Graduation Year

2005

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biochemistry and Molecular Biology

Major Professor

Daniel M. Sullivan.

Keywords

Etoposide, Mitoxantrone, Leptomycin B, Protein trafficking, Cancer

Abstract

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 IIalpha subcellular localization and further demonstrate that topo IIalpha translocates to the cytoplasm in a cell-density dependent manner.

We provide experimental data that supports the nuclear export of topo IIalpha 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 IIalpha, 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 IIalpha is likely to be exported from the nucleus at plateau cell densities when exportin-1 binds topo IIalpha. We confirmed that the nuclear export signals identified in topo IIalpha are functional when expressed in human myeloma cells transfected with an epitope-tagged topo IIalpha gene. Furthermore we demonstrate that the nuclear export signals can be abolished by site-directed 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 IIalpha. 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.

Share

COinS