Glutamine synthetase is essential for proliferation of fetal skin fibroblasts
Section snippets
Skin fibroblasts
Cultured skin fibroblasts were obtained from an aborted fetus after prenatal diagnosis by molecular genetic means, which had revealed a homozygous mutation (R324C) of the GS gene. The index patient of this family suffered from congenital GS deficiency and has been reported earlier [9]. As a result of the prenatal diagnosis, the gestation was terminated at 16+4 weeks of gestational age and a fetal skin biopsy was taken. The skin biopsy was initially cultured in standard medium containing a
Mutation analysis
Sequencing of the coding exons of the GS gene confirmed the homozygous mutation c.970C>T (R324C) in the fetal GS− skin fibroblasts. Fetal and mature control cells displayed wild type sequences in the GS gene.
Level of GS-expression in cells cultured in standard medium
When fibroblasts were cultured in standard medium containing 2 mM glutamine, Western blot analysis revealed a strong upregulation of GS in the fetal GS− fibroblasts, compared to the GS+ and GS control cells (Fig. 1a).
For analysis of the results of the RT-PCR the calculated relative
Discussion
There is only scarce knowledge on the role of GS during fetal development. We took advantage on the availability of cultured homozygous GS mutant fetal skin fibroblasts and determined their proliferation and viability both in the presence and absence of the GS inhibitor MSO and under the influence of different concentrations of extracellular glutamine.
GS− cells exhibited a significant upregulation of the GS protein, which is in accordance with the findings in immortalized lymphocytes from other
Acknowledgments
The authors are grateful to the technical help of I. Neumann, M. Grüneberg, M. Steinert, T. Janssen, and J. Beckstedde. The work has been supported in part by a grant from the Deutsche Forschungsgemeinschaft to J.H. (HA 4376/1-2) and by the Sonderforschungsbereich 575 “Experimental Hepatology”, Düsseldorf.
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