Requirement for proliferating cell nuclear antigen expression during stages of the Chinese hamster ovary cell cycle.
Proliferating cell nuclear antigen (PCNA/cyclin) is a nuclear protein that can stimulate purified DNA polymerase delta in vitro, and its synthesis correlates with the proliferation rate of cells. We have attempted to determine whether synthesis of PCNA/cyclin in Chinese hamster ovary cells is necessary to regulate entry into S phase. We have measured cellular PCNA/cyclin concentration of the mRNA or protein throughout the cell cycle. Cells were separated by centrifugal elutriation into populations enriched for G-1, S, and G-2/M phases. Quantitative Northern hybridization analysis was performed on RNA isolated from each cell population by using a cDNA clone of PCNA/cyclin as a probe. Results demonstrated that although intact PCNA/cyclin mRNA is present during all phases of the cell cycle, an induction of about 3-fold occurs during S phase. Two-parameter staining for PCNA/cyclin and DNA, and analysis by flow cytometry, confirmed that the quantity of PCNA/cyclin protein in the cells increases severalfold in G-1 or early S phase but generally is invariant in S and G-2/M phases. This cell cycle dependence of PCNA/cyclin expression suggests that the observed synthesis is a prerequisite for initiation of DNA replication. Introduction of an antisense oligonucleotide complementary to the PCNA/cyclin mRNA to inhibit PCNA/cyclin synthesis effectively prevented entry of G-1 phase cells into S phase. A complementary sense oligonucleotide used as a control did not have an inhibitory effect. This result suggests that a threshold concentration of PCNA/cyclin is necessary for entry into S phase.
Liu
Y C
YC
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Marraccino
R L
RL
Keng
P C
PC
Bambara
R A
RA
Lord
E M
EM
Chou
W G
WG
Zain
S B
SB
D000818:Animals; D001324:Autoantigens / Q000235:genetics*; D015152:Blotting, Northern; D002453:Cell Cycle; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000235:genetics; D005260:Female; D007399:Interphase; D009687:Nuclear Proteins / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010053:Ovary; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger / Q000235:genetics
D001324:Autoantigens; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D012333:RNA, Messenger; D004247:DNA