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Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard
P M O'Connor1, D K Ferris, G A White
1National Cancer Institute, NIH, Bethesda, Maryland 20892.
Abstract:
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.
Insights
Nitrogen mustard causes G2 arrest by inhibiting M phase-promoting factor (MPF) activation in lymphoma cells. This arrest is linked to increased MPF component phosphorylation, impacting cell cycle progression and drug sensitivity.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- M phase-promoting factor (MPF), a complex of p34cdc2 kinase and cyclin B, drives the G2 to M cell cycle transition.
- The precise role of MPF in G2 arrest following DNA damage remains incompletely understood.
Purpose of the Study:
- To investigate if nitrogen mustard (HN2) affects MPF formation or activation.
- To compare the effects of HN2 on cell cycle progression and MPF regulation in differentially sensitive human lymphoma cell lines.
Main Methods:
- Assessed cdc2 kinase activity, cdc2 and cyclin B protein turnover, and cdc2 phosphorylation status.
- Utilized two human lymphoma cell lines (CA46 and JLP119) with varying HN2 sensitivity but similar DNA damage.
- Quantified DNA interstrand and DNA-protein cross-link exposure.
Main Results:
- HN2 treatment induced G2 arrest and inhibited cdc2 kinase activity in CA46 cells, correlating with increased tyrosine-phosphorylated cdc2 and cyclin B.
- JLP119 cells, despite similar DNA damage, evaded G2 checkpoint control and continued division.
- JLP119 cells showed increased S phase delay and greater susceptibility to HN2 cytotoxicity compared to CA46 cells.
Conclusions:
- G2 arrest in response to HN2 is mediated by down-regulation of MPF activation processes, specifically through increased inhibitory phosphorylation of cdc2 and cyclin B.
- Differential evasion of cell cycle checkpoints contributes to varying HN2 sensitivity and cytotoxicity in lymphoma cells.