title: Efficient nucleofection of primary human B cells and B-CLL cells induces apoptosis, which depends on the microenvironment and on the structure of transfected nucleic acids.
abstract: Accumulation of neoplastic cells in B-cell chronic lymphocytic leukemia (B-CLL) is thought to be due to intrinsic defects in the apoptotic machinery of the leukemic cells or to an altered, survival-stimulating microenvironment in vivo. Despite their long survival in vivo, B-CLL cells undergo rapid spontaneous apoptosis ex vivo. To maintain survival in vitro, we established a coculture system using the human bone marrow-derived stromal cell line HS-5. The microenvironment in these cocultures lead to B-CLL cell survival for at least several months and therefore provided a tool for valid in vitro analysis, mimicking the in vivo situation. Although primary B lymphocytes are notoriously resistant to most gene transfer techniques, we achieved high transfection efficiency and cell viability in this coculture system by using a nucleofection-based strategy. Surprisingly, the introduction of circular plasmid DNA into B cells and B-CLL cells induced rapid apoptosis, which was independent of the type of transgene used, but dependent on the DNA concentration. However, transfection of these cells with mRNA was highly efficient and resulted in sustained cell viability and potent transgene expression. The described procedure represents a new approach to study gene function in primary B cells and B-CLL cells.
authors.affiliation: Department for Molecular Genetics, German Cancer Research Center, Heidelberg, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Seiffert
authors.forename: S
authors.initials: S
authors.lastname: Stilgenbauer
authors.forename: H
authors.initials: H
authors.lastname: Döhner
authors.forename: P
authors.initials: P
authors.lastname: Lichter
mesh_terms: D017209:Apoptosis / Q000502:physiology*; D001402:B-Lymphocytes / Q000166:cytology*; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival / Q000502:physiology; D018920:Coculture Techniques; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000473:pathology*; D009696:Nucleic Acids / Q000235:genetics; D010957:Plasmids / Q000493:pharmacokinetics; D012333:RNA, Messenger / Q000493:pharmacokinetics; D034741:RNA, Small Interfering / Q000493:pharmacokinetics; D017154:Stromal Cells / Q000166:cytology; D014162:Transfection / Q000379:methods*; D019076:Transgenes / Q000502:physiology; D014407:Tumor Cells, Cultured
chemical_list: D009696:Nucleic Acids; D012333:RNA, Messenger; D034741:RNA, Small Interfering