Date of Award
Doctor of Philosophy (PhD)
Programmed cell death (PCD), an active process that leads to cell suicide, is a critical mechanism in eukaryotes and prokaryotes. In eukaryotes, apoptosis, a well-characterized form of PCD, is a gene-directed program that can be inhibited by some oncogenic mutations, resulting in the development of cancer. Toxin-antitoxin (TA) is one example of an intracellular death program that is present in almost all bacteria and comprises a stable toxin and a labile antitoxin that blocks the lethal function of the toxin. MazEF, one of the best studied bacterial PCD systems, includes a toxin, namely, MazF that cleaves mRNAs at ACA sequences leading to the inhibition of protein synthesis. Although the exact mechanism remains unknown, it seems the MazF-mediated apoptosis depends on the presence of BAK, a pro-apoptotic protein in mammalian cells. Since cancer is a heterogeneous group of diseases with various sensitivities to different treatments, we examined the impact of MazF proteins on the growth and viability of three cancer cell lines: MCF7, HT29, and AGS. These cells lines were transfected with ACA-less mazF mRNAs, and the cells were evaluated for development of MazF-mediated cell death. Our data illustrated that expression of MazF proteins in the aforementioned cells leads to significant reductions in cell viability. In order to confirm the occurrence of apoptosis, the activation of caspase-3 and -7 and the presence of caspase-3’s substrate were evaluated in transfected cells. Our observations suggested that in the presence of MazF, the levels of activated caspase-3 and -7 were significantly elevated in transfected cells. In addition, the inhibition of protein synthesis by MazF in MCF7 and AGS cell lines were examined via quantifying mRNA translation on a single-cell basis. Our data confirmed that the expression of MazF causes a significant drop in the levels of protein translation in the examined cell lines. Recently, it has been reported that the DNA delivery system does not seem to be a suitable approach to transfer mazF in tumors due to the lack of continuous expression of MazF, degradation, and the incidence of mutations in mazF gene. To address these challenges, we developed an mRNA delivery system to transfer ACA-less mazF mRNA exclusively into cancer cells. To examine the expression and stability of mazF mRNA in cells, we synthesized mazF and GFP mRNA for IRES- or cap- dependent translation. Our results suggest that cap/IRES-dependency, poly-A tails, and modified/unmodified nucleotides played important roles in efficient expression of MazF proteins in transfected cell lines but is dependent on the cell line.
MazF is a nonspecific ribonuclease that can induce death in both normal and cancer cells. Thus, the application of MazF is dependent on efficient expression and delivery system to transfer MazF into tumors. We developed a listerial bi-vector expression and delivery system to transfer mazF mRNA into specific HER2-positive SKBR3 human breast cancer cells. Our results showed that following the infection of cells by functionalized bacterial vectors, caspase activities elevated in SKBR3 cells 48 hours post-transfection. In this research, this expression and delivery system for the delivery of mazF mRNA as a potential therapeutic mRNA in cancer cells was reported for the first time.
Saffarian Abbas Zadeh, Maryam, "Bacteria-Mediated Delivery of mazF mRNA into Cancer Cells for Induction of Apoptosis" (2018). All Dissertations. 2557.