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• W1993352077 abstract "The features and functions of prostatic neuroendocrine (NE) cells remain ill-defined. Neuroendocrine differentiation (NED) in adenocarcinoma of the human prostate (CaP) is associated with more aggressive disease, but the underlying mediators are poorly understood. We examined these issues in transgenic mice that utilize regulatory elements from the cryptdin-2 gene (Defcr2) to express simian virus 40 large T antigen (TAg) in prostatic NE cells. CR2-TAg mice develop prostatic intraepithelial neoplasia at 8 weeks of age, 1 week after the onset of TAg expression. An invasive phase follows 2–4 weeks later, with lymph node, liver, lung, brain, and bone metastases appearing within 16 weeks. DNA microarray studies revealed 122 mRNAs that were increased ≥2-fold in duplicate assays of 16-week-old CR2-TAg versus normal prostates. Thirty two transcripts encode proteins associated with neurons and endocrine cells (e.g. basic helix loop helix, SRY-related high mobility group box and sine-oculis homeobox transcription factors, Hu RNA-binding proteins, neuronatin, Racgap1, collapsin response mediator protein-1, synaptotagmin-1, proprotein convertase, and secretogranins). Follow-up studies of candidate mediators and biomarkers of differentiation/growth in the microarray data set involved real time quantitative reverse transcriptase-PCR assays of laser capture microdissected NE cells from CR2-TAg prostates plus liver metastases, and immunohistochemical comparisons of transgenic mouse prostates and 35 human CaP samples. Our findings include (a) expression of the bHLH mouse achaete-scute homolog (mASH1) in normal and CR2-TAg NE cells and foci of NED in human CaP, (b) glutamic acid decarboxylase and its product (γ-aminobutyric acid) in neoplastic NE cells juxtaposed next to cohorts of normal γ-aminobutyric acid receptor expressing secretory cells (a potential route for paracrine interactions between these two epithelial lineages), and (c) aromatic l-amino-acid decarboxylase, but not its dopamine/serotonin products, in CR2-TAg NE cells and NED. These results underscore the value of CR2-TAg mice for characterizing normal NE cell biology and tumorigenesis. The features and functions of prostatic neuroendocrine (NE) cells remain ill-defined. Neuroendocrine differentiation (NED) in adenocarcinoma of the human prostate (CaP) is associated with more aggressive disease, but the underlying mediators are poorly understood. We examined these issues in transgenic mice that utilize regulatory elements from the cryptdin-2 gene (Defcr2) to express simian virus 40 large T antigen (TAg) in prostatic NE cells. CR2-TAg mice develop prostatic intraepithelial neoplasia at 8 weeks of age, 1 week after the onset of TAg expression. An invasive phase follows 2–4 weeks later, with lymph node, liver, lung, brain, and bone metastases appearing within 16 weeks. DNA microarray studies revealed 122 mRNAs that were increased ≥2-fold in duplicate assays of 16-week-old CR2-TAg versus normal prostates. Thirty two transcripts encode proteins associated with neurons and endocrine cells (e.g. basic helix loop helix, SRY-related high mobility group box and sine-oculis homeobox transcription factors, Hu RNA-binding proteins, neuronatin, Racgap1, collapsin response mediator protein-1, synaptotagmin-1, proprotein convertase, and secretogranins). Follow-up studies of candidate mediators and biomarkers of differentiation/growth in the microarray data set involved real time quantitative reverse transcriptase-PCR assays of laser capture microdissected NE cells from CR2-TAg prostates plus liver metastases, and immunohistochemical comparisons of transgenic mouse prostates and 35 human CaP samples. Our findings include (a) expression of the bHLH mouse achaete-scute homolog (mASH1) in normal and CR2-TAg NE cells and foci of NED in human CaP, (b) glutamic acid decarboxylase and its product (γ-aminobutyric acid) in neoplastic NE cells juxtaposed next to cohorts of normal γ-aminobutyric acid receptor expressing secretory cells (a potential route for paracrine interactions between these two epithelial lineages), and (c) aromatic l-amino-acid decarboxylase, but not its dopamine/serotonin products, in CR2-TAg NE cells and NED. These results underscore the value of CR2-TAg mice for characterizing normal NE cell biology and tumorigenesis. The prostate is a tubuloalveolar gland composed of three epithelial lineages. Members of the secretory cell lineage (also known as luminal cells) predominate. Basal cells are the principal proliferating cell type and are located between the basement membrane and the secretory cell layer (1Bonkhoff H. Stein U. Remberger K. Hum. Pathol. 1994; 25: 42-46Crossref PubMed Scopus (199) Google Scholar). Neuroendocrine (NE) 1The abbreviations used are: NE, neuroendocrine; NED, neuroendocrine differentiation; PIN, prostatic intraepithelial neoplasia; CaP, conventional adenocarcinoma of the human prostate; CR2, mouse cryptdin-2 gene; TAg, simian virus 40 large T antigen; qRT-PCR, real time quantitative reverse transcriptase-PCR; LCM, laser capture microdissection; AFC, average fold change in mRNA level as determined by duplicate GeneChip studies; Cg, chromogranin; mASH1, mouse homolog of the Drosophila proneural gene complexachaete-scute, hASH1, human ortholog of mASH1; bHLH, basic helix loop helix; SCLC, small cell lung carcinoma; GAD, glutamic acid decarboxylase; GABA, γ-aminobutyric acid; AADC, aromaticl-amino-acid decarboxylase; CNS, central nervous system; HPLC, high pressure liquid chromatography; mAb, monoclonal antibody; PBS, phosphate-buffered saline; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 4-morpholinepropanesulfonic acid; DOPA, 3,4-dihy- droxyphenylalanine cells are least abundant and interposed between the other two cell types (2di Sant'Agnese P.A. Hum. Pathol. 1992; 23: 287-296Crossref PubMed Scopus (216) Google Scholar). NE cells have irregular dendritic-like extensions that allow them to contact neighboring epithelial cells over a distance of several cell diameters (2di Sant'Agnese P.A. Hum. Pathol. 1992; 23: 287-296Crossref PubMed Scopus (216) Google Scholar, 3di Sant'Agnese P.A. De Mesy Jensen K.L. Hum. Pathol. 1984; 15: 1034-1041Crossref PubMed Scopus (69) Google Scholar). The contributions of NE cells to normal prostatic development and physiology remain poorly defined. Prostate cancer is the second leading cause of cancer deaths among American men (4Jemal A. Thomas A. Murray T. Thun M. CA-Cancer J. Clin. 2002; 52: 23-47Crossref PubMed Scopus (2934) Google Scholar). NE cell carcinoma of the prostate is rare. Like NE cell cancers in other tissues (e.g. lung), it is highly invasive (5Cohen R.J. Glezerson G. Haffejee Z. Br. J. Urol. 1991; 66: 405-410Crossref Scopus (89) Google Scholar). Conventional adenocarcinoma of the prostate (CaP) can exhibit features of neuroendocrine differentiation (NED). Studies indicate that development of NED heralds a phase of more aggressive, androgen-independent growth (6Bohrer M. Schmoll J. Verh. Dtsch. Ges. Pathol. 1993; 77: 107-110PubMed Google Scholar, 7Dema A. Raica M. Tudose N. Rom. J. Morphol. Embryol. 1996; 42: 83-88PubMed Google Scholar, 8Speights V. Cohen M. Riggs M. Coffield K. Keegan G. Arber D. Br. J. Urol. 1997; 80: 281-286Crossref PubMed Google Scholar). The molecular mediators underlying this behavior are largely undefined. Moreover, variations in the reported incidence of NED in patients with CaP likely arise from sampling methods and from the limited number of NE cell biomarkers available to score neuroendocrine features. Animal models are needed to define the molecular features of prostatic NE cells so that their biological properties can be better characterized, new biomarkers of NED identified for more accurate phenotyping of patient populations, and new therapeutic targets uncovered. We have created one such genetically defined model. It is based on the finding that nucleotides −6500 to +34 of the mouse cryptdin-2 gene (Defcr2, abbreviated CR2) can be used to express foreign gene products, such as simian virus large T antigen (SV40 TAg), in a subset of mouse prostatic NE cells (9Garabedian E.M. Humphrey P.A. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15382-15387Crossref PubMed Scopus (139) Google Scholar). Multiple pedigrees of FVB/N mice containing a CR2-SV40 TAg transgene develop the presumptive precursor to CaP, prostatic intraepithelial neoplasia (PIN), by 8–9 weeks of age (9Garabedian E.M. Humphrey P.A. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15382-15387Crossref PubMed Scopus (139) Google Scholar). The onset of PIN follows the onset of transgene expression by ≤1 week, indicating that the NE cell population is exquisitely sensitive to transformation by this viral oncoprotein. These initiated NE cells expand rapidly within ducts and acini. Invasion into the underlying stroma is first apparent by 10 weeks of age and progresses quickly from 12 to 16 weeks. Unlike most transgenic mouse models of cancer, metastatic spread is a hallmark. The majority of males die by 6 months of age with metastases in their regional lymph nodes, liver, lung, brain, and bone (9Garabedian E.M. Humphrey P.A. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15382-15387Crossref PubMed Scopus (139) Google Scholar). Immunohistochemical studies have shown that the transformed NE cells do not contain detectable levels of the androgen receptor. Castration at 4 weeks has no effect on the course of the disease (9Garabedian E.M. Humphrey P.A. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15382-15387Crossref PubMed Scopus (139) Google Scholar). Female CR2-TAg mice have a normal lifespan and no evidence of cancer (9Garabedian E.M. Humphrey P.A. Gordon J.I. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15382-15387Crossref PubMed Scopus (139) Google Scholar). The CR2-TAg mouse offers an opportunity to obtain a molecular description of prostatic NE cells. A functional genomics approach is feasible because of the stereotyped pattern of evolution of this cancer, and because of the ability to recover amplified, neoplastic NE cell populations from discrete tumor foci using laser capture microdissection. In the present report, we describe the results of such an analysis. We have focused on identifying candidate mediators of NE cell differentiation and new biomarkers of this epithelial lineage. We show that data obtained from this mouse model are applicable to human CaP. Mice hemizygous for the CR2-TAg transgene and their normal littermates were housed in microisolater cages in a barrier facility, under a strict 12-h light cycle, and given free access to a standard chow diet (Lab Diet 5053, Purina Mills, St. Louis, MO). All mice were specified pathogen-free (defined as negative for hepatitis, Minute, lymphocytic choriomeningitis, ectromelia, polyoma, Sendai, pneumonia, and mouse adenoviruses, enteric bacterial pathogens, and parasites). CR2-TAg animals (n = 9) were sacrificed at 8 weeks of age, and their prostates were removed, fixed in 10% buffered formalin, washed in 70% ethanol, embedded in paraffin, and each gland serially sectioned in its entirety (section thickness = 5 μm). Every 10th section was stained with hematoxylin and eosin and examined by light microscopy. A total of 749 foci of PIN were evaluated for architectural pattern using standard criteria applied to the human prostate (10Bostwick D.G. Amin M.B. Dundore P. Marsh W. Schultz D.S. Hum. Pathol. 1993; 24: 298-310Crossref PubMed Scopus (218) Google Scholar). Prostates were removed from transgenic mice and their normal littermates after they were sacrificed at 10, 16, and 24 weeks of age (n = 8 mice/group/time point). Macroscopic liver metastases were dissected from five 24-week CR2-TAg males (n = 2–8 metastases/mouse). Samples, similar in size to the dissected metastases, were taken from comparable lobes of the livers of five age-matched normal male littermates. Total RNA was extracted from prostates, liver metastases, and normal liver samples using the RNeasy Midi kit (Qiagen, Valencia, CA). Equal sized aliquots of RNAs from a given type of tissue were pooled, and an aliquot of each pooled sample (2 μg) was used for random hexanucleotide-primed cDNA synthesis (11Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar). cDNAs were added to 25-μl reaction mixtures containing 12.5 μl of 2× SYBR Green master mix (Applied Biosystems, Foster City, CA), 0.25 units of UDP-N-glycosidase (Invitrogen), and 900 nmgene-specific primers (for a list of these primers, see Table A in the Supplemental Material). A melting curve was used to identify a temperature where only the amplicon, and not primer dimers, accounted for the SYBR green-bound fluorescence (12Hooper L.V. Mills J.C. Roth K.A. Stappenbeck T.S. Wong M.H. Gordon J.I. Methods Microbiol. 2002; 31: 559-589Crossref Google Scholar). Assays were performed in triplicate with an ABI Prism 7700 Sequence Detector (Applied Biosystems). All data were normalized to an internal standard (18 S ribosomal RNA; ΔΔC T method, User Bulletin 2, Applied Biosystems). Prostates were recovered from 16- and 24-week-old transgenic animals and macroscopic liver metastases from 24-week-old animals (n = 3 mice/time point). Tissues were immediately frozen in OCT (Sakura Finetek, Torrance, CA) and 8-μm-thick cryosections were prepared. Sections were stained briefly with hematoxylin and eosin and NE cells harvested by LCM using the PixCell II system (15-μm diameter laser spot; Arcturus; Mountain View, CA) and CapSure HS LCM Caps (Arcturus) (11Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar). RNA was isolated from 50,000 dissected cells/sample using the PicoPure isolation kit (Arcturus) and analyzed by qRT-PCR (11Stappenbeck T.S. Hooper L.V. Manchester J.K. Wong M.H. Gordon J.I. Methods Enzymol. 2002; 356: 168-196Google Scholar). CR2-TAg mice and their normal littermates were sacrificed at 16 weeks of age (n = 8 mice/group). Intact prostates were excised, snap-frozen in liquid nitrogen, and total cellular RNA was extracted (RNeasy Midi Kit). Equal amounts of RNA from each tissue in each group were pooled. Two cRNAs were then independently generated from each pooled RNA preparation. Each cRNA was hybridized to a set of Mu11K GeneChips (Affymetrix, Santa Clara, CA). Following hybridization and washing, the overall fluorescence intensity across each chip was scaled to a target intensity of 1500 using proprietary GeneChip software, and pairwise comparisons of mRNA levels were performed with the normal prostate GeneChips designated as “base line.” Data sets were generated by extracting all mRNAs that were called “Present” in either the normal or transgenic prostate GeneChip, and whose levels were ≥2-fold increased or decreased in transgenicversus normal prostates in duplicate GeneChip comparisons. Transgenic mice and their normal littermates were sacrificed at 10, 12, 16, and 24 weeks of age (n = 2–5 mice/time point/group/pedigree; n = 2 pedigrees). Dorsal, ventral, anterior, and lateral lobes of the prostate were removeden bloc. Liver and abdominal lymph nodes were also retrieved. All tissues were fixed for 6 h at room temperature in 10% neutral-buffered formalin, washed in 70% ethanol, and embedded in paraffin. Sections were deparaffinized in xylenes, rehydrated in isopropyl alcohol, microwaved for 25 min in 10 mm sodium citrate buffer, pH 6 (1500-watt GE Sensor Microwave), washed in PBS, placed in PBS-blocking buffer (1% bovine serum albumin, 0.3% Triton X-100 in PBS) for 30 min at 23 °C, and incubated overnight at 4 °C with one or more of the following antibodies: (i) goat anti-SV40 TAg (generated against the purified intact viral oncoprotein; final dilution in blocking buffer = 1:1000); (ii) mouse monoclonal antibody (mAb) to mouse achaete-scute homolog (mASH1; Clone 24B7.2D11.1; Pharmingen; diluted 1:10 when fluorescence detection methods were used, 1:250 for chromogenic development); (iii) rabbit anti-human synaptophysin (1:100, DAKO, Carpinteria, CA); (iv) mouse mAb to rat synaptophysin (clone SVP-38; 1:100, Sigma); (v) mouse mAb raised against human chromogranin A (CgA; clone LK2H10 (2di Sant'Agnese P.A. Hum. Pathol. 1992; 23: 287-296Crossref PubMed Scopus (216) Google Scholar); Chemicon, Temecula, CA; 1:1000); (vi) rabbit anti-γ-aminobutyric acid (GABA, Sigma; 1:100); (vii) mouse mAb against bovine GABAAreceptor β-chain, (clone bd17; Chemicon; 1:50); (viii) guinea pig anti-GABAB receptor (raised against a peptide whose sequence is shared by both GABAB R1a and GABABR1b and should therefore recognize both isoforms; Chemicon; 1:500); (ix) rabbit anti-bovine aromatic l-amino-acid decarboxylase (AADC; Protos Biotech, New York, (13Cho S. Hwang O. Baker H. Baik H.H. Volpe B.T. Son J.H. Joh T.H. Synapse. 1999; 34: 135-144Crossref PubMed Scopus (2) Google Scholar); 1:100 (immunofluorescence) or 1:250 (chromogenic detection)); (x) rat anti-serotonin (Chemicon; 1:100), (xi) mouse mAb raised against the C-terminal domain of mouse tyrosine hydroxylase (clone 1B5; Novocastra; 1:100); (xii) rabbit anti-mouse Nov (a generous gift from P. Ellis, University of Cambridge, Cambridge, UK; 1:500); (xiii) rabbit anti-human Nov (obtained from R. Rosenfeld, Oregon Health and Science University, Portland, OR; 1:2500); and (xiv) rabbit anti-mouse laminin (Chemicon; 1:1000). To evaluate integrin expression, prostates were fixed in periodate/lysine/paraformaldehyde for 45 min, washed in PBS, and frozen in OCT. Frozen sections were thawed at room temperature (20 min), washed in PBS followed by PBS-blocking buffer (20 min, room temperature), and then incubated overnight at 4 °C with one of the following antibodies: (i) hamster anti-mouse integrin α2 (Pharmingen, 1:500); (ii) rabbit anti-mouse integrin α3 (Chemicon, 1:500); (iii) rabbit anti-mouse integrin α5 (Chemicon, 1:500); (iv) rat anti-mouse integrin α6 (Pharmingen, 1:500); (v) mouse anti-human integrin αv (Chemicon, 1:500); (vi) rabbit anti-mouse integrin β1 (Chemicon, 1:500); and (vii) rat anti-mouse integrin β4 (1:500, Pharmingen). Antigen-antibody complexes were detected with fluorescein isothiocyanate-, indocarbocyanine (Cy3)-, Alexa fluor 488-, or Alexa fluor 633-conjugated antibodies to goat, mouse, rabbit, hamster, or guinea pig Ig (Jackson ImmunoResearch, West Grove, PA, or Molecular Probes, Eugene, OR; 1:500). In some cases, bound mouse antibodies were detected using the M.O.M. kit (Vector Laboratories), together with the Vectastain ABC elite kit and NovaRED (Vector Laboratories, Burlingame, CA). mASH1-antibody complexes were identified using the tyramide signal amplification indirect cyanine-3 kit (PerkinElmer Life Sciences). Stained slides were viewed and photographed using a Zeiss Axioscope or a Bio-Rad MRC 1024 confocal microscope. A tissue panel, consisting of 35 prostates with conventional adenocarcinoma, 8 lymph node metastases from a subset of these patients, 2 NE cell cancers of the prostate, and 2 small cell lung carcinomas (SCLC), was obtained, using an institutional human studies review board-approved protocol, from the archives of the Department of Pathology and Immunology at Washington University School of Medicine. Serial sections were cut from each block of paraffin-embedded tissue. Hematoxylin and eosin-stained sections were used to assign a histopathologic grade (Gleason score) to each sample. Gleason scores ranged from 5 to 10 (see Ref. 14Gleason D. Urologic Pathology: The Prostate. Lea and Febiger, Philadelphia1977: 171-198Google Scholar for details of the scoring system). Adjacent sections were incubated with a subset of the primary antibodies described above. Antigen-antibody complexes were detected using Vectastain ABC elite kit and NovaRED or 3,3′-diaminobenzidine/metal substrate (Pierce). Sections were surveyed, in a single-blinded fashion, for cells containing immunoreactive protein. Tissues were Dounce-homogenized in ice-cold homogenization buffer (50 mm Tris, pH 6.0, 0.2% Triton X-100, 1× complete protease inhibitor (Roche Molecular Biochemicals)) (15Lamprecht F. Coyle J.T. Brain Res. 1972; 41: 503-506Crossref PubMed Scopus (92) Google Scholar). The homogenate was centrifuged at 10,000 × g for 10 min at 4 °C. The supernatant was collected; protein concentrations were determined (Bio-Rad protein assay kit), and the material employed for immunoblot or enzyme activity assays. 25 μg of protein from three pooled 24-week normal prostates, three pooled 24-week CR2-TAg prostates, or normal mouse kidney (positive control) were separated by electrophoresis under reducing conditions using a 10% NuPAGE BisTris gel and MOPS-SDS buffer (Invitrogen). Following electrophoresis, proteins were transferred from the gel to a polyvinylidene difluoride membrane (Invitrogen) by electroblotting. The resulting blot was incubated with rabbit anti-AADC (see above; diluted 1:2500 in blocking buffer (0.05% Tween 20, 0.1% bovine serum albumin in PBS)) or a mouse mAb raised against tyrosine hydroxylase (also see above; 1:100). AADC-antibody complexes were detected using alkaline phosphatase-conjugated goat anti-rabbit Ig (Jackson ImmunoResearch; 1:10,000) and the Tropix Western-Light kit. Tyrosine hydroxylase-antibody complexes were visualized using alkaline phosphatase-conjugated goat anti-mouse Ig (Jackson ImmunoResearch). Control experiments were performed (with duplicate blots) where primary antibodies were omitted. Total cellular RNA isolated from 24-week-old CR2-TAg prostates and from brain (positive control) were used for RT-PCR assays. Four sets of primer pairs were used to generate amplicons representing various sets of exonic sequences from the Ddcgene: 5′-TTTTCAACATGGATTCCCGTG (forward) and 5′-ATCAGATGTGTAAGCAACCAGCTT (reverse); 5′-TACTGGCTGCTCGGACTAAAGTTA (forward) and 5′-CACCATTCAGAAGATACCGGAATT (reverse); 5′-GGGTCCCATCTGCAACCA (forward) and 5′-AAACCACATTTTCAAAGAGCGAAAT (reverse); 5′-GCCTTTAATATGGAGCCTGTTTATC (forward) and 5′-GTGGTAGTTATTTTTCTC (reverse). The amplicons were analyzed by agarose-gel electrophoresis. In addition, a single amplicon, containing the open reading frame that encodes the active form of AADC, was purified from the gel and its nucleotide sequence determined. AADC activity was assayed using the procedure of Lamprecht and Coyle (15Lamprecht F. Coyle J.T. Brain Res. 1972; 41: 503-506Crossref PubMed Scopus (92) Google Scholar) with modifications. Reaction mixtures (250 μl) contained 10 μg/ml pyridoxal phosphate (Sigma), 100 μg/ml ascorbic acid (Sigma), 600 μml-DOPA (Sigma), 30 mm sodium phosphate, pH 7.2, and dl-[14C]DOPA (American Radiolabeled Chemicals, St. Louis, MO; diluted to a final specific activity of 1 μCi/μmol; 0.15 μCi/assay). The reaction mixture was incubated for 15 min at 37 °C and then terminated by adding 1 ml of a solution of 4% perchloric acid and 2 mm4,5-dihydroxy-1,3-benzenedisulfonic acid (Tiron; Sigma). [14C]CO2 generated during the course of the assay was collected on Whatman filter paper (affixed to the top of the reaction vessel after being soaked in a solution of 20% α−phenylethylamine (Sigma) and 80% methanol). Radioactivity was measured by scintillation counting. Controls studies established that the assay was linear with respect to time (0–20 min) and input lysate protein concentration (0–5 μg). Prostates from three 24-week normal and three CR2-TAg mice were assayed separately in duplicate. Prostates were harvested from two 24-week-old normal mice and two age-matched CR2-TAg littermates. Liver metastases (carefully dissected from normal surrounding tissue) and lymph nodes containing metastases were obtained from one of the transgenic animals. Each sample was weighed, homogenized (separately) in 50 volumes of 0.1 m perchloric acid, 0.4 mmsodium metabisulfite, and centrifuged at ∼20,000 × gfor 10 min at 4 °C. The supernatant fraction was diluted 20-fold in HPLC buffer (1.2 mm heptanesulfonic acid, 0.2 mm EDTA, 100 mm sodium phosphate, pH 2.5, plus 6% methanol) and sterile-filtered through a 0.2-μm filter (Nylon Acrodisc 13, Pall Life Sciences, Ann Arbor, MI). 100-μl aliquots of each sample were analyzed for dopamine by chromatography on a HR-80 column (ESA, Chelmsford, MA) with the mobile phase consisting of the HPLC buffer described above (n = 3 assays/sample). Compounds were detected with a Coulochem Electrochemical Detector (model 5100A) fitted with a model 5011 analytical cell (ESA). Concentrations were calculated using dopamine standards run in parallel assays. Four architectural patterns of PIN are common in human CaP: tufted, micropapillary, cribriform, and flat. Bostwick et al. (10Bostwick D.G. Amin M.B. Dundore P. Marsh W. Schultz D.S. Hum. Pathol. 1993; 24: 298-310Crossref PubMed Scopus (218) Google Scholar) defined the frequency of these patterns in 60 patients. Micropapillary was most common (54.3% of fields examined per tissue sample), followed by tufted (32.5%), cribriform (9.8%), and flat (3.4%). We performed an analogous quantitative histopathologic analysis of nine 8-week-old CR2-TAg mouse prostates. This time point represents the initial week of onset of transgene expression, with the concomitant appearance of PIN. Each of the four patterns was observed (see Fig. 1S in the Supplemental Material). Tufted and cribriform patterns were encountered at a similar frequency in CR2-TAg mice as in men with PIN (50 and 7%, respectively). However, the flat pattern was very frequent in mice (43% of fields) rather than being least frequent, while micropapillary architecture was least frequent in mice (0.3%) rather than being most frequent as in humans. As the area occupied by PIN within an acinus or duct increases, different architectural patterns appear next to one another (Fig.1 A). Together, these findings indicate that a specific architectural pattern of PIN cannot be used as a diagnostic criterion to include or exclude a NE cell origin, at least in mice. Further histologic studies of 10–12-week-old CR2-TAg mice (n = 24) revealed that growth of PIN prior to invasion resembles the ductal/acinar spreading pattern observed in humans. In the dorsal, ventral, and paired lateral lobes of the mouse prostate, secretory cells were progressively replaced as transformed NE cells expanded. The underlying basal cell layer and basement membrane were preserved. A second pattern of cellular spreading was encountered in the anterior lobes of the mouse prostate (also called the coagulating glands). Here, transformed NE cells spread along the ducts between the basal cell layer and the overlying layer of normal secretory cells (Fig. 1 B). This undermining pattern of spread is not present in human prostates. By using a panel of integrin antibodies (see “Experimental Procedures”), we found that loss of β4is a sensitive marker of early invasion (Fig. 1 C). The progressive amplification of NE cells in CR2-TAg prostates offers an opportunity to use DNA microarrays to profile their molecular properties. Therefore, total cellular RNA was isolated from eight 16-week normal prostates and eight age-matched CR2-TAg prostates. Equal sized aliquots of RNA obtained from each prostate in a given group were pooled. Two cRNA targets were independently synthesized from each pooled RNA sample, and each cRNA was used to interrogate commercial, high density, oligonucleotide-based microarrays (GeneChips) containing probe sets representing 11,000 mouse genes and EST clusters. A total of 123 mRNAs (including 22 ESTs) were identified that satisfied the following selection criteria: the transcript was called Present by proprietary GeneChip software in either normal or transgenic prostatic RNAs; its levels were at least 2-fold different between transgenic versus normal prostates (base line); the difference was in the same direction (increased or decreased) in duplicate GeneChip comparisons. 122 transcripts were increased ≥2-fold in transgenic prostates, whereas one (encoding annexin A1) was decreased. The genes were sorted into several categories. The largest category (32 genes) was defined as “Neural/Endocrine.” An annotated list of all genes is provided in Table B of the Supplemental Material. Nine of the 32 genes in the Neural/Endocrine category specify transcription factors. They belong to the basic helix-loop-helix (bHLH), SRY-related high mobility group box (Sox), sine-oculis homeobox (Six), and hepatocyte nuclear factor (HNF) families. There were two bHLH proteins: mouse homolog of achaete-scute (mASH1; average fold change (AFC) as calculated by GeneChip software from duplicate DNA microarray assays = 38)) and Hes6 (AFC = 11). mASH1 forms a heterodimer with the ubiquitously expressed bHLH factor Tcfe2a (E2A immunoglobulin enhancer-binding factors E12/E47) to effect neural differentiation (16Johnson J.E. Birren S.J. Saito T. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3596-3600Crossref PubMed Scopus (134) Google Scholar, 17Tomita K. Nakanishi S. Guillemot F. Kageyama R. Genes Cells. 1996; 1: 765-774Crossref PubMed Scopus (138) Google Scholar). mASH1 is also important in differentiation of several NE cell populations (see below). Hes1 antagonizes the E box-binding ability of the mASH1-Tcfe2a heterodimer complex (18Sasai Y. Kageyama R. Tagawa Y. Shigemoto R. Nakanishi S. Genes Dev. 1992; 6: 2620-2634Crossref PubMed Scopus (583) Google Scholar) and can repress mASH1 transcription (19Chen H. Thiagalingam A. Chopra H. Borges M.W. Feder J.N. Nelkin B.D. Baylin S.B. Ball D.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5355-5360Crossref PubMed Scopus (233) Google Scholar). Hes6, in turn, functionally antagonizes the action of Hes1, thereby relieving Hes1 inhibition of bHLH proteins and promoting neural differentiation (20Bae S. Bessho Y. Hojo M. Kageyama R. Development. 2000; 127: 2933-2943Pu" @default.
• W1993352077 created "2016-06-24" @default.
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• W1993352077 creator A5010479640 @default.
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• W1993352077 creator A5062287369 @default.
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• W1993352077 date "2002-11-01" @default.
• W1993352077 modified "2023-10-17" @default.
• W1993352077 title "Molecular Characterization of a Metastatic Neuroendocrine Cell Cancer Arising in the Prostates of Transgenic Mice" @default.
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