Lectin binding in tissues from hydatidiform mole, invasive mole and choriocarcinoma to concanavalin-A, wheat germ agglutinin and peanut agglutinin.

A light microscopic analysis of lectin receptors in normal placenta and trophoblastic disease was performed utilizing biotinylated Concanavalin-A (Con-A), wheat germ agglutinin (WGA), and peanut agglutinin (PNA), in conjunction with an avidin-biotin peroxidase complex. Hydatidiform mole, invasive mole and choriocarcinoma exhibited increased receptors to Con-A and WGA compared to normal placenta. Increased reactivity to Con-A and WGA was associated merely with increased growth and proliferation of trophoblasts rather than a malignant transformation. Normal placenta, partial and complete mole generally showed moderate to strong binding with PNA after neuraminidase treatment, while invasive mole and choriocarcinoma (11 of 15 cases) generally showed minimal to absent reaction with PNA. Heterogeneity of PNA binding in choriocarcinoma was manifested by the presence of PNA reactivity in the trophoblast membrane in 2 cases wherein no prior neuraminidase treatment was given. This suggests that in some malignant trophoblasts, there is absence of sialic acid in the terminal cell surface carbohydrate groups resulting in the exposure of N-acetylgalactoseamine.

[Trace elements in the serum and cancerous tissue in patients with trophoblastic carcinoma].

In the serum and cancer tissue of 155 patients with trophoblastic tumor and in 114 patients with benign or malignant gynecologic tumor, 22 elements (17 trace and 5 major elements) were determined by neutron activation analysis. The serum and tissue level of Cr, the serum Zn level in patients with hydatidiform mole was significantly higher than that of the control group (P less than 0.01). The serum Se level in Patients with invasive mole and choriocarcinoma was lower (less than 1.0 mumol/l), but Cu/Zn ratio was higher (greater than 2.0) in comparison with that of the controls (P less than 0.01). With remission of the disease levels of the essential trace elements went up approaching the normal levels. There was a negative relationship between the serum Zn level and hCG content. The authors point out that the low level of Zn and Se, the high level of Cr may be related to the development of trophoblastic tumors.

[The DNA content in the cell nuclei of trophoblastic tumors]. / Soderzhanie DNK v iadrakh kletok opukholei trofoblasta.

The DNA content in cytotrophoblast (CTB) and syncytiotrophoblast (STB) cell nuclei was assayed in tissue sections of 7 hydatidiform moles (HM) and 27 choriocarcinomas (CH). The procedure involved Feulgen's reaction and scanning cytophotometry. The analysis of summarized histograms showed the DNA distribution in CTB cell nuclei, on the one hand, and that in STB, on the other, to differ significantly in both the tumors. The HM studied cases were referred to as two subtypes on the basis of such parameters as modal class value, its ploidy and degree of nuclear poly- and heteroploidy of CTB and STB. These characteristics were used to identify three patterns of CH. A pronounced modal class (2c--4c) was typical of type 1. A wider range of modal class (2c--10c or 4c--8c) was observed in type 2. Type 3 of tumor was characterized by a pronounced polyploidy with the absence of the modal class. The analysis of individual CTB and histograms showed no significant differences between HM and CH with respect to the DNA content. An increase in the share of highly polyploid cells was associated with a shorter survival of patients.

Identification and characterization of subpopulations of the free alpha-subunit that vary in their ability to combine with chorionic gonadotropin-beta.

The free (uncombined) alpha-subunit of hCG is secreted in excess over alpha beta dimer from both malignant and nonmalignant trophoblast cells and is secreted ectopically from a variety of other malignant cell types. The free alpha-subunits from various sources are distinguishable from those that combine because they migrate more heterogeneously and more slowly on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) than dimer alpha. We have previously identified three posttranslational modifications that may contribute to the altered mobility of the free alpha-subunit and to its inability to combine with the beta-subunit: 1) preferential phosphorylation of the free alpha-subunit, 2) O-glycosylation of free alpha, and 3) differences in the processing of the asparagine-linked oligosaccharides between the free and combinable forms. We have purified three populations of the alpha-subunit from the JAR choriocarcinoma cell line and from ChaGo, a bronchogenic carcinoma cell line that ectopically synthesizes only the alpha-subunit, in order to identify the posttranslational modifications that contribute to the altered mobility on SDS-PAGE. Fractionation of the oligosaccharides released from the alpha forms with peptide N-glycosidase has shown that the faster migrating alpha forms on SDS-PAGE have less completely processed oligosaccharide chains. Twenty-two to 25% of the JAR free alpha and 35-41% of the ChaGo alpha forms that migrate the fastest on SDS-PAGE recombine with beta in an in vitro recombination assay under conditions where 62% of the dimer alpha form recombines. In contrast, only 5-12% and 16-21% of the JAR free alpha and ChaGo alpha forms, respectively, that migrate the slowest on SDS-PAGE recombine with beta. The form of JAR free alpha least capable of combining with beta contains on O-linked glycan on Thr-39. This same site is a substrate for phosphorylation by JAR cells. However, most of ChaGo alpha fails to recombine with beta even though ChaGo alpha contains little O-linked carbohydrate. These results suggest that the larger asparagine-linked complex glycans on the slower migrating alpha forms are the major limiting factor for subunit combination. Although these modifications may not be rate limiting for combination in the rough endoplasmic reticulum, they may prevent dimerization of the free subunits later in the secretory pathway.

Endothelium and thrombomodulin: expression and distribution of thrombomodulin on altered endothelium and neoplastic syncytiotrophoblast.

We investigated the expression of thrombomodulin (TM) on endothelium in some pathologic states. We used the cultured endothelial cells treated with interleukin-1 (IL-1) and propagated cells by serial subculture for extended periods of time and assessed cell-surface TM molecules. We also studied the distribution of TM on surgical specimens of chorionic diseases, angiosarcoma and on several established cell lines of human choricarcinoma. Subculture of human umbilical vein endothelial cells (HUVE) up to 2 months (approximately 16 subcultures) decreased the number of cell-surface TM molecules by approximately 20% compared to the primary culture. The number of TM molecules also decreased on HUVE treated by IL-1. The treatment of the cells with IL-1 also induced change of shape. TM was found not only normal syncytiotrophoblast but also on neoplastic syncytiotrophoblast of choriocarcinoma and hydatidiform mole. However, TM was not expressed on the three established cell lines. TM was found on various types of vascular tumors, including angiosarcoma.

Comparative study of placental protein 19, human chorionic gonadotrophin and pregnancy-specific beta 1-glycoprotein as immunohistochemical markers for extravillous trophoblast in pregnancy and trophoblastic disease.

PP19, a new placental tissue protein, has alpha 1-beta 1 electrophoretic mobility, a molecular weight of 36,500 and 3.9% carbohydrate. To study immunocytochemical PP19 localization in extravillous trophoblast, we obtained formalin-fixed specimens from extravillous tubal pregnancy at gestational weeks (GW) 7-9 (12 blocks); four early intrauterine pregnancies at GW 7-13 (12 blocks); four late pregnancies at GW 28-38 complicated with intramural uterine myoma, placenta increta and abruptio placenta (8 blocks); four invasive complete moles (9 blocks); and seven primary and metastatic gestational choriocarcinomas (12 blocks). Immunohistochemical staining was done for PP19, pregnancy-specific beta 1-glycoprotein (SP1) and human chorionic gonadotrophin (hCG) using the indirect-labeled antibody method [purified PP19 (Lot no. 225/242) and antibody against PP19 (Lot no. 632ZA) prepared by H. Bohn, antibodies against hCG (Behringwerke, Marburg, FRG) and SP1 (Dakopatts, Copenhagen, Denmark)]. In both early and late intrauterine pregnancies, the extravillous syncytiotrophoblastic cell (XST) showed positive staining for hCG and SP1 in the cytoplasm, as well as for PP19, which stained more intensively in the nucleus than in the cytoplasm. The three proteins were not seen in the evtravillous cytotrophoblastic cell (XCT) in the trophoblastic cell column and shell. The interstitial cytotrophoblast-like cell (ICT), which infiltrated into the decidua and myometrium, and their blood vessels, was immunoreactively positive for PP19 but negative for hCG and SP1 with the exception of SP1-positive ICT in the myometrium in late pregnancy. XST and ICT in the endosalpinx of tubal pregnancy stained for all three proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

[Human chorionic prealbumin: its identification, physicochemical properties and detection in the blood serum in trophoblastic disease]. / Khorionicheskii preal'bumin cheloveka: identifikatsiia, fiziko-khimicheskie svoistva i ego obnaruzhenie v syvorotke krovi pri trofoblasticheskoi bolezni.

Human chorionic prealbumin (HCPA) was immunochemically identified and isolated in pure form from immature placenta, particularly, from chorion. Comparative immunodiffusion analysis shows that HCPA differs from the known placenta-specific proteins. HCPA is a glycoprotein with MW about 100 KD determined by gel-filtration and 45 KD by SDS polyacrylamide gel electrophoresis. By immunodiffusion HCPA was not found in fetal and adult tissues and in biological fluids. HCPA was discovered in patients with trophoblastic diseases but was not revealed in other oncological situations.

[Morphological and immunohistochemical study on specimens of trophoblastic diseases in which the presence of a villous formation could not be established].

The histopathological discrimination between malignant trophoblastic diseases and benign trophoblastic diseases depends on the presence or absence of a villous structure. However, molar extravillous trophoblasts and cells in some placental site trophoblastic tumors (PSTT) of a benign nature, lack a villous structure. We therefore observed the morphology of trophoblastic cells which do not constitute a villous structure, including choriocarcinoma cells, and analyzed the location of placental proteins in these cells immunohistochemically. The results were as follows: 1. Molar extravillous trophoblasts were composed of large mononuclear cells and multinuclear cells. Most of them were positive for hPL and negative for hCG and SP1. 2. Choriocarcinoma consisted of cytotrophoblast-like cells, syncytiotrophoblast-like cells, large mononuclear cells and multinuclear cells resembling large mononuclear cells. HCG was noted in syncytiotrophoblast-like cells and large mononuclear cells, while hPL and SP1 were found only in syncytiotrophoblast-like cells. 3. PSTT was made up of large mononuclear cells and multinuclear cells which contained abundant hPL and very little hCG and SP1 or none at all. Molar extravillous trophoblasts were clearly distinguishable from choriocarcinoma cells in terms of their morphology and the location of placental proteins. In contrast, it seemed difficult to distinguish cells of PSTT from molar extravillous trophoblasts on a cell level.

Structures, function, and transformational changes of the sugar chains of glycohormones.

Human chorionic gonadotropin (hCG), human luteinizing hormone, human thyroid-stimulating hormone, and human follicle-stimulating hormone are closely related family of proteins which share a common alpha-subunit. However, their sugar moieties are quite different. hCG contains five acidic asparagine-linked sugar chains. These five sugar chains are derived by sialylation from three neutral oligosaccharides: two biantennary (N-1 and N-2) and one monoantennary (N-3) complex-type oligosaccharides. Although hCG purified from the urine of pregnant women is more enriched in sialylated sugar chains than that purified from placenta, the molar ratio of N-1, N-2, and N-3 of these two hCGs are the same (1:2:1). Comparative study of the sugar moieties of the alpha- and beta-subunits of hCG revealed that alpha contains 1 mol each of N-2 and N-3, while beta contains 1 mol each of N-1 and N-2. This specific distribution of oligosaccharides at the four asparagine loci of the hCG molecule is now helping us to consider the functional role of the sugar moiety of glycohormones. hCG is produced not only by the trophoblast but also by various trophoblastic diseases. The hCGs purified from the urine of patients with hydatidiform mole contain the same oligosaccharides as normal hCG. However, those from the urine of choriocarcinoma patients contain five additional neutral oligosaccharides. In contrast, hCGs from invasive-mole patients contain three of the five oligosaccharides, specifically found in choriocarcinoma hCGs. The malignant transformational change of the sugar moiety of hCG can be explained by an increase of a fucosyltransferase, which forms the Fuc alpha 1----6GlcNAc group and by ectopic expression and subsequent modification of N-acetylglucosaminyltransferase IV. The appearance of tumor-specific sugar chains of hCG has been used to develop a new diagnostic method for invasive mole and choriocarcinoma.

[Characteristics of the production of chorionic gonadotropin by the cells of trophoblastic tumors (immunohistochemical research)]. / Osobennosti produktsii khorionicheskogo gonadotropina kletkami trofoblasticheskikh opukholei (immunogistokhimicheskoe issledovanie).

An immunohistochemical investigation of chorionic gonadotropin (CGT) in normal placental, hydatidiform mole and choriocarcinoma tissues was undertaken. Cyto- and syncytiotrophoblast both were shown to produce the hormone in normal and tumor trophoblast; the rate of synthesis increased in proportion to the degree of cell elements differentiation, except for very large elements of syncytiotrophoblast with numerous pycnotic nuclei which were found to contain either only traces of response to CGT or none at all. The percentage of syncytiotrophoblast--produced hormone progressively increased in the succession of the placenta--hydatidiform mole--choriocarcinoma. No significant difference in the levels of immunohistochemically--identified hormone was found in the hydatidiform mole and choriocarcinoma.

Biological characterization of 10-(2-propynyl) estr-4-ene-3, 17-dione (MDL 18,962), an enzyme-activated inhibitor of aromatase.

MDL 18,962 was shown to be a highly specific, potent (Ki = 3-4 nM), enzyme-activated inhibitor of aromatase with minimal intrinsic endocrine properties. The affinity of MDL 18,962 was higher for human and baboon placental aromatase than for rhesus placental or rodent ovarian aromatase. These species differences necessitated the development of a novel model of peripheral aromatase utilizing human enzyme. Human choriocarcinoma trophoblast xenografts in athymic nude mice were used for pharmacologic and pharmacokinetic evaluation of MDL 18,962. The ED50 for inhibition of aromatase activity in these trophoblast tumors at 6 h post-treatment was 1.4 mg/kg, s.c. and 3.0 mg/kg, oral. Preliminary results indicated that the ED50 for inhibition of peripheral aromatization of androgen by MDL 18,962 in female baboons was 0.01 mg/kg, i.v. and 4 mg/kg, oral.

A single amino acid substitution in an ectopic alpha subunit of a human carcinoma choriogonadotropin.

Human choriogonadotropin [hCG] has two dissimilar noncovalently associated subunits, designated alpha and beta. An ectopically secreted hCG alpha subunit that fails to associate with the beta subunit and displays an anomalously high molecular weight on molecular sieve chromatography but not on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been sequenced. A single substitution of Glu56 by Ala56 has been found in the altered subunit. No evidence for conformational differences between normal and ectopic alpha could be found using circular dichroism or intrinsic fluorescence as measures of secondary and tertiary structure, respectively. Hydrophobicity profiles as determined by the method of Kyte and Doolittle (Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132) predicted, however, that the hydrophilic segment, Thr54-Ser55-Glu56-Ser57-Thr58, becomes an extension of the preceding hydrophobic segment when Glu56 is substituted with Ala. This solitary hemoglobin S-like mutation may lead to an altered tertiary structure, self dimerization, or an alteration in glycosylation that could be responsible for the ectopic alpha subunit's failure to associate with the beta subunit.

Thyrotropic activity of acidic isoelectric variants of human chorionic gonadotropin from trophoblastic tumors.

Previous studies have indicated that hCG has a weak intrinsic thyroid-stimulating activity. Differences in the molecular composition and biological activity of hCG in patients with trophoblastic diseases and pregnant women occur, but are not well defined. Therefore, we have studied the effect of serum samples and purified hCG preparations from patients with trophoblastic diseases on T3 release from human and porcine thyroid slices in vitro. We examined 30 serum samples from 13 patients with nonseminomatous testicular germ cell tumors, 3 from women with choriocarcinoma, and 5 from patients with hydatidiform moles. In all but 1 serum sample from the tumor patients, but in none of 11 serum samples of pregnant women, T3-releasing activity was found. Two patients with testicular cancer and 1 patient with molar pregnancy experienced episodes of frank hyperthyroidism. Isoelectric focusing on polyacrylamide gels of tumor sera (n = 15) revealed substantial amounts of acidic isoelectric variants, pI 3.3-3.9, which were only barely detectable in pregnancy sera. The percentage of acidic hCG variants with pI 3.3-4.0 to total hCG with pI 3.3-5.2, as determined by hCG (+hCG beta) RIA of the eluted fractions of polyacrylamide gel isoelectric focussing, varied from 12-45% in sera of tumor patients and from 0-4% in pregnant sera. We purified the acidic variants of hCG with pI 3.6-3.8 (hCGav) from the urine of our patients. The beta-subunit of purified hCGav had a slightly higher mol wt (35,750) than that of hCG CR 119 (34,190) on polyacrylamide gel electrophoresis. The hCGav showed a dose-dependant stimulation of T3 release and cAMP generation from human thyroid slices, whereas the other hCG fractions on isoelectric focussing had no thyrotropic effect in similar dose levels. The TSH-like activity of hCGav could be roughly estimated as 10 mIU TSH/IU hCGav. Anti-hCG (+hCG beta) antiserum, but not anti-hTSH antiserum, neutralized the biological activity of hCGav. These findings strongly suggest that acidic hCG variants act as functional stimulators of the human thyroid in vitro. Since these molecular variants of hCG can exist in patients with trophoblastic diseases in significant amounts, they could be responsible for some cases of hyperthyroidism in trophoblastic diseases.

Tumor markers: value and limitations in the management of cancer patients.

Sixteen tumor markers are reviewed, and measured to the ideal: produced by the tumor cell alone absent in health and in benign disease present in all patients with a given malignancy level in the blood representative of tumor mass detectable in occult disease. The only marker that approaches the ideal is human chorionic gonadotropin (HCG) in gestational trophoblastic tumors. In this malignancy, the HCG level suggests the diagnosis and stage, confirms response to therapy, and predicts relapse. The three most widely used and intensely studied tumor markers are carcinoembryonic antigen (CEA), alphafetoprotein (AFP), and HCG. CEA cannot be used in screening for cancer, but in carcinoma of the colon its elevation preoperatively increases the likelihood of advanced disease and postoperative recurrence. Postoperatively, elevated titers are often but not invariably associated with recurrent disease. AFP and HCG are useful in the management of nonseminomatous germ cell testicular tumors. Like CEA, they cannot be used for screening. They are more likely to be increased with advancing stage, and after therapy rising levels almost always mean recurrent disease. Some markers are valuable in specific circumstances, such as calcitonin in screening for familial medullary carcinoma of the thyroid. In multiple myeloma, immunoglobulins are useful in determining the tumor mass and response to therapy. In neuroblastoma, catecholamine metabolites are useful primarily in making the diagnosis. In some malignancies, the absence of effective therapy lowers the value of the marker, as for AFP in hepatoma. The remaining markers are too unreliable or too little studied to be useful in the management of an individual patient with cancer. The purpose of this paper is to provide the clinician with an understanding of the limitations of the present tumor markers that will lead to wiser use of the tests, and to provide standards to which future tumor markers should be measured.

Expression of chorionic gonadotropin alpha- and beta-genes in normal and neoplastic human tissues: relationship to deoxyribonucleic acid structure.

We have investigated whether the expression of hCG genes can be attributed to changes in the structure of the alpha- and beta hCG genes, such as rearrangements, duplications, or methylation patterns. Various tissues and cell lines were studied: two term placentae, three trophoblastic tumor cell lines, two tumor cell lines ectopically producing alpha-subunit, normal cells not producing hCG or subunits, and a nonproducing malignancy. Gene structure was explored by restriction enzyme analysis and Southern blotting of DNA, using as probes 32P-labeled plasmids containing alpha- and beta hCG cDNAs. Similarly, methylation was evaluated using the restriction enzymes Msp I, Hpa II, and Hha I, each sensitive to a different pattern of cytosine methylation. No structural changes were observed in alpha- and beta hCG genes, although certain polymorphisms were observed. Analysis of methylation patterns revealed variation of the methylated cytosines; however, no clear correlation was seen between overall methylation or a specific pattern of methylation of these genes and their expression. Although specific methylated nucleotides of regulatory importance may not have been detected by our methods, we can still conclude that neither DNA structural alterations nor patterns of cytosine methylation appear to be major determinants of hCG expression.

Polyamines and nucleic acids in gestational trophoblastic tumors.

To test the relationship between tumor malignancy and content and distribution of polyamines and nucleic acids, 2 forms of human gestational trophoblastic tumors were examined: the hydatidiform mole (self-limited form) and the human chorio-carcinoma (invasive form) xenografted into nude mice. The results indicate that there are 2 significant differences between the choriocarcinoma and the mole: 1) the choriocarcinoma is characterized by increased polyamine and nucleic acid levels, 2) tissues differ in their putrescine:spermidine and spermidine:spermine ratios. There is an increase in polyamines in the urine of mole-bearing patients over that of normal controls. The correlation between putrescine and spermidine with the chorionic gonadotropin indicates that these 2 polyamines reflect the biological activity of the mole.

[The determination of the recognition site of an antiserum detecting desialylation on O-glycosidic carbohydrates of hCG and its clinical applications. The relationship between the presence of asialo-hCG and malignant change in trophoblastic disease].

Human chorionic gonadotropin (hCG) is a specific tumor marker glycoprotein hormone for trophoblastic diseases. It contains 4 asparagine-linked and 4 serine-linked carbohydrate units. Recently, variations in the carbohydrate moieties of hCG in chorio-carcinoma have been suggested. However, the immunological method of detecting these malignant transformational changes of carbohydrate units in hCG have not been investigated. We therefore attempted to assess the possibility of establishing a radioimmunoassay system which can detect these transformational changes in serine-O-glycosidically linked carbohydrate units of hCG. HCG-specific hCG beta COOH-terminal portion contains all 4 O-glycosidically linked sugar chains (positions 121, 127, 132 and 138). An antiserum (R141) generated against the enzymatically cleaved, desialylated hCG beta COOH-terminal peptide (residue 123-145) by toepad immunization method was extensively characterized. This antiserum reacts with asialo-hCG better than with native hCG. It does not bind with synthetic COOH-terminal peptides nor monosaccharides such as N-acetyl-D-galactosamine and D-galactose, which are sugar components in O-glycosidic carbohydrate chains. The HF-treated asialo-hCG, in which galactose residues are further removed, still reacts with the antiserum. The antiserum requires both the peptide sequence and N-acetyl-D-galactosamine residues for its binding. Glycophorine and fetuin, which also contain the same O-glycosidic carbohydrate structure, do not react with the antiserum. Beta subunit gains its binding capability to the antiserum only upon desialylation, while native alpha and beta subunits do not react. The antiserum has high specificity toward asialo-hCG in which particularly O-glycosidic carbohydrate units are desialylated. With a specific radioimmunoassay system using this antiserum (R141), urinary hCG preparations from 29 patients with various trophoblastic diseases were analyzed. Asialo-hCG immunoactivity was insignificant in all patients with hydatidiform mole, invasive hydatidiform mole and persisted trophoblastic disease. However, asialo-hCG was found in 2 out of 6 cases of choriocarcinoma. The existence of asialo-hCG may be related to the advancement of disease.(ABSTRACT TRUNCATED AT 400 WORDS)