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We used sequentially transformed mesenchymal stem cells to investigate how the events that lead to tumorigenicity influence the cellular response to radiation. Bone marrow derived SH2(+), SH4(+), Stro-1(+) mesenchymal stem cells (MSC) were transformed stepwise by retroviral transfection of hTERT, HPV-16 E6 and E7, SV40 small T antigen and oncogenic H-ras. Cells at three different stages of transformation were irradiated and compared using assays for cytotoxicity, apoptosis, DNA double-strand break (DSB) repair and checkpoint signaling. The effects of inhibition of cell cycle checkpoint signaling on radiosensitivity were investigated using RNA interference. During stepwise transformation, specifically after HPV-16 E6 and E7 transduction, MSCs became more sensitive to radiation. This was associated with increased residual DNA DSB at 24 h and increased apoptosis. Enhanced checkpoint signaling occurred during transformation and there was a differential effect of checkpoint targeting in cells at different stages; Chk1 knockdown enhanced radiosensitivity in all cells while Chk2 knockdown only affected non-transformed cells. These data show that transformation of MSC is associated with a reduction in DNA DSB repair capacity and increased radiosensitivity. Up-regulation of checkpoint signaling does not overcome this and the effect of checkpoint inhibition may change with transformation status.

Increased cell killing after exposure to low acute doses of X rays (0-0.5 Gy) has been demonstrated in cells of a number of human tumor cell lines. The mechanisms underlying this effect have been assumed to be related to a threshold dose above which DNA repair efficiency or fidelity increases. We have used cells of two radioresistant human tumor cell lines, one that shows increased sensitivity to low radiation doses (T98G) and one that does not (U373), to investigate the DNA damage response at low doses in detail and to establish whether there is a discontinuous dose response or threshold in activation of any important mediators of this response. In the two cell lines studied, we found a sensitive, linear dose response in early signaling and transduction pathways between doses of 0.1 and 2 Gy with no evidence of a threshold dose. We demonstrate that ATM-dependent signaling events to downstream targets including TP53, CHK1 and CHK2 occur after doses as low as 0.2 Gy and that these events promote an effective damage response. Using chemical inhibition of specific DNA repair enzymes, we show that inhibition of DNA-PK-dependent end joining has relatively little effect at low (

We have used DNA microarrays to identify changes in gene expression in cells of the radioresistant human glioma cell lines T98G and U373 after low radiation doses (0.2-2 Gy). Using Bayesian linear models, we have identified a set of genes that respond to low doses of radiation; furthermore, a hypothesis-driven approach to data analysis has allowed us to identify groups of genes with defined non-linear dose responses. Specifically, one of the cell lines we have examined (T98G) shows increased radiosensitivity at low doses (low-dose hyper-radiosensitivity, HRS); thus we have also assessed sets of genes whose dose response mirrors this survival pattern. We have also investigated a time course for induction of genes over the period when the DNA damage response is expected to occur. We have validated these data using quantitative PCR and also compared genes up-regulated in array data to genes present in the polysomal RNA fraction after irradiation. Several of the radioresponsive genes that we describe code for proteins that may have an impact on the outcome of irradiation in these cells, including RAS homologues and kinases involved in checkpoint signaling, so understanding their differential regulation may suggest new ways of altering radioresistance. From a clinical perspective these data may also suggest novel targets that are specifically up-regulated in gliomas during radiotherapy treatments.

Redpath, J. L., Short, S. C., Woodcock, M. and Johnston, P. J. Low-Dose Reduction in Transformation Frequency Compared to Unirradiated Controls: The Role of Hyper-radiosensitivity to Cell Death. Radiat. Res. 159, 433–436 (2003). Calculations based on plausible parameters taken from the existing experimental database, and new measurements on the cell cycle dependence of low-dose hyper-radiosensitivity (HRS) of non-tumorigenic HeLa × skin fibroblast human hybrid cells, provide the first experimental evidence that the selective killing of a transformation-sensitive G2/M-phase subpopulation as a consequence of low-dose HRS could account in part for the observed reduction of induced transformation frequencies at low doses to values below that observed spontaneously. However, it is clear that other mechanisms associated with classical adaptive response, such as induced DNA repair, are also likely to be involved.