A lectin histochemistry comparative study in human normal prostate, benign prostatic hyperplasia, and prostatic carcinoma

The partial oligosaccharide sequences of glycoconjugates and the nature of their glycosidic linkages were investigated in normal human prostate, benign prostatic hyperplasia (BPH) and prostatic carcinoma by means of lectin histochemistry, using light microscopy and Western blot analysis. The labeling pattern of BPH differed from that of normal prostate in having more intense staining with DSA, HPA, UEA-I and AAA, and in showing lesser staining with WGA and SBA. Prostatic carcinoma differed from normal prostates in displaying the more intense labeling with PNA, DSA, SBA, DBA, UEA-I and AAA, and in having lesser labeling with WGA. The main differences in labeling pattern between prostatic carcinoma and BPH were that the latter specimens showed more marked staining with PNA, DSA, DBA, SBA, UEA-I and AAA, and lesser staining with WGA and HPA. The staining patterns of SNA, MAA, ConA, LCA and GNA were similar in all three groups of specimens. For most of the lectins studied, including those showing a similar immunohistochemical staining in the three groups of specimens studied, the Western blot analysis showed differences in the banding pattern among normal, hyperplastic, and carcinomatous prostates. Present results suggest that the glycosylation of proteins was modified in both BPH and prostatic carcinoma. In BPH a strong expression of N-acetylgalactosamine residues occurred, while in prostatic carcinoma an increase of sialic aci, galactose and fucose residues was observed. No changes in mannose residues were detected.     

Characteristics of long-term human epithelial cell cultures derived from normal human fetal kidney

Summary Epithelial cell cultures were prepared from normal human fetal kidney and established in long-term culture. The growth characteristics and production of keratin, and alkaline phosphatase (AP) and gamma-glutamyl transpeptidase (GGT) activities were compared in a modified minimal essential medium (mMEM),d-valine-containing modified alpha-MEM (mALPHA) andl-valine mALPHA. The mean number of cumulative population doublings (CPDL) was significantly (P<0.001) enhanced with thel-valine mALPHA (40.8 CPDL) over that achievable in mMEM (14.2 CPDL) ord-valine mALPHA (18.3 CPDL) media. In all three media, greater than 95% of the cells in culture produced keratin throughout the life span of these cultures. Surface-associated fibronectin was absent in these cell cultures. AP and GGT activities increased as a function of subpassage and time in culture, with the greatest activity in thel-valine mALPHA. The expression of these renal cell-associated functions suggests that these cells in culture are proximal tubule epithelial cells. The conditions and procedures described in this paper can provide a human kidney epithelial cell culture system for studying human renal function, metabolism, cytotoxicity, genotoxicity, and transformation. Research was supported by a NIEHS (ES 3101) grant to S. M. D’Ambrosio and a NCI grant (CA21104) to J. E. Trosko.     

Plasticity of epithelial cells derived from human normal and ADPKD kidneys in primary cultures

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst formation initiated by dedifferentiation and proliferation of renal tubular epithelial cells. Renal tubular epithelial cells (RTC, derived from normal kidney tissue) in primary cultures exhibit both homogeneous expression of γ-glutamyl transferase and low molecular weight cytokeratin, two different markers for proximal and distal renal epithelial cells, respectively. RTC in cultures also abnormally express the dedifferentiation markers vimentin and PAX-2, which are proteins normally expressed in epithelial cells lining cysts in ADPKD kidneys but not tubular cells in normal kidneys. In contrast, different cultures of cystic epithelial cells (CEC, derived from the cysts walls of polycystic kidneys) display variable expression of cytokeratin, γ-glutamyl transferase, and PAX-2, but a constant level of vimentin. Importantly, RTC and CEC exhibit the capacity to convert to their respective original structures by forming tubules and cysts, respectively, when cultured in a three-dimensional gel matrix, whereas HK-2, LLC-PK1, and MDCK renal epithelial cell lines form cell aggregates or cysts. Our study demonstrates that the marker expression of the various epithelial cell types is not highly stable in primary cultures. Their modulation is different in cells originating from normal and ADPKD kidneys and in cells cultured in monolayer and three-dimensions. These results indicate the plasticity of epithelial cells that display a mixed epithelial/dedifferentiated/mesenchymal phenotype during their expansion in culture. However, RTC and CEC morphogenic epithelial properties in three-dimensional cultures are similar to those in vivo. Thus, this model is useful for studying the mechanisms leading to tubulogenesis and cystogenesis. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by a grant from The Polycystic Kidney Foundation. We gratefully acknowledge the support of the Children’s Medical Research Institute and Children’s Miracle Network Foundation.     

Diabetes, growth hormone-insulin-like growth factor pathways and association to benign prostatic hyperplasia

Diabetes significantly increases the risk of benign prostatic hyperplasia (BPH) and low urinary tract symptoms (LUTS). The major endocrine aberration in connection with the metabolic syndrome is hyperinsulinemia. Insulin is an independent risk factor and a promoter of BPH. Insulin resistance may change the risk of BPH through several biological pathways. Hyperinsulinemia stimulates the liver to produce more insulin-like growth factor (IGF), another mitogen and an anti-apoptotic agent which binds insulin receptor/IGF receptor and stimulates prostate growth. The levels of IGFs and IGF binding proteins (IGFBPs) in prostate tissue and in blood are associated with BPH risk, with the regulation of circulating androgen and growth hormone. Stromal-epithelial interactions play a critical role in the development and growth of the prostate gland and BPH. Previously, we have shown that the expression of c-Jun in the fibroblastic stroma can promote secretion of IGF-I, which stimulates prostate epithelial cell proliferation through activating specific target genes. Here, we will review the epidemiologic, clinical, and molecular findings which have evaluated the relation between diabetes and development of BPH.     

Epidermal growth factor binding and steroid receptor content in human benign prostatic hyperplasia

The receptor for epidermal growth factor (EGF-R) was characterized on membrane fractions from human benign prostatic hyperplasia (BPH). Specific binding of [125I]EGF reached equilibrium after 40 min at 25 degrees C and was stable for up to 120 min. Saturation analysis of EGF-R, performed by incubating the membranes with 0.0156-15 nM [125I]EGF in the presence and in the absence of 100-fold excess of cold EGF for 60 min, revealed the presence of two classes of binding sites with high and low affinities (Kd = 0.35 +/- 0.23 and 9.60 +/- 2.87 nM respectively). Competition experiments revealed that FSH, insulin and calcitonin did not compete with [125I]EGF. The simultaneous determination of EGF-R and that of estradiol (ER), progesterone (PR) and androgen receptors (AR) was performed using the same buffer to homogenate the tissues and to obtain cellular membranes. The steroid receptors (SR) were determined by means of the dextran-coated charcoal method. There was a significant negative correlation between nuclear SR and binding capacity of EGF-R. The presence of specific and high affinity binding sites for EGF and the modulation of the level of these sites by steroid receptors suggest a possible role of EGF in prostatic hyperplasia.     

Differential responses to growth factors by normal human mesothelial cultures from individual donors

A significant interindividual variation in the growth rates is found in normal cultured human mesothelial (NHM) cells derived from different donors. This variation is observed when the mesothelial cells are incubated in medium containing serum and when the potencies of several separate growth factors are measured by using defined media. Depending on the donor, gamma-interferon and interleukin-2 can be toxic, have no effect, or stimulate the growth rate of NHM cells. Cultured NHM cells can be induced to multiply by growth factors that are released by activated macrophages. Thus, interindividual variation in NHM cell growth control could play a role in the pathogenesis of mesothelioma for a person exposed to asbestos.     

Complete primary structure of prostatropin, a prostate epithelial cell growth factor

Bovine brain prostatropin is a potent and essential mitogen for prostate epithelial cell growth. The major form of prostatropin contains 154 amino acid residues in a single amino terminally blocked chain corresponding to a molecular weight of 17,400. The amino acid sequence of the 150 carboxy-terminal residues of prostatropin was derived by Edman degradation of overlapping peptides primarily generated by cleavage at lysyl and glutamyl residues. Analysis of the amino-terminal tetradecapeptide by fast atom bombardment mass spectrometry identified the blocking group as an acetyl moiety, and tandem mass spectrometry provided the sequence of the first 12 residues. Prostatropin residues 15-154 contain the sequence of bovine brain polypeptides recently described as acidic fibroblast growth factor and class I heparin-binding growth factor. The sequence of the first 25 residues of prostatropin is acetyl-Ala-(Gly, Glu)-Glu-Thr-Thr-Thr-Phe-Thr-Ala-Leu-Thr-Glu-Lys-Phe-Asn-Leu-Pro-Leu-Gly -Asn-Tyr-Lys-Lys-Pro. Reduced and carboxymethylated prostatropin exhibits mitogenic activity, suggesting that disulfide bonds among cysteine residues 30, 61, and 97 are not functionally essential. These results demonstrate by rigorous structural analysis that the brain-derived polypeptide previously described only as a mesenchymal and neuroectodermal cell mitogen is also an epithelial cell growth factor that may be involved in support of prostate hyperplasia and adenocarcinoma.     

Serum vascular endothelial growth factor C level in patients with prostate cancer and benign prostatic hyperplasia

OBJECTIVE: To compare serum vascular endothelial growth factor C (VEGF-C) levels in patients with benign prostatic hyperplasia (BPH) and prostate cancer (PCa) and analyze VEGF-C levels in relation to clinicopathologic parameters. STUDY DESIGN: Fifty-eight patients with PCa and 61 patients with BPH were included in this study. Serum VEGF-C levels were assessed by enzyme-linked immunosorbent assay. RESULTS: The serum VEGF-C level for patients with PCa was 3,432.06 +/- 1,851.07 as opposed to 3,166.68 +/- 1,921.2 for patients with BPH. There was no statistically significant difference between the 2 groups (p = 0.4448). There was no correlation of VEGF-C to tumor stage, grading or the preoperative prostate-specific antigen values. CONCLUSION: We cannot recommend VEGF-C serum level as a marker for tumor growth in PCa.     

Characterization of epithelial cell cultures derived from human tracheal glands

Summary Cultures of normal human tracheal gland epithelial cells that exhibit functional differentiation have been propagated in serum-free medium supplemented with insulin (5 μg/ml), epidermal growth factor (10 ng/ml), hydrocortisone (0.5 μg/ml), and bovine pituitary extract (25 μg/ml). The cells retain many characteristics of epithelial cells including microvilli on cell surfaces, desmosomes between cells, and tonofilaments in the cytoplasm. In addition, they exhibit keratin-positive titers and react positively with Peanut agglutinin, which is specific for the disaccharide β-d-galactose-(l→ 3)N-acetyld-galactosamine, a major component of mucin glycoprotein. The cells also exhibit normal Cl⁻ channel activity which was enhanced by the cAMP agonist Forskolin. The major component of the cellular secretion was hyaluronic acid; approximately 10% of the void volume material was resistant to hyaluronidase and may contain material similar to mucin glycoprotein. Some of the cell cultures have been maintained in serum-free conditions for 6 to 7 passages. This model will be important to study regulation of ion-channel activities and mucous glycoprotein secretion and to compare such regulations with the tracheal mucosal epithelial cells already established. This research was supported by USPHS grants HL 41979 and HL 33142 from the National Heart, Lung and Blood Institutes.     

表皮生长因子与前列腺增生及前列腺癌的关系

195 9年Ｃｏｈｅｎ[1] 在纯化神经生长因子 (ＮＧＦ)的过程中 ,首先从小鼠颌下腺中发现了一种对外胚层、中胚层和内胚层细胞具有促分裂活性的多肽生长因子 ,并能明显地促进表皮细胞的生长与角化 ,命名为表皮生长因子 (ＥｐｉｄｅｒｍａｌＧｒｏｕｔｈＦａｃｔｏｒＥＧＦ)。人的表皮生长因子以ｈＥＧＦ - β和ｈＥＧＦ -γ两种形式存在。由于它对胃酸具有很强的抑制作用 ,故又命名为尿抑胃素 (ｕｒｏｇａｓｔｒｏｎｅ)。 1975年Ｓｔａｒｋｅｙ等人[2 ] 从尿中纯化获得了ＥＧＦ的纯品 ,并确定其结构。ｈＥＧＦ - β和ｈＥＧＦ -γ具有高度的同…     

Spontaneous cell transformation: karyoplasts derived from multinucleated cells produce new cell growth in senescent human epithelial cell cultures

Previously, it was shown that SV40-induced cell transformation of human diploid (2N), epithelial cells was a dynamic process of nuclear and cellular events. In this process, nuclei of polyploid (above 2N) cells broke down into multinucleated cells (MNCs) by amitotic division. An induced mass karyoplast (i.e., small cell with reduced amount of cytoplasm) budding process from the MNCs produced transformed cells with extended life span (EL) and altered morphology. In this study, without the use of SV40 and no induction of karyoplast budding, the same sequence of cellular events was found to occur spontaneously for the same type of cells at replicative senescence (no mitosis). These cell transformation events were followed by phase-contrast photography of living cell cultures. Primary, diploid, epithelial cell cultures grew for two to three passages and then entered senescence. Cells remaining in the cultures after widespread cell death (mortality stage 1; M1) developed the typical large, flat-cell morphology of senescence with increased cytoplasmic volume. Some of these cells were MNCs, mostly with two to four nuclei. Cytokinesis in MNCs and spontaneous karyoplast budding from MNCs were observed, and new, limited EL cell growth was present either in foci of cells or as prolonged cell growth over one to two passages. At the end of their replicative phase, the EL cells entered another death crisis (M2) from which no cells survived. In M2-crisis, rarely transformed cells appear with immortal cell growth characteristics (i.e., cell lines). Numerous examples of fragmentation or amitosis of polyploid nuclei in the production of multinucleated cells (MNCs) are presented. Such nuclear divisions produced nuclei with unequal sizes, which suggest unbalanced chromosomal segregations. The nuclear and cellular events in cell transformation are compared with a natural (no induction) occurrence of MNC-offspring cells in mammalian placentas. The possibility of a connection between these two processes is discussed. And finally the difference in the duration of EL cell growth from SV40-MNCs versus from senescent-MNCs is ascribed to increased mutational load in SV40-induced MNCs as compared with that in senescence MNCs.     

Production of progenitor cells from primary human epithelial cell monolayer cultures

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70–80% confluence, they begin to float up into the overlying medium and are called “epithelial pop-up keratinocytes (ePUKs)” allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.     

Morphology and growth potential of stromal cell cultures derived from human endometrium

Summary Propagable cell cultures derived from human endometrial tissue were determined to contain cells predominantly of stromal cell origin based on their morphologic resemblance to endometrial stromal cells. These features included nexi, solitary cilia, and predecidual cytology. In addition to morphology the cell cultures retained a normal karyotype and responded to steroid hormones as evidenced by cellular aggregation. The stromal cells were evaluated for a variety of characteristics associated with transformed cells and seemed to be biologically normal without neoplastic phenotypes. Growth potential of the stromal cell cultures was also characterized in normal maintenance medium, in nutritionally depleted medium with reduced levels of calcium or serum, and in medium with increased levels of serum. The prolonged survival of the stromal cells in vitro coupled with the retention of in vivo characteristics and an absence of neoplastic phenotype provides a human cell system that is amenable to a variety of long-term experimental analyses. This work was supported by contract CP75956 and grant CA31733 from the National Cancer Institute. B. Hugh Dorman was the recipient of a predoctoral scholarship from the Chemical Industry Institute of Toxicology. Jill M. Siegfried was supported by National Research Service Award CA09156. David G. Kaufman is the recipient of a Research Career Development Award (CA00431) from the National Cancer Institute.     

EPN: a novel epithelial cell line derived from human prostate tissue

This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo "Hayflike crisis" and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high-molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Summary Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 μg/ml); ascorbic acid, 40 μg/ml;l-isoleucine, 50 μg/ml; epidermal growth factor, 20 ng/ml; insulin, 5 μg/ml; cholera toxin, 5 ng/ml; transferrin, 1 μg/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37°C in humidified gas containing 5% CO₂: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm. This study was supported by USPHS Grant CA-30185 from the National Large Bowel Cancer Project, National Cancer Institute.     

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

Primary and long term epithelial cell cultures from human fetal normal colonic mucosa

Primary and passaged cultures of normal colon epithelial cells, derived from human fetuses (13 to 17 wk of conceptual age) have been established. These cultures have been passaged 16 times thus far. The cultures have been initiated and maintained in medium consisting of 50% Dulbecco's minimum essential medium and 50% Ham's F12 medium and supplemented with antibiotics (penicillin, 100 U/ml; streptomycin, 100 micrograms/ml); ascorbic acid, 40 micrograms/ml; L-isoleucine, 50 micrograms/ml; epidermal growth factor, 20 ng/ml; insulin, 5 micrograms/ml; cholera toxin, 5 ng/ml; transferrin, 1 microgram/ml; fetal bovine serum (10%); and HEPES, 25 mM final concentration, and incubated at 37 degrees C in humidified gas containing 5% CO2: 95% air. The cellular and subcellular characteristics of primary and passaged cultures were defined using light microscopy and scanning and transmission electron microscopy. The cells exhibited microvilli on cell surfaces and showed junctional complexes and interdigitations between cells. Indented nuclei with dense chromatin and marginated heterochromatin, numerous mitochondria, rough endoplasmic reticulum, polysomes, and extensive Golgi zones were conspicuous. Also, periodic acid Schiff's reagent-positive staining of the cells suggests the active synthesis of complex mucopolysaccharides in the cytoplasm.     

Polymorphisms of MLH1 in benign prostatic hyperplasia and sporadic prostate cancer

Mismatch repair is one of several DNA repair pathways of which defects may lead to cancer. We hypothesize that polymorphisms of the MLH1 gene can be a risk factor for benign prostatic hyperplasia (BPH) and prostate cancer. The genetic distribution of MLH1 polymorphisms that lead to amino acid changes at codons 132, 219, 384, and 723 were analyzed in BPH and sporadic prostate cancer patients, and compared to healthy controls from an Asian population. These experiments demonstrate a protective role for the codon 384 variant allele against prostate cancer (P = 0.031) but not BPH when compared to normal controls and furthermore, an inverse association was observed with stage (P = 0.074) and grade (P = 0.056) of cancer. This is the first report that demonstrates a protective effect for the race-related MLH1 polymorphism at codon 384 against prostate cancer and these results are important in understanding their role in this disease.