Dibutyryl cAMP stimulates the proliferation of SH-SY5Y human neuroblastoma cells by up-regulating Skp2 protein.
OBJECTIVE
We previously found that the proliferation of SH-SY5Y neuroblastoma cells is stimulated when cAMP is up-regulated by stable expression of stimulatory G protein. Therefore, this study was performed to investigate the mechanism whereby cAMP stimulates the proliferation of SH-SY5Y cells.


METHODS
To investigate the effect of cAMP on cellular proliferation, SH-SY5Y neuroblastoma cells were treated with dibutyryl cAMP (dbcAMP), and then cell growth, thymidine incorporation and cell cycle phase distribution were analyzed. The expression and the activity of the molecules that regulate cell cycle progression were monitored by Western blot, RT-PCR, and kinase activity assay.


RESULTS
Treatment with dbcAMP produced a biphasic effect on cellular proliferation; especially treatment with low concentration of dbcAMP (0.5 mM) showed a higher cellular proliferation rate and promoted G1/S transition in cell cycle. The dbcAMP (0.5 mM) treatment increased CDK2 activity, and it significantly decreased p27Kip1 expression with a decreased half-life of p27Kip1 protein. Moreover, dbcAMP (0.5 mM) increased the protein level and the stability of Skp2 with a concomitant decrease in its ubiquitination.


CONCLUSIONS
cAMP up-regulates Skp2 protein by reducing its degradation probably through decreasing the ubiquitination of Skp2, which might result in accelerated degradation of p27Kip1, increase in CDK2 activity, and stimulation of SH-SY5Y cell proliferation in sequence.
Cho
Chin-Ho
CH
Department of Biochemistry and Molecular Biology, Laboratory of Cellular Signaling, Cancer Research Institute, Seoul National University College of Medicine, 28 Yongon-dong, Jongno-gu, Seoul, 110-799, South Korea.
Seo
Miran
M
Lee
Yun-Il
YI
Kim
So-Young
SY
Youn
Hong-Duk
HD
Juhnn
Yong-Sung
YS
D003994:Bucladesine / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D016193:G1 Phase; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism*; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D015854:Up-Regulation
D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D016213:Cyclins; D044786:S-Phase Kinase-Associated Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003994:Bucladesine; D051357:Cyclin-Dependent Kinase 2