Pseudomonas aeruginosa bacterial infections are a common problem in immune-deficient patients, such as burn victims, cancer patients, and cystic fibrosis patients. Treating these infections generally involves using an aminoglycosidic antibiotic, such as neomycin, which functions by blocking bacterial protein synthesis. Unfortunately, the range between effective treatment and toxicity is narrow. Patients treated long-term for P. aeruginosa infections often experience renal failure and deafness. The goal of this project is to develop the fruit fly, Drosophila melanogaster, as a model to understand the effects of neomycin treatment and to identify genes whose altered expression can help the fruit fly cope with the side effects of this drug. In order to develop this model, I established a dose-response curve of neomycin concentration to survivorship. I have created a strain that expresses neomycin resistance (neo^R) via the GAL4-UAS system, a transgenic tool that allows Drosophila researchers to control when, where, and what is expressed. Using this system, I will systematically drive this antibiotic resistance gene through various tissues in the flies to determine which tissues are affected by high doses of neomycin treatment. Finally, I will perform a genetic screen using interference RNA to knock down expression of genes potentially involved in aiding aminoglycoside toxicity. Knowledge gained from this type of study may help ameliorate the side effects of aminoglycosides or facilitate development of a drug that targets P. aeruginosa infections with the same specificity but without the long-term side effects.

The proper development of B lymphocytes, or B cells, is essential for the production of infection-fighting antibodies. In this study, we sought to examine the role of the c-Myc transcription factor in B cell development. The Myc family of oncoproteins (c-, N-, and L-Myc) are deregulated in various cancers, yet when properly functioning are essential for embryonic development. Our previous studies indicate that mice deficient in c-and N-Myc have a block in B cell development at the pre-B cell stage, while constitutive expression of c-Myc (Eμ-myc transgene) on certain B cell development-impaired genetic backgrounds can partially rescue development (Habib et al., Journal of Cell Biology 2007). In this study, we sought to determine if constitutive expression of c-Myc could drive B cell development in the μMT^-/- background, a special knockout mouse strain engineered with a deletion of the immunoglobulin heavy-chain (IgH) locus transmembrane region, impairing development of pro-B cells into pre-B cells. Our strategies include harvesting and sorting B220+ B cells from μMT^-/- and EμMyc x μMT^-/- mice bone marrow, and staining them for specific B cell development markers, as well as looking at immunoglobulin light chain (IgL) germline transcription and recombination via RT-PCR on cDNA and DNA, respectively. Our results suggest the expression of the Eμ-myc transgene partially rescues B cell development and proliferation, and promotes IgL transcription in the absence of IgH chain expression. This study further implicates Myc-family members in controlling B cell development at the pre-B cell stage. Defining Myc’s role in B cell development may contribute to developing therapeutics to treat B lymphoid cancers and B cell-mediated autoimmune diseases.

Melanoma is the most lethal form of skin cancer and the seventh most commonly diagnosed cancer in the United States, with increasing incidence in the young population. Currently, there is no effective treatment for metastatic melanoma. Previous studies have shown that DNA damage plays a role in tumorigenesis. The XRCC1 gene encodes a protein required for efficient repair of DNA damage and is an essential element to recruit and activate other proteins, including poly (ADP-ribose) polymerase (PARP), to initiate the repair. The XRCC1L360R point mutation is of interest because it occurs in the critical PARP binding site, and the mutation interferes with proper binding. Because PARP inhibitors are currently being evaluated in clinical cancer studies, an XRCC1L360R gene-targeted mutant mouse line was used to determine melanoma susceptibility. L360R mutant mice implanted with melanoma tumor cells had significantly smaller tumor volume than non-mutant littermates. These findings indicate that the XRCC1L360R point mutation is capable of suppressing tumor progression, possibly through inhibition of PARP activity. We suggest that XRCC1L360R may be a novel therapeutic target for melanoma and possibly other types of cancer, and is of interest for further preclinical investigations.

It has been theorized that mutation rates in the cancer cells are much greater than those in normal cells. Therefore, it is likely that an optimal mutation rate for cellular evolution exists. We hypothesized that moderately higher mutation rates in cells growing in a competitive environment facilitate adaptation and allow them to increase their fitness, a measure of offspring production. To demonstrate this, various strains of Escherichia coli, a Gram-negative bacterium and model organism were used. Most strains harbored a mutated DNA polymerase I (Pol I), an enzyme involved in chromosomal DNA replication, conferring different replication fidelities. As a control, two wild-type strains were competed together in replicate cultures and in the end, both strains won at about the same frequency. Each mutant was then competed in equal ratios against a wild-type strain, again in replicates, in an environment with limiting resources. In most cases, a moderate mutator strain with a mutation rate of 10- to 47-fold higher than wild-type totally overtook the culture. However, since the mutators won most of the time instead of every time, these results suggest that moderate mutators have a greater probability of acquiring advantageous mutations, rather than an initial growth advantage. As we learn more about mutation rate as a determinant of the ability to evolve, we hope to someday find a way to force mutators to lose and therefore decrease their evolutionary fitness. If we can model this in bacteria, the next step would be to utilize these methods in tissue culture and eventually in humans. In the future, this could be a promising route for drug development to target mutator pathways and delay the progression of cancer.

Merkel cell carcinoma (MCC) is a rare but aggressive skin cancer. The newly discovered human Merkel cell polyomavirus (MCPyV) is found in ~80% of MCC tumors but is undetectable in 20%. Genome-wide copy number analysis (n=26) and MCC miR-ome sequencing (n=7) were carried out on both virus-positive and virus-undetectable MCCs. Using high-throughput sequencing, we obtained 28.3 million miRNA sequences and found a striking upregulation of miR-16 in tumors as compared to perilesional skin. High miR-16 supports Rb function loss in both MCPyV-positive and MCPyV-undetectable tumors. In virus-positive tumors, high miR-16 expression is consistent with Rb inactivation via MCPyV large T antigen. In virus-undetectable tumors, high miR-16 expression persists due to focal Rb deletion, which occurred in 9 of 9 MCPyV-undetectable MCCs, but only in 1 of 9 MCPyV-high MCCs (p<0.001). In addition to miR-16 upregulation, we observed high levels of miR-21, an onco-miR that may synergize with MCPyV small t antigen. These cellular miRNA expression results were validated by qPCR. Furthermore, sequencing data revealed a novel MCPyV-encoded miRNA expressed in vivo in a subset of MCC. We detected this MCPyV-miRNA, via a custom qPCR, in 19 of 38 MCPyV-positive tumors but in 0 of 13 MCPyV-undetectable tumors. Different from that reported by Seo et al., a unique seed region allowed us to identify putative human target genes of MCPyV-miRNA. As one of the first viral encoded miRNAs found to express in human cancer, the MCPyV-miRNA enhances current understanding of MCPyV regulation of host genes in MCC. Having two pathways of Rb inactivation, MCPyV-undetectable MCC tumors may not need persistent polyomavirus to disable Rb if Rb deletion is present.