Immortalization of normal human cytotrophoblast cells by reconstitution of telomeric reverse transcriptase activity

Placental trophoblast cells are unique endocrine cells that play vital roles during the processes of embryonic implantation and placentation. However, research into the function of human trophoblast has been largely restrained mainly due to a lack of adequate cell models. A normal placenta-origin cytotrophoblast cell line (NPC) was previously established by our group, but these cells showed replicating senescence after 50 population doublings (PDs). In this study, the human telomerase catalytic subunit gene (htert) was transferred into B6 strain of NPC cells, and strains with reconstituted telomerase activity (B6Tert) were established. It was shown that B6Tert-1 cells produce various biomarkers of normal extravillous cytotrophoblasts during the early weeks of gestation. Meanwhile, the cell invasiveness was inhibited by transforming growth factor beta (TGFbeta). However, their ability to form syncytium was relatively low when stimulated with fetal calf serum (FCS). The cells maintained normal cell growth properties and failed to elicit tumours in nude mice. They proliferated continuously with no signs of senescence until the final count at 210 PDs. The growth rate of B6Tert-1 cells was increased when compared with the parental cells, which results, at least partly, from facilitating release of the G1/S checkpoint during the cell-cycle regulation. This is the first report of immortalizing human normal cytotrophoblast (CTB) cells by activation of telomerase activity. The cells will provide an ideal in vitro model for the study of human extravillous trophoblast (EVT) functions and consequently the mechanisms of embryonic implantation and placentation.     

Establishment of AIDS-related primary-effusion lymphoma (PEL) cell lines that proliferate continuously without serum

The gammaherpesvirus dually-infected (HHV8/EBV) PEL cell line, BC-1, was weaned gradually from fetal bovine serum (FBS) during successive feedings with RPMI 1640 medium containing human transferrin and selenium dioxide as the only additives. A serum-free cell line (sfBC-1) emerged that was 100% major histocompatibility complex (MHC) class II negative, compared with 10% MHC class II-negative cells before serum removal. In contrast, MHC class I expression by sfBC-1 cells slightly exceeded that of BC-1 cells. BC-1 and sfBC-1 cells were indistinguishable in six polymorphic genetic loci, confirming their relatedness and sfBC-1 cells contained HHV8 and EBV. These findings were not attributable to dual infection because the PEL cell line, BCBL-1, which is infected with HHV8 but not EBV, also contained MHC class II positive (45%) and class II negative (55%) cells. Moreover, a serum-free BCBL-1 (sfBCBL-1) cell line was established and the sfBCBL-1 cells were MHC class I up modulated and 100% MHC class II negative. The serum-free cell lines established in this study may be useful for exploring PEL-cell autocrine-growth pathways and for assessing MHC class II-negative PEL cells for tumorigenesis in animal model systems.     

Fertilization and early embrology: The secretion of gonadotrophin-releasing hormone by peri-implantation embryos of the rhesus monkey: comparison with the secretion of chorionic gonadotrophin

Chorionic gonadotrophin (CG) is the first clear embryonic signalduring early pregnancy in primates. CG has close structuraland functional similarities to pituitary luteinizing hormone(LH) which is regulated by gonadotrophin releasing hormone (GnRH).Tostudy the regulatory mechanism of CG secretion in primate embryos,we examined the production and timing of secretion of GnRH inperi-implantation embryos of the rhesus monkey. In-vivo fertilized/developedmorulae and early blastocysts, recovered from non-superovulated,naturally-bred rhesus monkeys by non-surgical uterine flushing,were cultured in vitro to hatched, attached and post-attachedblastocyst stages using a well-established culture system. Wemeasured GnRH and CG in media samples from cultured embryoswith a sensitive radioimmunoassay and bioassay, respectively.The secretion of GnRH (pg/ml; mean ± SEM) by embryos(n = 20) commenced from low levels (0.32 ± 0.05) duringthe pre-hatching blastocyst stage to 0.70 ± 0.08 at 6–12days and 1.30 ± 0.23 at 13 days of hatched blastocystattachment and proliferation of trophoblast cells. GnRH concentrationsin culture media obtained from embryos (n = 5) that failed tohatch and attach were mostly undetectable (0.1). Samples thatdid not contain detectable GnRH failed to show detectable CG.Immunocytochemical studies, using a specific monoclonal anti-GnRHantibody (HU4H) as well as polyclonal antisera (LR-1), revealedthat immunopositive GnRH cells were localized in pre-hatchingblastocysts (n = 4), in blastocysts (n = 2) after 5–10days of attachment and in monolayer cultures (n = 4) of well-establishedembryonic trophoblast cells. GnRH positive staining was seenonly in cytotrophoblasts but not in syncytiotrophoblasts. Similarly,cytotrophoblast, but not syncytiotrophoblast, cells of the rhesusplacenta were immunopositive. In controls, either in the absenceof antibody or in the presence of antibody pre-absorbed withGnRH, these cells failed to show stain. These observations indicate,for the first time, that an immunoreactive GnRH is producedand secreted by blastocysts during the peri-attachment periodand by embryo-derived cytotrophoblast cells in the rhesus monkey.     

Establishment of immortalized Schwann cells from Sandhoff mice and corrective effect of recombinant human β-hexosaminidase A on the accumulated GM2 ganglioside

We have established spontaneously immortalized Schwann cell lines from dorsal root ganglia and peripheral nerves of Sandhoff mice. One of the cell lines exhibited genetically and biochemically distinct features of Sandhoff Schwann cells. The enzyme activities toward 4-methylumbelliferyl N-acetyl--D-glucosamine (-hexosaminidases A, B, and S) and 4-methylumbelliferyl N-acetyl--D-glucosamine-6-sulfate (-hexosaminidases A and S) were decreased, and GM2 ganglioside accumulated in lysosomes of the cells. Incorporation of recombinant human -hexosaminidase isozymes expressed in Chinese hamster ovary cells into the cultured Sandhoff Schwann cells via cation-independent mannose 6-phosphate receptors was found, and the incorporated -hexosaminidase A degraded the accumulated GM2 ganglioside. The established Sandhoff Schwann cell line is useful for investigation and development of therapies for Sandhoff disease.     

Immunosuppression by Conditioned Media Derived From a Cloned Choriocarcinoma Cell Line in Serum-Supplemented and Defined Media

PROBLEM: Immunosuppressive factor(s) of trophoblast origin may contribute to the immunological privilege afforded the fetal allograft. Characterization of these immunoregulators in humans has been impeded by a lack of sufficient quantities of early gestational trophoblast for experimentation. METHOD: In this study, a cloned choriocarcinoma cell line (BeWo) was evaluated as an experimental model of trophoblast-derived immunoregulation. BeWo cells were cultured in both serum-supplemented (15% fetal bovine serum; FCS-CM) and serum-free (10% bovine serum albumin, BSA-CM; 0.01% gelatin, Gel-CM) media. Immunosuppressive activity was determined through the use of interleukin-2-dependent (CTLL-2) and -independent (LBRM) cell lines. Human chorionic gonadotropin (hCG) levels were determined by an immunoradiometric assay, and cellular morphology was assessed by light microscopy. RESULTS: In the serum-supplemented cultures, a portion of cells underwent transformation from single nucleated cytotrophoblast to multinucleated syncytiotrophoblast during days 1 to 5 of culture and was accompanied by a rise in hCG. Serum-free cultures were characterized as islands of cytotrophoblast and did not exhibit differentiation. FCS-CM suppressed CTLL-2 and LBRM proliferation with estimated EC₅₀ values of 415 and 280 μg protein/ mL, respectively. Gel-CM suppressed CTLL-2 and LBRM proliferation with EC₅₀ values of 12 and 7 μg protein/mL, respectively. BSA-CM suppressed CTLL-2 proliferation with an EC₅₀ of 132 μg protein/mL, but failed to suppress LBRM proliferation below 50% of control. CONCLUSION: These results suggest that the BeWo cell line is a promising model for the study of trophoblast-derived suppressive factors and that these factors can be generated in serum-free medium.     

Establishment of a pure culture of distinct cell types from bovine placental cotyledon

Methods are described to establish distinct cell cultures from bovine placental cotyledon. The villous tissue of the bovine placental cotyledon is collected and dissociated with 0.125% trypsin. The cells are then cultured in three different media: a serum-free medium, a growth factor supplemented medium, and a medium with 10% serum. A polygonal cell line grew out of the serum-containing medium, a fan-shaped cell line grew out of the serum-free medium, and an epitheloid cell line grew out of the growth factor supplemented medium. These cells maintained their morphology when grown in serum containing medium. The preference of distinct cells for different media in vitro reflects the in vivo physiological regulation of these cells. These distinct cultures re ideal to study the extrinsic and interactive factorsin bovine placenta.     

Diagnosing and preventing inherited disease: Detection of sickle gene by coelocentesis in early pregnancy: a new approach to prenatal diagnosis of single gene disorders

Coelomic fluid, placental tissue and maternal blood were collectedat 7–10 weeks gestation from each of 58 women undergoingelective termination of pregnancy for psychological indications.In all samples, a 364 bp fragment of the human -globin genespanning positions –23 to 341 was amplified. The restrictionendonuclease Ddel was used to detect the sickle mutation whichabolishes its restriction site. -Globin DNA was successfullyamplified from all samples. In 53 cases a normal maternal -globingenotype was detected. In three out of five cases, where thematernal haemoglobin phenotype was HbAS, heterozygosity forthe sickle mutation was demonstrated on analysis of coelomicfluid. In the remaining two cases a normal -globin genotypewas observed. Three further coelomic fluid samples were foundto be heterozygous for the sickle mutation. In these instancesthe maternal haemoglobin phenotype was normal, indicating paternaltransmission of the sickle gene. The results of the presentstudy have established that the diagnosis of sickle cell anaemia,and potentially other human single gene disorders, is feasibleby coelocentesis.     

Human globin gene expression in hybrid 2S MEL × human fibroblast cells

A somatic cell hybrid line, called M11-X, was developed in order to study the expression and regulation of the human -like globin genes in a mouse erythroid environment. M11-X cells were obtained by fusing the human fibroblast cell line GM3552 (which contains the translocation chromosome t(11;X) that carries the human -like globin genes) with hypoxanthine phosphoribosyltransferase (HPRT)-negative tetraploid (2S) mouse erythroleukemia (MEL) cells. After induction with 5 mM hexamethylene bisacetamide (HMBA), these cells contain approximately 300–600 copies per cell of correctly initiated, processed, and terminated human -globin mRNA; however, neither human -nor -globin mRNAs were detected. Carboxymethylcellulose chromatography followed by SDS-polyacrylamide gel electrophoresis and Western blotting revealed that normal human -globin protein was also present. These results suggest that the human -globin gene, when present in mouse erythroid cells, can be transcribed and its mRNA translated into normal products, but at a much lower level than the mouse -globin genes. Analysis of the frequency of cytosine methylation near the human -globin genes indicated that these genes are heavily methylated in M11-X cells. The inability to express the human -globin genes of these cells might be accounted for, at least in part, by DNA methylation.     

Synthesis of gonadotrophins and its regulation in the pituitary gland

Regulation of the synthesis of pituitary gonadotrophins LH andFSH has been studied in the rat using either cell-free translationof pituitary mRNAs, or hybridization techniques with syntheticoligodeoxynucleotides or cloned complementary DNAs. Gonadectomygreatly increases and supplementing gonadectomized rats withgonadal steroids diminishes the rate of synthesis of the gonadotrophinsubunits. Hybridization experiments suggest that gonadal steroidsregulate the expression of the genes coding for pituitary gonadotrophinsubunit precursors. Using the incorporation of labelled methionineby pituitary cells in culture, followed by specific immunoprecipitationof LH-related subunits and SDS-poly-acrylamide gel analysisof immunoprecipitated peptides, there was evidence that gonadotrophinreleasing hormone (GnRH) significantly enhances the radioactivityincorporated into both - and LH-subunits. This effect is specific,it is not a secondary effect due to the release of LH. A cyclicAMP (cAMP) analogue, 8-Br-cAMP, as well as forskolin and choleragen,which are cAMP generators and a diacylglycerol analogue, tetradecanoylphorbolacetate (TPA), mimic the stimulatory action of GnRH on the synthesisof the polypeptide chains of LH. However, no evidence has beenobtained that either cAMP or diacylglycerols mediate this GnRHeffect. These results suggest that the synthesis of pituitarygonadotrophins is under a double control of gonadal steroidsand GnRH which exert opposite effects, inhibitory for steroidsand stimulatory for GnRH. The negative control by steroids occursat the genomic level, while the positive effect of GnRH proceedsvia different mechanisms which remain to be elucidated.     

Hyperplasia of epithelium adjacent to transitional cell carcinoma can be induced by growth factors through paracrine pathways

Hyperplasia of transitional cell epithelium adjacent to human transitional cell carcinomas (TCC) is a common finding in pathology. This hyperplasia may be a precancerous aberration. Alternatively, it has been suggested that the hyperplasia is due to paracrine action of tumour-derived growth factors. In this study we tested the latter hypothesis using the mouse tumorigenic TCC cell line NUC-1. Transplantation of NUC-1 tumour cells into the urinary bladder submucosa of syngeneic mice in vivo induced hyperplasia of normal adjacent urothelium in all tested mice. Implantation of normal mouse bladder mucosa did not induce urothelial hyperplasia. In vitro, conditioned medium of NUC-1 cells induced the proliferation of the mouse urothelial cell line g/G, which closely resembles normal urothelial cells. This induction was inhibited by transforming growth factor ₁ (TGF₁). Similarly, TGF₁ inhibited the fibroblast growth factor-1 (FGF-1) and FGF-2 induced proliferation of g/G cells. Chemico-physical examination, bioassays with conditioned media, and RNA analysis of NUC-1 cells revealed that these cells secreted a growth factor with FGF-like properties. These results indicate that epithelial hyperplasia surrounding carcinomas is not necessarily a precancerous aberration, but may result from direct paracrine action of tumour-derived growth factors.     

Integrin-mediated entry into S phase of human gastric adenocarcinoma cells

The integrins are a family of integral membrane receptors that participate in binding to various extracellular and cell surface proteins during adhesion, migration, and homing of normal and neoplastic cells. In this study, we characterized the involvement of integrins in mediating the growth of an adhesion-dependent gastric adenocarcinoma line, ST2. This line was distinguished and selected for study based on its inability to grow when suspended in soft agar or plated on poly(2-hydroxyethyl methacrylate)-coated dishes. ST2 cells arrested in G0/G1 of the cell cycle when deprived of adhesion to substrate. Using purified matrix components, collagen was found to be highly active in promoting 1 integrin-mediated cell attachment and spreading. Subsequent to spreading on collagen, the cells were released from G0/G1 block and progressed into S phase. Monoclonal antibodies to 2 or 1 integrin blocked the reinduction of both cell spreading and entry into S phase. These studies suggest that during the metastatic process, integrin receptor interaction with the insoluble matrix may be an important step leading to proliferation of some tumors.     

Isolation,cultivation, and characterization of normal human esophageal epithelial cells

Summary Normal human esophageal epithelial cells can now be grown and serially propagated in cell culture either using serum-containing medium and lethally irradiated 3T3 fibroblasts to support epithelial cell growth or using hormone-supplemented, serum-free medium without a 3T3 feeder layer. The cells continue to express many differentiated functions characteristic of the native epithelium. When grown in serum-containing medium, the esophageal epithelial cell colonies are stratified and consist of a basal layer of dividing cells giving rise to the more differentiated cells in the upper cell layers. The cells contain keratin proteins and desmosomes between adjacent cells. The cells express other markers of terminal squamous differentiation, namely, involucrin and cross-linked envelopes. When grown in serum-free, hormone-supplemented medium, the colonies remain as a monolayer rather than stratifying because of the low levels of calcium required for optimal growth. Increased levels of calcium or the presence of serum (2.5% or greater) lead to stratification. The cells grown under serum-free conditions continue to express squamous differentiated properties as keratins, desmosomes (decreased numbers in low-calcium medium), involucrin, and cross-linked envelopes.     

Expression of melatoninergic receptors in human placental choriocarcinoma cell lines

BACKGROUND: Melatonin crosses the placenta and enters the fetalcirculation. Moreover, experimental data suggest a possibleinfluence of melatonin on placental function and fetal developmentin humans. To date, the expression and role of melatonin receptorsin human placenta choriocarcinoma cell lines and in human termplacental tissues remain to be elucidated. METHODS AND RESULTS:Results from RT–PCR, western blotting and confocal microscopydemonstrated that the MT1, MT2 and ROR1 melatonin receptorsare expressed in the human term placental tissues and in choriocarcinomacell lines JEG-3 and BeWo. Furthermore, enzyme-linked immunosorbentassay showed that 6-chloromelatonin (a melatonin agonist) inhibits,in a dose-dependent manner, forskolin-stimulated hCG- secretionin JEG-3 (P < 0.001) and BeWo (P < 0.05) cells but hadno effect on basal human chorionic gonadotrophin (hCG-) levels.This effect of 6-chloromelatonin on forskolin-stimulated HCG-secretion was abolished by pertussis toxin (PTX), suggestingthat melatonin regulates hCG- production by an action involvingan inhibitory Gi/o protein. In PTX-treated BeWo cells, 6-chloromelatoninstimulated basal hCG- secretion (P < 0.001). CONCLUSION:These results demonstrate, for the first time, the expressionof melatonin receptors in human term placental tissues and inchoriocarcinoma cells and suggest a possible paracrine/autocrinefunction for melatonin in human placenta.     

Coelomic fluid chorionic gonadotrophin and protein concentrations in normal and complicated first trimester human pregnancies

Coelomic fluid and maternal serum samples were collected from43 normal pregnancies and 18 missed abortions between 7 and12 weeks of gestation. The samples were analysed for the concentrationsof intact human chorionic gonadotrophin (HCG), free HCG, freeHCG and total protein. The relationships between the biologicalfindings and the ultrasound and pathological features were assessedby regression analysis. In normal pregnancies, intact HCG, freeHCG and free HCG concentrations were respectively 1.3, 185 and33 times higher in coelomic fluid than in maternal serum. Thecoelomic concentrations of intact HCG and free HCG decreasedsignificantly with advancing gestation. No relationship wasfound between coelomic fluid and maternal serum concentrationsof the different variables. These findings suggest that in normalpregnancies, the concentration of HCG in the coelomic fluid,as in maternal serum, is mainly influenced by cytotrophoblasticdifferentiation and that the metabolic clearance of HCG moleculesis slower in the coelomic cavity than in maternal serum. Inmissed abortions, the serum concentrations of intact HCG, HCGand free HCG and the coelomic concentration of total proteinwere significantly lower than in normal pregnancies. In threeout of nine anembryonic pregnancies diagnosed by ultrasound,embryonic remnants were present at histological examination.The coelomic concentration of total protein was extremely lowin all missed abortions with advanced trophoblastic necrosis,whereas the HCG concentration was low when embryonic remnantswere absent. These findings support the concept that embryonicand placental development are closely related in the first trimesterof human pregnancy, placental biological functions persistingonly for a limited period of time after embryonic demise.     

An Autocrine Activity Capable of Substituting for Serum in Cell Cultures

Abstract

Provided that high cell densities (above 10⁶/ml) are maintained, a factor-dependent murine hemopoietic progenitor cell line (FDC-PI) will proliferate in serum-free medium. The conditioned medium (CM) from high-density FDC-PI cells permits the serum-free survival of FDC-PI cells even at low density, indicating the existence of a diffusible autocrine factor. The requirement of FDC-PI for a colony-stimulating factor (either IL-3 or GM-CSF) is not abrogated by culturing the cells at high cell density or in the conditioned medium. Furthermore, the CM from FDC-PI enhances the mitogenic stimulation of normal human skin fibroblasts (HSF) by epidermal growth factor (EGF): i.e., the lag period before entry into the cell cycle is shortened by up to 6 hr. The fibroblasts themselves secrete an activity into serum-free medium that appears to be required during mitogenic stimulation by EGF. The HSF-CM also allows FDC-PI cells to survive and proliferate serum-free at low cell densities. Low concentrations of fetal calf serum or human plasma (0.2-2%) have the same effect as FDC-PI-CM and HSF-CM. We have tested many of the known growth factors, and none of them mimicked the autocrine serum replacing activity (ASRA). The activity in human plasma elutes from a gel-filtration column with an apparent molecular weight of 60,000. It appears as if cultured normal cells and cell lines produce molecules capable of complementing the growth factors required for the survival and proliferation of a range of cells in serum-free cultures.     

Development and characterisation of pilchard (Sardinops sagax neopilchardus) cell lines derived from liver and heart tissues

Two cell lines have been established from juvenile pilchards (Sardinops sagax neopilchardus) caught in waters off the Victorian coast of Australia. Following establishment of primary cultures derived from different pilchard tissues, using various cell culture media, a pilchard liver (PL) cell line and a pilchard heart (PH) cell line have been maintained in Eagles minimal essential medium supplemented with 10% foetal bovine serum for over four years. The cell lines have been cryopreserved in liquid nitrogen and can be recovered from storage with good cell viability. Stock cell cultures have been maintained at 20–22 °C on a continuous basis in normal atmosphere (100% air), with weekly subculture at a split ratio of 3:1. The origin of the cell cultures was confirmed by PCR analysis using primers designed to be specific for pilchard mitochondrial DNA. In addition, the liver cell line was cloned and both the parental cell line and clones thereof were shown to be susceptible to a broad range of marine and freshwater viral pathogens of fish.     

Transforming growth factor β1 acts as an inducer of matrix metalloproteinase expression and activity in human bone-metastasizing cancer cells

Bone metastases are a common complication in prostate and breast cancer patients. It leads to extensive morbidity and eventually mortality. Matrix metalloproteinases (MMPs) are known to be involved in the metastatic process. MMP activity can be down-regulated by transforming growth factor 1 (TGF-1), a growth-modulating factor, found in high concentrations in the bone. TGF-1 acts through the TGF-1 inhibitory element (TIE) element, a cis-acting element found in the promoter region of most MMP genes, with the exception of MMP-2. We used three human cell lines relevant for bone metastases, namely prostate adenocarcinoma PC-3, breast adenocarcinoma MDA-MB-231, and adenocarcinoma cells of unknown origin, Hs696, and one human osteosarcoma cell line, SAOS-2, and showed that in these cell lines TGF-1 partially lost its repressing action on MMP expression. TGF-1 was able to induce MMP-9 activity and protein expression in all three bone-metastatic tumour cell types, whereas MMP-9 protein levels were repressed in SAOS-2 cells. In PC-3 cells, TGF-1 repressed MMP-1 expression, whereas in MDA-MB-231 and SAOS-2 cells, an increase in the expression of MMP-1 protein was detected. Additionally, an increase in MMP-3 expression was observed in Hs696 cells. Expression and activity of the tissue inhibitors of matrix metalloproteinases, TIMP-1 and TIMP-2, were found increased in both PC-3 and MDA-MB-231 cells. With respect to cell proliferation, TGF-1 was able to induce a dose-dependent growth inhibition of up to 50% in primary human mammary epithelial cells. However, in none of the tumour cell lines was TGF-1 able to suppress growth substantially. Data presented in this paper support the hypothesis that TGF-1 can potentially disrupt the balance existing between osteoclast- and osteoblast-derived MMP activity by inducing altered expression of matrix metalloproteinases and their tissue inhibitors derived from bone-metastasizing cancer cells. This could eventually lead to skeletal destruction in patients with advanced metastatic disease.     

Adhesion polypeptides are useful for the prevention of peritoneal dissemination of gastric cancer

We examined the effect of adhesion polypeptides on the adhesion and invasiveness of gastric cancer cell lines. We previously reported the establishment of an extensively peritoneal-seeding cell line, OCUM-2MD3, from a poorly seeding human scirrhous gastric carcinoma cell line, OCUM-2M. Both 21 and 31 integrin expression was markedly increased on OCUM-2MD3 cells compared with OCUM-2M cells, and the ability of OCUM-2MD3 cells to bind to the extracellular matrix (ECM) was also significantly higher than that of OCUM-2M cells. The adhesion polypeptides, YIGSR and RGD, and two RGD derivatives significantly inhibited the adhesion of OCUM-2MD3 cells to the submesothelial ECM, while not inhibiting the adhesiveness of OCUM-2M cells and two well differentiated human gastric cell lines, MKN-28 and MKN-74. The YIGSR and RGD peptides also significantly inhibited the invasiveness of OCUM-2MD3 cells. The survival of nude mice with peritoneal dissemination given YIGSR sequenc e intraperitoneally was obviously longer than that of untreated mice. The survival of mice treated with RGD was also improved, and this effect was increased using the RGD derivatives, poly(CEMA-RGDS) and CM-chitin RGDS. These polypeptides appear to block the binding of integrins, which are expressed on OCUM-2MD3 cells, to the submesothelial ECM, and consequently inhibit peritoneal implantation. The peritoneal injection of adhe-sion polypeptides may be a new therapy against the dissemination of scirrhous gastric cancer, and may be useful for the prevention of dissemination in high-risk patients. © Rapid Science Ltd.     

Immunohistochemical study of cytochrome P-45011β -hydroxylase in human adrenal cortex with mineralo- and glucocorticoid excess

Summary Cytochrome P-450 specific for steroid 11-hydroxylation (P-450₁₁ ) was immunohistochemically demonstrated in the adrenal glands of human, pig and bovine and of mineralo- and glucocorticoid excess using a specific monoclonal antibody against P-450₁₁ of bovine adrenocortical mitochondria. P-450₁₁ was present in all three cortical zones of the histologically normal adrenal glands of bovine, pig and human, particularly in the zona fasciculata (ZF) and reticularis (ZR). The P-450₁₁ immunoreactivity was intensive in cortical micronodules and inner ZF and ZR in Cushing's disease, and relatively intensive in the zona glomerulosa (ZG) and outer ZF in idiopathic hyperaldosteronism (IHA), corresponding to the sites of active steroidogenesis. In adenomas with Cushing's syndrome and primary aldosteronism, compact cells were generally stained well. In the adrenal glands attached to the adenomas, immunoreactivity was observed only focally in ZG cells but not in ZF and ZR cells.This study was in part supported by a Grant from the Ministry of Health and Welfare Disorders of adrenal hormone Research Committee, Japan