SNAI2 as a novel radioprotector of normal tissue by gene transfer using a lentiviral bicistronic SIN vector.
Tumor radiotherapy with large-field irradiation results in an increase of p53-dependent apoptosis of the radiosensitive hematopoietic stem cells. Proapoptotic PUMA is a transcriptional target of p53. Thus suppression of PUMA expression by gene therapy with the transcription repressor SNAI2 as transgene might be a potential approach for normal tissue protection during radiotherapy. SNAI2 cDNA was cloned in a lentiviral SIN vector in a bicistronic expression cassette followed by a floxed IRES-EMCV linker and EGFP as selection gene. Wild-type p53 TK6 cells were used as the cellular model system. We could demonstrate the significant radioprotective effect of SNAI2 overexpression in a cytotoxicity assay after irradiation with 0-5 Gy compared with untransduced or control vector (inverse oriented SNAI2 cDNA)-transduced cells. Additionally, TK6-SNAI2 compared to TK6-SNAI2inv cells showed a survival advantage in a clonogenic assay after irradiation with 0-3 Gy. Determination of the proportion of sub-G(1) cells in TK6-SNAI2 cells revealed an approximately 50% reduction in apoptosis compared with both control entities. In this study using a bicistronic lentiviral vector, we were able to provide proof of principle that lentiviral overexpression of SNAI2 might be used for radioprotective gene therapy to widen the therapeutic range in radiotherapy.
Maier
Patrick
P
Department of Radiation Oncology, Mannheim Medical Center, University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim, Germany. patrick.maier@medma.uni-heidelberg.de
Herskind
Carsten
C
Barzan
David
D
Zeller
W Jens
WJ
Wenz
Frederik
F
D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation; D017931:DNA Primers; D018076:DNA, Complementary; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D011832:Radiation Injuries / Q000517:prevention & control; D011836:Radiation Tolerance / Q000502:physiology*; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; D017931:DNA Primers; D018076:DNA, Complementary; D011518:Proto-Oncogene Proteins; C000604408:SNAI2 protein, human; D000071250:Snail Family Transcription Factors; D014157:Transcription Factors; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins