Quantification of DNA synthesis from different pathways in cultured human fibroblasts and myocytes.
We have quantified DNA synthetic rates from different pathways in cultured cells using a new stable isotope technique. Human fibroblasts and myocytes were grown in culture media supplemented with [U-(13)C(6)]glucose and [(15)N]glycine. The cells were sampled daily from day 1 to day 5. A portion of the cells harvested at day 5 was subcultured for an additional 3 passages to reach isotopic plateau. In both cell types total DNA fractional synthetic rate (FSR) was found to agree closely with the rate of cell proliferation determined by cell counting (FSR = 0.94%. h(-1) v 0.92%. h(-1) for DNA synthesis and cell count, respectively, in myocytes and 0.85%. h(-1) v 0.91%. h(-1) for DNA synthesis and cell count, respectively, in fibroblasts). In fibroblasts the deoxyribonucleoside salvage pathway accounted for over 70% of total DNA synthesis. In myocytes the deoxyribonucleoside salvage pathway was minimal, whereas the de novo base synthesis pathway accounted for almost 80% of total DNA synthesis. We conclude that the contributions of various pathways to DNA synthesis are highly dependent on cell type. This new stable isotope technique can be modified for application in in vivo studies.
Martini
Wenjun Z
WZ
Metabolism Unit, Shriners Hospital for Children and Department of Surgery, The University of Texas Medical Branch, Galveston, USA.
Chinkes
David L
DL
Wolfe
Robert R
RR
D002247:Carbon Isotopes; D002452:Cell Count; D002455:Cell Division; D002460:Cell Line; D004247:DNA / Q000096:biosynthesis*; D005347:Fibroblasts / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D032342:Muscle Cells / Q000378:metabolism*; D009587:Nitrogen Isotopes
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D004247:DNA; D005947:Glucose; D005998:Glycine