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We have developed a high-throughput radiosensitivity screening method based on clonogenicity and screened a siRNA library against kinases. TPK1 knockdown caused significant radiosensitization in cancer but not normal tissue cell lines. Other means of blocking this pathway, knockdown of thiamine transporter-1 THTR1 or treatment with the thiamine analogue pyrithiamine hydrobromide PyrH caused significant tumor specific radiosensitization.
Thus this screen allowed the identification of thiamine metabolism as a novel radiosensitization target that affects DNA repair. Short-term modulation of thiamine metabolism could be a clinically exploitable strategy to achieve tumor specific radiosensitization. Surrogate markers and viability assays evaluating tumor cell survival following radiation have previously been used in radiation sensitivity screens [ 1 - 3 ] but have several limitations.
In particular cell viability assays such as the 3- 4,5-Dimethylthiazolyl -2,5-diphenyl tetrazolium bromide MTT assay, which measure the number of cells in active metabolism are unreliable for radiation studies for several reasons. Radiation induces cell cycle arrest slowing proliferation, without altering overall survival to the same extent [ 4 , 5 ]. Cells that ultimately show clonogenic failure often temporarily continue to proliferate for several generations yielding false negatives [ 6 ].
Moreover, cell viability assays can be affected by non-proliferating yet metabolically active senescent cells. Although we have successfully used this approach previously [ 8 ], it may only be suitable for identifying targets that directly interfere with DNA repair.
The gold standard for assessing cell radiosensitivity is the colony formation assay CFA as it evaluates the true clonogenic potential of a cell following irradiation and is thus the closest in vitro equivalent to the clinical use of radiation.
We adapted this assay so it can be performed in well plates, enabling us to screen for novel radiosensitizing targets in a high-throughput approach. Using this technique we screened a siRNA library targeting kinases. In addition to known expected targets, we identified a series of novel targets including thiamine pyrophosphokinase-1 TPK1 , a key component of the thiamine metabolism pathway.
The discovery that thiamine metabolism is associated with tumor radiosensitivity was an unanticipated finding, and may represent a new clinical strategy for augmenting radiotherapy. RESULTS A high-throughput colony formation screen identifies TPK1 as a radiosensitizing target HeLa cells were chosen for adaptation of the clonogenic assay to a well format because they form distinct, tight and countable colonies even after irradiation in well plates Fig.
By optimizing cell seeding density and colony growth times, we obtained colonies that were large enough to accurately assess clonogenic survival yet small enough to fit up to colonies per well so that a surviving fraction SF could be calculated reliably and reproducibly. The 7 Gy radiation dose provided a maximal difference between surviving fractions for the positive and negative controls. This yielded a Z-factor of 0.
A radiosensitization score, R-score, was calculated for each target, with low values indicating greater radiosensitization. The coefficient of determination R2 , a measurement for inter-assay variability, comparing the R-score values between the two runs was 0. Targets from the primary screen were ordered using rank product analysis, minimizing variability in R-scores between screening runs.
A full list of the targets with radiosensitivity scores and heatmap can be found in Table S1 and Supplementary Figure S2 , respectively.
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