The Nuclear Export of DNA Topoisomerase IIα in Hematological Myeloma Cell Lines as a Function of Drug Sensitivity: Clinical Implications and a Theoretical Approach for Overcoming the Observed Drug Resistance
Degree Granting Department
Biochemistry and Molecular Biology
Daniel M. Sullivan, M.D.
Duane C. Eichler, Ph.D
Kenneth Wright, Ph.D
Edward Seto, Ph.D
Srikumar Chellappan, Ph.D
Etoposide, Mitoxantrone, Leptomycin B, Protein trafficking, Cancer
The focus of this investigation is about DNA topoisomerases, the molecular targets of clinically important chemotherapy, and mechanisms of drug resistance in human myeloma and leukemia cell lines. The ultimate goal of this investigation was to identify mechanism(s) of drug resistance to anticancer agents so that a strategy to overcome drug resistance could be conceived. We established an in vitro cell model by using human leukemia and myeloma cell lines to investigate possible mechanisms of drug resistance that are observed in confluent cells. Plateau cell densities demonstrated de novo drug resistance to commonly used chemotherapeutic agents that was independent of altered drug transport. We established that cellular drug resistance in these cells is a function of topo IIα subcellular localization and further demonstrate that topo IIα translocates to the cytoplasm in a cell-density dependent manner. We provide experimental data that supports the nuclear export of topo IIα as the most likely event contributing to drug resistance to topoisomerase II inhibitors, which occurs when transformed cells transition from log to plateau cell density. We provided a plausible nuclear export pathway for topo IIα, by identifying two Leptomycin B sensitive nuclear export signals, which are homologous to the binding sites recognized by the nuclear export receptor, exportin-1. Thus, topo IIα is likely to be exported from the nucleus at plateau cell densities when exportin-1 binds topo IIα. We confirmed that the nuclear export signals identified in topo IIα are functional when expressed in human myeloma cells transfected with an epitope-tagged topo IIα gene. Furthermore we demonstrate that the nuclear export signals can be abolished by sitedirected mutagenesis of specific amino acids residues found in the nuclear export signal. Our data may have clinical relevance because plasma cells obtained from bone marrow aspirates of patients with multiple myeloma contain a cytoplasmic distribution of topo IIα. The potential implications of a functioning nuclear enzyme located in the cytoplasm of cells and theoretical mechanisms for overcoming the observed drug resistance are considered.
Scholar Commons Citation
Engel, Roxane, "The Nuclear Export of DNA Topoisomerase IIα in Hematological Myeloma Cell Lines as a Function of Drug Sensitivity: Clinical Implications and a Theoretical Approach for Overcoming the Observed Drug Resistance" (2005). Graduate Theses and Dissertations.