Andrew Kardana

In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing müllerian system

Diethylstilbestrol (DES) was widely used to treat pregnant women through 1971. The reproductive tracts of their female offspring exposed to DES in utero are characterized by anatomic abnormalities. Here we show that DES administered to mice in utero produces changes in the expression pattern of several Hox genes that are involved in patterning of the reproductive tract. DES produces posterior shifts in Hox gene expression and ho‐meotic anterior transformations of the reproductive tract. In human uterine or cervical cell cultures, DES induces HOXA9 or HOXA10 gene expression, respectively, to levels approximately twofold that induced by estradiol. The DES‐induced expression is not inhibited by cyclohexamide. Estrogens are novel morphogens that directly regulate the expression pattern of posterior Hox genes in a manner analogous to retinoic acid regulation of anterior Hox genes. Alterations in HOX gene expression are a molecular mechanism by which DES affects reproductive tract development. Changes in Hox gene expression are a potential marker for the effects of in utero drug use that may become apparent only at late stages of development.—Block, K., Kardana, A., Igarashi, P., Taylor, H. S. In utero diethylstilbestrol (DES) exposure alters Hox gene expression in the developing mullerian system. FASEB J. 14, 1101–1108 (2000)

Carbohydrate and peptide structure of the α- and β-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma

Human chorionic gonadotropin (hCG), purified from the urine of 14 individuals with normal pregnancy, diabetic pregnancy, hydatidiform mole, or choriocarcinoma, plus two hCG standard preparations, was examined for concurrent peptide-sequence and asparagine (N)- and serine (O)-linked carbohydrate heterogeneity. Protein-sequence analysis was used to measure amino-terminal heterogeneity and the “nicking” of internal peptide bonds. The use of high-pH anion-exchange chromatography coupled with the increased sensitivity of pulsed amperometric detection (HPAE/PAD) revealed that distinct proportions of both hCG α- and β-subunits from normal and aberrant pregnancy are hyperglycosylated, and that it is the extent of the specific subunit hyperglycosylation that significantly increases in malignant disease.Peptide-bond nicking was restricted to a single linkage (β47–48) in normal and diabetic pregnancy, but occurred at two sites in standard preparations, at three sites in hydatidiform mole, and at three sites in choriocarcinoma β-subunit. In the carbohydrate moiety, α-subunit from normal pregnancy hCG contained non-fucosylated, mono-and biantennary N-linked structures (49.3 and 36.7%, means); fucosylated biantennary and triantennary oligosaccharides were also identified (7.3 and 6.9%). In choriocarcinoma α-subunit, the level of fucosylated biantennary increased, offset by a parallel decrease in the predominant biantennary structure of normal pregnancy (P<0.0001). The β-subunit from normal pregnancy hCG contained fucosylated and nonfucosylated biantennary N-linked structures; however, mono- and triantennary oligosaccharides were also identified (4.6 and 13.7%). For O-linked glycans, in β-subunit from normal pregnancy, disaccharidecore structure predominated, whereas tetrasaccharide-core structure was also detected (15.6%). A trend was demonstrated in β-subunit: the proportions of the nonpredominating N- and O-linked oligosaccharides increased stepwise from normal pregnancy to hydatidi-form mole to choriocarcinoma. The increases were: for monoantennary oligosaccharide, 4.6 to 6.8 to 11.2%; for triantennary, 13.7 to 26.7 to 51.5% and, for O-linked tetrasaccharide-core structure, 15.6 to 23.0 to 74.8%. For hCG from individual diabetic pregnancy, the principal N-linked structure (34.7%) was consistent with a biantennary oligosaccharide previously reported only in carcinoma; and sialylation of both N- and O-linked antennae was significantly decreased compared to that of normal pregnancy.Taken collectively, the distinctive patterns of subunit-specific, predominant oligosaccharides appear to reflect the steric effect of local protein structure during glycosylation processes. The evidence of alternative or “hyperbranched” glycoforms on both α- and β-subunits, seen at low levels in normal pregnancy and at increased or even predominant levels in malignant disease, suggests alternative substrate accessibility for Golgi processing enzymes, α1,6fucosyltransferase andN-acetylglucosaminyltransferase IV, in distinct proportions of subunit molecules.

Development and characterization of antibodies to a nicked and hyperglycosylated form of hCG from a choriocarcinoma patient: generation of antibodies that differentiate between pregnancy hCG and choriocarcinoma hCG.

Human chorionic gonadotropin (hCG) exists in blood and urine as a variety of isoforms one of which contains peptide bond cleavages within its β-subunit loop 2 and is referred to as nicked hCG (hCGn). This hCG isoform appears to be more prevalent in the urine of patients with certain malignancies and possibly in some disorders of pregnancy. Until now, only indirect immunoassays could be used to quantify hCGn. We report the development of two monoclonal antibodies (MAbs) to a form of hCGn isolated from a choriocarcinoma patient. This hCG isoform was not only 100% nicked, but also contained 100% tetrasaccharide-core O-linked carbohydrate moieties in its β COOH-terminal region. Two-site immunometric assays have been developedusing these new antibodies, B151 and B152. The former exhibits good specificity for hCGn independent of the source of the hCGn, the form excreted by choriocarcinoma patients or the form of hCGn from normal pregnancies. The latter antibody, B152, is sensitive to the carbohydrate moieties and possibly other differences in hCG isoforms, but is not for nicking of the β-subunit. These two immunometric assays provide potential novel diagnostic tools for direct measurement of hCG isoforms which could not be accurately quantified earlier before development of the assays using these newly generated antibodies.

Selecting human chorionic gonadotropin immunoassays: Consideration of cross-reacting molecules in first-trimester pregnancy serum and urine

OBJECTIVE We investigated the variation in human chorionic gonadotropin results found with different commercial kits. Levels of human chorionic gonadotropin and related molecules were determined in pregnancy serum and urine and compared with the specificities of different laboratory, office, and home test kits. STUDY DESIGN Total human chorionic gonadotropin (nicked+nonnicked), nonnicked human chorionic gonadotropin, free beta subunit, and beta core fragment were measured in 242 serum samples and 125 urine samples from early pregnancies. RESULTS In serum, in the 2 weeks after the missed period when most pregnancy tests are performed, median levels of total, nonnicked, and beta human chorionic gonadotropin (total+free beta+beta core) were similar (< or = 12% difference). Individual values, however, varied significantly. For nonnicked human chorionic gonadotropin, values ranged from 41% to 145% and for beta from 101% to 145% of the total human chorionic gonadotropin level. In urine individual nonnicked values varied from < 1% to 148% and beta values from 102% to 547% of the total human chorionic gonadotropin level. A survey of 29 kits revealed that 10 were types detecting total human chorionic gonadotropin, five detecting nonnicked only, and 14 were beta assays. CONCLUSIONS Results from total, nonnicked, and beta human chorionic gonadotropin kits are not necessarily interconvertible. Individual variations in levels of nicked human chorionic gonadotropin, free beta and beta core, and differences in their recognition by immunoassays causes discordant results.

THE STABILITY OF hCG AND FREE β‐SUBUNIT IN SERUM SAMPLES

Human chorionic gonadotropin (hCG) free β‐subunit measurement is used as a screening test for Down syndrome pregnancies. Use, shipping, and storage conditions have, however, to some extent been limited by a stability problem; the swamping of serum free β‐subunit levels by new molecules coming from the dissociation of hCG. We examined the stability of free β‐subunit levels in six fresh serum samples from the first trimester of pregnancy. The mean hCG level in the fresh serum samples was 3710±886 μg/l; this included a 3·0±0·39 per cent nicked hCG component. The mean free β‐subunit level was 0·27±0·04 per cent and the nicked free β‐subunit level was 0·10±0·04 per cent of the hCG concentration. Samples were incubated for 0, 0·5, 1, 2, and 4 weeks at 21°C, with no additives. After half a week, the free β‐subunit level rose to 137±17 per cent, and after 4 weeks to 360±53 per cent of the starting level (ANOVA P⩽0·05). Parallel, but greater, increases were observed in nicked hCG and nicked free β‐subunit levels. The experiment was repeated with the addition of penicillin–streptomycin–fungizone to fresh serum. After half a week, the free β‐subunit level increased to only 101±3·0 per cent (t‐test, with/without additive, P⩽0·05), and after 4 weeks to only 136±7·8 per cent (P⩽0·05) of the starting level. At 4 weeks, nicked hCG production was reduced from 499±83 to 158±15 per cent of the starting level. We infer that nicking of hCG and dissociation of unstable nicked hCG may be pathways supplementing free β‐subunit in serum samples. We further infer that hCG nicking activity comes from microbes, and that free β‐subunit levels can be stabilized for shipping and longer storage by antibiotic/antimycotic additives.

Formation of a hCG variant with truncated peptide moiety as a biosynthetic product in vitro

Summary In serum of tumor patients variants of human chorionic gonadotropin (hCG) or of the free β-subunit with lower molecular weights may be present as well as proteolytically nicked forms. The origin of these variants, secreted by cells or formed in the periphery, is unknown. In the present study, brefeldin A (BFA) was used to block transiently the anterograde transport from the ER to the Golgi apparatus in JAr cells to increase the residency of hCG in the ER and thus to amplify possible proteolytic processes taking place in this cellular compartment. In JAr cells, BFA (2 hours, 0.05-0.4 μg/ml) diminished the amount of secreted free β-subunit (up to 90%) in a dose-dependent and reversible manner; hCG secretion was less affected. In the BFA cultures, the molar ratio hCG/free was increased up to five-fold. Pulse-chase experiments, using [ 3 H]-Leu for labeling of newly synthesized protein, showed the presence of lower molecular weight variants of the hCG-α and β subunits when hCG was arrested in the ER in presence of BFA. This could be observed most clearly in 8-bromo-cAMP (1 mM) stimulated cultures. In BFA treated cultures, the free β-subunit as well as the β-subunit contained in hCG showed and about 3 kD lower apparent molecular weight in comparison to the β-subunits of controls (34 kD). The lower molecular weight variants of the β-subunits still contained the carboxy-terminal peptide (CTP) since they were bound to anti-CTP-Sepharose. The carbohydrate part could also not account for the difference in the apparent molecular weight since it was not abolished by removal of the N-linked carbohydrate residues. The results demonstrate the capability of the ER to produce truncated variants of the free β-subunit and a truncated β-subunit contained in hCG which are secreted in vitro . During a transient arrest of the de novo synthesized proteins in the ER, the free β-subunit as well as hCG may be most probably proteolytic cleaved in the amino-terminal domain of the β-subunit or in the core part of the molecule.