Mitosis is the process whereby a cell divides to produce two identical copies of itself. One mechanism which ensures that these copies are identical is the mitotic checkpoint. This safeguard works by preventing division until all chromosomes have achieved proper attachment to mitotic spindles. In cancer cells, mitosis--and the mitotic checkpoint--are frequently disturbed. One way this occurs is through prolonged activation of the checkpoint. Increasingly, evidence indicates that prolongation of mitosis may be a cause, rather than just a consequence, of tumorigenesis. At the same time, antimitotic chemotherapeutics are used to deliberately prolong mitosis, which, when severe, can be cytotoxic to cancer cells. Thus, prolongation of mitosis appears to play important roles in the biology of both the formation and treatment of cancer. Despite identification of these roles, our knowledge of the mechanisms underlying them remains limited. In this dissertation, we present work which investigated two ideas relevant to understanding the consequences of prolonged activation of the mitotic checkpoint. First, we tested the hypothesis that human cells acquire DNA damage during prolonged mitotic arrest. This investigation revealed that, during mitotic arrest, human cells can indeed acquire DNA damage. Second, we tested the hypothesis that the p53 tumor suppressor inhibits the propagation of DNA damage which is acquired during mitotic arrest. This investigation revealed that p53 indeed inhibits the propagation, but not the acquisition, of DNA damage incurred during mitotic arrest. Taken together, the findings in these studies describe a novel source of DNA damage in human cells, suggest that prolonged mitosis may promote tumorigenesis and antimitotic toxicity through infliction of DNA damage, and propose a novel anticancer role for the p53 tumor suppressor.
Table of Contents
Table of Contents PageGeneral Introduction_____________________________________________________ 1 Mitosis is frequently abnormal in cancer cells_______________________________ 2 The mitotic checkpoint__________________________________________________ 3 Weakening of the mitotic checkpoint in cancer cells__________________________ 4 Prolonged activation of the mitotic checkpoint in cancer cells___________________ 6 Cell fate after mitotic arrest_____________________________________________ 10 p53 and the postmitotic response________________________________________ 12 Antimitotic chemotherapy and the mitotic checkpoint________________________ 16 Goals of this Dissertation_______________________________________________ 17 Figure 1. The five phases of mitosis.______________________________________ 19 Figure 2. The mitotic checkpoint.________________________________________ 20 Figure 3. Cancer-associated causes of prolonged mitotic checkpoint activation.___ 22 Figure 4. Cell fates following mitotic arrest.________________________________ 23 Human Cancer Cells Commonly Acquire DNA Damage During Mitotic Arrest____ 24 Abstract____________________________________________________________ 25 Introduction_________________________________________________________ 25 Materials and Methods________________________________________________ 26 Results and Discussion________________________________________________ 28 Figure 1. γ-H2AX foci accumulate during drug-induced mitotic arrest in HCT116 cells. 35 Figure 2. γ-H2AX foci acquired during mitotic arrest arise independently of cell death in HCT116 cells. 37 Figure 3. HCT116 cells released from mitotic arrest harbor chromosome aberrations. 39 Figure 4. Prometaphase elevation of γ-H2AX in HCT116 cells occurs both upon knockdown of CENP-E and in untreated cells with spontaneous spindle defects.___________________________ 40 Supplementary Methods_______________________________________________ 42 Supplementary Table. Quantification of chromosome aberrations._______________ 43 Supplementary Figure S1. Nocodazole induces transient mitotic arrest in HCT116 cells. 44 Supplementary Figure S2. Nocodazole does not induce γ-H2AX foci in premitotic HCT116 cells. 45 Supplementary Figure S3. Prophase HCT116 cells with spindle abnormalities do not contain elevated γ-H2AX.___________________________________________________________________ 47 Supplementary Figure S4. Acquisition of γ-H2AX foci during mitotic arrest is a common occurrence in human cell lines.____________________________________________________________ 48 Supplementary Figure S5. Anti-γ-H2AX antibody recognizes a single protein in HCT116 cells. 49 p53 suppresses structural chromosome instability following mitotic arrest in human cells 50 Abstract____________________________________________________________ 51 Introduction_________________________________________________________ 52 Results_____________________________________________________________ 54 Discussion___________________________________________________________ 61 Materials and methods_________________________________________________ 67 Figure 1 Both p53+/+ and p53-/- HCT116 cells acquire DNA damage during mitotic arrest. 71 Figure 2 p53 inhibits the polyploidization and survival of HCT116 cells following prolonged mitotic arrest.74 Figure 3 p53 knockdown phenocopies the response of p53-/- cells to nocodazole._ 76 Figure 4 p53 suppresses structural chromosome instability following mitotic arrest in HCT116 cells. 78 Figure 5 Polyploidization through inhibition of cytokinesis does not elicit significant DNA damage or p53-dependent postmitotic response._________________________________________ 80 Figure 6 p53 suppresses polyploidization and structural chromosome instability following mitotic arrest in IMR90 human diploid fibroblasts (HDFs)._________________________________ 82 Figure 7 p53 suppresses the frequency of, and structural chromosome instability in, spontaneous polyploid HCT116 cells.________________________________________________________ 84 Supplementary Figure S1 Cells staining entirely negative for MPM-2 have apoptotic nuclear morphology. 88 Supplementary Figure S2 Chromosome pulverization occurs in a fraction of nocodazole-treated HDFs. 89 Supplementary Figure S3 p53 knockdown does not influence cell death following mitotic arrest in HDFs. 90 Supplementary Figure S4 Mitosis is spontaneously prolonged in p53-/- HCT116 cells which fail to divide. 91 Discussion____________________________________________________________ 92 Conclusions_________________________________________________________ 93 Future Directions_____________________________________________________ 95 Figure 1. Working model for the consequences of DNA damage during mitotic arrest.105 References___________________________________________________________ 106
About this Dissertation
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|Committee Chair / Thesis Advisor|
|DNA Damage During Mitotic Arrest: A Novel, p53-Regulated Sourceof Structural Chromosome Instability in Human Cells ()||2018-08-28 12:19:59 -0400||