Regulation of the Cell Cycle
DNA damage caused by numerous alkylating agents or ionizing radiation in various cell types has been associated with a G2 phase delay or arrest. This G2 phase delay or arrest can occur by a variety of mechanisms. Nitrogen mustard induced alkylation of CA46 cells is associated with the accumulation of cyclin B protein (to levels seen at mitosis), but there is no accumulation of activated cdc2 during the G2 arrest due to the inability of cdc25 to become activated. High doses of ionizing radiation of mammalian cells in S phase is associated with G2 arrest and a decreased amount of cyclin B mRNA and protein. Both nitrogen mustard and ionizing radiation result in the accumulation of inactive cdc2, and consequently an inability of the arrested cells to progress into M phase without a delay to repair the DNA damage.
Proliferative inhibition of cells exposed to pyrrolizidine alkaloids has been previously shown in vivo and in vitro. Crosslinking of DNA induced by MCTP in porcine pulmonary endothelial cells was associated with; megalocytosis, and loss of proliferative ability, but a retention of the ability to synthesize DNA, RNA and proteins (Hoorn 1992; Wagner 1993). Macrocyclic pyrrolizidine alkaloids cause cross linking of macromolecules through bifunctional reactive sites which might be related to cell cycle disturbances leading to megalocytosis. We demonstrated that the majority of cdc2 in MCTP treated BPAEC is in the inactive triphosphorylated form definitively localizing the MCTP induced blockade to G2 phase (Thomas 1998). We also showed by flow cytometry that a second population of cells expressing cyclin B1 has continued incorporation of BrdU and DNA content consistent with octaploid cells. MCTP treated bovine endothelial cells had limited expression of p53 and p53 expression was not coordinated with G2 arrest or quadraploidy. It appears that endothelial cells treated with MCTP develop G2 arrest in association with persistent cyclin B1 expression, cdc2 inactivation and continued DNA synthesis through a pathway that is unrelated to altered expression of p53.
Flow Cytometric Analysis of DNA content in Human Pulmonary Artery Endothelial Cells treated with Monocrotaline Pyrrole
Cell Cycle Regulation and DNA Damage
