Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1.

Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.

Functional interaction of human Cdc37 with the androgen receptor but not with the glucocorticoid receptor.

Cdc37 is a molecular chaperone closely associated with the folding of protein kinases. Results from studies using a yeast model system showed that it was also important for activation of the human androgen receptor (AR). Based on results from the yeast model system (Fliss, A. E., Fang, Y., Boschelli, F., and Caplan, A. J. (1997) Mol. Biol. Cell 8, 2501-2509), we initiated studies to address whether AR and Cdc37 interact with each other in animal cell systems. Our results show that Cdc37 binds to AR but not to glucocorticoid receptors (GR) synthesized in rabbit reticulocyte lysates. This binding occurs via the ligand-binding domain of the AR in a manner that is partially dependent on Hsp90 and the presence of hormone. Further studies using the yeast system showed that Cdc37 is not interchangeable with Hsp90, suggesting that it functions at a distinct step in the activation pathway. Expression of a dominant negative form of Cdc37 in animal cells down-regulates full-length AR but has very little effect on an AR truncation lacking the ligand-binding domain or full-length GR. These results reveal differences in the mechanisms by which AR and GR become active transcription factors and strengthen the notion that Cdc37 has a wider range of polypeptide clients than was realized previously.

Control of estrogen receptor ligand binding by Hsp90.

The molecular chaperone Hsp90 interacts with unliganded steroid hormone receptors and regulates their activity. We have analyzed the function of yeast and mammalian Hsp90 in regulating the ability of the human estrogen receptor (ER) to bind ligands in vivo and in vitro. Using the yeast system, we show that the ER expressed in several different hsp82 mutant strains binds reduced amounts of the synthetic estrogen diethylstilbestrol compared to the wild type. This defect in hormone binding occurs without any significant change in the steady state levels of ER protein. To analyze the role of mammalian Hsp90, we synthesized the human ER in rabbit reticulocyte lysates containing geldanamycin, an Hsp90 inhibitor. At low concentrations of geldanamycin we observed reduced levels of hormone binding by the ER. At higher concentrations, we found reduced synthesis of the receptor. These data indicate that Hsp90 functions to maintain the ER in a high affinity hormone-binding conformation.

Interaction of a self-emulsifying lipid drug delivery system with the everted rat intestinal mucosa as a function of droplet size and surface charge.

PURPOSE: To investigate the interaction of positively charged self-emulsifying oil formulations (SEOF) following aqueous dilution as a function of resulting emulsion droplet charge and size with rat everted intestinal mucosa, adherent mucus layer and Peyer's patches, using cyclosporine A (CsA) as a lipophilic model drug. METHODS: Droplet size determination (TEM technique) and zeta-potential measurements were used to characterize the resulting emulsions. For the ex vivo interaction study, the well-known rat intestine everted sac technique was used in combination with confocal microscopy. RESULTS: The positively charged oil droplets formed by SEOF dilutions at ratios of 1/50 and 1/10 elicited the stronger interaction with the mucosal surface. The positive charge of the smaller droplets was more readily neutralized, and even reversed in aqueous solutions containing moderate subphysiological mucin concentrations. Parameters such as droplet size, negativity of the epithelial mucosa potential and presence of the mucus layer on the epithelial surface affected drug mucosa uptake and the adhesion of the positively charged droplets to the rat intestinal mucosa. CONCLUSIONS: The enhanced electrostatic interactions of positively charged droplets with the mucosal surface are mostly responsible for the preferential uptake of CsA from the positively charged droplets as compared to negatively charged droplets irrespective of the experimental conditions used. The increased uptake of the CsA from the negatively charged oil droplets was consistent with the dilution extent, as expected, whereas in the positively charged droplets, an intermediate droplet size range was identified resulting in optimum drug uptake and clearly suggesting that drug uptake was not consistent with either dilution extent or droplet size.