Model cells cells mixed with an equal volume of Matrigel were implanted sub cutaneously into the flanks of six to seven week old female nude mice as described. For MCF7 HER2 xenografts, nude mice were also Sunitinib VEGFR subcutaneously implanted with Inhibitors,Modulators,Libraries estrogen pellets, whereas nude mice with MCF7 TamR and MCF7 LTLTca xenografts were given subcutaneous injection of tamoxifen or 4 androstenedione, respectively, as described. Roscovitine treatment was initiated after three weeks of inoculation when tumors reached measurable size. Roscovitine suspension was prepared in 50 mM HCl solution as described earlier and admi nistered orally at a dose of 100 mg kg body weight for 10 consecutive days. Tumor volumes were measured with a vernier caliper at weekly intervals.

After the 25th day, the mice were euthanized, and the tumors were removed, weighed Inhibitors,Modulators,Libraries and processed for immunohistochemistry staining. Tumor volume calculation was performed using modified ellipsoidal formula, tumor volume 1 2, where Inhibitors,Modulators,Libraries L is the longitudinal diameter and W is the transverse diameter. Body weight was measured at weekly intervals to rule out the drug toxicity. Student Inhibitors,Modulators,Libraries t test was used to assess the statistical difference between control and roscovitine treated groups. The level of significance was set at P 0. 05. Immunohistochemistry and TUNEL assay IHC analysis was performed as described. The dilu tion of the PCNA antibody used for IHC was 1 in 100. After washing with 1 �� PBST, slides were further incu bated in DAKO universal secondary LINK solution for 30 minutes and horseradish peroxidase labeled streptavi din for 20 minutes at room temperature.

Successful staining was visualized using the DAB substrate, followed by counterstaining Inhibitors,Modulators,Libraries using Mayers hematoxylin. TUNEL staining was performed following manufacturers instructions. Results Roscovitine suppresses proliferation and survival of therapy resistant cells To examine whether deregulation of CDK2 signaling occurs in therapy resistant cells, we initially determined the status of CDK2 activation in therapy resistant model cells that exhibit therapy resistance to AE and AI. CDK2 acti vation was determined by measuring the levels of phos pho CDK2 by using western blot analysis. Phospho CDK2 levels Nilotinib FDA were enhanced in all three ther apy resistant model cells but not in the hormone sensi tive breast cancer cells such as MCF7 and ZR 75 1. We then tested whether roscovitine sup presses the growth of therapy resistant cells by exposing them to increasing concentrations of roscovitine and determined their rate of proliferation using a lumines cence based cell proliferation assay. Hormonal therapy sensitive MCF7 cells were used as a positive control and these cells had a dose dependent reduction in cell prolif eration.