T oligos have been shown to inhibit growth and induce apoptosis, autophagy Alisertib FDA and or senescence in human pancreatic, ovarian, breast cancer, melanoma, fibrosarcoma, and glioblastoma. Inhibitors,Modulators,Libraries Our data indicate that pretreatment of mammary tumor cells with T oligo but not a control oligo sensitizes the tumor cells to radiation in vitro and in an in vivo tumor model. Female C57BL 6 mice, six to eight weeks old, were pur chased from Taconic Farms. MMT mice were generated by breeding MUC1 trans genic mice with polyomavirus middle T oncogene expressing MT mice that develop spontaneous mammary carcinomas. Animals were maintained in microisolator cages under specific pathogen free conditions. The study of mice was approved by the Inhibitors,Modulators,Libraries Institutional Animal Care Inhibitors,Modulators,Libraries and Use Committee of Boston University Medical Center.

Oligonucleotides A 16 base phosphodiester linked oligonucleotide Inhibitors,Modulators,Libraries with 56% homology to the human telomere G rich sequence, and a con trol oligo were synthesized by the Midland Certified Reagent Company and resuspended in H20 to give a 2 mM stock solution. For the in vitro studies, the stock solution was diluted into culture med ium, and added to cells at a final concentration of 40 uM. In all experiments, cells were given medium con taining oligonucleotide once and not refed. For the in vivo studies, 2 mM of T oligo and control oligo were diluted in sterile PBS to make a 1. 2 mM concentration and 50 uL of this solution was injected into each mouse. Earlier studies of T oligos employed 100% homologs, establishing the efficacy for telomere homologs in comparison to inactive complementary and unrelated control sequences.

However, further work revealed that G rich oligos with substantial but less than 100% homology to telomeres were also effective in activation of the DNA damage signaling pathway Inhibitors,Modulators,Libraries leading to apop tosis of malignant cells and that some were even more effective than the same length 100% homologs. One of these 16 base T oligos was selected for the present studies. Cell yield and counting Primary mammary tumor cells from MMT mice were harvested and cultured in Dulbeccos Modified Eagles Medium with 10% heat inactivated fetal calf serum, 2 mM L glutamine, 100 U ml penicillin and 100 ug ml streptomycin. A triplicate set of cultured cells was pre treated with T oligo or control oligo at a final concentration of 0, 10, 20, 30 or 40 uM in DMEM for 24 hours, and then irradiated with 0, 3, 6, 9, or 12Gy.

The cells were trypsinized and collected at 0, 24, 48, 72 and 96 hours after irradiation for cell count using a cell counter. Mammary tumor cells were trypsinized to a single cell suspension and seeded into 10 inhibitor Imatinib cm tissue culture dishes. After the cells were treated with 40 uM T oligo or control oligo for 24 hours, they were irradiated at different dose levels and placed thereafter in an incubator until cells in the control groups formed multiple large clones.