U373-MG cells express PepT2 and accumulate the fluorescently tagged dipeptide-derivative β-Ala-Lys-N(ε)-AMCA.
Aim of this study was to examine the dipeptide transport of β-Ala-Lys-N(ɛ)-AMCA in the human glioma cell line U373-MG and its potential regulation by diverse hormones and culture media. A mixed glial primary cell culture of the newborn rat served as reference cell system. β-Ala-Lys-N(ɛ)-AMCA (β-Ala-Lys-N(ɛ)-7-amino-4-methyl-coumarin-3-acetic acid) is a highly specific reporter substrate to investigate the dipeptide transport system PepT2. We were able to demonstrate that U373-MG cells express PepT2-mRNA and translocate β-Ala-Lys-N(ɛ)-AMCA via PepT2 into the cytoplasm. Previous results demonstrated that β-Ala-Lys-N(ɛ)-AMCA specifically accumulates in differentiated and dedifferentiated astrocytes but neither in differentiated nor dedifferentiated oligodendrocytes and in neurons. U373-MG cells were incubated with estradiol, testosterone, thyronine, dexamethasone, dibutyryl cyclic adenosine monophosphate and tetradecanoylphorbol acetate in order to detect potential substance-dependent changes in dipeptide uptake. There was no significant increase or decrease of β-Ala-Lys-N(ɛ)-AMCA-uptake after stimulation. Northern blot analyses confirmed that PepT2-mRNA is expressed in U373-MG and glial cells but showed no regulation of PepT2-mRNA expression in both cell types. Future investigations might offer the opportunity of an anti-tumor therapy with cytotoxic agents linked to a dipeptide-derivative such as β-Ala-Lys.
Zimmermann
Mathias
M
Zentralinstitut für Laboratoriumsmedizin und Pathobiochemie, Charité-Universitätsmedizin Berlin, Campus Virchow Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany. mathias.zimmermann@charite.de
Kappert
Kai
K
Stan
Alexandru Constantin
AC
D000818:Animals; D001932:Brain Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D018920:Coculture Techniques; D003374:Coumarins / Q000378:metabolism*; D004354:Drug Screening Assays, Antitumor / Q000379:methods / Q000592:standards; D005456:Fluorescent Dyes / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D005910:Glioma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D009842:Oligopeptides / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D027981:Symporters / Q000096:biosynthesis* / Q000235:genetics
D003374:Coumarins; D005456:Fluorescent Dyes; D009842:Oligopeptides; D027981:Symporters; C118033:alanyl-lysyl-amca; C093694:hydrogen-coupled oligopeptide transporter PepT2