Ionizing radiation induces both single- and double-strand DNA breaks (DSB) in cells that then trigger DNA damage responses characterized by the recruitment of DNA-repair proteins to γH2AX foci at sites of DNA damage and the activation of checkpoint proteins that arrest cell cycle progression [
29]. Cell cycle arrest is a protective cellular response understood to block cell cycle progression and to permit DNA damage repair [
29]. An increase in DNA damage, reduced ability to repair DNA damage, and/or prolonged checkpoint activation can cause apoptosis [
30] or cause cells to undergo permanent cell cycle arrest (senescence) [
31]. We show here that pretreatment with T-oligo sensitizes mammary tumor cells to radiation, promoting growth inhibition and death of tumor cells
in vitro and in an
in vivo mouse model.
The mechanism by which T-oligo sensitize tumor cells remains to be fully elucidated. Although T-oligos do not act as telomerase inhibitors [
12] or cause digestion of the 3' telomere overhang [
5,
25,
32], T-oligos have been shown to rapidly concentrate in nuclei when added to cultured cells and the subsequent responses require WRN [
25], the protein mutated in the progeroid cancer-prone Werner syndrome. T-oligo/WRN interaction results in formation of DNA γH2AX damage-like foci at the telomeres [
25] with activation of ATM [
8,
32] and its many downstream effector proteins, leading to apoptosis and senescence [
5,
7,
8,
32]. In the present study, pretreatment with T-oligo enhances the formation of γH2AX foci (Figure
2) that customarily form at sites of DNA damage but after T-oligo treatment form at telomeres in the absence of detectable DNA damage [
25]. Activating the DNA damage response pathways by T-oligo treatment, as demonstrated to occur over several days in multiple cell types including breast carcinoma cells [
5‐
7,
9], could render tumor cells more apt to undergo apoptosis or senescence when exposed to IR. Alternatively, tumor cell inactivation could be due to the impairment of DNA repair by the pretreatment with T-oligo as demonstrated by slower decay of γH2AX foci and increased fragmentation of DNA in the comet assay (Figure
2). Increased radiosensitivity has been found in cells from patients with DDR or DNA-repair disorders such as Ataxia Telangiectasia (defect in ATM), Nijmegen Breakage Syndrome (defect in NBS1), Fanconi anemia, defective Artemis, DNA ligase I and DNA ligase IV [
33]. We favor a model of radiosensitization by T-oligos that encompasses the known and hypothesized effects of both IR and T-oligos: After 24 hours pretreatment, T-oligo-treated cells have entered an S-phase arrest [
5‐
8,
11] mediated by p95/Nbs1 [
10], presumptively due to G-quadruplex formation between single stranded telomeric DNA and the G-rich T-oligos [
12] with consequent stalling of replication forks [
34]. Without further intervention, malignant cells then begin to undergo apoptosis [
5‐
8,
11] or to enter senescence [
8,
35] or both, as in the case of breast carcinoma cells in this study and a previous one [
8], presumably in response to the collapse of their stalled replication forks. If such cells are then irradiated, the introduction of even modest numbers of DSBs and other DNA damage greatly enhances the replication stress and the processes of apoptosis and senescence.
Regardless of its mechanism of action, T-oligo pretreatment increased tumor cell sensitivity to radiation as demonstrated by the clonogenic assay (Figure
1). The present data suggest that combining T-oligos with low dose IR may permit safer and more effective radiotherapy of breast cancer and potentially other malignancies. T-oligo adjuvant therapy would thus be very beneficial to patients otherwise at risk of short-term and long-term adverse effects of IR, including radiation dermatitis, fibrosis, compromised wound healing, and secondary malignancies [
36]. T-oligos when applied alone are without detectable adverse effects on normal tissues after either local or systemic administration in multiple mouse models [
6,
8,
9,
11,
12] including the MMT mice. In accordance with this, we did not observe adverse effects in mice exposed to this agent and 3Gy IR including lethargy, anorexia, inactivity, ruffled fur coat or diarrhea.
The murine mammary tumor induced by PyMT shares many features with poor-prognosis human breast cancer such as a high frequency of distant metastases, persistent expression of biomarkers, ErbB2/Neu and cyclin D1, and loss of estrogen and progesterone receptor expression [
28]. In addition, the tumors develop in multiple stages amid a competent immune system, a trait also shared by human breast cancer [
22]. These advantages would appear to outweigh the greater individual variation in mammary tumor development in MMT mice versus mice bearing xenografts of established breast cancer cell lines. MMT mice thus provide a reliable model for the study of tumorigenesis in breast cancer as well as a useful tool for the evaluation of treatment modalities.