Consequences of Chromosome Instability in Mammalian Cells In Vitro and In Vivo
Andriani, Grasiella Angelina
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Chromosomal instability (CIN) is a cellular state with high propensity for chromosome mis-segregation. Ploidy changes, a direct consequence of CIN, have been correlated with impaired cellular fitness and a transcriptional signature associated with stress. Mounting evidence suggests that CIN plays a role in the etiology of neurological and neurodegenerative diseases and in the aging process, however the precise mechanisms involved are not completely elucidated. The general goal of the present work was to shed light into the cellular consequences of CIN, both in vitro and in vivo. In the first part of this thesis we asked whether the age-related accumulation of aneuploidy observed during normative aging in the mammalian cerebral cortex was recapitulated in murine models of accelerated aging. To this end, we collected brain and cerebellum samples from BUB1BH/H mice, which have a faulty mitotic checkpoint and ERCC1-/Δ7 mice, defective in nucleotide excision and interstrand crosslink repairs. Both murine cohorts, which exhibit features of premature aging, were analyzed at their maximum life span time point. Ploidy analysis carried on by Fluorescent In Situ Hybridization revealed no difference in the levels of not-diploid cells between progeroid and WT age-matched control mice during adulthood, suggesting that not all aging phenotypes are recapitulated in these mice models. Surprisingly, we found significantly high levels of aneuploidy in BUB1B H/H mice during embryonic development, which was accompanied by increased apoptosis and microglia staining, suggesting that not-diploid cells are eliminated, likely through immune-mediated processes. Therefore, the lack of aneuploid cells in the adult brain of progeroid mice possibly reflects the efficient clearance of these cells at earlier time points. However, exacerbated depletion of aneuploid cells within the brain is likely detrimental, as both mice models present markers of age-related neurodegeneration. In the second part of this thesis we investigated the fate of cells undergoing CIN in vitro. For this purpose, we knocked down genes important for faithful chromosome segregation (BUB1 and SMC1A) in human fibroblasts to induce CIN, and then followed growth, morphology and cellular outcomes. While apoptosis was a minor consequence of CIN under these experimental conditions, significant high levels of premature senescence were observed upon CIN generation, as assessed by decreased BrdU incorporation and expression of proliferation marker MKI67, increased SA- Rgalactosidase staining and cellular size and up-regulation of CDKN1A and CDKN2A. Genomic instability, through the generation of micronuclei and DNA double strand breaks, was observed in not-diploid cells. Oxidative stress was also a confirmed feature of CIN cells, as assessed by increased production of reactive oxygen species (ROS), elevated cellular autofluorescence and oxidative damage to DNA and RNA. In addition, we showed that CIN-induced senescent cells activate a unique SASP, which includes a novel growth factor CLEC1IA, which has not been associated with senescence before. These results showed that CIN is sufficient to trigger premature senescence in vitro , which is accompanied by SASP activation, suggesting that accumulation of CIN cells during aging potentially contributes to age-related inflammation through secretion of SASP components. Collectively, the findings presented in this thesis contribute to the field of aneuploidy and CIN because they demonstrate how cellular responses to ploidy changes can vary, depending on the experimental system, the cellular type and function and the genetic background.