Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard.
M phase-promoting factor (MPF) consists of a p34cdc2 (cdc2) kinase and cyclin B complex which in its active form promotes G2 to M transition. The role of MPF in G2 arrest following DNA damage, however, has remained largely uncharacterized. We have investigated whether nitrogen mustard (HN2) interfered with either the formation of MPF or its activation. For this purpose, we measured cdc2 kinase activity relative to cdc2 and cyclin B protein turnover and the phosphorylation status of cdc2. Studies were performed in two exceptional human lymphoma cell lines, which differed in HN2 sensitivity by 5-fold (CA46, 50% growth-inhibitory dose = 1.0 microM; JLP119, 50% growth-inhibitory dose = 0.2 microM) but exhibited virtually identical DNA interstrand and DNA-protein cross-link exposure. Following HN2 treatment, CA46 cells ceased to enter mitosis and exhibited a marked delay in G2 phase. Failure to enter mitosis paralleled inhibition of cdc2 kinase. Inhibition was not due to decreased levels of cdc2 or cyclin B protein; rather, G2 arrest correlated with the accumulation of both tyrosine-phosphorylated cdc2 and cyclin B. These findings implied that G2 arrest resulted from a down-regulation of the processes that activate MPF. We also found that JLP119 cells, within a few hours of mitosis at the time of drug treatment, evaded checkpoint control and continued cell division unabated by DNA damage. Furthermore, despite similar DNA cross-link exposure, JLP119 cells within the window of checkpoint control were more susceptible to S phase delay than CA46 cells. Altered cell cycle responses correlated with the greater susceptibility of JLP119 cells to the cytotoxic effects of HN2.
O'Connor
P M
PM
National Cancer Institute, NIH, Bethesda, Maryland 20892.
Ferris
D K
DK
White
G A
GA
Pines
J
J
Hunter
T
T
Longo
D L
DL
Kohn
K W
KW
D016203:CDC2 Protein Kinase / Q000187:drug effects*; D003432:Cross-Linking Reagents; D016213:Cyclins / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics*; D016195:G2 Phase / Q000187:drug effects*; D006801:Humans; D008466:Mechlorethamine; D010766:Phosphorylation; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D016213:Cyclins; D008466:Mechlorethamine; D016203:CDC2 Protein Kinase