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- Babiker, Adil A., et al.
(author)
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Mapping pro- and antiangiogenic factors on the surface of prostasomes of normal and malignant cell origin
- 2010
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In: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 70:8, s. 834-847
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Journal article (peer-reviewed)abstract
- BACKGROUND: Angiogenesis is the formation of new blood vessels by capillary sprouting from pre-existing vessels. Tumor growth is angiogenesis-dependent and the formation of new blood vessels is associated with the increased expression of angiogenic factors. Prostasomes are secretory granules produced, stored and released by the glandular epithelial cells of the prostate. We investigated the expression of selected angiogenic and anti-angiogenic factors on the surface of prostasomes of different origins as well as the direct effect of prostasomes on angiogenesis. METHODS: VEGF, endothelin-1, endostatin, and thrombospondin-1 were determined on prostasomes from seminal fluid and human prostate cancer cell lines (DU145,PC-3,LNCaP) using different immunochemical techniques. Human dermal microvascular endothelial cells were incubated with seminal and DU145 cell-prostasomes and with radioactive thymidine. The effect of prostasomes on angiogenesis was judged by measuring the uptake of labeled thymidine. The presence of any deleterious effects of prostasomes on the endothelial cells was investigated using thymidine assay and confocal laser microscopy. RESULTS: VEGF and endothelin-1 were determined on malignant cell-prostasomes (no difference between cell lines) but not determined on seminal prostasomes. The same applies for the expression of endostatin but with much higher expression on malignant cell-prostasomes with obvious differences between them. Seminal and DU145 cell-prostasomes were found to have anti-angiogenic effect which was more expressed by DU145 cell-prostasomes. No deleterious effect of prostasomes on endothelial function was detected using either thymidine assay or microscopy. CONCLUSIONS: Prostasomes contain pro- and anti-angiogenic factors that function to counteract each other unless the impact from one side exceeds the other to bring about dysequilibrium.
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| 2. |
- Babiker, Adil A., et al.
(author)
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Overexpression of ecto-protein kinases in prostasomes of metastatic cell origin
- 2006
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In: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 66:7, s. 675-686
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Journal article (peer-reviewed)abstract
- BACKGROUND:Prostasomes are secretory granules produced, stored, and released by the glandular epithelial cells of the prostate. They express numerous enzymes whose physiological roles have so far not been fully evaluated. In this study, we investigated the expression and function of prostasomal protein kinases and ATPase.METHODS:The protein kinase activities of prostasomes isolated from seminal fluid and malignant prostate cell lines (PC-3, DU145, and LNCaP) were investigated using the model phosphorylation substrates histone and casein, as well as the plasma proteins C3 and fibrinogen, in combination with specific protein kinase inhibitors. The prostasomal ATPase activity was also evaluated. The expression of protein kinases and ATPase on prostasomes was verified by flow cytometry.RESULTS:Prostasomes (intact or solubilized with octylglucoside or saponin) from prostate cancer cells had higher expression of protein kinases A, C, and casein kinase II compared to prostasomes isolated from seminal plasma, resulting in higher phosphorylation of both exogenous and endogenous substrates. Using intact prostasomes, it was found that prostasomes of metastatic origin had lower ATPase activity, resulting in higher residual ATP available for the phosphorylation reaction. Finally, complement component C3 and fibrinogen (two proteins whose activities are modulated by phosphorylation) were identified as physiologically relevant phosphorylation substrates.CONCLUSIONS:These results indicate that prostasomes are capable of modifying proteins possibly involved in the innate response by extracellular phosphorylation mediated by ecto-kinases. This is a novel mechanism by which prostatic malignant cells may interact with their environment.
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| 3. |
- Babiker, Adil A., et al.
(author)
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Prothrombotic effect of prostasomes of metastatic cell and seminal origin
- 2007
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In: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 67:4, s. 378-388
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Journal article (peer-reviewed)abstract
- BACKGROUND. Prostasomes are secretory granules produced by the glandular epithelial cells of the prostate. Seminal prostasomes contain high amounts of Tissue Factor (TF) but no studies of TF on malignant cell prostasomes have been made. Here we compare the expression, phosphorylation, and function of TF on prostasomes of different origin. METHODS. TF was detected on prostasomes isolated from seminal fluid and human prostate cancer cell lines (PC-3, DU145, and LNCaP) using FACS and enzyme immunoassay (EIA). Incubation of prostasomes with radioactive ATP under conditions favoring protein kinase A activity led to phosphorylation of TF as detected by immunoprecipitation and SDS-PAGE. The prothrombotic effect of prostasomes was investigated in whole blood and recalcified plasma. Blocking experiments were performed using anti-TF antibodies and corn trypsin inhibitor. RESULTS. TF was expressed on all tested prostasome preparations with lowest values found for seminal ones. Prostasomal TF was the main endogenous substrate for prostasomal protein kinase A. All tested prostasome preparations greatly enhanced the rate of clot formation in a dose-dependent fashion, that is, the clotting capability of prostasomes seemed to be related to the extent of their expression of TF. In addition, the density of the clot varied between different prostasome preparations. When incubated in whole blood, prostasomes were found to associate to WBC thereby inducing them to express and release TF. CONCLUSIONS. These data show that TF is overexpressed and also subjected to phosphorylation by malignant cell prostasomes. This suggests major roles for prostasomes in thrombotic events that occur in some advanced cases of prostate cancer.
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| 4. |
- Babiker, Adil A., et al.
(author)
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Transfer of functional prostasomal CD59 of metastatic prostatic cancer cell origin protects cells against complement attck
- 2005
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In: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 62:2, s. 105-114
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Journal article (peer-reviewed)abstract
- BACKGROUND: Prostasomes are secretory granules produced, stored, and released, by the glandular epithelial cells of the prostate. They express the glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein CD59, which has been shown to be transferred to spermatozoa and erythrocytes.METHODS: The CD59 content of prostasomes isolated from seminal fluid and malignant prostate cells (PC-3, DU145, and LNCaP) and the transfer of prostasomal CD59 to rabbit erythrocytes (RE) and to PIPLC-treated and unmanipulated cancer cells were investigated using FACS. All prostasomes were also incubated with RE and tested in a hemolytic assay.RESULTS: Prostasomes from cancer cells had higher expression of CD59 than those of normal cells. Prostasomal CD59 of different origin could be transferred to RE, malignant cell lines stripped of CD59 by PIPLC, or unmanipulated LNCaP cells. Malignant cell prostasomes had an increased ability to inhibit complement-mediated lysis compared to those from non-malignant cells.CONCLUSIONS: These results point to a novel mechanism by which prostasomes can protect prostatic malignant cells from complement attack.
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| 6. |
- Ronquist, Göran, et al.
(author)
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Human Prostasomes Contain Chromosomal DNA
- 2009
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In: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 69:7, s. 737-743
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Journal article (peer-reviewed)abstract
- BACKGROUND. The aim of this study was to perform a comprehensive evaluation of the occurrence of DNA in human prostasomes. METHODS. Prostasomes were purified from seminal fluid (seminal prostasomes) and from PC-3-cells (PC-3 cell prostasomes). DNA extracted from both sources of prostasomes was visualized on agarose gels. Further, theDNAwas cloned and sequenced (13 clones from seminal prostasomal DNAand 16 clones from PC-3 cell prostasomal DNA) and identified by alignment in the BLAST-nucleotide search database. In order to decide if the DNA was internally or externally located in/on prostasomes, prostasomes were treated with nuclease (DNase) and A260 was measured before and after treatment. Additionally, flow cytometric studies were performed with membrane permeable and membrane impermeable DNA stains. RESULTS. We identified human chromosomal DNA in purified prostasomes from both sources and treatment with DNase demonstrated that the prostasome-shielded DNA was protected from enzyme attack. Membrane-permeable DNA stain raised the fluorescence contrary to membrane-impermeable stain. Clearly discernible nucleic acid of prostasomes was separated on 1% agarose gel yieldingDNAfragments of about 13 kbp and below with a marked band at about 1 kbp. Cloning and sequencing of 13 fragments from seminal prostasomes and 16 from PC-3 cell prostasomes revealed a chromosomal origin of the DNA. In purified seminal prostasomes, 4 out of 13 DNA clones featured gene sequences (31%). The corresponding figure for PC3-derived prostasomes was 4 out of 16 clones featuring gene sequences (25%). CONCLUSION. Human prostasomes contain chromosomal DNA. Both nuclease treatment and differential DNA stainings indicated an inside location of the prostasomal DNA. Our findings suggest a DNA-delivery function of prostasomes to sperm cells.
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| 7. |
- Ronquist, Göran K, et al.
(author)
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Prostasomes are heterogeneous regarding size and appearance but affiliated to one DNA-containing exosome family
- 2012
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In: The Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 72:16, s. 1736-1745
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Journal article (peer-reviewed)abstract
- BACKGROUND:Prostate acinar epithelial cells release microvesicles (prostasomes) that possess pleiotropic biological effects relevant for successful fertilization. Prostasomes are formed in a similar way as exosomes but are heterogeneous as regards size and appearance. Like exosomes they are thought to be mediators of intercellular communication.METHODS:We prepared seminal prostasomes in accordance with the prevailing protocol for exosome preparation including passage through a 0.2 µm filter and centrifugation in a sucrose gradient.RESULTS:We compared the "filterable prostasomes" with those trapped on the filter ("nonfilterable prostasomes") and, qualitatively, no conspicuous differences were apparent regarding ultrastructure and SDS-PAGE banding pattern. Moreover, both types of prostasomes contained DNA fragments and Western blot revealed presence of prostate specific membrane antigen (PSMA), CD38, and annexin A1.CONCLUSIONS: Reasonably, prostasomes could be included in the exosome family and be regarded as one entity containing chromosomal DNA.
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