PepT2 transporter protein expression in human neoplastic glial cells and mediation of fluorescently tagged dipeptide derivative beta-Ala-Lys-Nepsilon-7-amino-4-methyl-coumarin-3-acetic acid accumulation

• Published in: Journal of neurosurgery Vol. 112; no. 5; p. 1005
• Main Authors: Zimmermann, Mathias, Stan, Alexandru Constantin
• Format: Journal Article
• Language: English
• Published: United States 01.05.2010
• Subjects: Acetic Acid - metabolism; Animals; Astrocytes - metabolism; Astrocytes - pathology; Astrocytoma - metabolism; Astrocytoma - pathology; Blotting, Northern; Brain Chemistry; Carrier Proteins - biosynthesis; Carrier Proteins - metabolism; Cell Line, Tumor - metabolism; Cell Line, Tumor - pathology; Coumarins - chemical synthesis; Coumarins - metabolism; Dipeptidases - genetics; Fluorescence; Immunohistochemistry; Oligopeptides - chemical synthesis; Oligopeptides - metabolism; Rats; RNA, Messenger - metabolism; Symporters - genetics
• ISSN: 1933-0693
• DOI: 10.3171/2009.6.JNS08346

The present study was aimed at analyzing the accumulation of the fluorescently tagged dipeptide derivative, beta-Ala-Lys-N(epsilon)-7-amino-4-methyl coumarin-3-acetic acid (AMCA), in primary cultures of human neoplastic glial cells. This molecule is a highly specific reporter used to investigate the dipeptide transport system hPepT2. In this study the authors used immunocytochemical methods to determine the cell-specific accumulation of a small and fluorescently tagged reporter molecule named beta-Ala-Lys-N(epsilon)-AMCA to detect dipeptide transport capacity of neoplastic glial cells. Furthermore, specific mRNA levels were quantified using Northern blot analysis and the tissue distribution of hPepT2 mRNA transcripts was demonstrated with in-situ hybridization histochemical analysis. Recent fluorescent immunocytochemical analyses have revealed that beta-Ala-Lys-N(epsilon)-AMCA specifically accumulates within anaplastic cells of astrocytic lineage but not in anaplastic oligodendrocytes or neurons. Northern blot analysis demonstrated that human hPepT2 mRNA is specifically detected in primary cell cultures of human glioblastoma but not in oligodendroglioma. Moreover, in situ hybridization analyses revealed an astrocytic localization of hPepT2 transcripts in human glioblastoma and astrocytoma cells. The hPepT2 transcription levels were clearly dependent on the grade of glial cell differentiation: within low-grade gliomas (WHO Grade II), more hPepT2 mRNA was detected compared with tumors of a higher grade of dedifferentiation (WHO Grade IV). Analysis of expression levels of hPepT2 mRNA in human neoplastic glial cells xenografted into the brains of athymic rats (han rnu(+/+)) showed a markedly increased expression of hPepT2 after 2 weeks of growth in vivo compared with the primary counterparts grown in vitro. The authors concluded that expression of the hPepT2 transporter protein is a characteristic of glial cells of astrocytic lineage, and is dependent on the grade of astroglial cell differentiation and the extracellular matrix (here brain neuropil). The authors found that beta-Ala-Lys-N(epsilon)-AMCA is as an excellent reporter molecule for assessing neoplastic glial cell function and physiological characteristics.