The mitotic spindle checkpoint is a constitutively active mechanism that protects cells from aneuploidy. Cell lines exhibit variable capacities to activate this checkpoint and rodent cells, such as CHO-K1, are well known to possess a less stringent checkpoint than human cells. Preliminary analysis of a panel of CHO-K1-human chromosome somatic cell hybrids, identified 15A (retaining human chromosome 15) as having an augmented checkpoint. Reanalysis of the two cell lines here, using both microscopy and flow cytometry, found 15A to have a higher mitotic index during an unperturbed cell cycle; but they were more unstable when their mitotic spindle checkpoint was hyper-activated with nocodazole. The complex 15A phenotype was hypothesised to result from: (1) a gene on human chromosome 15 complementing a putative CHO-K1 mitotic spindle checkpoint genetic lesion and (2) 15A cells being intrinsically "primed" for mitotic slippage by virtue of deregulation of pro-mitotic slippage mediators. The first hypothesis was addressed using a candidate gene approach. A putative CHO-K1 mitotic spindle checkpoint lesion was mapped to bubr1 at human 15q15. CHO bubr1 was cloned de novo and we have identified a putative novel heterozygous kinetochore-binding domain mutation in CHO- K1. In an attempt to validate that human BubR1 can complement the putative CHO-K1 BubR1 defect, the propensity to tolerate BubR1 overexpression was shown to be cell type/sub-clone-specific. CHO-AAS are a derivative of CHO- K1 and human BubR1 was transiently and constitutively expressed in these cells, where it increased the mitotic index and induced mitotic slippage. The second hypothesis was addressed by carrying out Western blots for markers of mitotic spindle checkpoint activation, mitotic slippage and apoptosis in basal and nocodazole treated CHO-K1 and 15A cells. Most notably, TAp73 protein (a transcriptionally active p53-family member protein) is specifically and constitutively highly upregulated in 15A under basal conditions. Downstream TAp73 targets, such as Cdk inhibitors p21 and p57, create a pro-mitotic slippage milieu in 15A. TAp73 was shown to be post-transcriptionally upregulated in 15A; to be localised in the nucleus and to be stabilised independent of a canonical DNA- damage response (as evidenced by Western blotting for y-H2AX and PML proteins). Ectopic TAp73 overexpression in CHO-K1 induced apoptosis. Thus, the highly pro-apoptotic TAp73 is held in check in 15A. This warrants further investigation that may elucidate novel mechanisms for p73 regulation.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:588491 |
| Date | January 2011 |
| Creators | McAllister, Roisin |
| Publisher | University of Ulster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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