Cellular interactions of the phosphorylated form of AKT in prostate cancer.

Phospho-Akt (P-Akt1) promotes proliferation and increased survival in vitro and plays an important role in prostate cancer (PCa) progression as well as the prediction of the probability of recurrence. In this study, the goal was to demonstrate the involvement and impact of P-Akt1 on cellular interactions, biomechanisms, and pathways in PCa. Tissue microarrays from 640 PCa patients were immunostained with various antibodies. Ki-67 was used to measure proliferation index, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling was used for apoptotic index. Increased expression of P-Akt1 was associated with an increased proliferation but inversely correlated with apoptotic index. Higher levels of P-Akt1 are associated with both higher levels of cytoplasmic p27 and higher levels of nuclear p27, suggesting an involvement in both cytoplasmic entrapment and phosphorylation of p27. P-Akt1 expression significantly correlated with nuclear and cytoplasmic staining of FHKR and GSK. The strongest correlations were found with the P- forms of both, suggesting enzyme kinetics in the latter. Here, phosphorylation is the principal method of FHKR and GSK inactivation. P-Akt1 correlated with nuclear transcription factor kappa B, suggesting a role in the inhibition through phosphorylation of nuclear transcription factor kappa B. The results of the current study are unique because of the scope of the markers and the size of the population used. In vitro- and in vivo-derived information of P-Akt1 and its downstream effectors demonstrates significant involvement in PCa. Our data suggest that PCa uses multiple mechanisms to regulate this pathway and substantiate the concept of redundancy in cancer pathway regulation. Consequently, new hypothesis-driven studies can be derived from this information.

Measurement of effector protein injection by type III and type IV secretion systems by using a 13-residue phosphorylatable glycogen synthase kinase tag.

Numerous bacterial pathogens use type III secretion systems (T3SSs) or T4SSs to inject or translocate virulence proteins into eukaryotic cells. Several different reporter systems have been developed to measure the translocation of these proteins. In this study, a peptide tag-based reporter system was developed and used to monitor the injection of T3S and T4S substrates. The glycogen synthase kinase (GSK) tag is a 13-residue phosphorylatable peptide tag derived from the human GSK-3beta kinase. Translocation of a GSK-tagged protein into a eukaryotic cell results in host cell protein kinase-dependent phosphorylation of the tag, which can be detected with phosphospecific GSK-3beta antibodies. A series of expression plasmids encoding Yop-GSK fusion proteins were constructed to evaluate the ability of the GSK tag to measure the injection of Yops by the Yersinia pestis T3SS. GSK-tagged YopE, YopH, LcrQ, YopK, YopN, and YopJ were efficiently phosphorylated when translocated into HeLa cells. Similarly, the injection of GSK-CagA by the Helicobacter pylori T4SS into different cell types was measured via phosphorylation of the GSK tag. The GSK tag provides a simple method to monitor the translocation of T3S and T4S substrates.

Uterine response to estradiol under action of chorionic gonadotropin in mice.

This work examined the effect of chorionic gonadotropin on proliferative and morphogenetic reactions in the uterus under short- and long-term estrogen treatments. Ovariectomized mice received a single injection with estradiol dipropionate (2 micro g per 100 g; subcutaneously, sc) or vehicle and injections with human chorionic gonadotropin (10 IU per 100 g; sc) or vehicle twice a day for 2 days. Other groups of animals received injections with estradiol once a week or vehicle and injections with chorionic gonadotropin or vehicle once a day for 30 days. The uteri were removed 48 h after the last estradiol or vehicle injection. In animals treated with estradiol and chorionic gonadotropin for a month, the incidence of atypical endometrial hyperplasia was significantly higher. In animals treated with estradiol and chorionic gonadotropin for 2 days or for a month, uterine mass was slightly increased, the number of mitotic cells and BrdU-labeled cells was greater in luminal epithelium, glandular epithelium, stromal and myometrial cells, whereas the expression of estrogen receptors-alpha was lower in all uterine compartments, than in control. In mice who received estradiol and chorionic gonadotropin for 2 days, levels of beta-catenin and glycogen synthase kinase-3beta in luminal and glandular epithelia were lower. In animals treated with estradiol and chorionic gonadotropin for a month, the level of beta-catenin was slightly higher, and the expression of glycogen synthase kinase-3beta was lower in luminal and glandular epithelia. Thus, chorionic gonadotropin exerts estradiol-induced proliferative and morphogenetic changes in the uterus. This action of chorionic gonadotropin is associated with decreased expression of estrogen receptors-alpha and with changes in expression of beta-catenin and glycogen synthase kinase-3beta in the uterus.