1. Volume 54, Issue 6, Pages 1344-1353 (December 2008)
Delta-Like 1 (Dlk-1), a Novel Marker of Prostate Basal and Candidate Epithelial Stem Cells, Is Downregulated by Notch Signalling in Intermediate/Transit Amplifying Cells of the Human Prostate 
Jens A. Ceder, Linda Jansson, Leszek Helczynski, Per-Anders Abrahamsson 
European Urology 
Volume 54, Issue 6, Pages (December 2008)
DOI: /j.eururo
Copyright © 2008 European Association of Urology Terms and Conditions

2. Fig. 1
Expression of Notch-related proteins in the human adult prostate. The luminal cell layer was found to strongly express Notch-1 receptors (A), but did not exhibit nuclear Notch-1 immunoreactivity (see inset in A: inset does not show the nuclear DAPI stain). In contrast, the basal cell layer was negative for Notch-1 immunoreactivity. However, note a rare basal 34ßE12-cytokeratin phenotype cell that extends to the lumen and co-expresses Notch-1 (arrow in A), suggesting an intermediate phenotype. The neuroendocrine cell (NE) lineage (positive for chromogranin A; CgA) (arrow in B) was found to be negative for Notch-1 receptors (arrow in inset of B: inset shows only Notch-1 and DAPI staining). Staining specifically against cleaved Notch-receptors (cNICD) revealed that Notch-signalling is confined to rare blood vessels in the adult prostate (arrow in C). Note absence of cNICD in nuclei of epithelial cells (inset in C: without DAPI staining) and presence of cNICD in nuclei of blood vessels (arrow in inset of C). A fraction of CD34-positive blood vessels co-expressed the Notch-ligand Delta1 (D), whereas the luminal cell layer of glands and acini expressed the Notch-ligand Jagged1 (E). Note that, whereas the basal cell layer is negative for Jagged1, few 34ßE12-positive cells reaching the lumen stained positively for Jagged1 (inset in E). The potential Notch-inhibitor Delta-like 1 (Dlk-1) was found to be strongly expressed in the basal cell layer (F) and in a few cells reaching the lumen (arrow in F). Dlk-1 was further found to be expressed in most blood vessels (stars in G) and in round scattered cells of the stroma (arrow in G), negative for tyrosine-kinase KIT (arrowhead in G). However, note that KIT-positive candidate epithelial stem cells co-expressed Dlk-1 (see arrow in H and inset of H). Rare NE cells co-stained for CgA and Dlk-1 (see arrow in I and inset of I). The majority of CgA-positive (J) NE cells, however, were negative for Dlk1 expression (arrow in inset of J: inset shows only the Dlk-1 and DAPI staining). K shows unspecific signals and artefacts in the red and green channels of negative control reactions omitting primary antibodies. Pre-absorption of the Notch-1 antibodies using the immunizing Notch-1 peptide confirmed the specificity of the Notch-1 immunoreactivity (L). Scale bar: 20μm.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

3. Fig. 2
Tissue culture of human prostate explants leads to upregulation and activation of Notch-1, and to downregulation of basal cell proteins in intermediate cells. Jagged1 expression was lost (A) together with luminal cell markers as exocrine cells became fragmented in cultured explants. In contrast, cells with a basal 34ßE12-cytokeratin phenotype expanded extensively in response to tissue culture (A, B, D). However, some cells in 3-d tissue cultures exhibited slightly weaker 34ßE12 staining. Importantly, activate Notch signalling (cNICD) was found in these weakly 34ßE12-cytokeratin positive cells (B). Note that in 7-d cultured explants, Notch-1 is expressed in virtually all epithelial cells, and that the majority of cells show nuclear Notch-1 localisation (eg, arrows in C). Also note the absence of Dlk-1 in the bulk of epithelia in cultured explants (C), but that stromal Dlk-1 is not modulated (arrowhead in C). In 7-d explant cultures, 34ßE12-positive cells were found to have spread to the surface, where they often heavily downregulated 34ßE12 expression (D). Note the inverse correlation between 34ßE12-cytokeratin expression and cleaved active Notch-1 immunoreactivity, suggesting engagement in differentiation towards the luminal cell lineage. Scale bar: 20μm.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

4. Fig. 3
Notch inhibition in primary prostate epithelial cell cultures leads to suppressed downregulation of Dlk-1 expression. Notch receptors are cleaved and activated (cNICD) in primary prostate epithelial cell cultures (A), and Notch-1 immunoreactivity is predominantly nuclear (B), suggesting that the Notch-1-pathway is active, although Jagged1 protein expression was undetectable (A). Dlk-1 immunoreactivity was negative in most cell cultures. However, in one culture, a small cluster of Dlk-1–positive cells was found, and Notch-1 was accumulated in a perinuclear fashion, with low or no Notch-1-immunoreactivity in the nuclei (arrows in B). Administration of the Notch inhibitor L-685,458 to established cultures led to lost cNICD immunoreactivity, but Dlk-1 was not upregulated (C: inset shows DAPI staining in C). However, administration of the Notch inhibitor at the initiation and during culture of primary cells led to suppressed downregulation of Dlk-1 (D), and to a perinuclear localisation of Notch-1 (inset in D), with no nuclear Notch-1 immunoreactivity. Scale bar: 20μm.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

5. Fig. 4
Notch-inhibition in primary prostate epithelial cell cultures leads to decreased cell proliferation. Whereas L-685,458 treatment did not lead to a change in number of floating apoptotic cells, administration of L-685,458 led to significantly (p<0.001) decreased cell densities, as measured in sulforhodamine B assays. Error bar=average absolute deviation from the median, dimethyl sulfoxide (DMSO)=controls, L-685,458=cultures treated with gamma-secretase and Notch cleavage inhibitor.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions

6. Fig. 5
Hypothetical prostate epithelial cell lineage differentiation model. CD133/KIT/Dlk-1-positive stem cells self-renew and give rise to Dlk-1–expressing daughter cells. Proliferating daughter cells in turn gives rise, depending on intrinsic and extrinsic signals, to early intermediate luminal cells, NE cells, or p63-positive basal cells that can revert to a transit-amplifying intermediate phenotype. We suggest a dual role for Notch signalling in the human prostate: (a) to allow early luminal commitment and inhibit NE differentiation, in part by downregulating Dlk-1 and upregulating the Notch target Hes1 [29] and (b) to inhibit terminal luminal differentiation by interfering with AR-regulated transcription through upregulation of Hey1 [24]. We suggest that Jagged1 is upregulated by stromal factors, and that Notch-activity and proliferation is downregulated as cells terminally differentiate and start to express PSA. A possible mechanism for inactivation of Notch receptors is Fringe expression, as Fringe proteins have been reported to shunt Jagged1 activation of Notch-1 [30]. We propose that, if mutations in genes that inactivate the Notch pathway occur in stem or intermediate cells, this will abrogate terminal luminal differentiation and cause hyperplasia.
European Urology  , DOI: ( /j.eururo )
Copyright © 2008 European Association of Urology Terms and Conditions