson, utilizes GSEs, which are short (150-500bp) cDNA fragments which encode dominantly acting peptides or inhibitory antisense RNA. Peptides will interact with, and block intracellular signalling pathways which result in cytotoxicity. Antisense RNAs will prevent the translation of proteins integral to induction of cytotoxicity. GSEs are obtained from a normalized cDNA HeLa cell library which possesses approximately equal representation of both high and low abundance mRNAs. GSEs are expressed within host cells using a retroviral expression vector, and are selected for in the presence of LPS. Those cells which become LPS resistant may contain retrovirus containing a GSE.
The specific aims of this project are (1) to transduce HT1080 fibrosarcoma cells with a normalized retroviral library of random cDNA fragments from HeLa cells.
In aim 2, library-transduced cells will be selected for increased resistance to Aa LPS. Integrated proviruses will be recovered from LPS-selected cells and used to transfer LPS resistance to wild type HT1080 cells (aim 3). These studies will identify an array of GSEs which can induce LPS resistance.
Aim 5 will determine which of the cloned GSEs transduce LPS resistance to human gingival fibroblasts.
Aim 6 will sequence the GSEs inducing LPS resistance, and Aim 7 will seek to identify the genes from which GSEs are derived by homology searches for known genes, and by full-length sequence analysis for unknown genes. The ultimate goal of these studies is to develop novel therapeutic approaches based on increasing the resistance of host cells to the cytotoxic effects of LPS.
| Chang, B D; Watanabe, K; Broude, E V et al. (2000) Effects of p21Waf1/Cip1/Sdi1 on cellular gene expression: implications for carcinogenesis, senescence, and age-related diseases. Proc Natl Acad Sci U S A 97:4291-6 |
