Most cancer cells exhibit genomic instability — that is, a sustained increase in the rate of genetic alterations relative to that observed in normal, non-neoplastic cells. Genomic instability seems to be characteristic of most human cancers and is widely regarded as a driving force for their development.
In the vast majority of solid tumors, genomic instability manifests itself as gross structural and numerical abnormalities affecting chromosomes. This phenotype, termed chromosomal instability (CIN), accounts for the aneuploidy and chromosomal rearrangements found in virtually all cancer cells at the cytogenetic level. However, the molecular basis for CIN remains poorly understood.
Our lab is broadly interested in the mechanisms that control the fidelity of chromosome transmission in human cells, which we suspect may be disrupted in some CIN cancers. To do this, we exploit novel methods that we recently developed to delete genes in cultured human cells. We can thus rapidly generate isogenic cell lines in which a particular gene has been 'knocked out,' either constitutively or conditionally. Such somatic cell knockouts provide a rigorous system for understanding the function of genes within the human genome, without the unwanted complications sometimes seen with other approaches that have been used in the past.
While similar strategies have been exploited with great success in many model organisms, our ability to work with cultured human cells has certain practical benefits. First, the large size of human cells makes it considerably easier to monitor the spatial dynamics of whole chromosomes through the light microscope, particularly in live cells expressing green fluorescent protein (GFP)-tagged proteins. Second, our somatic cell knockout lines can be cultured in large quantities, providing a valuable source of material for biochemical assays of key enzymes involved in chromosome segregation. Third, our approach takes full advantage of the recently completed sequencing of the human genome, as well as the tremendous amount of gene expression data provided by microarrays and SAGE. Fourth, the ability to rigorously test gene function directly in human cells brings us closer to understanding the mechanistic basis of genomic instability, and ultimately, to identifying molecules that can selectively kill cells in which these pathways have become corrupted during tumorigenesis.
Imaging Mitosis in Human Cells
A histone H2B-GFP fusion protein was expressed in human cells containing (A) or lacking (B) hSecurin. Progress through mitosis was monitored in living cells by timelapse fluorescence microscopy.
These experiments reveal a dramatic defect in anaphase chromosome segregation in human cells that lack securin.