scovitinetreated embryos exhibited approximately 40% reduction in pituitary POMC eGFP expression compared with controls. A modest, approximately 20%, reduction of POMCeGFP expression was also observed in the olomoucine treated group, whereas PD 0332991 and CAY10572 caused no significant change in pituitary POMC eGFP expression compared Tie-2 with controls. To determine the specificity of R roscovitine action against zPttg overexpressing POMC cells, we generated another double transgenic line by breeding Tg:Pomc Pttg fish with a previously generated PRL RFP transgenic line, in which RFP was targeted to pituitary lactotrophs by a zebrafish Prolactin promoter. In vivo treatment between 18 and 48 hpf of Tg:Pomc Pttg,Prl RFP and Tg:Pomc Pttg,POMC eGFP embryos with R roscovitine revealed no effect on Prl RFP expression, but a greater than 50% reduction of POMC eGFP expression compared with control groups.
R Roscovitine Action in Mouse Corticotroph Tumor Cells. Olomoucine and roscovitine are structurally related 2,6,9 trisubstituted purines, which cause G1/S or G2/M arrest by competing for ATP binding sites on CDK1 and CDK2. The R isomer of roscovitine is a more potent and selective inhibitor of CDK2/cyclin E, and murine corticotrophs are highly sensitive to disrupted CDK2/cyclin E mediated cell cycle pathways. Cyclin E up regulation leads to cell cycle reentry of differentiated POMC cells and also inactivates p27kip1, further enhancing cell cycle progression. In addition, p27kip1 protects differentiated pituitary POMC cells from reentering the cell cycle, whereas p57Kip2 is required for cell cycle exit of pituitary precursor cells.
Given the in vivo potency of R roscovitine against zebrafish Pttg overexpressing corticotrophs, we studied its effect on CDK2/cyclin E mediated cell cycle pathways in mouse ACTH secreting pituitary tumor cells. Treatment with R roscovitine led to decreased cell number by 24 h. Western blot analysis of protein extracts derived from R roscovitine treated cells revealed evidence for cell cycle arrest, including decreased cyclin E, increased p27Kip1, p57Kip2, and p21Cip1 expression, as well as reduced Thr821 phosphorylation of Rb. R roscovitine treatment also induced senescent features by 48 h as evidenced by increased gal expression. Consistent with decreased cell viability, we detected decreased ACTH concentrations in culture medium derived from R roscovitine treated AtT20 cells.
Western blot analysis of protein extracts derived from R roscovitine treated AtT20 cells showed suppressed ACTH expression. These results indicate that R roscovitine targets cdk2/cyclin E mediated cell cycle progression, and also inhibits corticotroph ACTH protein expression. R Roscovitine Inhibits in Vivo Corticotroph Tumor Growth and ACTH Expression. To further establish R roscovitine action on corticotroph tumors in vivo, we injected athymic nude mice s.c. with AtT20 corticotroph tumor cells. Three days after tumor cell injection, 29 of 30 mice had developed small but visible s. kg or vehicle via oral gavage twice daily for 5 d each week. After 3 wk, R roscovitine caused approximately 50% weight reduction of dissected tumor xenografts. Consistent with the in vitro observations, Western blot and immunohistochemistry analysis of tumor specimens showe