Influence Of HCMV Infection On MCM-loading Factors Cdc6 And Cdt1 In HEL Cells

Objective To demonstrate the influence of HCMV infection onMCM-loading factors Cdc6 and Cdt1 in human embryonic lungfibroblastic(HEL)cells and investigate the pathogenesis of HCMVinfection from molecular level.Method To synchronize the HEL cells in G₀/G₁ by serum starvationand measure cell cycle of HEL cells by flow cytometry analysis.Synchronized HEL cells were infected with HCMV, at the same time themock-infected group was set up as control group. During the time,observation of the pathomorphological change of infected HEL cells wasperformed until 7 days post infection by inverted phase contrastmicroscope. Infected HEL cells were harvested at 12 hours postinfection (h p.i.), 24h p.i., 48h p.i., 72h p.i. and 96h p.i.. To extract thetotal RNA and detect the expressions of Cdc6 mRNA withsemiquantitative reverse transcription-polymerase chainreaction(RT-PCR) technique, Cdt1 protein was detected byimmunocytochemistry method.Results1.The change of morphology was not observed in themock-infected cells. In HCMV-infected group, the partial HEL cellsbecame round, their cytoplasm became large and bulgy at 36h p.i.Cytopathic effect(CPE) was observed in all infected HEL cells at 7daysp.i..2. There were significant difference in expression levels of Cdc6mRNA in two groups (P＜0.05) at 12h p.i., 24h p.i., 48h p.i., 72hp.i. and96hp.i. The expression levels of Cdc6 mRNA in HCMV-infected groupwere obviously higher than the ones in the mock-infected group at 12h p.i., 24h p.i., 48h p.i., but at 72h p.i. and 96hp.i. the expression levels ofCdc6 mRNA in HCMV-infected group were lower than the ones in themock-infected group. In the HCMV-infected group the expression levelsof Cdc6 mRNA reached at a peak at 12h p.i., then decreased gradually,and reached a nadir at 72h p.i., but then returned a little at 96h p.i.; whilein the mock-infected group the expression levels of Cdc6 mRNAreached at a peak at 12h p.i., then decreased gradually, and reached anadir at 48h p.i., but then returned a little at 72h p.i. and continuedincreasing at 96h p.i.3. There were significant difference in expression levels of Cdt1protein in two groups (P＜0.05) at 12h p.i., 24h p.i., 96h p.i., but not at48h p.i., 72h p.i (P＞0.05). Cdt1 protein in HCMV-infected group werestatistically lower than the ones in the mock-infected group at 12hp.i.and, 96h p.i, but at 24h p.i. the expression levels of Cdt1 protein inHCMV-infected group were statistically higher than the ones in themock-infected group. The expression levels of Cdt1 protein reached at apeak at 12h p.i., reached a nadir at 24h p.i., but then returned a little at96h p.i. in the mock-infected group; while the expression levels of Cdt1protein were low at 12h p.i., reached a peak at 24h p.i., and thendecreased sharply at 48h p.i, maintained constantly lower stable level inHCMV-infected.Conclusion1. HCMV can induce the over expressions of Cdc6 mRNA andaccumulation of Cdc6 by the HCMV infection, cell cycle progressionwas affected at last.2. HCMV can induce the expressions delaying of Cdt1, lead toaffect the loading of MCM compounds and the formation of pre-RC. Cellcycle was arrested at G₁ phase, replication of DNA was forbidden.