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See detailIntermediate basal cells of the prostate: In vitro and in vivo characterization
Garraway, Levi; Lin, Douglas; Signoretti, Sabina et al

in Prostate (2003), 55(3), 206-218

BACKGROUND. Progenitor cells within the prostate basal layer may play important roles in differentiation and carcinogenesis; however, prostate stem cell populations remain uncharacterized. METHODS ... [more ▼]

BACKGROUND. Progenitor cells within the prostate basal layer may play important roles in differentiation and carcinogenesis; however, prostate stem cell populations remain uncharacterized. METHODS. Immunohistochemical and immunoblot analyses were used to characterize prostate epithelial cells (PrEC), a commercially available prostate basal cell isolate. RESULTS. Proliferating PrECs exhibited immunophenotypic characteristics most consistent with basal cells, but during senescence PrECs up-regulated androgen receptor (AR) mRNA, p27, and low-molecular-weight cytokeratin (LMWCK) expression, suggestive of partial differentiation. PrECs also stained strongly for involucrin, which marked a subset of intermediate prostate basal cells in vivo. Basal hyperplasia consisting of involucrin-positive cells was prevalent in prostate tissue from androgen-ablated patients, and formed epithelial clusters flanked by involucrin-negative basal and luminal monolayers. Cultivation of PrECs on matrigel together with androgen-treated stromal conditioned media resulted in dense aggregates, with a peripheral rim of basal-like cells expressing p63 and basal cytokeratins. CONCLUSIONS. PrEC represents an epithelial population whose basal characteristics are modified in response to matrigel, stromal factors, and senescence, consistent with a transient amplifying population. These cells may derive from a previously unrecognized, involucrin-positive subset present in vivo. (C) 2003 Wiley-Liss, Inc. [less ▲]

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See detailGene transcript quantitation by real-time RT-PCR in cells selected by immunohistochemistry-laser capture microdissection.
Lindeman, Neal; Waltregny, David ULg; Signoretti, Sabina et al

in Diagnostic Molecular Pathology : The American Journal of Surgical Pathology, Part B (2002), 11(4), 187-92

Studying tissue-based gene expression in different cell populations often requires immunohistochemistry-guided microdissection. However, mRNA degradation occurs during long staining procedures. We ... [more ▼]

Studying tissue-based gene expression in different cell populations often requires immunohistochemistry-guided microdissection. However, mRNA degradation occurs during long staining procedures. We combined a novel rapid immunoperoxidase technique with laser capture microdissection (LCM) and real-time quantitative RT-PCR to compare p27 mRNA expression in prostatic basal/secretory cells. Eight frozen prostate sections were immunostained with antibody 34betaE12 (high-molecular-weight keratin). Secretory and basal cells were separately collected by LCM. p27 transcripts from each cell group were quantitated by real-time RT-PCR, with GAPDH as standard. Immunostaining took 22 minutes, with RNA extraction from approximately 40 dissected cells from each compartment initiated within 40 minutes. Qualitative RT-PCR gave a product of the expected size from each sample. Quantitative RT-PCR gave basal/secretory p27/GAPDH ratios of 0.99-16.24 (mean 5.53 +/- 0.643). Immunostaining for keratin 34betaE12 can be done on frozen sections in approximately 20 minutes, and mRNA from pure cell populations can be quantitated by RT-PCR. We used this technique to show that p27 transcript levels are greater in basal than in secretory prostate cells, suggesting, when combined with prior studies, that regulation of p27 occurs at the protein level in normal cells. This technique may have wide applicability to studies of gene expression in distinct cell populations in heterogeneous tissues. [less ▲]

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See detailAndrogen-driven prostate epithelial cell proliferation and differentiation in vivo involve the regulation of p27.
Waltregny, David ULg; Leav, Irwin; Signoretti, Sabina et al

in Molecular Endocrinology (2001), 15(5), 765-82

Androgens control both growth and differentiation of the normal prostate gland. However, the mechanisms by which androgens act upon the cell cycle machinery to regulate these two fundamental processes are ... [more ▼]

Androgens control both growth and differentiation of the normal prostate gland. However, the mechanisms by which androgens act upon the cell cycle machinery to regulate these two fundamental processes are largely unknown. The cyclin-dependent kinase (cdk) inhibitor p27 is a negative cell cycle regulator involved in differentiation-associated growth arrest. Here, we investigate the role and regulation of p27 in the testosterone proprionate (TP)-stimulated regeneration of the ventral prostate (VP) of castrated rats. Continuous TP administration to castrated rats triggered epithelial cell proliferation, which peaked at 72 h, and then declined despite further treatment. Castration-induced atrophy of the VP was associated with a significant increase in p27 expression as compared with the VP of intact animals. Twelve hours after the initiation of androgen treatment, total p27 levels as well as its fraction bound to cdk2, its main target, significantly dropped in the VP of castrated rats. Thereafter, concomitantly to the induction of epithelial cell proliferation, the glandular morphology of VP was progressively restored at 48-96 h of TP treatment. During this period of the regenerative process, whereas both proliferating basal and secretory epithelial cells did not express p27, the protein was selectively up-regulated in the nonproliferating secretory epithelial compartment. This up-regulation of p27 expression was coincident with an increase in its association with, and presumably inhibition of, cdk2. At each time point of TP treatment, p27 abundance in the VP was inversely correlated with the level of its proteasome-dependent degradation activity measured in vitro in VP lysates, whereas only slight changes in the amount of p27 transcripts were detected. In addition, the antiandrogen flutamide blocked maximal TP-induced p27 degradation completely. Finally, the expression of skp2, the ubiquitin ligase that targets p27 for degradation, was seen to increase with androgen administration, preceding maximal proliferation and concomitantly to augmented p27 degradation activity. Taken together, our data indicate that androgens mediate both proliferation and differentiation signals in normal prostate epithelial cells in vivo, through regulation of p27. [less ▲]

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See detailRapid immunostaining of frozen sections: a tool for laser capture microdissection and quantitative RT-PCR
Lindeman, Neal; Waltregny, David ULg; Signoretti, Sabina et al

in Laboratory Investigation : Journal of Technical Methods & Pathology (2001), 81

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See detailThe p27 ubiquitin ligase skp2 is overexpressed in breast cancer
Signoretti, Sabina; Monti, Francesca; Isaac, Beth et al

in Laboratory Investigation : Journal of Technical Methods & Pathology (2001), 81

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See detailp63 is a prostate basal cell marker and is required for prostate development.
Signoretti, Sabina; Waltregny, David ULg; Dilks, James et al

in American Journal of Pathology (2000), 157(6), 1769-75

The p53 homologue p63 encodes for different isotypes able to either transactivate p53 reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63). p63 is expressed in the basal cells of many ... [more ▼]

The p53 homologue p63 encodes for different isotypes able to either transactivate p53 reporter genes (TAp63) or act as p53-dominant-negatives (DeltaNp63). p63 is expressed in the basal cells of many epithelial organs and its germline inactivation in the mouse results in agenesis of organs such as skin appendages and the breast. Here, we show that prostate basal cells, but not secretory or neuroendocrine cells, express p63. In addition, prostate basal cells in culture predominantly express the DeltaNp63alpha isotype. In contrast, p63 protein is not detected in human prostate adenocarcinomas. Finally, and most importantly, p63(-/-) mice do not develop the prostate. These results indicate that p63 is required for prostate development and support the hypothesis that basal cells represent and/or include prostate stem cells. Furthermore, our results show that p63 immunohistochemistry may be a valuable tool in the differential diagnosis of benign versus malignant prostatic lesions. [less ▲]

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