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Molecular and cellular characterization of ABCG2 in the prostate - PubMed

  • ️Mon Jan 01 2007

Molecular and cellular characterization of ABCG2 in the prostate

Laura E Pascal et al. BMC Urol. 2007.

Abstract

Background: Identification and characterization of the prostate stem cell is important for understanding normal prostate development and carcinogenesis. The flow cytometry-based side population (SP) technique has been developed to isolate putative adult stem cells in several human tissue types including the prostate. This phenotype is mainly mediated by the ATP-binding cassette membrane transporter ABCG2.

Methods: Immunolocalization of ABCG2 was performed on normal prostate tissue obtained from radical prostatectomies. Normal human prostate SP cells and ABCG2+ cells were isolated and gene expression was determined with DNA array analysis and RT-PCR. Endothelial cells were removed by pre-sorting with CD31.

Results: ABCG2 positive cells were localized to the prostate basal epithelium and endothelium. ABCG2+ cells in the basal epithelium constituted less than 1% of the total basal cell population. SP cells constituted 0.5-3% of the total epithelial fraction. The SP transcriptome was essentially the same as ABCG2+ and both populations expressed genes indicative of a stem cell phenotype, however, the cells also expressed many genes in common with endothelial cells.

Conclusion: These results provide gene expression profiles for the prostate SP and ABCG2+ cells that will be critical for studying normal development and carcinogenesis, in particular as related to the cancer stem cell concept.

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Figures

Figure 1
Figure 1

ABCG2+ cells in the prostate epithelium. Serial sections of normal human prostate were stained for ABCG2 (A – C) or CD138 (D – F). A subpopulation of cells in the basal epithelium is ABCG2+. Endothelial cells of capillaries (black arrow) are also ABCG2+ but are CD138- (red arrow). All basal cells of a particular small gland were ABCG2+/CD138+ (B). Original magnification is 100×, magnification for C and F is 200×.

Figure 2
Figure 2

Prostate epithelial side population. Prostate cells prepared from tissue specimens were analyzed for Hoechst 33342 dye efflux. The cytograms show (A) forward and side light scatter of the SP and non-SP populations as defined by (B) the dye efflux characteristics of the cells. Cytograms show the dye efflux characteristics of (C) the prostate cancer cell line C4-2 and (D) the breast cancer cell line MCF-7 used as positive controls.

Figure 3
Figure 3

Flow analysis of prostate epithelial cells. Epithelial cells collected on a Percoll density gradient were stained with CD31 and ABCG2. Cells in R3 of each cytogram were sorted for further analysis.

Figure 4
Figure 4

Overlap of prostate cell transcriptomes. The Venn diagram depicts shared genes expressed by each cell type: ABCG2+ (5D3) are red, endothelial (CD31+) are green, and SP are blue. Other colors show genes that are shared between the cell types, and white shows genes shared by all three.

Figure 5
Figure 5

Gene expression analysis of prostate SP cells. (A): Expression of various stem cell genes in human prostate SP (SP+) vs. non-SP (SP-), by reverse transcription polymerase chain reaction (RT-PCR). The ribosomal protein-encoding gene RPL13a served as the reaction control. Nestin, telomerase, Bmi-1 are reported in the literature to be stem cell genes. CD133 has been reported as a prostate stem cell gene. Androgen receptor (AR) is known to be expressed by differentiated cells. The SP+ and SP-cells were also CD31+, indicating possible endothelial contamination. (B): Prostate endothelial cells (CD31+) also expressed these genes.

Figure 6
Figure 6

Expression of stem cell genes in ABCG2+ cells. Expression level is relative to CD31+ cells. Enrichment of TERT, CD133, BMI-1, NES and ABCG2 in the ABCG2+ cells vs. CD31+ was verified.

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