Closing the phenotypic gap between transformed neuronal cell lines in culture and untransformed neurons.
Studies of neuronal dysfunction in the central nervous system (CNS) are frequently limited by the failure of primary neurons to propagate in vitro. Neuronal cell lines can be substituted for primary cells but they often misrepresent normal conditions. We hypothesized that a three-dimensional (3D) cell culture system would drive the phenotype of transformed neurons closer to that of untransformed cells, as has been demonstrated in non-neuronal cell lines. In our studies comparing 3D versus two-dimensional (2D) culture, neuronal SH-SY5Y (SY) cells underwent distinct morphological changes combined with a significant drop in their rate of cell division. Expression of the proto-oncogene N-myc and the RNA-binding protein HuD was decreased in 3D culture as compared to standard 2D conditions. We observed a decline in the anti-apoptotic protein Bcl-2 in 3D culture, coupled with increased expression of the pro-apoptotic proteins Bax and Bak. Moreover, thapsigargin (TG)-induced apoptosis was enhanced in the 3D cells. Microarray analysis demonstrated significantly differing mRNA levels for over 700 genes in the cells of the two culture types, and indicated that alterations in the G1/S cell-cycle progression contributed to the diminished doubling rate in the 3D-cultured SY cells. These results demonstrate that a 3D culture approach narrows the phenotypic gap between neuronal cell lines and primary neurons. The resulting cells may readily be used for in vitro research of neuronal pathogenesis.
Myers
Tereance A
TA
Division of Bacteriology & Parasitology, Tulane National Primate Research Center, 18703 Three Rivers Road, Covington, LA 70433, USA.
Nickerson
Cheryl A
CA
Kaushal
Deepak
D
Ott
C Mark
CM
Höner zu Bentrup
Kerstin
K
Ramamurthy
Rajee
R
Nelman-Gonzalez
Mayra
M
Pierson
Duane L
DL
Philipp
Mario T
MT
D000818:Animals; D017209:Apoptosis / Q000235:genetics; D051017:Apoptosis Regulatory Proteins / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D048430:Cell Shape / Q000502:physiology; D051959:ELAV Proteins / Q000235:genetics; D000067779:ELAV-Like Protein 4; D020869:Gene Expression Profiling; D018816:Genes, cdc / Q000502:physiology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D009924:Organ Culture Techniques / Q000379:methods; D016716:PC12 Cells; D010641:Phenotype; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D051381:Rats
D051017:Apoptosis Regulatory Proteins; D018797:Cell Cycle Proteins; D051959:ELAV Proteins; D000067779:ELAV-Like Protein 4; C498010:ELAVL4 protein, human; D047908:Intracellular Signaling Peptides and Proteins; C000715470:MAS1 protein, human; C099927:NMI protein, human; D000090063:Proto-Oncogene Mas; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger