Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

Investigation of androgen receptor-dependent alternative splicing has identified a unique subtype of lethal prostate cancer.

A complete proteomics study characterizing active androgen receptor (AR) complexes in prostate cancer (PCa) cells identified a diversity of protein interactors with tumorigenic annotations, including known RNA splicing factors. Thus, we chose to further investigate the functional role of AR-mediated alternative RNA splicing in PCa disease progression. We selected two AR-interacting RNA splicing factors, Src associated in mitosis of 68 kDa (SAM68) and DEAD (Asp-Glu-Ala-Asp) box helicase 5 (DDX5) to examine their associative roles in AR-dependent alternative RNA splicing. To assess the true physiological role of AR in alternative RNA splicing, we assessed splicing profiles of LNCaP PCa cells using exon microarrays and correlated the results to PCa clinical datasets. As a result, we were able to highlight alternative splicing events of clinical significance. Initial use of exon-mini gene cassettes illustrated hormone-dependent AR-mediated exon-inclusion splicing events with SAM68 or exon-exclusion splicing events with DDX5 overexpression. The physiological significance in PCa was investigated through the application of clinical exon array analysis, where we identified exon-gene sets that were able to delineate aggressive disease progression profiles and predict patient disease-free outcomes independently of pathological clinical criteria. Using a clinical dataset with patients categorized as prostate cancer-specific death (PCSD), these exon gene sets further identified a select group of patients with extremely poor disease-free outcomes. Overall, these results strongly suggest a nonclassical role of AR in mediating robust alternative RNA splicing in PCa. Moreover, AR-mediated alternative spicing contributes to aggressive PCa progression, where we identified a new subtype of lethal PCa defined by AR-dependent alternative splicing.

Phosphorylation of prion protein at serine 43 induces prion protein conformational change.

The cause of the conformational change of normal cellular prion protein (PrP) into its disease-associated form is unknown. Posttranslational modifications, such as glycosylation, acetylation, S-nitrosylation, and phosphorylation, are known to induce protein conformational changes. Here, we investigated whether phosphorylation could induce the conformational change of PrP because PrP contains several kinase motifs and has been found recently in the cytosol, in which kinases generally reside. Neuronal cyclin-dependent kinase 5 (Cdk5) phosphorylated recombinant PrP(23-231) at serine 43 (S43) in an in vitro kinase assay. Cdk5-phosphorylated PrP became proteinase K resistant, formed Congo Red-positive fibrils, and formed aggregates that were immunostained with anti-PrP and anti-phospho-PrP(S43) (anti-pPrP(S43)). pPrP(S43) was detected in PrP/Cdk5/p25 cotransfected N2a cells. Roscovitine inhibition of Cdk5 activity or transfection of N2a cells with mutant PrP S43A eliminated the anti-pPrP(S43)-immunopositive protein. Alkaline phosphatase-sensitive and proteinase K-resistant pPrP(S43) immunoreactivity was observed in scrapie-infected but not control-injected mice brains. These results raise the possibility that phosphorylation could represent a physiological mechanism of PrP conversion in vivo.

Transcription, translation, and immunolocalization of ODVP-6E/ODV-E56 and p74 proteins: two highly conserved ODV-associated envelope proteins of Choristoneura fumiferana Granulovirus.

Choristoneura fumiferana granulovirus (ChfuGV) infection results two types of enveloped virions: Occlusion-derived virus (ODV) and budded virus (BV). Structural proteins ODVP-6E/ODV-E56 and p74 are two major conserved ODV-associated proteins that may be involved in the initiation of viral infection cycle in susceptible host insect larvae. This study presents the characterization of ChfuGV odvp-6e/odv-e56 and p74 transcription and translation as well as immunolocalization of these proteins in the occluded ChfuGV virion. Our results revealed that the transcription of odvp-6e/odv-e56 and p74 genes, both, start at 24 hours post infection (h p.i.). Using monospecific polyclonal antibodies made against ODVP-6E/ODV-E56 and p74 we demonstrated that these proteins are both expressed late in infection (24 h p.i.). Immunogold labeling using antisera against ODVP-6E/ODV-E56 and p74 proteins demonstrated that ODVP-6E/ODV-E56 and p74 proteins are both associated with the ODV envelop of ChfuGV.

Choristoneura fumiferana Granulovirus pk-1: a baculoviral protein kinase.

Open reading frame (ORF) 3 on the Choristoneura fumiferana granulovirus (ChfuGV), located in the 11 kb fragment of the BamHI genomic bank encodes a predicted 32-kDa putative kinase protein. Bioinformatics analysis on the predicted amino acid sequence of ChfuGV PK-1 revealed the existence of 11 catalytic subdomains. Sequence analysis within the 5'-untranslated region (5'-UTR) of ChfuGV pk- 1 indicates the presence of both putative early and late promoter motifs, indicating that pk-1 may be expressed throughout the infection cycle. Promoter sequence analysis reveals that pk-1 is deprived of a TATA box and appears instead to be regulated by other cis-acting transcriptional regulatory elements. Temporal transcription analysis by RT-PCR confirms the appearance of transcripts detected from 2 h p.i. until 72 h p.i. Northern blot hybridization characterizes pk-1 transcription as a 1.2 kb transcript. Homology comparisons reveal that ChfuGV PK-1 protein is most closely related to Phthorimaea operculalla granulovirus (PoGV) with 80 % amino acid identity.

Identification and characterization of a conserved baculoviral structural protein ODVP-6E/ODV-E56 from Choristoneura fumiferana granulovirus.

A gene that encodes a homologue to baculoviral ODVP-6E/ODV-E56, a baculoviral envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). The ChfuGV odvp-6e/odv-e56 gene was located on an 11-kb BamHI subgenomic fragment using different sets of degenerated primers, which were designed using the results of the protein sequencing of a major 39 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1062 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 353 amino acids with a predicted molecular mass of 38.5 kDa. The amino acid sequence data that was derived from the nucleotide sequence in ChfuGV was compared to those of other baculoviruses. ChfuGV ODVP-6E/ODV-E56, along with other baculoviral ODVP-6E/ODV-E56 proteins, all contained two putative transmembrane domains at their C-terminus. Several putative N- and O-glycosylation, N-myristoylation, and phosphorylation sites were detected in the ChfuGV ODVP-6E/ODV-E56 protein. A similar pattern was detected when a hydrophobicity-plots comparison was performed on ChfuGV ODVP-6E/ODV-E56 with other baculoviral homologue proteins. At the nucleotide level, a late promoter motif (GTAAG) was located at -14 nt upstream to the start codon of the ChfuGV odvp-6e/odv-e56 gene. A slight variant of the polyadenylation signal, AATAAT, was detected at the position +10 nt that is downstream from the termination signal. A phylogenetic tree for baculoviral ODVP-6E/ODV-E56 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV ODVP-6E/ODV-E56 is most closely related to those of Cydia pomonella granulovirus (CpGV) and Plutella xylostella granulovirus (PxGV).

Identification, characterization and phylogenic analysis of conserved genes within the p74 gene region of Choristoneura fumiferana granulovirus genome.

The genes located within the p74 gene region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing an 8.9 kb BamHI restriction fragment on the ChfuGV genome. The global guanine-cytosine (GC) content of this region of the genome was 33.02%. This paper presents the ORFs within the p74 gene region along with their transcriptional orientations. This region contains a total of 15 open reading frames (ORFs). Among those, 8 ORFs were found to be homologues to the baculoviral ORFs: Cf-i-p , Cf-vi, Cf-vii, Cf-viii (ubiquitin), Cf-xi (pp31), Cf-xii (lef-11), Cf-xiii (sod) and Cf-xv-p (p74). To date, no specific function has been assigned to the ORFs: Cf-i, Cf-ii, Cf-iii, Cf-iv, Cf-v, Cf-vi, Cf-vii, Cf-ix and Cf-x. The most noticeable ORFs located in this region of the ChfuGV genome were ubiquitin, lef-11, sod, fibrillin and p74. The phylogenetic trees (constructed using conceptual products of major conserved ORFs) and gene arrangement in this region were used to further examine the classification of the members of the granulovirus genus. Comparative studies demonstrated that ChfuGV along with the Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), Adoxophyes orana granulovirus (AoGV) and Cryptophlebia leucotreta granulovirus (ClGV) share a high degree of amino acids sequence and gene arrangement preservation within the studied region. These results support a previous report, which classified a granuloviruses into 2 distinct groups: Group I: ChfuGV, CpGV, PhopGV and AoGV and Group II: Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The phylogenetic and gene arrangement studies also placed ClGV as a novel member of the Group I granuloviruses.

Choristoneura fumiferana Granulovirus p74 protein, a highly conserved baculoviral envelope protein.

A gene that encodes a homologue to baculoviral p74, an envelope-associated viral structural protein, has been identified and sequenced on the genome of Choristoneura fumiferana granulovirus (ChfuGV). A part of the ChfuGV p74 gene was located on an 8.9 kb BamHI subgenomic fragment using different sets of degenerated primers. These were designed using the results of the protein sequencing of a major 74 kDa structural protein that is associated with the occlusion-derived virus (ODV). The gene has a 1992 nucleotide (nt) open-reading frame (ORF) that encodes a protein with 663 amino acids with a predicted molecular mass of 74,812 Da. Comparative studies revealed the presence of two major conserved regions in the ChfuGV p74 protein. This study also shows that all of the p74 proteins contain two putative transmembrane domains at their C-terminal segments. At the nucleotide sequence level, two late promoter motifs (TAAG and GTAAG) were located upstream of the first ATG of the p74 gene. The gene contained a canonical poly(A) signal, AATAAA, at its 3 non-translated region. A phylogenetic tree for baculoviral p74 was constructed using a maximum parsimony analysis. The phylogenetic estimation demonstrated that ChfuGV p74 is related the closest to those of Cydia pomonella granulovirus (CpGV) and Phthorimaea operculella granulovirus (PhopGV).

Identification, characterization and phylogenic analysis of conserved genes within the odvp-6e/odv-e56 gene region of Choristoneura fumiferana granulovirus.

The genes that are located within the odvp-6e/odv-e56 region of the Choristoneura fumiferana granulovirus (ChfuGV) were identified by sequencing the 11 kb BamHI restriction fragment on the ChfuGV genome. The global GC content that was calculated from the data obtained from this genomic region was 34.96%. The open-reading frames (ORFs), located within the odvp-6e/odv-e56 region, are presented and compared to the equivalent ORFs that are located at the same region in other GVs. This region is composed of 14 ORFs, including three ORFs that are unique to ChfuGV with no obvious homologues in other baculoviruses as well as eleven ORFs with homologues to granuloviral ORFs, such as granulin, CfORF2, pk-1, ie-1, odv-e18, p49, and odvp-6e/odv-e56. In this study, the conceptual products of seven major conserved ORFs (granulin, CfORF2, IE-1, ODV-E18, p49 and ODVP-6E/ODV-E56) were used in order to construct phylogenetic trees. Our results show that granuloviruses can be grouped in 2 distinct groups as follows: Group I; Choristoneura fumiferana granulovirus (ChfuGV), Cydia pomonella granulovirus (CpGV), Phthorimaea operculella granulovirus (PhopGV), and Adoxophyes orana granulovirus (AoGV). Group II; Xestia c-nigrum granulovirus (XcGV), Plutella xylostella granulovirus (PxGV), and Trichoplusia ni granulovirus (TnGV). The ChfuGV conserved proteins are most closely related to those of CpGV, PhopGV, and AoGV. Comparative studies, performed on gene arrangements within this region of genomes, demonstrated that three GVs from group I maintain similar gene arrangements.

Proteomic-coupled-network analysis of T877A-androgen receptor interactomes can predict clinical prostate cancer outcomes between White (non-Hispanic) and African-American groups.

The androgen receptor (AR) remains an important contributor to the neoplastic evolution of prostate cancer (CaP). CaP progression is linked to several somatic AR mutational changes that endow upon the AR dramatic gain-of-function properties. One of the most common somatic mutations identified is Thr877-to-Ala (T877A), located in the ligand-binding domain, that results in a receptor capable of promiscuous binding and activation by a variety of steroid hormones and ligands including estrogens, progestins, glucocorticoids, and several anti-androgens. In an attempt to further define somatic mutated AR gain-of-function properties, as a consequence of its promiscuous ligand binding, we undertook a proteomic/network analysis approach to characterize the protein interactome of the mutant T877A-AR in LNCaP cells under eight different ligand-specific treatments (dihydrotestosterone, mibolerone, R1881, testosterone, estradiol, progesterone, dexamethasone, and cyproterone acetate). In extending the analysis of our multi-ligand complexes of the mutant T877A-AR we observed significant enrichment of specific complexes between normal and primary prostatic tumors, which were furthermore correlated with known clinical outcomes. Further analysis of certain mutant T877A-AR complexes showed specific population preferences distinguishing primary prostatic disease between white (non-Hispanic) vs. African-American males. Moreover, these cancer-related AR-protein complexes demonstrated predictive survival outcomes specific to CaP, and not for breast, lung, lymphoma or medulloblastoma cancers. Our study, by coupling data generated by our proteomics to network analysis of clinical samples, has helped to define real and novel biological pathways in complicated gain-of-function AR complex systems.

Loss of anti-Bax function in Gerstmann-Sträussler-Scheinker syndrome-associated prion protein mutants.

Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine ((M)) or valine ((V)) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V), D202N(V), P102L(V) and Q217R(V) retained, whereas the P102L(M), P105L(V), Y145stop(M) and Q212P(M) PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V), none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.