Quantification of NGF-dependent neuronal differentiation of PC-12 cells by means of neurofilament-L mRNA expression and neuronal outgrowth.
We demonstrate that the degree of neuronal development of PC-12 cell differentiation can be quantified by the expression of neurofilament-L (NF-L) mRNA, when an optimal concentration of NGF (50 ng/ml) is used. During the first 7 days of NGF treatment, the relative amount of NF-L mRNA was found to increase continuously and to correlate with the outgrowth of neurites in a statistically significant way. Thus, mRNA expression is, under these conditions, a suitable means for reliably monitoring the differentiation of PC-12 cells as early as after 3 days of NGF treatment. The results obtained with 5 ng/ml NGF differ from those with 50 ng/ml: during the first 3 days of NGF treatment, neuronal outgrowth was less than with 50 ng/ml, although the NF-L mRNA levels did not depend significantly on NGF concentration. Beyond day 3, NF-L mRNA levels did not increase further at 5 ng/ml as opposed to 50 ng/ml NGF. These differences point to different signal transduction processes involved in neuronal differentiation at high and low NGF concentration. Expression of NF-L protein in response to NGF treatment was also demonstrated. In summary, our results stress that stable and sustained differentiation of PC-12 cells can only be achieved with 50 ng/ml NGF.
Schimmelpfeng
J
J
Forschungszentrum Karlsruhe, Institute for Medical Engineering and Biophysics, P.O. Box 3640, D-76021 Karlsruhe, Germany. schimmelpfeng@imb.fzk.de
Weibezahn
K-F
KF
Dertinger
H
H
D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D049109:Cell Proliferation; D005786:Gene Expression Regulation / Q000502:physiology; D020932:Nerve Growth Factor / Q000494:pharmacology*; D016900:Neurofilament Proteins / Q000096:biosynthesis*; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016716:PC12 Cells; D012333:RNA, Messenger / Q000096:biosynthesis*; D051381:Rats
D016900:Neurofilament Proteins; D012333:RNA, Messenger; C044882:neurofilament protein L; D020932:Nerve Growth Factor