Detection of a tyrosine-phosphorylated form of cyclin A during liver regeneration.
Cyclin A functions in both the S and G2-M phases of the cell cycle. The expression of cyclin A during liver regeneration was compared with that of cyclin B1 and p34cdc2. Liver regeneration was followed at 2-h intervals from 12 to 48 h after partial hepatectomy (PH). Immunohistochemical staining using proliferating cell nuclear antigen revealed DNA synthesis peaks at 18 h after PH. The most intense nuclear staining of hepatocytes with cyclins A and B1 and p34cdc2 antibodies occurred at 26 h post-PH, which corresponds with the onset of mitosis. Quantitative mRNA expression of cyclins A and B1 and p34cdc2 was determined by competitive reverse transcription-PCR. Construction of mRNA internal standards and coamplification during reverse transcription-PCR allowed quantitation of all three cell cycle genes. At 24 h post-PH, cyclin A mRNA levels were approximately 5 fg/100 ng total RNA. In contrast, cyclin B1 and p34cdc2 levels were 20-fold higher, 100 fg/100 ng total RNA. Cyclin B1 and p34cdc2 mRNA levels showed two peaks, at 26 and 38-44 h post-PH, whereas the levels of cyclin A were constant during this interval. Immunoblots revealed the presence of cyclin A in normal liver, and significant amounts were present as early as 12 h post-PH. At 26 h post-PH, tyrosine-phosphorylated forms of cyclin A were detected. Cyclin B1 and p34cdc2 protein were not present until 22-24 h post-PH, and two peaks were observed, at 26 and 38-44 h, coinciding with the mRNA pattern. Histone H1 kinase activity was associated with the two peaks of cyclin B1 and p34cdc2 expression. The unique pattern of cyclin A expression and detection of tyrosine-phosphorylated forms suggest a different mechanism for the regulation of cyclin A during liver regeneration.
Spiewak Rinaudo
J A
JA
Laboratory of Experimental Carcinogenesis, National Cancer Institute, NIH, Bethesda, Maryland 20892-4255, USA.
Thorgeirsson
S S
SS
D000818:Animals; D002453:Cell Cycle; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins / Q000737:chemistry / Q000378:metabolism*; D006498:Hepatectomy; D015151:Immunoblotting; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008115:Liver Regeneration / Q000502:physiology*; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011494:Protein Kinases / Q000032:analysis / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors
C534701:Ccnb1 protein, rat; D019926:Cyclin B; D056744:Cyclin B1; D016213:Cyclins; D010750:Phosphoproteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D011494:Protein Kinases; C102333:histone H1 kinase