FOXO3a mediates the androgen-dependent regulation of FLIP and contributes to TRAIL-induced apoptosis of LNCaP cells.

Androgen-withdrawal-induced apoptosis (AWIA) is deregulated in androgen refractory prostate cancer. Androgens have been shown to positively regulate expression of the antiapoptotic FADD-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (FLIP), and reduced FLIP expression precedes apoptosis after androgen withdrawal. Here, we show that FLIP protein expression is downregulated in castrated rats, while in LNCaP cells, androgens regulate FLIP in a manner that is dependent on phosphoinositol-3-kinase (PI3K) and Akt signaling. Specifically, treatment of LNCaP cells with LY294002, or expression of either PTEN or a non-phosphorylatable form of FOXO3a (FOXO3aTM), downregulates FLIP protein and mRNA. Conversely, treatment with androgens in the absence of PI3/Akt signaling, or following expression of FOXO3aTM, leads to increased FLIP expression. A FOXO3a binding site was identified in the FLIP promoter and shown necessary for the combined effects of androgens and FOXO3a on FLIP transcription. FOXO3a binds the androgen receptor, suggesting that the transcriptional synergy depends on an interaction between these proteins. Finally, LNCaP cells are sensitized to TRAIL-induced apoptosis by PTEN or LY294002, and rescued by androgens. FOXO3aTM also sensitizes cells to androgen-inhibited TRAIL apoptosis. Androgen rescue was diminished when either FOXO3a or FLIP was reduced by siRNA. These data support a role for FOXO3a in AWIA.

Common mutations in BRCA1 and BRCA2 do not contribute to early prostate cancer in Jewish men.

BACKGROUND: Families with a high incidence of hereditary breast cancer, and subsequently shown to have terminating mutations in BRCA1 or BRCA2, appear to have a higher incidence of prostate cancer among male relatives. We aimed to determine whether the common germline mutations of BRCA1 or BRCA2 in Ashkenazi Jewish men predisposed them to prostate cancer. METHODS: We examined genomic DNA from 83 (for BRCA1 185delAG) or 82 (for BRCA2 6174delT) Ashkenazi Jewish prostate cancer patients, most of whom were treated at a relatively young age, for the most common germline mutation in each gene seen in the Ashkenazi population. RESULTS: Our study should have been able to detect a 4-5-fold increase in the risk of prostate cancer due to mutation of BRCA1 or BRCA2. However, only one (1.15%; 95% confidence interval, 0-3.6%) of the patients was heterozygous for the BRCA1 mutant allele, and only two were heterozygous for the BRCA2 mutation (2.4%; 95% confidence interval, 0-6.2%). CONCLUSIONS: The incidence of each of the germline mutations in these prostate cancer patients closely matched their incidence (about 1%) in the general Ashkenazi Jewish population. This suggests that unlike cases of breast and ovarian cancers, mutations in BRCA1 or BRCA2 do not significantly predispose men to prostate cancer.

Convenient, nonradioactive, heteroduplex-based methods for identifying recurrent mutations in the BRCA1 and BRCA2 genes.

The ability to identify individuals who are predisposed to specific malignant tumors is a promising molecular diagnostic by-product of over two decades of intensive research into the genetic pathogenesis of human cancer. Approximately 2% of Ashkenazi Jews carry recurrent germline mutations in either the BRCA1 or BRCA2 genes that may predispose these individuals to the development of breast and ovarian cancer. We have developed a nonisotopic method, based on the formation of heteroduplexes between polymerase chain reaction (PCR) amplified wild-type and mutant alleles, which can be used to identify the BRCA1 185delAG and the BRCA2 6174delT mutations. The same assay can also be used to verify the loss of heterozygosity in a tumor sample arising in an individual with a germline mutation. The four steps described in this report (PCR amplification, heteroduplex formation, acrylamide gel electrophoresis, and ethidium bromide staining/UV-fluorescence photography) can be readily and reproducibly performed in the course of a single day, making this a useful method for the routine identification of these mutations.

Glutamate-dependent phosphorylation of elongation factor-2 and inhibition of protein synthesis in neurons.

Postischemic delayed neuronal death is attributed to excitotoxic activation of glutamate receptors. It is preceded by a persistent inhibition of protein synthesis, the molecular basis of which is not known. Here we have examined in cortical neurons in culture the regulation by glutamate of phosphorylation of eukaryotic elongation factor-2 (eEF-2) by eEF-2 kinase, a Ca2+/calmodulin-dependent enzyme. Using a phosphorylation state-specific antibody, we show that glutamate, which triggers a large influx of Ca2+, enhances dramatically the phosphorylation of eEF-2. On the basis of kinetic and pharmacological analysis, we demonstrate a close correlation among the increase in cytosolic Ca2+ concentration, the degree of eEF-2 phosphorylation, and the inhibition of protein synthesis. A 30 min treatment with NMDA induced a transient phosphorylation of eEF-2 and delayed neuronal death. However, pharmacological inhibition of protein translation was not neurotoxic by itself and protected neurons against the toxicity evoked by low concentrations of NMDA. Thus, phosphorylation of eEF-2 and the resulting depression of protein translation may have protective effects against excitotoxicity and open new perspectives for understanding long-term effects of glutamate.

The canary androgen receptor mRNA is localized in the song control nuclei of the brain and is rapidly regulated by testosterone.

Singing in canaries is an androgen-inducible behavior, under the control of an identified motor pathway, which includes several discrete "song nuclei" in the telencephalon. To determine whether the mRNA for the canary androgen receptor (cAR) is expressed in these song control nuclei, we synthesized probes from the recently cloned cAR cDNA and used in situ hybridization to examine spring male canary brain sections. Concentrations of cAR mRNA are detectable in several of the song control nuclei of the forebrain, including high vocal center (HVC), lateral magnocellular nucleus of the anterior neostriatum and robust nucleus of the archistriatum. In addition, we also show that testosterone treatment rapidly induces a significant reduction of cAR mRNA levels in nucleus HVC of females. Since the effects of androgen on singing behavior occur much more slowly, the behavioral effects are probably a secondary or independent result of androgen's primary and immediate action on target gene transcription.

Immediate-early gene responses in the avian song control system: cloning and expression analysis of the canary c-jun cDNA.

Previous studies have shown that song presentation results in a rapid rise in mRNA levels for the ZENK gene (the avian homologue of zif-268, Egr-1, NGFI-A, and Krox-24) in specific parts of the songbird forbrain. Metrazole-induced seizures also cause an increase in ZENK mRNA, even more widely throughout the telencephalon. Surprisingly, however, little or no ZENK induction by either stimulus was observed in several forebrain areas involved in auditory processing and song production. To learn whether this pattern of regulation is specific to ZENK, we examined the response of another 'immediate-early' gene, c-jun. Here we first describe the identification, cloning and sequence analysis of a canary cDNA encoding c-jun. Then, by in situ hybridization we show that c-jun is also induced by song or seizure, and in a pattern mostly similar to ZENK. As with ZENK, no induction of c-jun is observed in the androgen receptor-containing song nuclei or within the primary thalamo-recipient auditory area of the forebrain. Thus common immediate early gene responses appear to be selectively uncoupled from physiological activation in these specific forebrain regions, which are also characterized by tight developmental, hormonal and seasonal regulation.

Seasonal and tissue-specific regulation of canary androgen receptor messenger ribonucleic acid.

Natural seasonal fluctuations in androgen levels appear to cause changes in physiology and reproductive behavior, such as singing, in canaries. Little is known, however, about the cellular mechanisms underlying these changes. Because androgens act principally through nuclear receptors in other species, we have isolated and sequenced a cDNA likely to encode the canary androgen receptor and used this cDNA to examine the regulation of AR mRNA levels in the testis, kidney, and liver of the canary. The sequence corresponds to most of the coding portion of seven of the eight exons found in the homologous mammalian gene, including the domains that bind to DNA and androgen and affect transcription. Its mRNA is approximately 8 kilobases in length and is encoded by a single gene. In the testis, the transcript is expressed specifically in the Sertoli cells. The androgen receptor antagonist flutamide represses AR mRNA levels in kidney, but induces them in liver, indicating that androgen regulates its receptor, but does so in a tissue-specific manner, as is seen for the estrogen receptor in rodents. In addition, there are natural seasonal fluctuations in AR mRNA levels in testis and liver correlated with seasonal differences in the levels of circulating androgens. This is the first evidence of natural feedback regulation of AR mRNA levels.

The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator.

Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined.

Probes for rare mRNAs reveal distributed cell subsets in canary brain.

cDNA clones of 7 low-abundance canary brain RNAs hybridize in situ to different subsets of brain cells. Although these cell sets are distinct, they are dispersed in a variety of brain regions with overlapping anatomical distributions. These cDNA clones were initially selected by their relative hybridization to forebrain and rest-of-brain RNAs and represent a sampling of a much larger population of differentially expressed RNAs present at individual concentrations of 10(-7) to 10(-4) as a fraction of polyadenylated RNA mass. Our results suggest the existence of several thousand low-abundance brain mRNAs likely to be distributed in diverse and overlapping brain cell subsets. Furthermore, our experiments define a simple and general strategy for producing and analyzing molecular probes for subsets of brain cells and provide an initial set of useful reagents for further study of brain organization and development.

Long-term hyperalgesia induced by neonatal beta-endorphin and morphiceptin is blocked by neonatal Tyr-MIF-1.

Male rats were injected s.c. once daily during the first week of life with beta-endorphin (BE), morphiceptin, the antiopiate Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2), or one of the two opiate peptides in combination Tyr-MIF-1. Pups treated with neonatal BE removed their tails from a series of increasingly hot water baths significantly faster than controls on day 9, confirming our earlier studies. In addition, we found that Tyr-MIF-1 blocked this effect of BE. At 4.5 months, latency to lick a hindpaw in the hot-plate test was significantly faster in groups given BE alone, morphiceptin alone, or the control vehicle than in any of the 3 groups given Tyr-MIF-1. At 6 months the two groups given opiate peptides alone showed faster tail-flick latencies than the controls and the groups given Tyr-MIF-1. These results indicated that the long-term nociceptive changes induced by the opiate peptides were opposite to those induced by Tyr-MIF-1. Mean tail-flick latencies of the groups on day 9 correlated well with hot-plate and tail-flick scores in adulthood, indicating that the effects of the peptides were persistent. The neonatal peptide treatments did not differentially affect the analgesia induced by the stress of footshock or warm-water swim. Rats given either of the opiate peptides alone tended to fall off a rotorod faster than those in the other groups. These results support the role of Tyr-MIF-1 as an antiopiate and further illustrate the long-term effects of neonatally administered peptides.