gms | German Medical Science

Introduction: Tamoxifen (4-hydroxytamoxifen, 4OHT) is used as an activator of CreER recombinase in conditional mouse systems. Through this approach the possibility of a tissue and time dependent activation of the Cre recombinase is made possible. We used this approach in an in vitro culture system of primary neural stem and progenitor cells (NSC/NPC). In addition to the expected recombination of the conditional knock-out and knock-in alleles however we observed significant cytotoxicity of 4OHT on NSC/NPC.

Objectives: We are currently analyzing the effects of 4OHT on NSC/NPC and on HEK293 cells to better understand the non-estrogen receptor mediated effects of tamoxifen.

Materials and Methods: We are using primary NSC/NPC cultures derived from P0-P2 mice brains as well as HEK293 cells. For dose-response curves we performed MTT assays. Type of cell death was analyzed via flow cytometry measurements, western blots analysis and CaspaseGlo^® 3/7 assays. Alterations on RNA-level were detected by microarray analysis and verified via qRT-PCR. To identify possibly involved signaling pathways we are currently performing a high throughput kinase inhibitor screen.

Results: 24 h treatment of NSC/NPC with 0.5 µM 4OHT resulted in cytotoxicity. Western blot analyses of LC3 I/II protein levels, AnnexinV/7-AAD stainings and caspase 3/7 assays suggested a non-apoptotic autophagic cell death upon 4OHT treatment. Similar results were observed in HEK293 cells. Microarray data suggest an involvement of Socs2 in mediating autophagic cell death.

Conclusions: Our findings indicate that the use of tamoxifen-mediated activation of CreER recombinase in conditional knock-out and knock-in mouse systems has limitations in culture systems of NSC/NPC as tamoxifen treatment at concentrations required for CreER activation induces cytotoxicity in this culture system. We are currently further identifying the mechanisms underlying this cytotoxicity and whether these observations also have implications for in vivo analyses of conditional mouse models.