The long-range objective of this research is to establish pharmacological profiles based on individual organelle and single cell analyses as a means of defining subcellular drug distributions. These distributions will contribute to improved drug screening methods, more accurate prediction of the side effects of drug treatment, and ultimately, the development of personalized drug treatments. Doxorubicin, a fluorescent anti-cancer agent, is used as a model. It is hypothesized that the subcellular localization of this drug in a few drug-treated single cells will accurately represent drug localization in the original cell culture. Furthermore, the analysis of individual organelles released from a doxorubicin-treated single cell will reveal differences in subcellular drug distributions and organelle properties that are related to drug resistance and drug sensitivity. Current analysis of subcellular drug distributions is based on confocal microscopy or on chromatographic separation of drug-containing cell extracts. The proposed studies will determine subcellular drug distributions in single cells based on the analysis of individual organelles by capillary electrophoresis with laser-induced fluorescence detection. For the first time, this technique will be used to directly measure the drug content in individual intact organelles. This approach will also reveal an additional biochemical property of the individual organelle that may be involved in doxorubicin subcellular distribution. Such properties include mitochondial membrane potential, lysosomal pH, the presence of P.glycoprotein, and cell cycle.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM061969-01A1
Application #
6327220
Study Section
Special Emphasis Panel (ZRG1-BMT (01))
Program Officer
Okita, Richard T
Project Start
2001-04-01
Project End
2005-03-31
Budget Start
2001-04-01
Budget End
2002-03-31
Support Year
1
Fiscal Year
2001
Total Cost
$245,198
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Chen, Yun; Xiong, Guohua; Arriaga, Edgar A (2007) CE analysis of the acidic organelles of a single cell. Electrophoresis 28:2406-15
Chen, Yun; Arriaga, Edgar A (2006) Individual acidic organelle pH measurements by capillary electrophoresis. Anal Chem 78:820-6
Eder, Angela R; Chen, Jennifer S; Arriaga, Edgar A (2006) Separation of doxorubicin and doxorubicinol by cyclodextrin-modified micellar electrokinetic capillary chromatography. Electrophoresis 27:3263-70
Eder, Angela R; Arriaga, Edgar A (2006) Capillary electrophoresis monitors enhancement in subcellular reactive oxygen species production upon treatment with doxorubicin. Chem Res Toxicol 19:1151-9
Eder, Angela R; Arriaga, Edgar A (2005) Micellar electrokinetic capillary chromatography reveals differences in intracellular metabolism between liposomal and free doxorubicin treatment of human leukemia cells. J Chromatogr B Analyt Technol Biomed Life Sci 829:115-22
Chen, Yun; Walsh, Richard J; Arriaga, Edgar A (2005) Selective determination of the doxorubicin content of individual acidic organelles in impure subcellular fractions. Anal Chem 77:2281-7
Xiong, Guohua; Chen, Yun; Arriaga, Edgar A (2005) Measuring the doxorubicin content of single nuclei by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection. Anal Chem 77:3488-93
Gunasekera, Nilhan; Lee, Sang Won; Kim, Sunghoon et al. (2004) Nuclear localization of aminoacyl-tRNA synthetases using single-cell capillary electrophoresis laser-induced fluorescence analysis. Anal Chem 76:4741-6
Fuller, Kathryn M; Arriaga, Edgar A (2004) Capillary electrophoresis monitors changes in the electrophoretic behavior of mitochondrial preparations. J Chromatogr B Analyt Technol Biomed Life Sci 806:151-9
Anderson, Adrian B; Arriaga, Edgar A (2004) Subcellular metabolite profiles of the parent CCRF-CEM and the derived CEM/C2 cell lines after treatment with doxorubicin. J Chromatogr B Analyt Technol Biomed Life Sci 808:295-302

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