The known DNA repair defects that produce large differences in radiation sensitivity, are often associated with complex clinical syndromes such as ataxia-telangiectasia, Nijmegan breakage syndrome and Fanconi anaemia(28
). The proteins identified by these repair defects do not represent potential therapeutic targets due to lack of tumour specificity. Many of the key, therapeutically exploitable mechanisms that determine intrinsic tumour radiosensitivity are largely unknown. The clinical importance of these mechanisms is shown by the known correlation between increased tumour radioresistance and adverse patient outcomes(2
The EGFR pathway is the most widely studied contributor to tumour cell radioresistance. Recent trials have demonstrated the large benefits that can potentially be derived from biological treatments that selectively render tumour cells more sensitive to radiation by manipulation of this pathway and illustrate the need for greater understanding of the molecular basis for tumour radioresistance(5
This siRNA screen of genes involved in DNA repair was based on the critical role that unrepaired DSBs play in cell death following IR. Of the genes whose knockdown was associated with increased γH2AX foci in SQ20B cells following IR, several have already been shown to be associated with increased cell radiosensitivity (17
) thus validating the primary screening endpoint. The experimental design used both irradiated and unirradiated tumour cells as well as parallel siRNA screens in a tumour line and normal tissue line allowing the identification of siRNAs that cause tumour specific radiosensitisation. High throughput screens will inevitably feature both false positive and false negative results. Failure to cause sufficient gene knockdown is one of the most common causes for obtaining false negative results. ATM
was one of the genes included in this library which is known to be involved in cell radiosensitivity but which was not in the top 30 Z-scores of the irradiated cell screens. In this case it is probable this occurred because ATM
is one of the genes involved in causing H2AX phosphorylation in response to DSB formation(30
), and thus is a false negative in this assay.
Of the targets identified by this screen we elected to investigate POLQ
further as this gene has not previously been linked to tumour cell radiosensitivity and because a previous study has suggested a potentially exploitable difference in expression between normal tissues and tumour cells (25
POLQ is a member of the A family of DNA polymerases which, unusually for this class of polymerases, synthesises DNA with very low fidelity(31
). The normal physiological functions associated with this protein are currently unclear. It has previously been suggested that POLQ plays a dominant role in the process of somatic hypermutation of immunoglobulin genes. This suggestion arose from the observation that mice deficient in POLQ
had a substantially decreased frequency of mutations in immunoglobulin genes(33
). A separate group also found a decrease in mutation frequency in POLQ
deficient mice but to a lesser degree(34
). However a recent study found that mutation types and frequencies were similar in wild type, POLQ−/−
, and POLQ−/−
) Accordingly this group suggested that POLQ does not have a significant role in the hypermutation pathway.
It has been suggested that POLQ has a role in BER but this remains unresolved. Mutation of POLQ
in the DT40 chicken B cell lymphocyte line has been shown to increase sensitivity to H2
mutants had significantly higher sensitivity to methyl methanesulfonate than either single mutant. Extracts obtained from this cell line were used to show that POLQ
mutant cells have markedly reduced single nucleotide BER capacity in vitro and that this reduction was of a similar magnitude to cells deficient in POLβ
). These findings led to the suggestion that POLQ and POLβ cooperate in BER.
Recent biochemical analysis has looked at the in vitro activity of cloned human POLQ(37
). It was shown that full-length POLQ has 5′-deoxyribose phosphate (5′-dRP) lyase activity. A C-terminal fragment of POLQ was shown to carry 5′-dRP lyase activity and this appeared to be independent of polymerase activity. The full-length protein and the C-terminal fragment were shown to have BER activity in vitro. Although these findings have been used to support the argument that POLQ may have a role in BER in vivo it should be noted that the rate of 5′-dRP lyase activity of POLQ is approximately 40 fold slower than that of POLβ. We found that POLQ
knockdown did not alter the sensitivity of cells to temozolomide either with or without IR. We interpret this to mean that the mechanism by which POLQ
knockdown causes increased sensitivity to IR is independent of base excision repair although it remains possible that POLQ facilitates repair via BER of a lesion that is produced by IR but not by temozolomide.
expression was previously assessed by RT-PCR in a variety of different normal human tissues(25
). Interestingly, expression was primarily limited to lymphoid tissues such as the fetal liver, thymus and bone marrow. Critical normal tissues such as lung, liver, small intestine, kidney, heart, brain and spinal cord that typically limit the radiation dose that can be delivered to patients did not appear to express POLQ
. This would imply that inhibition of POLQ would not alter the intrinsic sensitivity of these tissues. Intriguingly this study also found that POLQ
was overexpressed in a large proportion of tumours derived from patients with colon, lung and gastric cancer. Given the findings presented here, POLQ inhibition in these tumours would be predicted to reduce their radiation survival.
This difference in expression of POLQ
in tumour cells and critical, radiosensitive normal tissues was central to our decision to investigate POLQ further. Although it is possible that depletion of POLQ
from the small number of normal tissues that express this protein may render these cells more sensitive to radiation(38
), the very restricted normal tissue expression means that POLQ inhibition may improve the therapeutic ratio of radiotherapy.
Our findings confirm that POLQ is overexpressed in tumour cells derived from a variety of primary sites and that POLQ knockdown causes increased intrinsic radiosensitivity. Our results are consistent with the limited normal tissue expression of POLQ, and therefore that depletion of POLQ might cause much less radiosensitisation of normal tissues compared with tumours. These findings raise the possibility that POLQ inhibition could be used clinically to cause tumour specific radiosensitisation. Additionally the technique used in this study successfully identified several other genes already known to play a role in intrinsic radiosensitivity thus validating its use in future screens of larger siRNA libraries.