Thioredoxin and Jab1 control estrogen- and antiestrogen-mediated progression of the cell cycle through p27 interactions

A major problem with breast cancer treatment is the prevalence of antiestrogen resistance, be it de novo or acquired after continued use. Many of the underlying mechanisms of antiestrogen resistance are not clear, although estrogen receptor-mediated actions have been identified as a pathway that is blocked by antiestrogens. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are capable of producing reactive oxygen species (ROS) through metabolic activation, and these ROS, at high levels, can induce irreversible growth arrest that is similar to the growth arrest incurred by SERMs. This suggests that SERM-mediated growth arrest may also be through ROS accumulation. Breast cancer receiving long-term antiestrogen treatment appears to adapt to this increased, persistent level of ROS. This, in turn, leads to the disruption of reversible redox signaling that involves redox-sensitive phosphatases and protein kinases and transcription factors. This has downstream consequences for apoptosis, cell cycle progression, and cell metabolism. For this dissertation, we explored if altering the ROS formed by tamoxifen also alters sensitivity of the drug in resistant cells. We explored an association with a thioredoxin/Jab1/p27 pathway, and a possible role of dysregulation of thioredoxin-mediated redox regulation contributing to the development of antiestrogen resistance in breast cancer. We used standard laboratory techniques to perform proteomic assays that showed cell proliferation, protein concentrations, redox states, and protein-protein interactions. We found that increasing thioredoxin reductase levels, and thus increasing the amount of reduced thioredoxin, increased tamoxifen sensitivity in previously resistant cells, as well as altered estrogen and tamoxifen-induced ROS. We also found that decreasing levels of Jab1 protein also increased tamoxifen sensitivity, and that the downstream effects showed a decrease p27 phosphorylation in both cases. We conclude that the chronic use of tamoxifen can lead to an increase in ROS that alters cell signaling and causing cell growth in the presence of tamoxifen, and that this resistant cell growth can be reversed with an alteration to the thioredoxin/Jab1 pathway.

Enhanced Growth of Tetracycline-susceptible Soil Bacteria Treated with Iron and Oxytetracycline

Antibiotic resistance has become an important area of research because of the excessive use of antibiotics in clinical and agricultural settings that are driving the evolution of antibiotic resistant bacteria. However, drug tolerance is a naturally occurring phenomenon in soil communities, and is often linked to those soils that are exposed to heavy metals as well as antibiotics. Resistance to antibiotics maybe coupled with resistance to heavy metals in soil bacteria through efflux pumps that can be regulated by iron. Although considered s a heavy metal, iron is an essential component of life that regulates gene expression through the Ferric Uptake Regulator (Fur) protein. This master regulator protein is known to control siderophore production, and other biological pathways. As a suspected controller of biofilm formation, the role of Fur in environmental antibiotic resistance may be greater than is currently realized. In this study, we sought to explore a potential Fur-regulated drug tolerance pathway by understanding the response of soil bacteria when stressed with oxytetracycline and iron. Bacteria were collected from two locations in Miami Dade County. Isolates were first tested using Kirby-Bauer Disk Diffusion tests for antibiotic resistance/susceptibility and identified by 16S rDNA sequencing. A 96-well growth assay was developed to measure planktonic cell growth with 3 mM FeCl3, Oxytetracycline HCl, and the combination treatments. A Microtiter Dish Biofilm Formation Assay was employed and Fur diversity was evaluated. Tetracycline-susceptible bacterial isolates developed drug resistance with iron supplementation, but iron did not enhance biofilm formation. Development of a Fur-dependent drug resistance may be selected for, but further study is required to evaluate Fur evolution in the studied isolates. Gene expression analysis is also needed to further understand the ecological role of Fur and antibiotic resistance.