Uptake, cellular distribution and novel cellular binding proteins of immunostimulatory CpG oligodeoxynucleotides in glioblastoma cells.
Glioblastomas are the most malignant and most frequent brain tumors and exciting targets of gene and immunotherapy. Despite rapid development of experimental therapy little is known about the cellular behaviour of therapeutic oligodeoxynucleotides (ODNs). Here we designed uptake, cellular distribution and cellular binding proteins of immunostimulatory CpG-ODNs in glioblastoma cells by flow cytometry, fluorescence microscopy and mass spectrometry. Our data show that the phosphorothioate (PS) CpG-ODNs uptake in T98G and C6 cells is dose-, time-, temperature-dependent and independent of the CpG dinucleotides. Uptake can be inhibited by sodium azide, polyanions but not by chloroquine. After internalisation FITC labelled CpG-ODNs showed a spotted distribution in cytoplasm. Dozens of cellular binding proteins were identified using mass spectrometry. The binding of ODNs to proteins is dependent on modification and sequence but independent on CpG motif. ODNs bind to cellular proteins that are important for RNA processing and transport. Furthermore, three novel membrane proteins were identified, which might contribute to uptake of ODNs. ODNs binding to these proteins might interfere with the physiological function and thus might cause unwanted effects. Such binding also might influence the uptake efficiency or cellular distribution of therapeutic ODNs.
Zhang
Zhiren
Z
Institute of Brain Research, University of Tuebingen, Tuebingen, Germany. zhangzhiren@yahoo.com
Weinschenk
Toni
T
Schluesener
Hermann J
HJ
D000276:Adjuvants, Immunologic / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D001932:Brain Neoplasms / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D005909:Glioblastoma / Q000378:metabolism*; D006801:Humans; D009363:Neoplasm Proteins / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D002352:Carrier Proteins; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides