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Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue.

Luo W, Hu Q, Wang D, Deeb KK, Ma Y, Morrison CD, Liu S, Johnson CS, Trump DL - Oncotarget (2013)

Bottom Line: The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs.Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer.Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York.

ABSTRACT
Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissue obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

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Quantitative real-time PCR validation of selected genes in endothelial cellsQuantitative real-time PCR was used to validate gene expression of AREG, JMY, EPB41, GMNN and FAM53C in endothelial cells derived from malignant lesions vs benign lesions in AA and CA patients with prostate cancer. Relative gene expression level for qRT-PCR was normalized to the reference gene GAPDH. Gene expression from microarray was plotted together with the qRT-PCR results. Results were shown as Mean ± SD (triplicate). “*” represents p value <0.05 by t-Test.
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Figure 4: Quantitative real-time PCR validation of selected genes in endothelial cellsQuantitative real-time PCR was used to validate gene expression of AREG, JMY, EPB41, GMNN and FAM53C in endothelial cells derived from malignant lesions vs benign lesions in AA and CA patients with prostate cancer. Relative gene expression level for qRT-PCR was normalized to the reference gene GAPDH. Gene expression from microarray was plotted together with the qRT-PCR results. Results were shown as Mean ± SD (triplicate). “*” represents p value <0.05 by t-Test.

Mentions: To validate the gene expression level measured by microarray, we selected 3 genes, AREG, JMY and FAM53C, which were differentially expressed in TdECs and NdECs of AA and CA, and measured their expression level by qRT-PCR. As shown in Figure 4, there is a good agreement of expression pattern between the microarray and qRT-PCR data.


Isolation and genome-wide expression and methylation characterization of CD31+ cells from normal and malignant human prostate tissue.

Luo W, Hu Q, Wang D, Deeb KK, Ma Y, Morrison CD, Liu S, Johnson CS, Trump DL - Oncotarget (2013)

Quantitative real-time PCR validation of selected genes in endothelial cellsQuantitative real-time PCR was used to validate gene expression of AREG, JMY, EPB41, GMNN and FAM53C in endothelial cells derived from malignant lesions vs benign lesions in AA and CA patients with prostate cancer. Relative gene expression level for qRT-PCR was normalized to the reference gene GAPDH. Gene expression from microarray was plotted together with the qRT-PCR results. Results were shown as Mean ± SD (triplicate). “*” represents p value <0.05 by t-Test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824530&req=5

Figure 4: Quantitative real-time PCR validation of selected genes in endothelial cellsQuantitative real-time PCR was used to validate gene expression of AREG, JMY, EPB41, GMNN and FAM53C in endothelial cells derived from malignant lesions vs benign lesions in AA and CA patients with prostate cancer. Relative gene expression level for qRT-PCR was normalized to the reference gene GAPDH. Gene expression from microarray was plotted together with the qRT-PCR results. Results were shown as Mean ± SD (triplicate). “*” represents p value <0.05 by t-Test.
Mentions: To validate the gene expression level measured by microarray, we selected 3 genes, AREG, JMY and FAM53C, which were differentially expressed in TdECs and NdECs of AA and CA, and measured their expression level by qRT-PCR. As shown in Figure 4, there is a good agreement of expression pattern between the microarray and qRT-PCR data.

Bottom Line: The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs.Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer.Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Therapeutics, Roswell Park Cancer Institute, Buffalo, New York.

ABSTRACT
Endothelial cells (ECs) are an important component involved in the angiogenesis. Little is known about the global gene expression and epigenetic regulation in tumor endothelial cells. The identification of gene expression and epigenetic difference between human prostate tumor-derived endothelial cells (TdECs) and those in normal tissues may uncover unique biological features of TdEC and facilitate the discovery of new anti-angiogenic targets. We established a method for isolation of CD31+ endothelial cells from malignant and normal prostate tissue obtained at prostatectomy. TdECs and normal-derived ECs (NdECs) showed >90% enrichment in primary culture and demonstrated microvascular endothelial cell characteristics such as cobblestone morphology in monolayer culture, diI-acetyl-LDL uptake and capillary-tube like formation in Matrigel®. In vitro primary cultures of ECs maintained expression of endothelial markers such as CD31, von Willebrand factor, intercellular adhesion molecule, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2. We then conducted a pilot study of transcriptome and methylome analysis of TdECs and matched NdECs from patients with prostate cancer. We observed a wide spectrum of differences in gene expression and methylation patterns in endothelial cells, between malignant and normal prostate tissues. Array-based expression and methylation data were validated by qRT-PCR and bisulfite DNA pyrosequencing. Further analysis of transcriptome and methylome data revealed a number of differentially expressed genes with loci whose methylation change is accompanied by an inverse change in gene expression. Our study demonstrates the feasibility of isolation of ECs from histologically normal prostate and prostate cancer via CD31+ selection. The data, although preliminary, indicates that there exist widespread differences in methylation and transcription between TdECs and NdECs. Interestingly, only a small proportion of perturbed gene was overlapped between American (AA) and Caucasian American (CA) patients with prostate cancer. Our study indicates that identifying gene expression and/or epigenetic differences between TdECs and NdECs may provide us with new anti-angiogenic targets. Future studies will be required to further characterize the isolated ECs and determine the biological features that can be exploited in the prognosis and therapy of prostate cancer.

Show MeSH
Related in: MedlinePlus