Molecular genetic profiling of Gleason grade 4/5 prostate cancers compared to benign prostatic hyperplasia.

PURPOSE: Because Gleason grade 4/5 cancer is the primary cause of failure to cure prostate cancer, we examined the molecular profiles of this high grade cancer in search of potentially new therapeutic interventions as well as better serum markers than prostate specific antigen. MATERIALS AND METHODS: We compared the gene expressions in fresh frozen tissues from 9 men with Gleason grade 4/5 cancer to 8 men with benign prostatic hyperplasia (BPH) treated with radical retropubic prostatectomy. Labeled complementary RNA from each of the 17 tissues was applied to HuGeneFL probe arrays representing approximately 6,800 genes (Affymetrix, Inc., Santa Clara, California). After removing all genes undetectable in BPH and grade 4/5 cancers, and transforming the data into a parametric distribution, we chose only those up and down-regulated genes with a p difference in fluorescence between grade 4/5 cancer and BPH of p <0.0005. This value reduced the data set to 40 up-regulated and 111 down-regulated genes. We then eliminated all genes that were not expressed in all 8 BPH and 9 grade 4/5 tissues, which produced a final set of 86 genes, of which 22 were up-regulated and 64 were down-regulated. RESULTS: Cluster analysis cleanly separated men with grade 4/5 cancers from those with BPH. Only 17 of the 86 candidate genes (20%) were known to be prostate cancer related and 42 (49%) were related to other cancers. The most up-regulated gene is Hepsin, a trypsin-like serine protease with its enzyme catalytic domain oriented extracellularly. Prostate specific membrane antigen is the second most up-regulated gene (all other reports on prostate specific membrane antigen have been at the protein level). The genes for prostate specific antigen (hK3) and human glandular kallikrein2 (hK2) showed equivalent expression levels 10 times the average of other genes. Complete lists of all 22 up-regulated genes and 64 down-regulated genes, together with their locus on the chromosome, are presented in rank order. CONCLUSIONS: We characterize for the first time 64 down-regulated and 22 up-regulated genes in Gleason grade 4/5 cancer, using the gene profile from BPH as control tissue. A number of interesting new genes, previously undescribed in prostate cancer, are presented as possibilities for further study.

Enhanced sensitivity and long-term G2 arrest in hydrogen peroxide-treated Ku80-null cells are unrelated to DNA repair defects.

While the Ku complex, comprised of Ku70 and Ku80, is primarily involved in the repair of DNA double-strand breaks, it is also believed to participate in additional cellular processes. Here, treatment of embryo fibroblasts (MEFs) derived from either wild-type or Ku80-null (Ku80(-/-)) mice with various stress agents revealed that hydrogen peroxide (H(2)O(2)) was markedly more cytotoxic for Ku80(-/-) MEFs and led to their long-term accumulation in the G2 phase. This differential response was not due to differences in DNA repair, since H(2)O(2)-triggered DNA damage was repaired with comparable efficiency in both Wt and Ku80(-/-) MEFs, but was associated with differences in the expression of important cell cycle regulatory genes. Our results support the notion that Ku80-mediated cytoprotection and G2-progression are not only dependent on the cell's DNA repair but also may reflect Ku80's influence on additional cellular processes such as gene expression.

Safety and immunogenicity profile of a recombinant outer-surface protein A Lyme disease vaccine: clinical trial of a 3-dose schedule at 0, 1, and 2 months.

OBJECTIVES: This study compared the tolerability of a Lyme disease vaccine administered intramuscularly at 0 and 1 months with that of a vaccine administered at 0, 1, and 2 months to determine (1) whether adding a third dose of vaccine 1 month after the second would affect the safety profile, and (2) whether a shortened vaccination schedule of 0, 1, and 2 months would provide an immune response similar to that obtained with vaccine administered at 0, 1, and 12 months. BACKGROUND: An efficacy trial of a Lyme disease vaccine had demonstrated safety and efficacy against definite (clinically manifested and laboratory-confirmed) Lyme disease after 3 doses at 0, 1, and 12 months and resulted in 90% of subjects having titers > or =1400 enzyme-linked immunosorbent assay units (EL.U)/mL (the proposed seroprotective level for 1 tick season). METHODS: This multicenter, open-label, prospective, randomized study assessed the safety and efficacy of different doses of a recombinant outer-surface protein A (OspA) vaccine in 956 volunteers aged 17 to 72 years from 3 Lyme disease-endemic sites. Blood samples were collected at months 0, 2, 3, 12, and 13 to assess total immunoglobulin-G anti-OspA titers. RESULTS: Most adverse events were transient and mild to moderate. The geometric mean antibody titer increased 2.8-fold from month 2 (1786 EL.U/mL to 4842 EL.U/mL), and approximately 90% of the volunteers had a titer > or =1400 and 99% had a titer > or =400 EL.U/mL (the mini- mum seroprotective level at any given time) after the third dose. An antibody kinetics model predicts that protection would last for a typical tick-transmission season. CONCLUSIONS: In volunteers aged 17 to 72 years, 3 doses of vaccine administered in 2 months was well tolerated, more immunogenic than 2 doses, and provided a higher probability of protection before exposure or travel to Lyme disease-endemic areas.

HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation.

Colorectal carcinoma RKO cells expressing reduced levels of the RNA-binding protein HuR (ASHuR) displayed markedly reduced growth. In synchronous RKO populations, HuR was almost exclusively nuclear during early G(1), increasing in the cytoplasm during late G(1), S and G(2). The expression and half-life of mRNAs encoding cyclins A and B1 similarly increased during S and G(2), then declined, indicating that mRNA stabilization contributed to their cell cycle-regulated expression. In gel-shift assays using radiolabeled cyclin RNA transcripts and RKO protein extracts, only those transcripts corresponding to the 3'-untranslated regions of cyclins A and B1 formed RNA-protein complexes in a cell cycle-dependent fashion. HuR directly bound mRNAs encoding cyclins A and B1, as anti-HuR antibodies supershifted such RNA-protein complexes. Importantly, the expression and half-life of mRNAs encoding cyclins A and B1 were reduced in ASHuR RKO cells. Our results indicate that HuR may play a critical role in cell proliferation, at least in part by mediating cell cycle-dependent stabilization of mRNAs encoding cyclins A and B1.

HuR regulates p21 mRNA stabilization by UV light.

Expression of the cyclin-dependent kinase inhibitor p21 is highly induced by many stresses, including exposure to short-wavelength UV light (UVC), which increases p21 mRNA stability. Investigation into the mechanisms underlying this stabilization process revealed that proteins present in cytoplasmic lysates of human RKO colorectal carcinoma cells formed complexes with p21 mRNA that were inducible by treatment with UVC and other stress agents. The ubiquitous Elav-type RNA-binding protein HuR was identified within the p21 mRNA-protein complexes, as antibodies recognizing HuR supershifted these complexes and revealed HuR-immunoreactive proteins complexing with p21 mRNA on Western blots. Lowering of endogenous HuR levels through expression of antisense HuR decreased p21 RNA-protein complexes, greatly reduced the UVC inducibility and half-life of p21 mRNA, and prevented UVC-mediated induction of luciferase activity in p21 3' untranslated region-containing reporter constructs. Our findings indicate that HuR plays a major role in regulating stress-induced p21 expression by enhancing p21 mRNA stability and that these effects are coupled to HuR's elevated presence in the cytoplasm.

Expression of cyclin E or DP/E2F rescues the G1 arrest of trol mutant neuroblasts in the Drosophila larval central nervous system.

The trol locus of Drosophila regulates the timing of neuroblast proliferation. In trol mutants, quiescent neuroblasts fail to begin division. We have investigated this cell cycle arrest to examine trol function. Induced expression of cyclin E or DP/E2F in trol mutants results in normal levels of dividing neuroblasts, while cyclin B expression has no effect. cyclin E expression is lower in the trol mutant larval CNS as assayed by quantitative RT-PCR, suggesting that trol neuroblasts are arrested in G1 due to lack of Cyclin E. Neither cyclin E nor E2F expression can phenocopy ana mutations, indicating that arrest caused by lack of Trol is different from Ana-mediated arrest.

Mutation of the central nervous system neuroblast proliferation repressor ana leads to defects in larval olfactory behavior.

In the developing nervous system, interactions between glia and immature neurons or neuroblasts regulate axon pathfinding, migration, and cell division, and therefore affect structure and function. Glial control of neuroblast cell division has been documented by studies of the anachronism (ana) gene of Drosophila melanogaster. ana encodes a glycoprotein which, in the developing larval central nervous system, is secreted by glia that neighbor regulated neuroblasts. Mutations in ana lead to premature neuroblast proliferation in the larval brain. Examination of lacZ expression from an ana enhancer trap line as well as detection of the ana protein show that ana is also expressed in the larval antennal-maxillary complex (AMC) at all larval stages. As previously reported for the central nervous system, ana expression in the AMC appears to be confined to glial cells. Larval olfactory system function in ana mutants was assayed in a behavioral paradigm. When tested with the three different chemoattractants, third instar ana9 mutant larvae showed diminished olfactory response compared to controls. Examination of a second ana allele revealed aberrant olfactory response to ethyl acetate, demonstrating that more than one mutation in ana can give rise to abnormal larval olfactory behavior. Assays of early first instar ana9 mutant larvae revealed defective olfactory behavior, implying that the olfactory phenotype stems from early larval AMC and/or embryonic origins. This is consistent with proliferation analysis in the early larval AMC region which uncovered a significantly higher number of S-phase cells in ana9 mutants.

Lobomycosis as a disease of the Atlantic bottle-nosed dolphin (Tursiops truncatus Montagu, 1821).

Skin lesions on an Atlantic bottlenosed dolphin, captured off the coast of Florida, were investigated and found to be histologically and microbiologically indistinguishable from those caused in humans by Loboa loboi. All attempts to isolate the etiologic agent or to transmit the infection to mice and monkeys ended in failure. Sight records of other suspected dolphin cases of lobomycosis in Florida waters are described along with citations of two previously confirmed and published dolphin infections.

Vocalization of naive captive dolphins in small groups.

Pure-tone whistles (2403) by four individual dolphins (Delphinus delphis bairdi) were analyzed for duration and the elapse of time before either response by another animal or a repeat whistle by the same animal. Only five major types of whistle emissions were recorded, all stereotyped and each characteristic of the animal emitting it. Only one of the four animals emitted two different whistles, one of which was rare and both of which were stereotyped. A pure-tone chirp and pulsed sounds are discussed. We found no evidence of a dolphin "language," but we present evidence of social response to acoustic signals.