Synchronization of normal human keratinocyte in culture: its application to the analysis of 1,25-dihydroxyvitamin D3 effects on cell cycle.
In this report, we applied the isoleucine-deprivation method to normal human keratinocytes in cultivation with serum-free MCDB153 medium. The growing cells were synchronized by transfer to MCDB153 medium prepared without isoleucine, and the degree of synchronization was analyzed by the cell cycle distribution and phosphorylation of retinoblastoma protein (pRB), one of the tumor suppressor gene products. 1,25-dihidroxyvitamin D3 (1,25(OH)2D3), a biologically active form of vitamin D3, is supposed to induce the differentiation and growth inhibition of human keratinocytes caused by cell cycle arrest. We examined its effect on cell cycle kinetics, especially progression from G1/G0 to S-phase, by using this synchronized system. We showed that 10(-6) M 1,25(OH)2D3 inhibited the progression from G1/G0 to S-phase strikingly in synchronized normal human keratinocytes.
Department of Dermatology, Osaka University School of Medicine, Japan.
T
T
Kobayashi
H
H
Okumura
K
K
Hashimoto
H
H
Asada
S
S
Inui
K
K
Yoshikawa
D015152:Blotting, Northern; D002117:Calcitriol / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D007532:Isoleucine / Q000172:deficiency; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D016192:Resting Phase, Cell Cycle / Q000187:drug effects; D016160:Retinoblastoma Protein / Q000378:metabolism; D016196:S Phase / Q000187:drug effects
D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D016160:Retinoblastoma Protein; D007532:Isoleucine; D002117:Calcitriol