Effect of serum starvation and chemical inhibitors on cell cycle synchronization of canine dermal fibroblasts.
The cell cycle stage of donor cells and the method of cell cycle synchronization are important factors influencing the success of somatic cell nuclear transfer. In this study, we examined the effects of serum starvation, culture to confluence, and treatment with chemical inhibitors (roscovitine, aphidicolin, and colchicine) on cell cycle characteristics of canine dermal fibroblast cells. The effect of the various methods of cell cycle synchronization was determined by flow cytometry. Short periods of serum starvation (24-72 h) increased (P<0.05) the proportion of cells at the G0/G1 phase (88.4-90.9%) as compared to the control group (73.6%). A similar increase in the percentage of G0/G1 (P<0.05) cells were obtained in the culture to confluency group (91.8%). Treatment with various concentrations of roscovitine did not increase the proportion of G0/G1 cells; conversely, at concentrations of 30 and 45 microM, it increased (P<0.05) the percentage of cells that underwent apoptosis. The use of aphidicolin led to increase percentages of cells at the S phase in a dose-dependent manner, without increasing apoptosis. Colchicine, at a concentration of 0.1 microg/mL, increased the proportion of cells at the G2/M phase (38.5%, P<0.05); conversely, it decreased the proportions of G0/G1 cells (51.4%, P<0.05). Concentrations of colchicines >0.1 microg/mL did not increase the percentage of G2/M phase cells. The effects of chemical inhibitors were fully reversible; their removal led to a rapid progression in the cell cycle. In conclusion, canine dermal fibroblasts were effectively synchronized at various stages of the cell cycle, which could have benefits for somatic cell nuclear transfer in this species.
Khammanit
R
R
Department of Anatomy, Faculty of Veterinary Medicine, Kasetsart University, Bangkok 10900, Thailand.
Chantakru
S
S
Kitiyanant
Y
Y
Saikhun
J
J
D000818:Animals; D016590:Aphidicolin / Q000494:pharmacology; D018929:Cell Culture Techniques / Q000662:veterinary; D002453:Cell Cycle / Q000187:drug effects* / Q000502:physiology*; D002478:Cells, Cultured; D003078:Colchicine / Q000494:pharmacology; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D020405:Dermis / Q000166:cytology*; D004285:Dogs; D005260:Female; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects*; D006131:Growth Inhibitors / Q000494:pharmacology*; D011687:Purines / Q000494:pharmacology; D000077546:Roscovitine
D016895:Culture Media, Serum-Free; D006131:Growth Inhibitors; D011687:Purines; D000077546:Roscovitine; D016590:Aphidicolin; D004247:DNA; D003078:Colchicine