R5.3: Chromosome Damage

Double strand breaks in DNA are the most lethal events to occur following irradiation of a cell. One reason for this is the possibility of significant chromosomal alterations caused by the presence of exposed ends of DNA.

Chromosome damage arises when a single chromosome is broken before duplication in S phase. Chromatid damage results from damage to one arm of the duplicated chromosome, with no damage to the other.

Hall describes three situations that may arise when double strand breaks are induced in a population of chromosomes:

  • The broken ends may rejoin to their original positions and no obvious chromosomal defects will be identifiable
  • A fragment of the chromosome may be lost if several double strand breaks occur within a single chromosome
  • Broken ends may attach to incorrect broken ends, leading to the formation of lethal abnormalities

Lethal Chromosomal Damage

Three lethal effects seen following radiation include:

  • A dicentric chromosome is formed when part of an arm of two separate chromosomes are separated. The two chromosomes then attach to each other rather than their separated arms. The new chromosomes contains two centromeres, making it impossible for the chromosome to divide normally at mitosis
  • An anaphase bridge is formed when a duplicated chromosome loses both ends of a paired arm. The arms then unite, and when the cell tries to divide at mitosis it is unable to separate the fused arms.
  • A ring is formed when both ends are lost from the same chromosome. The chromosome then attaches its new ends together, leading to formation of a ring.

Other chromosomal abnormalities

It is possible for chromosomes to reattach in a non-lethal fashion. For example, the ends of two chromosomes may become swapped. This may cause no effect, or may lead to abnormal gene expression if there is an alignment of promoter regions with incorrect genes. These translocations are seen in many malignancies, either causing deactivation of tumour suppressor genes or activation of oncogenes.

Assays for Chromosome Damage

Conventional Smear

Cells must be specially prepared to view the chromosomes.

  • Ideally a highly mitotic population is cultured (see lymphocytes below)
  • Cells are arrested in metaphase
  • The cells are treated to cause swelling of the nucleus and spreading out of the chromosomes
  • The cells are plated on a slide and left to try
  • The slide is stained for DNA molecules

This method allows chromosomes to be visualised under light microscopy, where they can be counted and observed for abnormalities. Abnormalities such as translocations are often difficult to visualise with this method, however lethal chromosome abnormalities are typically visible.

In Situ Hybridisation

In Situ Hybridisation describes a variety of techniques that have a similar process:

  • A probe is used to bind to a specific sequence of DNA, RNA or protein
  • If required, an antibody directed against the probe is added to the cell. This antibody is capable of creating a visible effect when bound to the probe.

Some probes can produce a visible effect without an antibody attached

Common in situ hybridisation techniques include:

  • Fluorescense In Situ Hybridisation (FISH) - DNA strands can be targeted by specific probes. These can either be 'chromosome painting', which cause each chromosome to fluoresce a difference colour; or can be directed against specific genes (such as ERBB2). Chromosome painting is particularly useful at detecting translocations, as the translocated arm will be a different colour to the host chromosome.
    • FISH requires a specific microscope which can cause the molecules to fluoresce.
  • Silver In Situ Hybridisation (SISH) - This is more commonly used for gene number counting. The antibody used in SISH causes silver atoms to collect in the region of the gene. The number of genes can then be counted using a normal light microscope (the silver appears as a dark spot).

In Vivo Lymphocyte Assay

Peripheral lymphocytes can be harvested after radiation exposure. They can be simulated to divide in culture (eg. with phytohaemagluttinin), and then arrested in metaphase. Chromosome smears can then be performed (including FISH) to judge the number of abnormalities present.