Molecular Mechanisms of Radiation-Induced Genomic Instability in Human Cells

Howard L. Liber
hliber@colostate.edu
Department of Environmental and Radiological Health Sciences
Colorado State University
Website: http://www.cvmbs.colostate.edu/erhs/faculty/liber/liber.htm

This research will be to investigate the condition known as genomic instability. This can be defined as a state in which genetic alterations, including chromosome aberrations and gene mutations, occur at rates that are much higher than normal. In fact, genomic instability is what allows a normal cell to accumulate the multiple genetic alterations that are required to convert it into a cancer cell. The chromosomes of human cells have structures at their ends called telomeres. Telomeres normally function to prevent chromosomes from fusing together end-to-end. An important quality of telomeres is that each time a cell replicates, i.e., divides, each telomere becomes shorter. It is thought that eventually, telomere shortening is a signal for cells to cease dividing. Radiation may act at the level of disrupting the signaling pathways that connect telomere metabolism to growth arrest and programmed cell death, "apoptosis". This project will focus on the relationships that exist between telomere structure and function and genomic instability to determine if telomere shortening may be a significant contributor to aging, and cancer cells need to develop the means to bypass this form of growth control.

Project Goals:

1. Determine if radiation alters genomic instability.
   
2. Investigate whether radiation-induced DNA double strand breaks produce alterations in these signaling pathways, which allow cells to continue to grow despite containing critically short telomeres.
   
3. Quantify the fractions of cells within a population that exhibit reduced telomere lengths and relate changes in telomere length to the genetic background of the cell, as well as to their response to ionizing radiation.

Experimental Approach:

Our overall strategy for the research is to create a series of human cell lines that differ in key elements of growth checkpoints, apoptosis, or DNA repair in response to radiation-induced damage. With these cell lines, we can test the instability that develops in a two-stage process. The first stage requires telomeres to be reduced in size to some critical length. This occurs in normal cells as a consequence of replication-dependent telomere shortening. Normally, when telomeres reach this critical size, they signal for either growth arrest or for programmed cell death via the p53 protein. In our model, failure to arrest or die leads to continued telomere shortening and ultimately telomere fusions. The second step in instability development is the loss or alteration in this p53-dependent control that then allows for cells with short telomeres to survive. The telomere fusions that subsequently develop lead to the various chromosome aberrations and gene mutations that characterize genome instability. By testing the model, it can be determined if instability develops after radiation-induced DNA damage [the signal] alters the normal p53-dependent controls that signal for elimination of cells with short telomeres [the target]. To test our two stage model, studies will be conducted to establish the associations between telomere length and degree of genomic instability. We will examine seven closely-related cell lines, that vary in p53 status to determine theit ability to undergo apoptosis, or ability to repair double strand breaks. For each line, cells will either be untreated or treated with a low or high dose of radiation; 50 independent surviving cells will be isolated from each condition. In each population, telomere lengths will be established, and instability will be assessed in two distinct ways, as (i) frequency of chromosome aberrations and (ii) spontaneous mutation frequency and rate.

Expected Outcomes:

1. Determine the frequency of both spontaneous and radiation-induced genomic instability as a function of radiation dose
   
2. Determine the cell's role in genetic background on apoptosis, mutation and chromosome damage, and ultimately on genomic instability
   
3. Establish the association between telomere length and the degree of genomic instabilit