F9 embryonal carcinoma cells fail to stop at G1/S boundary of the cell cycle after gamma-irradiation due to p21WAF1/CIP1 degradation.
We studied the ability of F9 teratocarcinoma cells to arrest in G1/S and G2/M checkpoints after gamma-irradiation. Wild-type p53 protein was rapidly accumulated in F9 cells after gamma-irradiation, however, this was followed not by a G1/S arrest but by a short and reversible delay of the cell cycle in G2/M. In order to elucidate the reasons of the lack of G1/S arrest in F9 cells, we investigated the expression of p53 downstream target Cdk inhibitor p21WAF1/CIP1. In spite of p53-dependent activation of p21WAF1/CIP1 gene promoter and p21WAF1/CIP1 mRNA accumulation upon irradiation, the p21WAF1/CIP1 protein was not detected by either immunoblot or immunofluorescence techniques. However, the cells treated with a specific proteasome inhibitor lactacystin revealed the p21WAF1/CIP1 protein both in non-irradiated and irradiated cells. Therefore we suggest that p21WAF1/CIP1 protein is degraded by a proteasome-dependent mechanism in F9 cells and the lack of G1/S arrest after gamma-irradiation is due to this degradation. We also examined the expression and activity of cell cycle regulatory proteins: G1- and G2-cyclins and cyclin-dependent kinases. In the absence of functional p21WAF1/CIP1 inhibitor, the activity of G1 cyclin/Cdk complexes was insufficiently inhibited to cause a G1 arrest, whereas a decrease of cdc2 and cyclin B1-associated kinase activities was enough to contribute to a reversible G2 arrest following gamma-irradiation. After gamma-irradiation, the majority of F9 cells undergo apoptosis implying that wt-p53 likely triggers pro-apoptotic gene expression in DNA damaged cells. Elimination of defected cells might ensure maintenance of genome integrity in the remaining cell population.
Malashicheva
A B
AB
Institute of Cytology, Laboratory of Molecular Basis of Cell Differentiation, Russian Academy of Sciences, St Petersburg, Tikhoretsky, Russia.
Kislyakova
T V
TV
Aksenov
N D
ND
Osipov
K A
KA
Pospelov
V A
VA
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D042846:CDC2-CDC28 Kinases; D018236:Carcinoma, Embryonal / Q000473:pathology / Q000532:radiotherapy; D002453:Cell Cycle / Q000502:physiology* / Q000528:radiation effects*; D019926:Cyclin B / Q000378:metabolism; D056744:Cyclin B1; D019938:Cyclin D1 / Q000378:metabolism / Q000528:radiation effects; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000378:metabolism / Q000528:radiation effects; D016213:Cyclins / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D003546:Cysteine Endopeptidases / Q000187:drug effects; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004249:DNA Damage / Q000528:radiation effects; D016193:G1 Phase / Q000528:radiation effects; D005720:Gamma Rays; D051379:Mice; D009097:Multienzyme Complexes / Q000187:drug effects; D015739:Nocodazole / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000528:radiation effects; D011518:Proto-Oncogene Proteins; D016196:S Phase / Q000528:radiation effects; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
C534700:Ccnb1 protein, mouse; C493957:Cdkn1a protein, mouse; D019926:Cyclin B; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D015853:Cysteine Proteinase Inhibitors; D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C067713:lactacystin; D019938:Cyclin D1; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495893:Cdk2 protein, mouse; C495902:Cdk4 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D015739:Nocodazole; D000111:Acetylcysteine