Minocycline-Mediated Improvement of Cognitive Impairment Induced by Irradiation Involves Preventing Immature Neurons From Apoptosis

Abstract

Purpose/Objective(s): Cancer cells exhibit high degree of genome instability including frequent micronuclei, extranuclear organelles containing DNA fragments or whole chromosomes that arise due to DNA damage or mitotic errors. Micronuclei exhibit abnormalities in DNA replication, DNA damage response and membrane stability, leading to accumulation of mutations that are subsequently passed on to the main genome. As a consequence, micronuclei may act as a “mutator” organelle in tumorigenesis, and may be responsible for chromothripsis, catastrophic event leading to a large number of clustered rearrangements observed in many malignancies. We performed studies to gain insight into the mechanism of microncuclear-mediated DNA damage and mutagenesis. Materials/Methods: Micronuclei were induced in human RPE-1 and U2OS cell lines by treatment with 100 ng/ml nocodazole for 6 hours, leading to increased rate of chromosome missegregation, followed by synchronization with mitotic shake-off to release cells into subsequent interphase. Cells were fixed at given intervals in G1, S and G2 phases of the cell cycle and indirect immunofluorescence, time-lapse microscopy and flow cytometry were performed per previously established protocols. Antibodies used included anti-RPA70 (Cell Signaling), anti-RPA32 (Abcam), antig-H2AX (Cell Signaling), anti-Mcm2 (Cell Signaling) and anti-Mcm3(Cell Signaling). Results: Sor G2phase micronculei with ruptured nuclear membranes exhibit massive DNA damage as evidenced by positive g-H2AX and TUNEL staining, but G0and G1synchronized micronuclei show no such damage. Loss of nuclear membrane integrity by itself is thus not sufficient to result in DNA damage, and can only lead to damage in the setting of ongoing DNA replication. Strikingly, micronuclei exhibit a dramatic reduction in nucleoplasmic levels of Replication Protein A complex (RPA70 and RPA32) and DNA helicase (Mcm 2 and Mcm3), which may account for the observed delay in DNA replication, replication stress and blunted DNA damage response. Further dilution of these factors in the setting of sudden nuclear membrane rupture leads to replication catastrophe and massive DNA damage. Conclusions: Nucleocytoplasmic transport abnormalities in micronuclei lead to inability to concentrate key replication and DNA damage response factors, including components of RPA and DNA helicase complexes, contributing to error-prone DNA replication and blunted DNA damage response. Sudden loss of micronuclear membrane integrity in the setting of ongoing DNA replication leads to replication catastrophe and accumulation of massive DNA damage that is subsequently passed on to the main tumor genome driving tumor progression. Author Disclosure: A. Spektor: None. S. Liu: None. E. Jackson: None. D. Pellman: None.