The mammalian target of rapamycin (mTOR) kinase forms two multiprotein complexes, mTORC1 and mTORC2, which regulate cell growth, cell survival, and autophagy. Allosteric inhibitors of mTORC1, such as rapamycin, have been extensively used to study tumor cell growth, proliferation, and autophagy but have shown only limited clinical utility. Here, we describe AZD8055, a novel ATP-competitive inhibitor of mTOR kinase activity, with an IC 50 of 0.8 nmol/L. AZD8055 showed excellent selectivity (∼1,000-fold) against all class I phosphatidylinositol 3-kinase (PI3K) isoforms and other members of the PI3K-like kinase family. Furthermore, there was no significant activity against a panel of 260 kinases at concentrations up to 10 μmol/L. AZD8055 inhibits the phosphorylation of mTORC1 substrates p70S6K and 4E-BP1 as well as phosphorylation of the mTORC2 substrate AKT and downstream proteins. The rapamycin-resistant T37/46 phosphorylation sites on 4E-BP1 were fully inhibited by AZD8055, resulting in significant inhibition of cap-dependent translation. In vitro, AZD8055 potently inhibits proliferation and induces autophagy in H838 and A549 cells. In vivo, AZD8055 induces a dose-dependent pharmacodynamic effect on phosphorylated S6 and phosphorylated AKT at plasma concentrations leading to tumor growth inhibition. Notably, AZD8055 results in significant growth inhibition and/or regression in xenografts, representing a broad range of human tumor types. AZD8055 is currently in phase I clinical trials. Cancer Res; 70(1); 288-98. ©2010 AACR.

Steroid hormones regulate cell function via specific receptors, members of a super family of ligand activated transcription factors, expressed in their target tissues. A second oestrogen receptor (ER beta) has recently been shown by RT-PCR to have a wide tissue distribution distinct from that of oestrogen receptor alpha (ER alpha). We have raised a polyclonal antiserum using a peptide specific for ER beta in order to determine the cellular sites of expression of the receptor. In the adult rat ER beta was localised to cell nuclei in a wide range of tissues including ovary, oviduct, uterus, lung, adrenal, seminal vesicle, bladder, heart, prostate and testis. In the ovary ER beta was present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea whereas ER alpha was undetectable in these cell types. In the uterus ER beta and ER alpha were both present in epithelial cells lining the lumen and glands. In the lung ER beta was present in the cells lining the bronchioles and alveoli as well as in smooth muscle. In bladder and seminal vesicle immunostaining was intense in epithelial cells but the receptor was also expressed in nuclei of smooth muscle cells. Cell nuclei of the heart ventricle were immunopositive for ER beta as were most cells of the adult rat adrenal. In the seminiferous epithelium of the testis, nuclei of Sertoli cells were immunopositive but expression was not stage dependent. In conclusion, immunohistochemistry has proved invaluable in visualising specific sites of expression of ER beta in complex tissues including those of the reproductive tract.

In a subset of infertile men, a spectrum of spermatogenic defects ranging from a complete absence of germ cells (sertoli cell only) to oligozoospermia is associated with microdeletions of the DAZ (deleted in azoospermia) gene cluster on human distal Yq. DAZ encodes a testis-specific protein with RNA-binding potential recently derived from a single-copy gene DAZL1 (DAZ-like) on chromosome 3. Y chromosomal DAZ homologues are confined to humans and higher primates. It remains unclear which function unique to higher primate spermatogenesis DAZ may serve, and the functional status of the gene recently has been questioned. To assess the extent of functional conservation we have tested the capacity of a human DAZ gene contained in a 225-kb yeast artificial chromosome to complement the sterile phenotype of the Dazl null mouse (Dazl ؊͞؊ ), which is characterized by severe germ-cell depletion and meiotic failure. Although Dazl ؊͞؊ mice remained infertile when the DAZ transgene was introduced, histological examination revealed a partial and variable rescue of the mutant phenotype, manifest as a pronounced increase in the germ cell population of the seminiferous tubules and survival to the pachytene stage of meiosis. As well as constituting definitive proof of the spermatogenic role of the DAZ gene product, these findings confirm the high degree of functional conservation between the DAZ and DAZL1 genes, suggesting they may constitute a single target for contraceptive intervention and raising the possibility of therapeutic up-regulation of the DAZL1 gene in infertile men.