Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma

BACKGROUND: The EphB4 receptor tyrosine kinase has been reported as increased in tumours originating from several different tissues and its expression in a prostate cancer xenograft model has been reported. METHODS: RT-PCR, western blotting and immunohistochemical techniques were used to examine EphB4 expression and protein levels in human prostate cancer cell lines LNCaP, DU145 and PC3. Immunohistochemistry was also used to examine localisation of EphB4 in tissue samples from 15 patients with prostate carcinomas. RESULTS: All three prostate cancer cell lines expressed the EphB4 gene and protein. EphB4 immunoreactivity in vivo was significantly greater in human prostate cancers as compared with matched normal prostate epithelium and there appeared to be a trend towards increased expression with higher grade disease. CONCLUSION: EphB4 is expressed in prostate cancer cell lines with increased expression in human prostate cancers when compared with matched normal tissue. EphB4 may therefore be a useful anti-prostate cancer target

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma-1

<p><b>Copyright information:</b></p><p>Taken from "Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma"</p><p>BMC Cancer 2005;5():119-119.</p><p>Published online 20 Sep 2005</p><p>PMCID:PMC1266025.</p><p>Copyright © 2005 Lee et al; licensee BioMed Central Ltd.</p>amplifications of and in three samples for each cell line were analysed to determine the relative levels of expression using the BioRad iCycler. All /ratios were close to 1 indicating that there are comparable amounts of both templates in each RNA samples. (B) Dilutions of a single RNA sample for each cell line were also amplified in triplicate with both gene primer sets to confirm that the ratio was consistent regardless of starting template concentration. (C) Triplicate amplifications of , and in three samples for each cell line using the Corbett Rotorgene confirmed the previous result for /and showed a similar result when expression was normalised to a second housekeeping gene

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma-3

<p><b>Copyright information:</b></p><p>Taken from "Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma"</p><p>BMC Cancer 2005;5():119-119.</p><p>Published online 20 Sep 2005</p><p>PMCID:PMC1266025.</p><p>Copyright © 2005 Lee et al; licensee BioMed Central Ltd.</p>n the surface and in the cytoplasm of LNCaP and DU145 and in the cytoplasm only of PC3. There was no reactivity to the primary antibody (EphB4 1° only) or secondary antibody (2° only) alone and little background fluorescence (No staining)

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma-4

<p><b>Copyright information:</b></p><p>Taken from "Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma"</p><p>BMC Cancer 2005;5():119-119.</p><p>Published online 20 Sep 2005</p><p>PMCID:PMC1266025.</p><p>Copyright © 2005 Lee et al; licensee BioMed Central Ltd.</p>staining of formalin fixed, paraffin-embedded tissue samples. Images (20× magnification) of normal (panel 2) and diseased tissue (panel 4) from eight different patient samples are shown with a Haematoxylin/eosin stained consecutive section (panels 1 and 3). The Gleason score for each disease focus is shown in the top left-hand corner of the image of each tumour focus. A single sample that contained a focus of basal cell hyperplasia, two samples with well-differentiated adenocarcinoma, two samples of moderately differentiated adenocarcinoma and three samples of poorly differentiated adenocarcinoma are shown. The brown stain from the biotinylated secondary antibody indicates the EphB4 protein. Nuclei are stained with Harris haematoxylin and appear blue. Staining of normal tissues was either absent or weak and diffuse. Increased staining of the tumour tissue appeared to correlate with increased Gleason score. There was no cross-reactivity with the secondary antibody alone (result not shown)

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma-5

<p><b>Copyright information:</b></p><p>Taken from "Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma"</p><p>BMC Cancer 2005;5():119-119.</p><p>Published online 20 Sep 2005</p><p>PMCID:PMC1266025.</p><p>Copyright © 2005 Lee et al; licensee BioMed Central Ltd.</p>stochemistry. Images from 10× magnification (A) and a region from this (as indicated by the box) at 20× magnification (B) are shown for 2 regions of normal prostate duct (1 and 2) and 8 regions with disease (3–10) from this single sample. A comparable level of EphB4 staining was seen in regions of high grade PIN (eg. focus 8 indicated PIN) and regions of poorly differentiated adenocarcinoma (eg. focus 7 indicated PDA). Regions of well-differentiated (eg focus 3) and moderately differentiated (eg focus 10) also show comparable staining

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma-0

<p><b>Copyright information:</b></p><p>Taken from "Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma"</p><p>BMC Cancer 2005;5():119-119.</p><p>Published online 20 Sep 2005</p><p>PMCID:PMC1266025.</p><p>Copyright © 2005 Lee et al; licensee BioMed Central Ltd.</p>ll lines LNCaP, DU145 and PC3. Sizes of the amplified products are shown on the right (bp). An RNA sample to which no reverse transcriptase was added and a PCR reagent only (containing no template) amplification were also performed as negative controls for each stage of the RT-PCR

Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma-2

<p><b>Copyright information:</b></p><p>Taken from "Investigation of the expression of the EphB4 receptor tyrosine kinase in prostate carcinoma"</p><p>BMC Cancer 2005;5():119-119.</p><p>Published online 20 Sep 2005</p><p>PMCID:PMC1266025.</p><p>Copyright © 2005 Lee et al; licensee BioMed Central Ltd.</p>and PC3 (P). (A) The immunoblot was incubated sequentially with antibodies specific to EphB4, α-actin and calnexin and exposed to autoradiographic film for the indicated times. MCF10A engineered to express EphB4 (5 μg protein lysate) was used as a positive control. The arrow indicates the expected size of each protein – 120 kDa for EphB4, 43.2 kDa for α-actin and 90 kDa for calnexin. The size of the marker proteins is shown on the left in kDa. (B) Coomassie stained duplicate gel for loading comparison