Objective    To investigate the effects of circadian clock gene Period2 (Per2) on the proliferation

differentiation and apoptosis of K562 cells and its probable molecular mechanism.     Methods    The Per2 expression plasmid pcDNA3.1-Per2 and empty control plasmid were respectively transfected into K562 cells with cationic liposome

and the resistan

t cells stably expressing Per2 gene were obtained by G418 selection. Their morphological changes were observed under light microscope following Wright-Giemsa staining. Trypan blue excluding staining and MTT assay were employed to evaluate cell proliferation. Flow cytometry was performed to analyze cell cycle distribution and cell apoptosis

and electron microscopy was used to detect cell apoptosis. Meanwhile

the expressions of proliferation and apoptosis associated proteins

such as P53

Cyclin B1 and C-Myc

were respectively detected by RT-PCR and Western blot analysis at mRNA and protein level.      Results    The K562/Per2 cell line stably expressing Per2 gene was screened out. As compared with either the empty plasmid transfected group (K562/empty) or the untreated group (K562/untreated)

K562/Per2 cells was smaller in volume and showed no obvious cellular differentiation. Circadian clock gene Per2 could significantly inhibit both growth and proliferation of K562 cells. The percentage of K562 cells in G

2

M phase increased [K562/Per2 group (36.1±5.5)%

K562/empty group (12.5±2.9)%

untreated group (9.7±2.3)%

P0.05

]

and more apoptotic cells were found in the transfected group (14.8%

P0.05). Nuclear condensation

fragmentation

karyopycnosis and apoptotic bodies were found under transmission electron microscope in the K562/Per2 group. P53 was significantly elevated at the mRNA and protein level

while Cyclin B1 and C-Myc were downregulated.     Conclusion    Expression of circadian clock gene Per2 can not only inhibit the growth and proliferation of K562 cells

but  also induce massive apoptosis possibly through its regulation on cell cycle associated genes and subsequent inhibition on cell cycle progression.