Control

Predictive assays based on measuring DNA damage in cells irradiated in vitro using standard techniques have yielded inconsistent results as discussed. Recently much has been discovered about the molecular processes involved in the recognition of DSBs within a cell and the subsequent signalling processes leading to repair of DNA damage or to cell death. The question arises as to whether this greater understanding can lead to the discovery of molecular markers of DNA damage and repair that might be quantified and may result in the development of more sensitive and reliable assays of DNA damage and repair than previously used cell-based techniques.

28 1.7.1 DNA DSBs and γH2AX induction

When a DSB is introduced into DNA the histone protein H2AX becomes rapidly phosphorylated at serine 139 within its COOH-terminal region involving hundreds to thousands of H2AX molecules in a megabase region on either side of the DSB (Rogakou, Pilch et al. 1998; Stiff, O'Driscoll et al. 2004) . A commercially available monoclonal antibody to the phosphorylated form of H2AX (γH2AX) has been developed and using immunocytochemistry it is possible to visualise these DNA DSBs as large foci within the cell nucleus (Rogakou, Boon et al. 1999). A single DSB is sufficient for the formation of a γH2AX focus and there appears to be a 1:1 correspondence between the number of DSBs and γH2AX foci after DNA damage induction (Sedelnikova, Rogakou et al. 2002). Immunofluorescent staining for γH2AX foci can detect DSB induction at much lower doses than established DNA DSB assays and has been reported to be sensitive enough to detect DSBs in cells after doses as low as 0.001Gy (Rothkamm and Lobrich 2003).

The phosphorylation of H2AX is thought to recruit DNA repair factors to the site of the DNA DSB (Paull, Rogakou et al. 2000) and may also be involved in the amplification of DNA damage signals that activate the G2/M checkpoint to prevent damaged cells from entering mitosis (Fernandez-Capetillo, Chen et al. 2002). Inability to form γH2AX foci has been correlated to radiosensitivity, genomic instability and other repair defects (Bassing, Chua et al. 2002; Celeste, Petersen et al. 2002; Kuhne, Riballo et al. 2004; Taneja, Davis et al. 2004). The mechanism by which γH2AX is removed following DNA repair is incompletely understood. In some studies the kinetics of γH2AX loss mirrors the kinetics of DNA DSB rejoining (Rothkamm and Lobrich 2003) whilst others have found that although the number of γH2AX foci formed after irradiation correlates with the number of double strand breaks formed the kinetics of foci development and loss differ from those characteristic of double-strand break re-joining, and loss of γH2AX may therefore be indicative of more than simple DSB re-joining..(MacPhail, Banath et al. 2003).

Clinically relevant ionizing radiation doses induce similar patterns of γH2AX focus formation in radiosensitive and radioresistant human tumour cell lines and xenografted tumours, but radiosensitive tumour cells and xenografts retain γH2AX for a greater duration than radioresistant cells and tumours (MacPhail, Banath et al. 2003; Taneja, Davis et al. 2004). There is evidence that the rate of loss of γH2AX after irradiation of cells in

29 culture correlates with clonogenic survival at 2Gy (MacPhail, Banath et al. 2003) and the rate of γH2AX disappearance is slower in radiosensitive tumour cells and radiosensitive murine normal tissue than radioresistant cell lines or normal tissue (Olive and Banath 2004). The techniques for immunofluorescent staining and γH2AX quantification in cultured cells are quick and yield results in a number of days rather than many weeks. Quantification of γH2AX induction and the kinetics of γH2AX loss in normal human cells or tissues after a test dose of radiation could therefore potentially form the basis of a predictive assay of human normal tissue radiosensitivity.

Normal cells sampled from patients and utilised for predictive testing should be plentiful and easily and rapidly accessible by non-invasive means. For γH2AX quantification the cells would ideally be non-cycling as γH2AX foci are also induced at collision of replication forks during DNA replication (Furuta, Takemura et al. 2003). Human peripheral blood lymphocytes (PBLs) obtained by venepuncture in the clinic fulfil both these criteria and could be used as a surrogate tissue to test if there is a relationship between cellular γH2AX induction and loss in vitro after irradiation and normal tissue radiosensitivity.

1.8 Aims

The aims of this project were therefore:

• To conduct a systematic review of the literature regarding functional cell-based assays in the predictive testing of normal tissue radiosensitivity to ascertain whether studies so far have been performed with sufficiently rigorous approach to assay quality control, avoidance of bias in study design, and statistical analysis of results, and to determine if the perception that cell-based assays are not helpful in predicting normal tissue radiosensitivity is really valid based on current literature.

• To develop a rapid, reliable cell-based assay measuring γH2AX kinetics in human PBLs in a methodical and systematic fashion with appropriate attention to issues of precision and quality control, which might have a potential role as a predictive assay for normal tissue radiosensitivity in the clinic

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2.

Materials and methods