Flow cytometric analysis of the expression of PCNA during the cell cycle in HeLa cells and effects of the inhibition of DNA synthesis on it.
Flow cytometric bivariate DNA/PCNA analysis was performed to investigate the expression of PCNA during the cell cycle and the implication in DNA replication in HeLa cells, using a monoclonal antibody (PC10) to PCNA. The expression of PCNA was evident in almost all cells growing exponentially, when cells were fixed in methanol. The total amount of PCNA altered a little during the cell cycle. However, the treatment with Triton X-100 extracted 80-89% of total PCNA from the cells, resulting in the dramatic change of bivariate DNA/PCNA distribution pattern. PCNA was completely removed from nuclei in both G1 and G2 phases by the detergent treatment, whereas a certain amount of PCNA remained in S phase nuclei. After the treatment of cells with Triton X-100, PCNA was detected exclusively in S phase cells. The bivariate DNA/PCNA distribution pattern in cells treated with Triton X-100 was strikingly so similar to the DNA/BrdUrd distribution pattern that it was unable to differentiate one from the other. It is concluded that the detergent treatment of cells allows the rapid analysis of the cell cycle. The inhibition of DNA synthesis with 10 mM hydroxyurea elevated cellular PCNA content mainly due to the increase in the fraction of the detergent extractable PCNA. It was apparent, however, that in cells incubated with Triton X-100, the pattern of the bivariate DNA/PCNA distribution was not basically different from that in cells without HU treatment. The level of PCNA bound to nuclear structures (PCNA not extracted with detergent) increased in cells arrested at the G1/S boundary with the time of hydroxyurea treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
Sasaki
K
K
Department of Pathology, Iwate Medical University School of Medicine, Morioka, Japan.
Kurose
A
A
Ishida
Y
Y
D000911:Antibodies, Monoclonal / Q000032:analysis / Q000276:immunology; D001973:Bromodeoxyuridine / Q000378:metabolism; D002453:Cell Cycle; D002467:Cell Nucleus / Q000737:chemistry; D004247:DNA / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry / Q000379:methods; D016193:G1 Phase; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology; D009687:Nuclear Proteins / Q000032:analysis* / Q000235:genetics* / Q000276:immunology; D017830:Octoxynol; D018809:Proliferating Cell Nuclear Antigen; D016196:S Phase
D000911:Antibodies, Monoclonal; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D017830:Octoxynol; D004247:DNA; D001973:Bromodeoxyuridine; D006918:Hydroxyurea