Expression of cyclins D1, D2 and E correlates with proliferation of rat stellate cells in culture.
OBJECTIVE
Regulation of cell cycle progression of cultured rat stellate cells was studied.


METHODS
DNA synthesis was determined by the uptake of [3H]thymidine or 5-bromo-2'-deoxyuridine. Cell cycle distribution was analyzed using FACScan of cellular DNA stained with propidium iodide. Expression of cyclins and cyclin-dependent kinase 4 was evaluated by Western and Northern blotting.


RESULTS
DNA synthesis of primary-cultured stellate cells was found to accelerate 48 h after plating. Cell cycle analysis revealed that more than 93% of the cells were in G0/G1 phase during the first 48 h after plating. The cell population in S phase abruptly increased to about 16% 72 h after culture and shifted to G2/M phase thereafter. The level of proteins and mRNAs for cyclins D1, D2 and E started to increase 48 h after culture with a concomitant expression of platelet-derived growth factor receptor beta, while the level of cyclin-dependent kinase 4 and its mRNA remained unchanged. On the other hand, stellate cells remained in G1 phase when they were cultured in the presence of 3-isobutyl-1-methylxanthine or dibutyryl cAMP after plating. Attenuation of the expression of cyclins D1, D2 and E and platelet-derived growth factor receptor beta, but not cyclin-dependent kinase 4 was found in stellate cells cultured with these agents. Further analysis revealed that LY294002, a selective inhibitor of phosphatidylinositol 3-kinase, suppressed DNA synthesis and cyclin D1 expression in a dose-dependent manner without affecting platelet-derived growth factor receptor beta expression.


CONCLUSIONS
Induction of G1 cyclins may play crucial roles in cell cycle transition of cultured stellate cells from G1 to S. Expression of platelet-derived growth factor receptor beta and activation of phosphatidylinositol 3-kinase may be involved in the process.
Kawada
N
N
Department of Internal Medicine, Osaka City University Medical School, Osaka, Japan. kawada-nori@mve.biglobe.ne.jp
Ikeda
K
K
Seki
S
S
Kuroki
T
T
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D003994:Bucladesine / Q000494:pharmacology; D002453:Cell Cycle; D002455:Cell Division; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D019938:Cyclin D1 / Q000032:analysis* / Q000235:genetics; D056742:Cyclin D2; D019927:Cyclin E / Q000032:analysis* / Q000235:genetics; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000032:analysis / Q000235:genetics; D016213:Cyclins / Q000032:analysis* / Q000235:genetics; D004247:DNA / Q000096:biosynthesis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D016193:G1 Phase; D007093:Imidazoles; D008099:Liver / Q000166:cytology*; D008297:Male; D009025:Morpholines / Q000494:pharmacology; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011725:Pyridines; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D017479:Receptors, Platelet-Derived Growth Factor / Q000032:analysis; D016196:S Phase; D013936:Thymidine / Q000378:metabolism; D014316:Tritium
C486197:Ccnd2 protein, rat; D002867:Chromones; D056742:Cyclin D2; D019927:Cyclin E; D016213:Cyclins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D011725:Pyridines; D012333:RNA, Messenger; D014316:Tritium; D019938:Cyclin D1; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D003994:Bucladesine; D004247:DNA; D017479:Receptors, Platelet-Derived Growth Factor; C495903:Cdk4 protein, rat; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D001973:Bromodeoxyuridine; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one; D015056:1-Methyl-3-isobutylxanthine; D013936:Thymidine