Although a causal role of genetic alterations in human cancer is well established, it is still unclear whether dietary fat can modulate cancer risk in a predisposed population. Epidemiological studies suggest that diets rich in omega-3 polyunsaturated fatty acids reduce cancer incidence. To determine the influence of fatty acids on prostate cancer risk in animals with a defined genetic lesion, we used prostate-specific Pten-knockout mice, an immune-competent, orthotopic prostate cancer model, and diets with defined polyunsaturated fatty acid levels. We found that omega-3 fatty acids reduced prostate tumor growth, slowed histopathological progression, and increased survival, whereas omega-6 fatty acids had opposite effects. Introducing an omega-3 desaturase, which converts omega-6 to omega-3 fatty acids, into the Pten-knockout mice reduced tumor growth similarly to the omega-3 diet. Tumors from mice on the omega-3 diet had lower proportions of phosphorylated Bad and higher apoptotic indexes compared with those from mice on omega-6 diet. Knockdown of Bad eliminated omega-3-induced cell death, and introduction of exogenous Bad restored the sensitivity to omega-3 fatty acids. Our data suggest that modulation of prostate cancer development by polyunsaturated fatty acids is mediated in part through Bad-dependent apoptosis. This study highlights the importance of gene-diet interactions in prostate cancer.

To delineate the DCC-induced apoptotic pathway, we have identified a protein, DIP13␣, which interacts with DCC. The DIP13␣ protein has a pleckstrin homology domain and a phosphotyrosine binding domain. It interacts with a region on the DCC cytoplasmic domain that is required for the induction of apoptosis. Although ectopic expression of DIP13␣ alone causes only a slight increase in apoptosis, co-expression of DCC and DIP13␣ results in an ϳ5-fold increase in apoptosis. Removal of the DCC-interacting domain on DIP13␣ abolishes its ability to enhance DCC-induced apoptosis. Inhibition of endogenous DIP13␣ expression by small interfering RNA blocks DCC-induced apoptosis. Our data suggest that DIP13␣ is a mediator of the DCC apoptotic pathway.The candidate tumor-suppressor gene deleted in colorectal cancer (DCC) 1 was first cloned from a locus on chromosome arm 18q where allelic deletions occur in over 70% of primary colorectal tumors (1). Since that time, loss of heterozygosity at the DCC locus and loss of DCC expression have been shown in many other tumor types (2) including prostate carcinomas (3). The loss of DCC expression has therefore been associated with tumor progression. Although the known tumor suppressor gene MADH4/DPC4/Smad4 is mapped in close proximity to the DCC locus (4, 5), re-evaluation of loss of heterozygosity at 18q in colon tumor samples indicated that DCC, but not Smad4, was the most frequently altered gene on chromosome 18q13.3-21.3 (6). In a study of 115 pancreatic and 14 biliary cancers for homozygous deletions of DCC exons and flanking 18q regions, seven homozygous deletions were seen in the region that includes the DCC gene. In fact, DCC was the only known gene affected by all seven deletions. In two tumors, the deletions inactivate DCC but not Smad4 (7). These loss of heterozygosity and mutational data support the hypothesis that DCC acts as a tumor suppressor.DCC encodes a type I membrane protein that falls into a subgroup of the immunoglobulin superfamily (8). We and others have shown that DCC may exert its tumor-suppression function through induction of apoptosis (9, 10). DCC and its orthologs, UNC-40 in Caenorhabditis elegans and frazzled in Drosophila, have been established as receptors for netrin-1 and play an important role in axon outgrowth and cell migration in the developing nervous system (11)(12)(13)(14). It has been shown that induction of apoptosis by DCC can be blocked by netrin-1 (10). Expression of DCC colocalizes with areas of apoptotic precerebellar neurons in netrin-1 Ϫ/Ϫ mice, whereas apoptosis is absent in the same DCC-expressing areas in wild-type mice (15). Therefore DCC may induce cell death in settings where ligand is unavailable. Consistent with this view, netrin-1 knockout mice grow fewer cells particularly in the developing brainstem (16, 17).Very little is known about the apoptotic signaling of DCC. Unlike the other well characterized receptors such as Fas and tumor necrosis factor receptor, no apparent death domain can be identified in the DCC cytoplasmic r...

Genetically engineered mice are being used increasingly for delineating the molecular mechanisms of prostate cancer development. Epithelium-stroma interactions play a critical role in prostate development and tumorigenesis. To better understand gene expression patterns in the normal sexually mature mouse prostate, epithelium and stroma were laser-capture microdissected from ventral, dorsolateral, and anterior prostate lobes. Genome-wide expression was measured by DNA microarrays. Our analysis indicated that the gene expression pattern in the mouse dorsolateral lobe was closest to that of the human prostate peripheral zone, supporting the hypothesis that these prostate compartments are functionally equivalent. Stroma from a given lobe had closer gene expression patterns with stroma from other lobes than epithelium from the same lobe. Stroma appeared to have higher expression complexity than epithelium. Specifically, stromal cells had higher expression levels of genes implicated in cell adhesion, muscle development, and contraction, in structural constituents of cytoskeleton and actin binding, and in components such as sarcomere and extracellular matrix collagen. Among the genes that were enriched in the epithelium were secretory proteins, including seminal vesicle protein secretion 2 and 5. Surprisingly, prostate stroma expressed many osteogenic molecules, as confirmed by immunohistochemistry. A "bone-like" environment in the prostate may predispose prostate cells for survival in the bone. Chemokine Cxcl12 but not its receptor, Cxcr4, was expressed in normal prostate. In prostate tumors, interestingly, Cxcl12 was up-regulated in epithelial cells with a concomitant expression of Cxcr4. Expression of both the receptor and ligand may provide an autocrine mechanism for tumor cell migration and invasion.Prostate cancer is a major health issue in the United States. Genetically engineered mice are an important model for studying the molecular mechanisms of prostate tumorigenesis (1). The usefulness of mice to model human prostate cancer is well accepted (1, 2). However, gene expression in mouse prostate has not been well characterized. More importantly, expression differences between the mouse prostate epithelium and stroma are largely unknown, despite the critical role of stromal-epithelial interactions in the development of normal prostate and prostate cancer. The mouse prostate consists of three distinct lobes (anterior, dorsolateral, and ventral), which differs anatomically from the human prostate (a single lobe with peripheral, transition, and central zones (3)).Gene expression profiling allows for global assessment of gene expression patterns and identification of differentially expressed genes. In human prostate cancer cells, differentially expressed genes have been analyzed by SAGE (serial analysis of gene expression) (4), expressed sequence tag library analysis (5), and DNA microarrays (6 -11). Some of the identified genes have been further characterized (12-15). Overall, this approach has identified many candi...

Human adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 (APPL1) and adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 2 (APPL2) are homologous effectors of the small guanosine triphosphatase RAB5 that interact with a diverse set of receptors and signaling proteins and are proposed to function in endosome-mediated signaling. Herein, we investigated the membrane targeting properties of the APPL1 and APPL2 Bin/ Amphiphysin/Rvs (BAR), pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains. Coimmunopre-cipitation and yeast two-hybrid studies demonstrated that full-length APPL proteins formed homooligomers and heterooligomers and that the APPL minimal BAR domains were necessary and sufficient for mediating APPL-APPL interactions. When fused to a fluorescent protein and overexpressed, all three domains (minimal BAR, PH and PTB) were targeted to cell membranes. Furthermore, full-length APPL proteins bound to phosphoinositides, and the APPL isolated PH or PTB domains were sufficient for in vitro phosphoinositide binding. Live cell imaging showed that full-length APPL-yellow fluorescent protein (YFP) fusion proteins associated with cytosolic membrane structures that underwent movement, fusion and fission events. Overexpression of full-length APPL-YFP fusion proteins was sufficient to recruit endogenous RAB5 to enlarged APPL-associated membrane structures, although APPL1 was not necessary for RAB5 membrane targeting. Taken together, our findings suggest a role for APPL proteins as dynamic scaffolds that modulate RAB5-associated signaling endosomal membranes by their ability to undergo domain-mediated oligomerization, membrane targeting and phosphoinositide binding. Human APPL1 (adaptor protein, phosphotyrosine interaction , PH domain and leucine zipper containing 1)/APPL (adaptor protein containing PH domain, PTB domain and Leucine zipper motif)/DIP13a (DCC-interacting protein 13a) and APPL2/DIP13b (referred to herein as APPL1 and APPL2, respectively) are two highly homologous proteins that contain three domains: an N-terminal Bin/Amphiphysin/ Rvs (BAR) domain, a central pleckstrin homology (PH) domain and a C-terminal phosphotyrosine binding (PTB) domain. APPL1 interacts with a diverse set of receptors, including the netrin-1 receptor DCC (deleted in colorectal carcinoma) (1), the nerve growth factor (NGF) receptor TrkA (2,3), the follicle-stimulating hormone (FSH) receptor (FSHR) (4,5) and the AdipoR1 and AdipoR2 adiponectin receptors (6,7). APPL1 also associates with signaling proteins, including AKT (v-akt murine thymoma viral oncogene homolog) proteins (5,8,9), phosphatidylinositol 3-kinase (PI3K) subu-nits (8) and the OCRL (oculocerebrorenal syndrome of Lowe) and INPP5B (inositol polyphosphate-5-phosphatase, 75 kDa) phosphatidylinositol 5-phosphatases (10). Many of these interactions are mediated by the APPL1 PTB domain, suggesting that APPL1 may function as an adaptor linked to distinct signaling pathways. APPL2 was originally iden...

Examination of a group of mutants of plasmid NR1 that had lost the expression of IncFII plasmid incompatibility (Inc-) revealed a group that had also lost replication proficiency (Rep-). These mutants were obtained from plasmids in which the NR1 replication control region was present in a cointegrate with plasmid pBR322. Whereas the wild-type parental cointegrate plasmid was capable of replicating in a polA host owing to the PolA independence of NR1 replication, the mutants were not able to transform a polA host. Losses of both expression of IncFII plasmid incompatibility and replication proficiency were found to result from the same single base-pair substitution in four independently isolated Inc- Rep- mutants. The mutation inactivates promoter PE for the transcription of RNA-E, a trans-acting repressor of translation of the essential RepA1 replication initiation protein of NR1. Although the loss of RNA-E synthesis had been expected to increase the expression of repA1, the efficiency of translation of repA1 mRNA from these mutants was at least 100-fold lower than that from the wild type, as revealed by repA1-lacZ translational fusions. The PE mutation introduced a stop codon into a 24-amino-acid reading frame that precedes the repA1 gene and terminates just 2 bp downstream from the repA1 start codon. This putative leader peptide was also expressed in a lacZ translational fusion, and its expression was reduced by a factor of 10(4) by the PE mutation. The expression of the leader peptide and the expression of repA1 were regulated by RNA-E. These results suggest that the expression of repA1 is coupled to the translation of the leader peptide and that the repression of repA1 translation by RNA-E may occur via inhibition of the translation of the leader peptide.

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Replication-proficient (Rep') revertants were isolated from mutants of IncFll plasmid NR1 that were replication defective (Rep-). The parental Rep-plasmids contained a mutation that inactivated promoter PE for transcription of RNA-E, a trans-acting repressor of translation of the essential RepAl replication initiation protein of NR1. The PE mutation also introduced a nonsense codon into a leader peptide gene that precedes and slightly overlaps the repAl translation initiation site in the mRNA. This reduced the rate of synthesis of RepAl by uncoupling its translation from that of the leader peptide. The reduced rate of RepAl synthesis was responsible for the Rep-phenotype. All Rep' revertants retained the PE mutation and contained second-site mutations responsible for suppression of the Rep-phenotype. One Rep+ revertant contained a second mutation adjacent to the Shine-Dalgarno sequence of repAl. Another Rep+ revertant contained a mutation in the repA2 gene, which encodes the trans-acting repressor of transcription of repAl. By using translational JacZ gene fusions, it was found that both kinds of suppressor mutation increased the expression of repAl to a level sufficient to support replication. In both cases, the synthesis of RepAl remained uncoupled from that of the leader peptide. The Shine-Dalgarno mutation increased the rate of leader peptide-independent translation of repAl mRNA and also reduced the sensitivity of repAl mRNA to inhibition by RNA-E. The repA2 mutation inactivated the RepA2 repressor and increased the rate of transcription ofrepAl mRNA. The translational lacZ gene fusions were used to assess the range of regulation of expression of repAl provided by each of the RNA-E and RepA2 regulatory circuits. By constructing miniplasmids that contained various combinations of the mutations, the contributions of the RNA-E and RepA2 regulatory circuits were assessed with respect to control of plasmid copy number and stable inheritance. Plasmids that lacked either circuit were less stable than wild-type plasmids.