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• W2023423534 abstract "In situ hybridization and immunohistochemistry were used to localize and compare the expression of the long form of the human prolactin receptor in fetal, prepubertal, and adult prostate. Results were then compared with hyperplastic, dysplastic, and neoplastic lesions. Both receptor message and protein were predominately localized in epithelial cells of the fetal, neonatal, prepubertal, and normal adult prostate. In hyperplastic lesions the expression of the receptor was unchanged with respect to normal epithelial cells. Irrespective of grade, markedly enhanced expression of the receptor was evident in dysplastic lesions. In lower Gleason grade carcinomas the intensity of receptor signal at the message and protein levels approximated that found in normal prostatic epithelium. However, in foci within higher grade cancers, receptor expression appeared diminished. Results from our study suggest that prolactin action plays a role in the development and maintenance of the human prostate and may also participate in early neoplastic transformation of the gland. Diminution of receptor expression in high grade neoplasms could reflect the emergence of a population of cells that are no longer responsive to the peptide hormone. In situ hybridization and immunohistochemistry were used to localize and compare the expression of the long form of the human prolactin receptor in fetal, prepubertal, and adult prostate. Results were then compared with hyperplastic, dysplastic, and neoplastic lesions. Both receptor message and protein were predominately localized in epithelial cells of the fetal, neonatal, prepubertal, and normal adult prostate. In hyperplastic lesions the expression of the receptor was unchanged with respect to normal epithelial cells. Irrespective of grade, markedly enhanced expression of the receptor was evident in dysplastic lesions. In lower Gleason grade carcinomas the intensity of receptor signal at the message and protein levels approximated that found in normal prostatic epithelium. However, in foci within higher grade cancers, receptor expression appeared diminished. Results from our study suggest that prolactin action plays a role in the development and maintenance of the human prostate and may also participate in early neoplastic transformation of the gland. Diminution of receptor expression in high grade neoplasms could reflect the emergence of a population of cells that are no longer responsive to the peptide hormone. It has long been thought that prolactin (PRL) influences normal growth, development, and function of the prostate.1Grayhack JT Pituitary factors influencing the growth of the prostate.Natl Cancer Inst Monog. 1963; 12: 159-199Google Scholar, 2Negro-Vilar A Saad WA McCann SM Evidence for a role of prolactin in prostate and seminal vesicle growth in immature animals.Endocrinology. 1997; 100: 729-737Crossref Scopus (121) Google Scholar, 3Webber M Murphy GP Sandberg AA Karr JP Polypeptide hormones and the prostate. The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 175B. Alan R. Liss, New York1981: 63-88Google Scholar, 4Sandberg AA Some experimental results with prolactin: an overview of effects on the prostate.in: Murphy GP Sandberg AA Karr JP The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 75B. Alan R. Liss, New York1981: 9-18Google Scholar, 5Keenan EJ Ramsey EE Kemp DD the role of prolactin in the growth of the prostate gland.in: Murphy GP Sandberg AA Karr JP The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 75B. Alan R. Liss, New York1981: 9-18Google Scholar, 6Costello LC Franklin RB Effect of prolactin on the prostate.Prostate. 1994; 24: 162-166Crossref PubMed Scopus (158) Google Scholar The possibility that the hormone exerts trophic effects on the prostate has been suggested by numerous past investigations, largely done in rats,1Grayhack JT Pituitary factors influencing the growth of the prostate.Natl Cancer Inst Monog. 1963; 12: 159-199Google Scholar, 2Negro-Vilar A Saad WA McCann SM Evidence for a role of prolactin in prostate and seminal vesicle growth in immature animals.Endocrinology. 1997; 100: 729-737Crossref Scopus (121) Google Scholar, 3Webber M Murphy GP Sandberg AA Karr JP Polypeptide hormones and the prostate. The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 175B. Alan R. Liss, New York1981: 63-88Google Scholar, 4Sandberg AA Some experimental results with prolactin: an overview of effects on the prostate.in: Murphy GP Sandberg AA Karr JP The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 75B. Alan R. Liss, New York1981: 9-18Google Scholar, 5Keenan EJ Ramsey EE Kemp DD the role of prolactin in the growth of the prostate gland.in: Murphy GP Sandberg AA Karr JP The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 75B. Alan R. Liss, New York1981: 9-18Google Scholar, 6Costello LC Franklin RB Effect of prolactin on the prostate.Prostate. 1994; 24: 162-166Crossref PubMed Scopus (158) Google Scholar and inferred by results from studies in humans which have shown that circulating PRL levels rise sharply around the time of sexual maturation and are significantly elevated in adult men when compared to prepubertal boys.7Bartke A Prolactin and physiological regulation of the mammalian testes. The testis in normal and infertile men. Raven Press, New York1977: 367-378Google Scholar In this regard PRL may indirectly influence proliferation in the gland by up-regulating the levels of prostatic androgen receptor.8Prins GS Prolactin influence on cytosol and nuclear androgen receptors in the ventral, dorsal, and lateral lobes of the rat prostate.Endocrinology. 1987; 120: 1457-1464Crossref PubMed Scopus (69) Google Scholar, 9Reiter E Bonnet P Sente B Dombrowicz D de Leval J Closset J Hennen G Growth hormone and prolactin stimulate androgen receptor, insulin-like growth factor-1 (IGF-1) and IGF-1 receptor levels in the prostate of immature rats.Mol Cell Endocrinol. 1992; 88: 77-87Crossref PubMed Scopus (67) Google Scholar Conversely, androgens may have a regulatory effect on intraglandular PRL synthesis by secretory epithelium as shown by both in vivo and organ culture studies of rat10Nevalainen MT Valve EM Ahonen T Yagi A Paranko J Harkonen PL Androgen-dependent expression of prolactin in rat prostate epithelium in vivo and in organ culture.FASEB J. 1997; 11: 1297-1330Crossref PubMed Scopus (70) Google Scholar and human prostate.11Nevalainen MT Valve EM Ingleton PM Nurmi M Martikainen PM Harkonen PL Prolactin and prolactin receptors are expressed and functioning in the human prostate.J Clin Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar Indirect evidence of possible PRL involvement in the development of benign prostatic hyperplasia (BPH) and/or carcinoma has come from reports that circulating hormone levels were significantly higher in older men when compared with those found in younger males.3Webber M Murphy GP Sandberg AA Karr JP Polypeptide hormones and the prostate. The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 175B. Alan R. Liss, New York1981: 63-88Google Scholar, 12Harper ME Peeling WB Cowley T Brownsey BG Phillips ME Groom G Fahmy DR Griffiths K Plasma steroid.Acta Endocrinol (Copenh). 1976; 81: 409-426PubMed Google Scholar, 13Saroff J Kirdoni RY Chu TM Chu TM Wajsman Z Murphy GP Measurements of prolactin and androgens in patients with prostatic disease.Oncology. 1980; 37: 46-52Crossref PubMed Scopus (47) Google Scholar Moreover, patients with prostate cancer have been reported to have higher levels of plasma PRL than did age-matched controls,3Webber M Murphy GP Sandberg AA Karr JP Polypeptide hormones and the prostate. The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 175B. Alan R. Liss, New York1981: 63-88Google Scholar, 12Harper ME Peeling WB Cowley T Brownsey BG Phillips ME Groom G Fahmy DR Griffiths K Plasma steroid.Acta Endocrinol (Copenh). 1976; 81: 409-426PubMed Google Scholar, 13Saroff J Kirdoni RY Chu TM Chu TM Wajsman Z Murphy GP Measurements of prolactin and androgens in patients with prostatic disease.Oncology. 1980; 37: 46-52Crossref PubMed Scopus (47) Google Scholar and high affinity PRL binding sites have been detected in normal, BPH, and neoplastic human prostate.3Webber M Murphy GP Sandberg AA Karr JP Polypeptide hormones and the prostate. The Prostate Cell: Structure and Function. Part B, Prolactin, Carcinogenesis, and Clinical Aspects. 175B. Alan R. Liss, New York1981: 63-88Google Scholar, 11Nevalainen MT Valve EM Ingleton PM Nurmi M Martikainen PM Harkonen PL Prolactin and prolactin receptors are expressed and functioning in the human prostate.J Clin Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 14Leake A Chisolm GD Habib FK Characterization of the prolactin receptor in human prostate.J Endocrinol. 1983; 99: 321-328Crossref PubMed Scopus (15) Google Scholar PRL, along with growth hormone, belongs to a superfamily of growth factors.15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar The peptide hormone is known to have highly pleiotropic actions including those related to regulation of growth and differentiation. These broad range of effects are now known to be mediated by the prolactin receptors (PRLr) present in a large number of tissues including the human prostate.11Nevalainen MT Valve EM Ingleton PM Nurmi M Martikainen PM Harkonen PL Prolactin and prolactin receptors are expressed and functioning in the human prostate.J Clin Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar, 15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar, 17Ben-Jonathan N Merson JL Allen DL Steinmetz RW Extra pituitary prolactin: distribution, regulation, function and clinical aspects.Endocr Rev. 1996; 17: 639-669PubMed Google Scholar PRLrs are devoid of intrinsic enzymatic activity15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar but are known to signal intracellularly via the JAK/STAT pathway, as well as the Ras/Raf/MAP kinase cascade.15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar Three isoforms of PRLr (long, intermediate, and short forms), which differ in the lengths of their cytoplasmic domains, have been identified in rat tissues,15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar, 18Clevenger CV Wan-Pin Chang Ngo W Expression of prolactin and prolactin receptor in human breast carcinoma.Am J Pathol. 1995; 146: 695-703PubMed Google Scholar but only the long and an analogous intermediate form of the receptor have been detected in human tissues.18Clevenger CV Wan-Pin Chang Ngo W Expression of prolactin and prolactin receptor in human breast carcinoma.Am J Pathol. 1995; 146: 695-703PubMed Google Scholar Interestingly, among the rat isoforms, both the long and intermediate forms are capable of transducing lactogenic as well as mitogenic signals.15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar, 18Clevenger CV Wan-Pin Chang Ngo W Expression of prolactin and prolactin receptor in human breast carcinoma.Am J Pathol. 1995; 146: 695-703PubMed Google Scholar, 19O'Neal KD, Yu Lee L: Differential signal transduction of the short, Nb2, and long prolactin receptors. J Biol Chem, 269:26076–26082Google Scholar In contrast, the short form does not transduce differentiation signals but can signal cell growth in NIH 3T3 cells.20Das Rina Vonderhaar BK Transduction of prolactin's (PRL) growth signal through both long, and short forms of the PRL receptor.Mol Endocrinol. 1995; 9: 1750-1759Crossref PubMed Google Scholar As reported for cells in the breast, brain, placenta, and lymphoid cells,17Ben-Jonathan N Merson JL Allen DL Steinmetz RW Extra pituitary prolactin: distribution, regulation, function and clinical aspects.Endocr Rev. 1996; 17: 639-669PubMed Google Scholar Nevalainen et al11Nevalainen MT Valve EM Ingleton PM Nurmi M Martikainen PM Harkonen PL Prolactin and prolactin receptors are expressed and functioning in the human prostate.J Clin Invest. 1997; 99: 618-627Crossref PubMed Scopus (174) Google Scholar recently demonstrated that PRL is produced locally by secretory epithelia in organ cultures of the human prostate. These findings indicate that an intraprostatic as well as a pituitary source for PRL exists and together with PRLr constitute an autocrine/paracrine pathway which likely mediates local hormone effects on the gland. In the current study, we used immunohistochemistry and in situ hybridization to localize PRLr in the developing and adult human prostate and in hyperplastic, dysplastic (also termed prostatic intraepithelial neoplasia), and carcinomatous lesions of the gland. Our goals were to investigate whether this key component of PRL action is present during prostatic organogenesis and to determine whether its expression is altered in BPH, prostatic intraepithelial neoplasia lesions, and carcinoma. To our knowledge this is the first comprehensive morphological investigation of PRLr expression in the human prostate across a wide spectrum of normal and pathological states. Overall, our findings indicate that PRL likely influences the development of the human prostate and contributes to the maintenance of the adult gland. Our results also suggest that PRL plays a role in early carcinogenesis of the human prostate and that diminished PRLr expression in poorly differentiated cancers may reflect a progressive loss of responsiveness to the peptide hormone in populations of neoplastic cells. The majority of specimens were selected from a pool of 40 radical prostatectomies done at Stanford Medical Center during the years 1994–1997. Patients ranged from 54 to 71 years of age. Specimens selected for study included 10 samples of lesion-free tissue from the peripheral, central, and transition zones,21McNeal JE Prostate: Histology for Pathologists.in: Sternberg SS Raven Press, New York1992: 749-763Google Scholar 5 BPH specimens, 20 examples of dysplasia of varying grades of severity, and 18 examples of Gleason grades 3–4 carcinoma. The methods used for the collection, fixation, sample selection, and processing of these specimens are the same as previously described.22Leav I McNeal JE Zair J Alroy J The localization of transforming growth factor α and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions.Hum Pathol. 1998; 29: 668-675Abstract Full Text PDF PubMed Scopus (40) Google Scholar In addition archival tissues obtained at autopsy from collections at the Department of Pathology at Tufts University were also studied. They included prostates from two fetuses at 29 and 34 weeks of gestation who died 1 and 7 days, respectively, after premature birth, 2 glands from neonates that were 3 hours old and 1 week of age, and one prostate from a prepubertal individual who was 11 years old. The sense and antisense probes used in this study were generated from the H1/H2 human prolactin receptor clone23Boutin JM Edery M Shirota M Jolicoeur C Lesueur L Ali S Gould D Djiane J Kelly PA Identification of a c-DNA encoding a long form of prolactin receptor in human hepatoma and breast cancer cells.Mol Endocrinol. 1989; 3: 1455-1461Crossref PubMed Scopus (236) Google Scholar which had been inserted into a pBlueScript vector. The entire 2556-bp sequence of the receptor was digested with the BamHI restriction enzyme to generate a 200-bp fragment which was subcloned into the BamHI site of the pBlueScript vector. The fragment is from the cytoplasmic domain of the long form of the human prolactin receptor, from nucleotides 1029 to 1233. This sequence was chosen because it has no homology with other members of the same family of receptors, such as the human growth hormone receptor.15Goffin V Kelly PA Prolactin, and growth hormone receptors.Clin Mol Endocrinol. 1996; 45: 247-255Crossref PubMed Scopus (52) Google Scholar, 16Bole-Feysot C Goffin V Edery M Prolactin (PRL), and its receptor: actions, signal transduction pathways and phenotypes observed in PRL receptor knockout mice.Endocr Rev. 1998; 19: 225-268Crossref PubMed Google Scholar, 18Clevenger CV Wan-Pin Chang Ngo W Expression of prolactin and prolactin receptor in human breast carcinoma.Am J Pathol. 1995; 146: 695-703PubMed Google Scholar One microgram of the recombinant plasmid vector was linearized by Xbal and EcoRI enzymes to generate antisense and sense templates, respectively. To generate labeled riboprobes the templates were transcribed using RNA polymerases T7 (antisense) or T3 (sense), NTP labeling mix (the UTP component was 2/3 normal UTP, 1/3 digoxigenin −11 UTP), and RNase inhibitor. In vitro transcription was carried out at 37°C for 1 hour in a 1× transcription buffer (Boehringer Mannheim, Indianapolis, IN). Formalin-fixed paraffin-embedded sections 5 μm thick were dewaxed, rehydrated, and washed in phosphate-buffered saline. In situ hybridization was carried out in an automated instrument (Gen II, Ventana Medical Systems, Tucson, AZ) in which all applications were standardized according to the manufacturer's protocols. Briefly, the sections were exposed to proteinase K (100 μg/ml in 1 mol/L Tris-EDTA buffer, pH 8) for 8 minutes at 37°C. Prehybridization was carried out in 2× saline sodium citrate (SSC) for 15 minutes at 45°C. Sense or antisense riboprobes in 100 μl of hybridization buffer (50% deionized formamide, 2× SSC, 10% dextran sulfate, 1% SDS and 250 μg/ml denatured herring sperm DNA) were manually applied to the sections. Optimal dilutions of riboprobe in the hybridization buffer generally ranged from 1:100 to 1:300 (concentration: 10–20 pmol/L riboprobe). The Ventana Automated System utilizes a unique “liquid coverslip.” Following a 1-hour hybridization at 42°C, the automated sequence continued with posthybridization washes. The stringency conditions were determined by the duration, temperature, and concentration of the SSC solutions. The highest stringency we used in this study was 0.5× SSC for 20 minutes at 65°C. A blocking solution, which included normal sheep serum and tetramisol (Sigma), was then applied to the sections. This was followed by 20 minutes' exposure to primary antibody (anti-digoxin; Sigma) diluted 1:500 in normal sheep serum/Tris-NaCl buffer. The detection steps used 5 reagents supplied by the manufacturer (Ventana Blue kit). The slides were removed from the machine, stained with nuclear fast red (Rowley Biochemical Institute, Danvers, MA), dehydrated, and coverslipped. Negative controls included sections incubated with the sense probe, pretreated with RNase A, or by omission of probe. Positive controls were surgical biopsy specimens of human breast. For these studies we used a monoclonal antibody, B.6, raised against a membrane-enriched fraction of a metastatic human breast cancer line (MCF-7) (a generous gift from Dr. B.K. Vonderhaar, National Cancer Institute). The specificity of this reagent both in its binding characteristics and immunostaining of T47-D human breast cancer cells have been reported.24Banerjee R Ginsburg E Vonderhaar B Characterization of a monoclonal antibody against human prolactin receptors.Int J Cancer. 1993; 55: 712-721Crossref PubMed Scopus (33) Google Scholar In addition, we also used a rabbit polyclonal antibody directed against the complete extracellular domain of the human PRLr, which we termed the CL-AB (a generous gift from Dr. Charles Clevenger, University of Pennsylvania Medical School). Six-micrometer-thick sections were dewaxed, placed in a 0.01 mol/L citrate buffer (pH 6), and then heated in a microwave oven at high power for 2 or 3 cycles at 5 minutes each. The B.6 antibody was then applied at a dilution of 1:250 and the polyclonal reagent at 1:100. Biotinylated horse anti-mouse or goat anti-rabbit were used as secondary antibodies with the B.6 and polyclonal antibodies, respectively. The remaining immmunohistochemical procedures were carried out as previously described.22Leav I McNeal JE Zair J Alroy J The localization of transforming growth factor α and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions.Hum Pathol. 1998; 29: 668-675Abstract Full Text PDF PubMed Scopus (40) Google Scholar For all omission controls, nonimmune or preimmune sera of mouse or rabbit origin were substituted for the primary antibodies at the appropriate dilutions. In addition, we preincubated the polyclonal reagent with the immunizing peptide as a blocking control. The blocking peptide is a chimera between glutathione-S-transferase and the extracellular domain of the human PRLr expressed in Escherichia coli (a gift from Dr. Charles Clevenger). Five μg of the peptide was incubated with 5 μl of the antibody overnight at 4°C and then applied to the sections. In addition, the specificity of the CL-AB for identifying PRLr in human prostate epithelium was determined by immunoblotting using the prostatic carcinoma cell lines LNCaP and PC-3 (see below). Positive controls were the same surgical biopsy specimens of human breast used for in situ hybridization studies. The intensity of signal at both the mRNA and protein levels were independently evaluated and semiquantitated using a grading scale of 1–4, with 4 representing the highest value, by three of us (I.L., F.M., and M.L.). LNCaP, PC-3, and MCF-7 cell lines (American Type Culture Collection) were grown in RPMI 1640 medium, F-12 nutrient mixture (HAM) culture medium, and Dulbecco's modified Eagle's (high glucose) culture medium, respectively, supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antimycotic (Gibco BRL, Gaithersburg, MD) at 37°C in a humidified atmosphere of 5% carbon dioxide and 95% air. Approximately 1 × 106 cells were trypsinized, washed with phosphate-buffered saline, and protein lysates were extracted with lysis buffer (10% sucrose, 1% Nonidet P-40, 20 mmol/L Tris, pH 8.0, 137 mmol/L NaCl, 10% glycerol, 2 mmol/L EDTA, 10 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, and 1 μg of leupeptin) agitated on ice every 10 minutes for 30 minutes, and centrifuged for 20 minutes at 14,000 rpm. The supernatant was removed and the concentration was determined with a spectrophotometer (Beckman DU 650) using the DC protein assay (BioRad, Hercules, CA). Western blotting was performed using the Novex transfer system (Novex, San Diego, CA), 12% Tris-glycine pre-cast Novex gel, and HyBond ECL nitrocellulose membrane (Amersham Life Science, Cleveland, OH). Four hundred μg of lysate was transferred to the membrane, blocked in 5% milk in TBST for 1 hour, and blotted overnight at 40°C with the CL-AB (1:3000 dilution in 5% milk). For detection, the membrane was incubated in horseradish peroxidase-steptavidin goat anti-rabbit secondary antibody (BioRad) (1:10000 dilution in 5% milk in TBST for 30 minutes at room temperature) and developed with the ECL detection system (Amersham). As previously described,22Leav I McNeal JE Zair J Alroy J The localization of transforming growth factor α and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions.Hum Pathol. 1998; 29: 668-675Abstract Full Text PDF PubMed Scopus (40) Google Scholar the fetal human prostate at 29–34 weeks of gestation is composed of immature stroma containing branching cords of epithelial cells, some of which are arranged in solid nests and others containing lumens (Figure 1, A and B). In that study, we used high molecular weight cytokeratin immunostaining to show that solid epithelial nests were entirely composed of basal cells.22Leav I McNeal JE Zair J Alroy J The localization of transforming growth factor α and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions.Hum Pathol. 1998; 29: 668-675Abstract Full Text PDF PubMed Scopus (40) Google Scholar With lumen formation basal cells were located along the perimeters of developing acini and ducts, a location found in the postnatal, prepubertal, and adult prostate glands.22Leav I McNeal JE Zair J Alroy J The localization of transforming growth factor α and epidermal growth factor receptor in stromal and epithelial compartments of developing human prostate and hyperplastic, dysplastic, and carcinomatous lesions.Hum Pathol. 1998; 29: 668-675Abstract Full Text PDF PubMed Scopus (40) Google Scholar At all stages of development uniformly strong signals for PRLr message (+4) was strikingly evident in epithelial cells irrespective of whether they were arranged in solid nests or formed lumens (Figure 1A). Faint signals (<1) were also detected in the stroma (Figure 1A). The immunolocalization of receptor protein in fetal, postnatal, and prepubertal prostates was identical to that found at the message level. Light intensity immunostaining (1–2+) for the receptor was detected in the cytoplasm of both immature luminal and basal cells in these glands (Figure 1B). Thus, when compared with transcript signals staining intensity was reduced in these tissues regardless of whether the B.6 or CL-AB antibody reagent was used (Figure 1B). Stromal staining was rarely seen in these glands and when it was found it was always faint (<1). Localization of PRLr mRNA was evident in all but one of the 25 adult prostate specimens we selected for in situ hybridization studies. In all instances hybridization signals were predominately found in the cytoplasm of epithelial cells and approximated the levels of expression intensity found in immature glands (1–2+) (Figure 1C). As was seen in the immature prostates, only a very faint signal was present in the stromal compartment, where it was exclusively localized in the cytoplasm of smooth muscle cells. No consistent differences in either the intensity or in the localization of PRLr mRNA expression was evident between the three anatomical zones of the adult gland (1–2+). In concert with these findings, the intensity of immunostaining in these three zones mirrored results seen at the message level (Figure 1, D and E). Differences were, however, evident in the localization of receptor staining with the two antibodies. Although both antibodies localized the receptor predominately in the cytoplasm of epithelial cells, the nuclei of basal cells were consistently stained with the CL-AB antibody, a feature less frequently found in secretory cells (Figure 1E). In contrast clear immunostaining of nuclei was not well delineated with the B.6 reagent. Light staining of smooth muscle cells (<1) was occasionally observed with the B.6 antibody, which rarely occurred when the CL-AB reagent was used. In the 5 cases of BPH studied we found that PRLr message and protein were predominately localized in epithelial cells and that signal intensities at both levels were comparable to what we observed in normal prostate cells (see above and Figure 2, A and B). As was the case for normal prostatic epithelia, nuclear immunostaining with the CL-AB also occurred in hyperplastic cells. In all BPH lesions, stromal expression of the receptor approximated the faint intensity seen in normal prostate tissue. With rare exception (2 lesions in 2 separate cases) PRLr expression was markedly increased (4+) in dysplastic epithelia when compared with normal adult glandular cells (Figure 3, A–C, E, and F). This finding was consistent at both the mRNA (Figure 3, A–C) and protein levels (Figure 3, E and F). The enhanced (4+) expression was observed with each antibody reagent and occurred irrespective of the grade of the dysplastic lesion (Figure 3," @default.
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• W2023423534 title "Prolactin Receptor Expression in the Developing Human Prostate and in Hyperplastic, Dysplastic, and Neoplastic Lesions" @default.
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