Metastatic phenotype correlates with high expression of membrane-associated complete beta-human chorionic gonadotropin in vivo

BACKGROUND: Investigations using living human cancer cells and the nude mouse model were conducted to evaluate the expression of human chorionic gonadotropin (hCG) in various cancers grown in vitro and in vivo. The aim was to determine whether membrane-associated hCG in any of its forms is a characteristic metastatic marker, and at what levels or ratios. METHODS: Human cancer cell lines known to produce tumors that metastasize spontaneously when grown in nude mice (n = 4) were compared with those that do not produce such tumors (n = 4) using analytical (quantitative) flow cytometry. Monoclonal antibodies directed to epitopes of intact hCG (hCG-holo) and its subunits, including beta-human chorionic gonadotropin with its carboxy-terminal peptide (hCG beta-CTP), allowed for the determination of hCG beta-CTP/hCG-holo ratios. RESULTS: No significant difference in hCG beta-CTP/hCG-holo ratios was found between the cultured human cancer cells that do not metastasize spontaneously (ratio = 2.39) and those that do (ratio = 2.13), and no difference was seen in their growth rate in nude mice. However, the cells isolated from tumors that do not metastasize spontaneously showed a decrease in their ratios to values less than 1. They reverted to their original values after reestablishment in culture and subsequent passages. In contrast, the ratios shown by cells isolated from tumors that metastasize spontaneously increased to 3 to 6 times their original values in culture, then reverted to their original values after reestablishment in culture and subsequent passages. CONCLUSIONS: To our knowledge, these data demonstrate the following for the first time: 1) There is a direct in vivo correlation between human cancer cells that metastasize spontaneously in nude mice and the expression of membrane-associated complete hCG beta (hCG beta-CTP); and the correlation identifies this molecule as a characteristic metastatic phenotype marker. 2) The marked ratio variations under different conditions indicate that the metastatic phenotype is an unstable event. 3) Growth and local invasion in vivo correlates with the expression of hCG-holo.     

Ruptured popliteal artery aneurysm

BACKGROUND: Previous studies in which monoclonal antibodies (MoAbs) were used against different epitopes of human chorionic gonadotropin (hCG) demonstrated the presence of membrane-associated hCG and its subunits by cancer cells of different types and origins and by human embryonic and fetal cells. To elucidate the mechanism of action of a synthetic vaccine against hCG, experiments were conducted to determine the presence or absence of direct dose dependent cytolytic activity by hCG MoAbs, including those elicited by the vaccine. METHODS: Human adenocarcinoma cells from the uterine cervix (ATCC HeLa CCL 2.0) grown in defined media at 37 degrees C were treated for 2-3 days with different selected doses of each of 12 MoAbs directed against different epitopes of hCG. Three of these MoAbs were against three different epitopes of the synthetic hCGbeta vaccine. RESULTS: There was a direct dose dependent effect by a MoAb directed against the natural hCGbeta carboxy terminal peptide (CTP), by a MoAb directed against hCGalpha, and by one of the three MoAbs produced by the synthetic hCGbeta-CTP, which is the main component of the World Health Organization (WHO) vaccine being tested for fertility control and for cancer treatment or prevention. CONCLUSIONS: For the first time (to the authors' knowledge), these results show the existence of hCG MoAbs that have direct dose related cytotoxicity at 37 degrees C and explain the mechanism of action of the WHO anti-hCG vaccine.     

Thyrotropic action of human chorionic gonadotropin

Hyperthyroidism or increased thyroid function has been reported in many patients with trophoblastic tumors. In these cases, greatly increased human chorionic gonadotropin (hCG) levels and suppressed TSH levels suggest that hCG has thyrotropic activity. Recent investigations have clarified the structural homology not only in the hCG and TSH molecules but also in their receptors, and this homology suggests the basis for the reactivity of hCG with the TSH receptor. The clinical significance of the thyrotropic action of hCG is now also recognized in normal pregnancy and hyperemesis gravidarum. Highly purified hLH binds to recombinant hTSH receptor and is about 10 times as potent as purified hCG in increasing cAMP. The beta-subunits of hCG and hLH share 85% sequence identity in their first 114 amino acids but differ in the carboxy-terminal peptide because hCG beta contains a 31-amino acid extension (beta-CTP). A recombinant mutant hCG that lacks beta-CTP showed almost identical potency to LH on stimulation of recombinant hTSH receptor. If intact hCG were as potent as hLH in regard to its thyrotropic activity, most pregnant women would become thyrotoxic. One of the roles of the beta-CTP may be to prevent overt hyperthyroidism in the first trimester of pregnancy when a large amount of hCG is produced by the placenta. Nicked hCG preparations, obtained from patients with trophoblastic disease or by enzymatic digestion of intact hCG, showed approximately 1.5- to 2-fold stimulation of recombinant hTSH receptor compared with intact hCG. This suggests that the thyrotropic activity of hCG may be influenced by the metabolism of the hCG molecule itself. Deglycosylation and/or desialylation of hCG enhances its thyrotropic potency. Basic hCG isoforms with lower sialic acid content extracted from hydatidiform moles were more potent in activating adenylate cyclase, and showed high bioactivity/immunoactivity (B/I) ratio in CHO cells expressing human TSH receptors. This is consistent with the finding that the beta-CTP truncated hCG with higher thyrotropic potency is substantially deglycosylated and desialylated in the beta-subunit relative to intact hCG because all four O-linked glycosylation sites occur within the missing C-terminal extension. The desialylated hCG variant also interacts directly with recombinant hTSH receptors transfected into human thyroid cancer cells. There is thyroid-stimulating activity in sera of normal pregnant women, and this correlates with serum hCG levels. The thyroid gland of normal pregnant women may be stimulated by hCG to secrete slightly excessive quantities of T4 and induce a slight suppression of TSH, perhaps being about 1 mU/L less than nongravid levels, but not high enough to induce overt hyperthyroidism. Maternal thyroid glands may secrete more thyroid hormone during early pregnancy in response to the thyrotropic activity of hCG that overrides the normal operation of the hypothalamic-pituitary-thyroid feedback system. Biochemical hyperthyroidism associated with hyperemesis gravidarum has been attributed to hCG. In patients with hyperemesis gravidarum, thyrotropic in serum correlated with hCG immunoreactivity, and the severity of vomiting as indicated by clinical and biochemical parameters correlated with the degree of thyroid stimulation. To understand the thyrotropic action of hCG, it is necessary to know whether hCG activates the same domain of the TSH receptor as does TSH. The identification of the molecular structure of the hCG isoform with the highest thyrotropic potency will resolve the enigma of gestational thyrotoxicosis and the hyperthyroidism associated with trophoblastic disease and hCG-producing tumors.     

Polypeptide pattern of human breast epithelial cells following human chorionic gonadotropin (hCG) treatment

Numerous attempts have made to describe the particular protein pattern of malignant cells by using high resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). The placental hormone human chorionic gonadotropin (hCG) inhibits tumor initiation and progression in experimental animals and has an inhibitory effect on the proliferation of human breast epithelial cells (HBEC) in vitro. The inhibitory effect on the immortalized HBEC MCF-10F is accompanied by the immunocytochemical expression of inhibin alpha and beta subunits by treated cells. With the purpose of clarifying the molecular mechanisms involved in this effect, the pattern of protein synthesis and mRNA were studied by 2-D PAGE in the immortalized HBEC MCF-10F cells treated in vitro 1001U for 24 h. The effect of hCG treatment on the synthesis of MCF-10F cells was monitored by labeling both control and treated cells with [S35]methionine and separation by 2-D PAGE. At least 11 proteins were preferentially synthesized and five specific polypeptides were decreased in hCG treated cells in comparison with controls. The hCG induced at least four new mRNAs which encoded protein in the molecular mass range of 24-72 kDa. It also increased the expression of at least six mRNAs and reduced the expression of least four mRNAs in comparison with control cells. The hCG-treated cells actively synthesized a 33-kDa polypeptide which was not present in control cells. The nature of this hCG-inducible 33 kDa protein elucidated by immunoprecipating [S35]methionine-labeled proteins with antisera directed against rat inhibin subunit alpha and beta b.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epidermal growth factor and laminin receptors contribute to migratory and invasive properties of gliomas

Gliomas are characterized by their extensive invasion into the brain parenchyma. Recently it has been shown that normal brain cells can produce laminin, fibronectin and collagen type IV when confronted by invading glioma cells. Laminin stimulates cell migration of several human glioma cell lines in vitro. This migration can be inhibited by adding blocking monoclonal antibodies (MAbs) against the most expressed integrin subunits, alpha3 and beta1. Previous studies have shown that glioma cell migration, invasion and growth are stimulated by epidermal growth factor (EGF). However, MAb directed against the EGF receptor (EGFR) did only partly inhibit the invasive process in vitro. Since laminin has regional peptide homology with EGF (EGF-like repeats), the present work was aimed at studying how two human glioma cell lines exposed to antibodies to the EGFR, reacted to laminin stimulated migration. Furthermore, we wanted to study which role the EGFR and the laminin receptor integrin subunits alpha3 and beta1 play during glioma cell invasion. EGFR expression of two glioma cell lines, AN1/lacZ and U-251/lacZ was studied by flow cytometry and immunofluorescence microscopy. A cell migration assay was used to study effects of MAbs against EGFR on migration from laminin-stimulated tumor spheroids. Tumor cell invasion was evaluated by using an in vitro co-culture model, where normal fetal brain cell aggregates were confronted with multicellular tumor spheroids. The results show that both cell lines expressed EGFR, AN1/lacZ 4-fold more than U-251/lacZ. MAb against EGFR inhibited the laminin-stimulated migration only from AN1/lacZ spheroids. MAbs against alpha3 and beta1 integrin subunits inhibited glioma cell invasion in vitro. The present work indicates possible connections between laminin-stimulated cell migration and the EGFR expression on glioma cells. These elements contribute to the characteristic features of glioma cells and may be an important part of the complex relationships between growth factors, integrins and extracellular matrix during glioma cell invasion.     

Immunochemical mapping of human lutropin: II. Characterization of two monoclonal antipeptide antibodies reacting with the native beta-subunit

To investigate the epitopes present on the beta-subunit of the human lutropin (hLHbeta) and their topographical relationship at the surface of the molecule, we produced two monoclonal antipeptide antibodies, designated LHP03 and LHP04, capable of binding to the radiolabeled 125I-hLHbeta and directed to the 43-52 and 110-117 regions of the hLHbeta, respectively. Analysis of the accessibility of the epitopes on hLH and on the beta-subunit of human chorionic gonadotropin (hCGbeta), equine LH (eLHbeta) and ovine LH (oLHbeta) indicated that: (i) LHP03 binds to both the free hLHbeta subunit and dimeric hLH whereas LHP04 binds preferentially to the free hLHbeta, (ii) LHP03 recognizes weakly the hCGbeta and oLHbeta in comparison to hLHbeta and (iii) LHP04 binds oLHbeta as well as hLHbeta but does not bind to hCGbeta and eLHbeta. The topographical relationship of epitopes recognized by LHP03 and monoclonal antibodies recognizing dimer specific epitopes on hLH allowed us to localize discontinuous antigenic sites that overlaps or are located outside the hHLbeta(43-52) region. Together, our results demonstrated that the hHLbeta(43-52) portion is accessible on both the free hLHbeta subunit and hLH whereas the COOH-terminal portion, hHLbeta(110-117), is probably buried at the alpha/beta interface of the hormone.     

Human luteinizing hormone and chorionic gonadotropin are targeted to a regulated secretory pathway in GH3 cells

LH is a dimeric glycoprotein hormone that is stored in the anterior pituitary and is released in response to GnRH, while the placental hormone, human CG (hCG), sharing the same alpha-subunit and a related beta-subunit, is secreted constitutively. In search of a determinant that allows sorting of LH into a regulated secretory pathway, the genes encoding the common alpha- and LH/CG beta-subunits were expressed in the GH3 rat pituitary tumor cell line, which contains a regulated secretory pathway. Steady state labeling and subsequent chase experiments showed that not only LH but also hCG can be sorted to a regulated secretory pathway; after an initial period of constitutive secretion, the mature forms of both hormones containing processed oligosaccharides were stored intracellularly, and their release was stimulated by either forskolin or KCl depolarization. In Chinese hamster ovary cells, which lack a regulated pathway and are devoid of storage granules, only hormones containing unprocessed N-linked oligosaccharides were found. In GH3 cells the LH beta-subunit was partially retained in an endoglycosidase H-sensitive form, presumably in the endoplasmic reticulum; the enzyme-resistant fraction was secreted through a regulated secretory pathway. A large fraction of the hCG beta-subunit was released constitutively, although some mature hCG beta-subunit accumulated in secretory granules and was released by forskolin. The common alpha-subunit was secreted constitutively with little intracellular accumulation of the mature forms. We conclude that the LH beta-subunit contains sufficient information to direct LH to a regulated pathway, and alpha:LH beta assembly is not a prerequisite for this targeting. The sorting of hCG to a regulated pathway in GH3 cells presumably reflects a structural similarity between LH and hCG. In addition, we have shown that GH3 cells can recognize the N-linked oligosaccharides on the gonadotropin subunits as substrates for sulfation.     

Effects of captopril on morphologic changes in kidney of spontaneously hypertensive rats with adriamycin nephropathy

Despite the fact that a number of alterations of the hypothalamic-pituitary-gonadal hormone axis have been identified in patients with testicular cancer, little is known about the gonadotrophin secretion pattern in such patients who have greatly increased human chorionic gonadotrophin (hCG) serum concentrations. The aim of this study was to assess this issue in detail using a longitudinal study design and a panel of highly sensitive and specific immunoassays. Eleven patients with non-seminomatous (n=11), and one with seminomatous testicular cancer with pretreatment hCG serum concentrations exceeding 10(5) pg/ml (>1000 mIU/ml) were selected and followed for a mean of 166 days (mean of 14 serum samples/patient) after initial diagnosis. Serum concentrations of hCG, its free alpha- (hCGalpha) and beta- (hCGbeta) subunits, human follicle-stimulating hormone (hFSH) and human luteinizing hormone (hLH) were determined by highly sensitive and specific enzymometric immunoassays based on a panel of monoclonal antibodies (MCA) established in our laboratory. A potential FSH-like activity (FSA) of hCG in the respective sera was determined by radioreceptor assays (RRA) for LH/CG and FSH. Specificity of FSA at the level of the receptor was assessed by MCA-based immunoabsorption studies. At diagnosis, hCG (9.8x10(7)+/-4.84x10(7) pg/ml; range 1.1x10(5)-5x10(8) pg/ml) was greatly increased and serum hFSH was undetectable (<9 pg/ml) in 11 patients, and one patient had very low, albeit detectable (approximately 30 pg/ml) hFSH concentrations. hLH was below the limit of detection (<2 pg/ml) in five individuals. During successful chemotherapy, hCG rapidly declined to physiological concentrations and hFSH/hLH returned to normal or even reached supraphysiological values. There was a highly significant negative correlation between hCG and hFSH (P=0.0001) and, to a lesser extent, hLH (P=0.0265). The ability of serum hCG to block the binding of [125I]rFSH (rat FSH) to its receptor was found to be 0.01-0.1% compared with the FSH standard; this could be reversed by an anti-hCG MCA. Addition of a specific MCA against hFSH blocked 3 microg/ml of the hFSH standard, but had no effect on the FSA of serum hCG in the FSH RRA. As observed during pregnancy, secretion of gonadotrophin -- particularly that of FSH -- is substantially or completely suppressed in patients with testicular cancer when serum hCG concentrations exceed 10(5)-10(6) pg/ml (approximately 10(3)-10(4) mIU/ml). As determined by RRA, the intrinsic FSA of tumour-derived hCG is most probably responsible for the suppression of hFSH in this group of patients with testicular cancer.     

Follicle stimulating activity of human chorionic gonadotropin: effect of dissociation and recombination of subunits

The follicle stimulating activity (FSA) and interstitial cell stimulating activity (ICSA) of highly purified human chorionic gonadotropin (hCG), its alpha and beta subunits, and hCG generated by subunit recombination were determined by ovarian weight and ventral prostate weight bioassays. Whereas highly purified hCG exhibited both FA and ICSA, its separated subunits were essentially devoid of both activities. ICSA and FSA, indistinguishable from that of the highly purified hCG, were restored by recombination of the hCG subunits. These observations are consistent with the hypothesis that the FSA and ICSA found in highly purified hCG preparations are properties of the hCG molecule.     

Differential effects of interleukin 1-alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNF-alpha) on motility of human melanoma cell lines on fibronectin

Interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) induce a motogenic response in a number of benign and malignant cells. We examined the chemokinetic effects of these cytokines on the cell migration of four melanoma cell lines on fibronectin using modified Boyden chambers and video-time lapse analysis. Flow cytometry analysis of IL-1 receptors, TNF receptors, and shifts in beta 1 integrin expression were correlated with the effects of these cytokines on cell migration on fibronectin. The four melanoma cell lines exhibited heterogeneous expression of types I and II IL-1 receptors as well as p60 TNF receptors. Scant p80 TNF receptor expression was detected on only one cell line. Three of four melanoma cell lines demonstrated type I IL-1 receptors by Western blotting. IL-1 alpha and TNF-alpha induced heterogeneous modulation of beta 1 integrin expression in the four melanoma cell lines tested; downward shift of the alpha 2, alpha 3, alpha 4, and beta 1 integrin subunits was detected among three of the melanoma cell lines as were upward shifts of the alpha 4, alpha 5, and alpha 6 integrin subunits among three of the melanoma cell lines. IL-1 alpha and TNF-alpha induced enhanced migration on fibronectin in one of the melanoma cell lines and were related to an upward shift in the alpha 4 and alpha 5 integrin subunit expression. Taken together, the findings indicate that expression of a particular receptor for IL-1 or TNF does not necessarily signal a motogenic response in melanoma cells, but induces heterogeneous shifts in beta 1 integrin expression. However, upregulation in alpha 4 and alpha 5 integrin subunits appears to relate to enhanced migration on fibronectin.     

Stem cell factor regulates human melanocyte-matrix interactions

Stem cell factor (SCF) is hypothesized to play a critical role in the migration of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, c-kit, result in defects in coat pigmentation in mice and in skin pigmentation in humans. In this report we directly show that SCF alters the adhesion and migration of human melanocytes to extracellular matrix (ECM) ligands and regulates integrin expression at the protein level. SCF decreased adhesion of neonatal and fetal cells to collagen IV, and increased attachment of fetal cells to laminin. Attachment of fetal cells to fibronectin was decreased, but was unchanged in neonatal cells. Flow cytometry analysis of neonatal melanocytes showed that SCF down-regulated the expression of the alpha 2 receptor, and up-regulated the expression of the alpha 3, alpha 5 and beta 1 integrin receptors. SCF down-regulated expression of alpha 2, alpha 5 and beta 1 integrins by fetal melanocytes, and up-regulated expression of the alpha v and alpha 3 integrin receptors. Analysis of melanocyte migration using time-lapse videomicroscopy showed that SCF significantly increased migration of neonatal, but not fetal, melanocytes on fibronectin (FN). We conclude that SCF regulates integrin expression at the protein level and that SCF has pleiotropic effects on melanocyte attachment and migration on ECM ligands. We suggest that this may be one mechanism by which SCF regulates melanocyte migration during development of the skin.     

The possible significance of parallel changes in plasma lutein and retinol in Pakistani infants during the summer season

We established new cell lines from head and neck cancer patients for studies of adhesion molecules and cellular behavior in nine patients with primary or metastatic cancer treated at the Asan Medical Center. Explant cultures of fresh tumor tissue were used to develop new permanent tumor cell lines. Lines were tested for tumor formation and histology in nude mice. Flow cytometry and indirect immunofluorescence were used to assess DNA content and expression of the alpha 6, beta 4, and beta 1 integrin subunits and the intercellular adhesion molecule 1 (ICAM-1). In vitro growth patterns and adhesion to plastic were assessed using phase contrast microscopy. AMC-HN-1 to -8 were derived from patients with squamous cell carcinoma. AMC-HN-9 was from an undifferentiated carcinoma of the parotid gland. The 8 lines we tested produced nude mouse tumors that are identical to the histology of the original tumors. AMC-HN-1, -2, -5, and -9 have epithelioid or spindle cell morphology with poor cell-to-cell and cell-to-substrate adhesiveness. AMC-HN-3, -4, -7, and -8 grow as adherent epithelioid monolayers. AMC-HN-6 exhibits multilayer stratification. Four lines are near diploid, 4 are hyperdiploid and 1 is hypodiploid. Only three express ICAM-1. All lines express the alpha 6, beta 4, and beta 1 integrin subunits but to different extent. Four, AMC-HN-1, -2, -5, and -6, express the beta 4 integrin at low levels, AMC-HN-3, -4, -7, and -9, have intermediate beta 4 expression, and AMC-HN-8 has extremely high beta 4 expression. The AMC-HN cell lines are representative in vitro models for the study of head and neck cancer biology. Our preliminary results indicate a close relationship between integrin expression and cell adhesion in vitro.     

Histo-blood group A/B versus H status of human carcinoma cells as correlated with haptotactic cell motility: approach with A and B gene transfection

In a search for the molecular basis of ABH status of tumors as correlated with malignancy, we studied various malignancy-related phenotypes of high H/Le(y)-expressing tumor cell lines in comparison with phenotypes of the same lines transfected with histo-blood group A or B genes. A and B gene transfectants, prepared independently from different H-active parental cells, showed A or B activity and abolition of H activity. All A and B gene transfectants, regardless of source, were characterized by significantly reduced Matrigel-dependent haptotactic motility. The level of haptotaxis of all transfectants was similar to that of parental cells in the presence of antibodies against human integrin subunits alpha3, alpha6, or beta1. These subunits showed high expression of A or B epitope in the A and B gene transfectants. Enhancement versus reduction of malignancy, associated with deletion versus induction of A/B epitopes, may be due in part to enhanced haptotaxis sustained by alpha3, alpha6, and beta1 integrin receptors, the activities of which are regulated by H or A/B glycosylation. These phenotypic changes provide a rationale for the deletion of A and B epitopes as one criterion defining human tumor malignancy.     

Dependence of mesenchymal cell responses on duration of exposure to bone morphogenetic protein-2 in vitro

Murine Leydig (TM3) and Sertoli (TM4) cell lines were studied as nonprofessional antigen-presenting cells using the antigen model of human choriogonadotropin (hCG alpha/beta) and specific T-cell hybridomas. Both cell lines were treated with IFN-gamma to induce I-A(d) and I-E(d) molecules expression. Only the TM3 cell line, which expressed MHC-class II molecules upon IFN-gamma stimulation, was able to uptake, process, and present the human choriogonadotropin beta subunit to related T-cell hybridomas. Interestingly, the TM3 cell line was incapable of presenting the human choriogonadotropin alpha subunit, the presentation of which, by classical APC, is highly efficient. Using T-cell hybridomas directed against the immunogenic regions of hCG alpha/beta previously described in BALB/c mice, we showed that the TM3 cell line generated a narrower peptide repertoire than classical APC (i.e., B cells, macrophages, and dendritic cells). This experimental system suggests that Leydig cells could initiate, in vivo, an autoimmune process directed against gonadal tissues. In particular, such a mechanism has been evoked in experimental autoimmune orchitis.