Regulation of p21 and p27 expression by the hepatitis B virus X protein and the alternate initiation site X proteins, AUG2 and AUG3.
BACKGROUND
The hepatitis B virus X gene has three in-frame start codons encoding the pX, AUG2 and AUG3 proteins. The AUG2 and AUG3 genes are 5'-truncated in respect to the full-length pX gene; however, all three genes terminate at the same stop codon. The activity of pX as an oncogene is well characterized; however, less is known about the AUG2 and AUG3 proteins.


METHODS
The effects of pX, AUG2 and AUG3 on p21Cip,1/WAF,1/MDA6 and p27Kip-1 cyclin kinase inhibitor (CKI) protein expression, and the impact they have on proliferation, were investigated in CHO K-1 cells. CHO K-1 cells were chosen because they can be transfected at 100% efficiency.


RESULTS
p21- and p27-luciferase reporter expression is modulated by increasing doses of the hepatitis B X proteins. At low concentrations of pX or AUG2, p21- and p27-luciferase activity was increased, and at high concentrations, p21- and p27-luciferase activity was decreased. Expression of the AUG3 gene showed a different profile: it was increasingly stimulatory with dose for both promoters. Western blot analyses demonstrated that p21 and p27 protein levels were modulated as predicted based on data generated in the promoter-luciferase experiments. Tritiated thymidine labeling of DNA showed biphasic kinetics of incorporation in the presence of varying pX and AUG2 concentrations, whereas labeling decreased with AUG3 concentration. The growth inhibitory effect of pX expression was reduced by antisense ablation of either p21 or p27.


CONCLUSIONS
The relative expression level of pX, AUG2, and AUG3 impacts on CKI expression and cell proliferation. Our findings may explain why divergent effects of pX expression on growth have been observed by different groups, which may be related to relative pX expression levels.
Leach
J Kevin
JK
Departments of Radiation Oncology, Richmond Commonwealth University, Richmond, USA.
Qiao
Liang
L
Fang
Youwen
Y
Han
Song L Y
SL
Gilfor
Dpnna
D
Fisher
Paul B
PB
Grant
Steven
S
Hylemon
Philip B
PB
Peterson
Darrell
D
Dent
Paul
P
D000818:Animals; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000032:analysis* / Q000235:genetics*; D018387:Codon, Initiator / Q000235:genetics; D006224:Cricetinae; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D016213:Cyclins / Q000032:analysis* / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000032:analysis*; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D006509:Hepatitis B / Q000235:genetics*; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D011401:Promoter Regions, Genetic / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics*; D024363:Transcription Initiation Site; D025521:Tumor Suppressor Proteins / Q000032:analysis* / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D018387:Codon, Initiator; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D015534:Trans-Activators; D025521:Tumor Suppressor Proteins; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D050760:Cyclin-Dependent Kinase Inhibitor p27