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Novel approaches for characterizing the riboflavin transport and trafficking mechanism and its potential as a target in breast cancer

Phelps, Mitch A.
http://rave.ohiolink.edu/etdc/view?acc_num=osu1133261831

Abstract Details

2005, Doctor of Philosophy, Ohio State University, Biophysics.
Transporter interactions may affect a drug’s overall disposition, pharmacodynamic and toxicologic effects. Riboflavin (RF, vitamin B2) is an essential nutrient required for normal growth and development of all known cell types. It is absorbed into cells and tissues through a saturable mechanism and subsequently converted into its cofactor forms, flavin mononucleotide and flavin adenine-dinucleotide, which serve as electron carriers for numerous redox enzymes. The proteins responsible for RF transport remain unidentified in eukaryotes, and only riboflavin kinase (RFK) and riboflavin carrier protein (RCP) are known to bind RF in animals. This work summarizes novel approaches to characterize and identify components of the RF transport mechanism. These approaches include the characterization of a rhodamine-riboflavin conjugate (RD-RF) for following RF transport and trafficking in cell culture. RD-RF exhibits FRET, which demonstrated potential use in live-cell imaging and RF binding assays. Unmodified RF was investigated as a photoaffinity probe for tagging RF transport proteins. Although RF covalently bonded to proteins during photoillumination, reaction rates were slow and led primarily to non-specific labeling. To identify mammalian RCP homologue sequences, a bioinformatics search strategy was employed revealing retbindin as the most likely RCP homologue. However, cloned and expressed human and mouse retbindin did not bind RF and were not recognized by antibodies previously shown to bind mammalian RCP. DNA microarrays and real-time PCR were used to evaluate expression profiles and determine if increased RF transport resulting from RF starvation was due to upreglation of RF transport proteins. Results indicated no genes were significantly up or downregulated. No changes in RF transport or cofactor formation were observed when RFK expression was diminished with interfering RNA, indicating RFK is not directly linked to RF transport. Seven breast cancer cell lines were evaluated for RF uptake and RCP expression. RCP was not detected, but differential uptake of RF was observed. RF pharmacokinetics and tumor distribution was evaluated in xenografted nude mice. Differential uptake of RF in cell culture did not translate into differential tumor absorption in vivo. This work adds to the body of research on RF transport and increases understanding of the complex RF transport mechanism.
James Dalton (Advisor)
riboflavin; transport; FRET; breast cancer; pharmacokinetics; xenograft; photoaffinity; retbindin; siRNA; flavokinase

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Citations

  • Phelps, M. A. (2005). Novel approaches for characterizing the riboflavin transport and trafficking mechanism and its potential as a target in breast cancer [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1133261831

    APA Style (7th edition)

  • Phelps, Mitch. Novel approaches for characterizing the riboflavin transport and trafficking mechanism and its potential as a target in breast cancer. 2005. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1133261831.

    MLA Style (8th edition)

  • Phelps, Mitch. "Novel approaches for characterizing the riboflavin transport and trafficking mechanism and its potential as a target in breast cancer." Doctoral dissertation, Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1133261831

    Chicago Manual of Style (17th edition)

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This open access ETD is published by The Ohio State University and OhioLINK.