You have accessJournal of UrologyStem Cell Research: Stem Cell Research II1 Apr 2018MP81-15 PROSTATE SMALL CELL CARCINOMA EXPRESS STEM CELL FACTORS THAT CAN TRANSFORM NORMAL AND CANCER CELLS INTO STEM-LIKE Adelle Kanan and Alvin Liu Adelle KananAdelle Kanan More articles by this author and Alvin LiuAlvin Liu More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2723AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer can be grouped into either luminal-like (differentiated) or stem-like (undifferentiated). The latter type includes small cell carcinoma. The LuCaP 145.1 small cell carcinoma xenograft line shows expression of stem cell transcription factors LIN28A, NANOG, POU5F1 and SOX2. Luminal-like adenocarcinoma LuCaP lines show expression of only one or two of these factors, while cancer cells of Gleason patterns 3 and 4 do not. Forced expression of these four genes in differentiated somatic cells produces induced pluripotent stem (iPS) cells. We want to determine if other LuCaP small cell carcinoma xenografts also express these genes, and whether these genes cloned from LuCaP 145.1 can also transform cell types, both normal and cancer, into stem-like. METHODS LuCaP small cell carcinoma harvested from mice were minced and digested by collagenase. The resultant cells were centrifuged on Percoll density gradients. Gene expression was analyzed by RT-PCR. Full-length LIN28A, NANOG, POU5F1 and SOX2 cDNA (Kozak box, ATG start to stop codon) were cloned from LuCaP 145.1 into mammalian expression vector pVITRO1-neo, and transfected by electroporation into HEK293F fibroblasts and luminal-like prostate cancer cells LNCaP and C4-2B. Individual colonies under drug G418 selection (1 mg/ml) were cloned, and analyzed for gene expression. Transfected cells were grown 1) in media supplemented with 10% fetal bovine serum under normoxia, 2) in serum-free media with KnockOut Serum Replacement, 3) on Matrigel-coated plates, 4) on irradiated mouse embryonic fibroblasts (MEF) under hypoxia. RESULTS The four stem cell factor genes were expressed by all established LuCaP small cell carcinoma examined: LuCaP 93, 145.1, 145.2, 173.1, 173.2A. LuCaP 145.1 cells also showed a stromal cell density (1.035). Transfection of plasmid pLP2 containing LIN28A/POU5F1 and pSN4 containing SOX2/NANOG into HEK293F, LNCaP and C4-2 produced cells displaying a stem cell-like colony morphology. Neo (G418)-resistant cells appeared in ~ 1 week, and showed expression of the stem cell factor genes as well as low expression of B2M (β 2-microglobulin) a characteristic of stem cells. The colony morphology was distinct from that of the untransfected cells, and showed differences among the culture conditions used. CONCLUSIONS Transcription factors cloned from small cell carcinoma are capable of transforming normal and cancer cells. The generated plasmid vectors are safer to use than lentiviral vectors; G418 allows selection of transfected cells, and deters microbial contamination. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1102 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Adelle Kanan More articles by this author Alvin Liu More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...