Presenting stage and risk group in men dying of prostate cancer.

Introduction: Prostate cancer remains the 3rd leading cause of cancer-related mortality in Canadian men, and yet screening for prostate cancer continues to be controversial because the majority of men diagnosed with prostate cancer do not die of the disease. It also remains uncertain whether treatment of cases that can be treated with curative intent alters the mortality rate. There are very few studies describing the presenting stage, risk groups, and survival after diagnosis for men dying of prostate cancer in the literature. In this study, we explored these characteristics for all men who died of prostate cancer in British Columbia between 2013 and 2015. Methods: The population-based BC Cancer databases were used to identify all patients diagnosed between January 2013 and December 2015 who died of prostate cancer. Patient, tumour, and treatment characteristics were collected, and the risk grouping for each tumour was determined. The proportion of cases in each risk group at the time of diagnosis was determined. Survival time from diagnosis to death was calculated for all patients and for each risk group using the Kaplan-Meier method. Results: A total of 1256 patients died of prostate cancer. Of patients who presented with metastatic disease, 57.2% presented with a Gleason score of 8 or more, compared with only 35.7% of patients who presented with nonmetastatic disease (p < 0.0001). The presenting stage and risk group of those dying of prostate cancer were as follows: 32% metastatic disease, 3% regional (defined as node-positive), 39% localized high risk, 9% localized intermediate risk, 4% localized low risk, 6% localized not otherwise specified, and 7% unknown. Therefore, 80.3% of those with a known risk group presented with either localized high-risk, regional, or metastatic disease at diagnosis. The median survival times from diagnosis to death were 12 years for localized low-risk, 10 years for localized intermediate-risk, 6.5 years for localized high-risk, 4 years for regional, and 1.7 years for metastatic disease at diagnosis. Conclusions: This population-based analysis demonstrates that patients with localized high-risk, regional, or metastatic disease at diagnosis constitute the overwhelming majority of patients who die of prostate cancer in British Columbia. Unless these disease states can reliably be identified at an earlier low- or intermediate-risk localized state in the future, it is unlikely that treatment of localized low- and intermediate-risk cancer will have an impact on survival. Furthermore, patients with de novo metastatic disease had identifiable risk factors of a higher prostate-specific antigen and Gleason score. Further studies are required to confirm these results.

A Phase II trial of 8 weeks of degarelix for prostate volume reduction: Efficacy and hormonal recovery.

PURPOSE: The purpose of this study was to determine the efficacy of 8 weeks of degarelix for prostate downsizing before interstitial brachytherapy. We also report associated toxicity and the time course of endocrine recovery over the following 12 months. METHODS AND MATERIALS: Fifty patients were accrued to an open-label Phase II clinical trial (www.clinicaltrials.gov ID NCT01446991). Baseline prostate transrectal ultrasound (TRUS) was performed on all patients followed by degarelix administration and a repeat TRUS at Week 8. Brachytherapy was performed within 4 weeks of the 8-week TRUS for all patients who achieved suitable downsizing. RESULTS: The median prostate volume was reduced from 65.0 cc (interquartile range [IQR]: 55.2-80.0 cc) to 48.2 cc at 8 weeks (IQR: 41.2-59.3 cc), representing a median decrease of 26.2% (IQR: 21-31%). Functional recovery of testosterone within an age-adjusted normal range occurred at a median of 34.1 weeks (IQR: 28.2-44.5 weeks) from the date of the final injection. Despite this recovery, follicle-stimulating hormone and luteinizing hormone levels remained abnormally elevated throughout 12 months. Quality-of-life implications are discussed. CONCLUSIONS: Degarelix is effective for prostate downsizing before prostate brachytherapy with a median volume decrease of 26.2% by 8 weeks. Despite the short course of treatment and eventual testosterone recovery, follicle-stimulating hormone and luteinizing hormone remain elevated beyond 12 months. Further investigation with randomized comparisons to other hormonal agents is warranted.

Human telomerase RNA-protein interactions.

Telomere length is maintained in most eukaryotic cells by telomerase. The core components of this ribonucleoprotein (RNP) enzyme include a protein catalytic subunit, composed of motifs conserved among reverse transcriptases (RT), and an RNA subunit that contains a short template sequence essential for the synthesis of telomeric repeats. We developed an electrophoretic mobility shift assay using active telomerase partially purified from 293 cells and radiolabeled, in vitro-transcribed human telomerase RNA (hTR) to investigate the molecular interactions of the human telomerase RT (hTERT) and telomerase-associated proteins with hTR. A specific hTR-protein complex was identified and shown to contain hTERT and human Staufen by antibody supershift assays. Variants of hTR altered in distinct structural elements were analyzed for their ability to competitively inhibit complex formation. Human telomerase RNAs lacking the CR4-CR5 domain were poor inhibitors of hTR-protein complex formation, suggesting that the CR4-CR5 domain of hTR is a potential protein-binding site. Furthermore, alterations in the telomerase RNA pseudoknot's P3 helix, the CR7 domain, or the H/ACA box efficiently inhibited formation of the complex, indicating that these domains are dispensable for the assembly of a telomerase RNP in vitro. Potential telomerase-associated proteins that bind hTR were also identified using a UV cross-linking assay.

Functional regions of human telomerase reverse transcriptase and human telomerase RNA required for telomerase activity and RNA-protein interactions.

Telomerase is a specialized reverse transcriptase (RT) that is minimally composed of a protein catalytic subunit and an RNA component. The RNA subunit contains a short template sequence that directs the synthesis of DNA repeats at the ends of chromosomes. Human telomerase activity can be reconstituted in vitro by the expression of the human telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. We analyzed telomerase activity and binding of hTR to hTERT in RRL by expressing different hTERT and hTR variants. hTRs containing nucleotide substitutions that are predicted to disrupt base pairing in the P3 helix of the pseudoknot weakly reconstituted human telomerase activity yet retained their ability to bind hTERT. Our results also identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures between nucleotides 208 and 330 of hTR (which include the conserved CR4-CR5 domain) were found to be important for hTERT-hTR interactions and for telomerase activity reconstitution. Human TERT carboxy-terminal amino acid deletions extending to motif E or the deletion of the first 280 amino acids abolished human telomerase activity without affecting the ability of hTERT to associate with hTR, suggesting that the RT and RNA binding functions of hTERT are separable. These results indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved RT motifs and (ii) bind nucleotides distal to the hTR template sequence.

Expression of hTERT and hTR in cis reconstitutes and active human telomerase ribonucleoprotein.

Telomeres in eukaryotic cells are generally synthesized and maintained by the ribonucleoprotein (RNP) telomerase. This enzyme is composed of at least two subunits, the telomerase reverse transcriptase (TERT) and the telomerase RNA. Human telomerase activity can be reconstituted in vitro by the expression of the telomerase protein catalytic subunit (hTERT) in the presence of recombinant human telomerase RNA (hTR) in a rabbit reticulocyte lysate (RRL) system. The hTERT and hTR subunits are independently expressed in vivo, and little is known about the mechanism of their assembly. To facilitate recombinant telomerase RNP formation and reconstitution, we engineered a construct, termed hTERT-hTR cis, in which the 3' end of the hTERT coding sequence was extended by the addition of the sequence encoding hTR. Expression of the hTERT-hTR cis construct in vitro (in RRL) and in vivo (in the yeast Saccharomyces cerevisiae) produced hTERT-hTR transcripts of the predicted size. Active human telomerase was reconstituted by hTERT-hTR cis expression in both RRL and S. cerevisiae. Assembly of functional human telomerase by the bicistronic expression of the protein and RNA components may facilitate the overexpression and reconstitution of this enzyme in heterologous systems.

Functional reconstitution of human telomerase expressed in Saccharomyces cerevisiae.

Telomerase is a ribonucleoprotein enzyme complex that adds DNA repeats at the ends of chromosomes. In an effort to establish an in vivo heterologous expression system for active human telomerase, we expressed human telomerase reverse transcriptase (hTERT) in Saccharomyces cerevisiae and affinity-purified the protein as a fusion with glutathione S-transferase (GST). Addition of the GST moiety to the N terminus of hTERT did not interfere with telomerase activity when GST-hTERT was expressed in rabbit reticulocyte lysate (RRL) in the presence of the human telomerase RNA (hTR). Active human telomerase was immunoprecipitated from yeast lysates that co-expressed GST-hTERT and hTR. In addition, telomerase activity could be reconstituted in vitro by the addition of hTR to GST-hTERT that was immunoprecipitated from either RRL or S. cerevisiae lysates. The expression and reconstitution of human telomerase activity in yeast will provide powerful biochemical and genetic tools to study the various components required for the assembly and function of this enzyme.

Human immunodeficiency virus type 1 (HIV-1) Vpr functions as an immediate-early protein during HIV-1 infection.

Human immunodeficiency virus type 1 (HIV-1) Vpr is a virion-associated protein which facilitates HIV-1 infection of nondividing cells by contributing to the nuclear transport of the preintegration complex (PIC). Vpr was also shown to induce a cell cycle G2 arrest in infected proliferating cells that optimizes HIV-1 long terminal repeat (LTR)-directed gene expression and viral production. However, it is unclear whether this activity is mediated primarily early by virion-associated Vpr or alternatively late during infection when Vpr is de novo expressed. We report here that in the absence of de novo expression, virion-associated Vpr induces a transient G2 arrest that can subsequently lead to cell killing by apoptosis. Interestingly, the induction of both cell cycle G2 arrest and apoptosis by virion-associated Vpr requires viral entry but not viral replication, since reverse transcriptase and protease inhibitor treatments do not prevent these Vpr effects. These results raise the possibility that in vivo both infectious and noninfectious viruses contribute to the dysfunction and killing of CD4(+) cells. In addition, our results reveal that virion-associated Vpr stimulates viral replication in proliferating cells after establishing a cell cycle G2 arrest by increasing LTR-directed gene expression. Importantly, this Vpr-mediated LTR activation appears to be a requirement for subsequent optimal Tat transactivation. Taken together, these results strongly suggest that in addition to participating in the HIV PIC nuclear transport in nondividing cells, virion-associated Vpr activates HIV-1 LTR-directed gene expression by manipulating the host cell cycle. From this, we conclude that Vpr functions as an immediate-early protein during HIV-1 infection.

Incorporation of Vpr into human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 gag precursor.

The 96-amino acid Vpr protein is the major virion-associated accessory protein of the human immunodeficiency virus type 1 (HIV-1). As Vpr is not part of the p55 Gag polyprotein precursor (Pr55(gag)), its incorporation requires an anchor to associate with the assembling viral particles. Although the molecular mechanism is presently unclear, the C-terminal region of the Pr55(gag) corresponding to the p6 domain appears to constitute such an anchor essential for the incorporation of the Vpr protein. In order to clarify the mechanism by which the Vpr accessory protein is trans-incorporated into progeny virion particles, we tested whether HIV-1 Vpr interacted with the Pr55(gag) using the yeast two-hybrid system and the maltose-binding protein pull-down assay. The present study provides genetic and biochemical evidence indicating that the Pr55(gag) can physically interact with the Vpr protein. Furthermore, point mutations affecting the integrity of the conserved L-X-S-L-F-G motif of p6(gag) completely abolish the interaction between Vpr and the Pr55(gag) and, as a consequence, prevent Vpr virion incorporation. In contrast to other studies, mutations affecting the integrity of the NCp7 zinc fingers impaired neither Vpr virion incorporation nor the binding between Vpr and the Pr55(gag). Conversely, amino acid substitutions in Vpr demonstrate that an intact N-terminal alpha-helical structure is essential for the Vpr-Pr55(gag) interaction. Vpr and the Pr55(gag) demonstrate a strong interaction in vitro as salt concentrations as high as 900 mM could not disrupt the interaction. Finally, the interaction is efficiently competed using anti-Vpr sera. Together, these results strongly suggest that Vpr trans-incorporation into HIV-1 particles requires a direct interaction between its N-terminal region and the C-terminal region of p6(gag). The development of Pr55(gag)-Vpr interaction assays may allow the screening of molecules that can prevent the incorporation of the Vpr accessory protein into HIV-1 virions, and thus inhibit its early functions.