G2 delay induced by nitrogen mustard in human cells affects cyclin A/cdk2 and cyclin B1/cdc2-kinase complexes differently.
We investigated the temporal regulation of cyclin A- and B1-dependent kinases in human lymphoma cells treated with nitrogen mustard (HN2) and pentoxifylline, to determine whether the activity of these complexes correlated with cell cycle arrest induced by DNA damage. Cells were synchronized in G1/S, treated with HN2, and then postincubated with pentoxifylline. HN2-induced a protracted delay in G2 phase. This delay correlated with suppression of cyclin B1- and cdc2-kinase activities, and stabilization of hyperphosphorylated-cdc2 in the presence of similar cyclin B1 levels to those found in mitosis. HN2 had no discernible effect on the S phase activity of cyclin A- or cdk2-immune complexes. Entry of control cells into mitosis correlated with destruction of cyclin A, disappearance of cyclin A-bound cdk2 and decreased cdk2 kinase activity. G2 delay induced by HN2 was associated with stabilization of cyclin A, increased abundance of cyclin A-bound cdk2, and increased cdk2 activity. Cyclin A was also associated with cdc2, which, contrary to complexes containing cdk2, were only activated upon entry into mitosis. Pentoxifylline abrogated cell cycle arrest induced by aphidicolin and HN2 in human lymphoma cells. Pentoxifylline also reverted the activity of cyclin A- and B1-kinases in HN2-treated cells to approximately that observed in controls. Our findings suggest that delayed entry into mitosis following DNA damage correlates with suppression of cyclin B1/cdc2 and cyclin A/cdc2 complexes, while maintaining cyclin A/cdc2 complexes in an active state. Furthermore, we found that pentoxifylline disrupts the signal transduction pathway that regulates these complexes when damaged DNA is present, resulting in abrogation of cell cycle arrest.
O'Connor
P M
PM
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Ferris
D K
DK
Pagano
M
M
Draetta
G
G
Pines
J
J
Hunter
T
T
Longo
D L
DL
Kohn
K W
KW
D000936:Antigen-Antibody Complex; D016590:Aphidicolin / Q000494:pharmacology; D016203:CDC2 Protein Kinase / Q000302:isolation & purification / Q000378:metabolism*; D042846:CDC2-CDC28 Kinases; D002453:Cell Cycle / Q000187:drug effects*; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D016213:Cyclins / Q000302:isolation & purification / Q000378:metabolism*; D005434:Flow Cytometry; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008223:Lymphoma; D008466:Mechlorethamine / Q000494:pharmacology*; D008940:Mitotic Index / Q000187:drug effects; D010431:Pentoxifylline / Q000494:pharmacology; D011485:Protein Binding; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases; D013997:Time Factors; D014407:Tumor Cells, Cultured
D000936:Antigen-Antibody Complex; D016213:Cyclins; D016590:Aphidicolin; D008466:Mechlorethamine; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D010431:Pentoxifylline