Expression of oncodevelopmental gene products by human tumor cells in culture.

Sixty-seven human tumor cell lines and 15 lines derived from normal tissue were examined for the production of the oncodevelopmental markers carcinoembryonic antigen, alpha and beta subunits of chorionic gonadotropin, placental and nonplacental forms of alkaline phosphatase, gamma-glutamyltransferase, cystyl aminopeptidase, and calcitonin. Both intracellular and extracellular levels of these markers were determined at three phases during the growth of each culture. Sixty-eight percent of the cell lines produced elevated levels (greater than or equal to 90th percentile) of at least one marker. Of those, 46% produced elevated levels of one marked only, 29% produced two, 22% produced three, and 4% produced four markers. No cell line produced more than four markers at elevated levels. In most instances, however, the expression of any two particular markers was discordant. For approximately 50% of the possible marker pairs, Spearman rank-ordered correlation analyses showed significant negative correlations, indicating that when one marker was produced at elevated levels by a given cell line, other markers were usually absent ot produced at relatively low levels. In no instance was a significant positive correlation found between two markers. These data indicated that, although most human malignant cells examined produced one or more oncodevelopmental gene markers at elevated levels, no predictable coexpression of any two of the markers was seen.

Stimulation of synthesis and secretion of chorionic gonadotropin subunits by eutopic and ectopic hormone-producing human cell lines.

The effects of dibutyryl cyclic adenosine 3'3':-5'-monophosphate (AMP) and sodium butyrate on the synthesis and secretion of human chorionic gonadotropin (HCG) by trophoblastic and nontrophoblastic human cell lines were studied by radioimmunoassay and pulse-chase labeling techniques. Dibutyryl cyclic AMP stimulated synthesis and secretion of HCG-alpha and HCG-beta subunits by the trophoblastic cell lines JAR and BeWo, whereas butyrate had no effect or decreased secretion. On the other hand, a number of nontrophoblastic cell lines (including the breast carcinoma lines ZR-75-31, BT-20, and MCF-7; the bronchogenic carcinoma line ChaGo; and the cervical carcinoma line HeLa S3) were induced to synthesize and secrete increased amounts of HCG subunits by butyrate, but dibutyryl cyclic AMP had less or no stimulatory effect. The nontrophoblastic brain tumor line CBT was an exception to this general rule in that HCG-beta production was stimulated by dibutyryl cyclic AMP but not by butyrate. In all cases, the drug-induced increase in HCG subunit secretion was directly proportional to the elevation of HCG subunit synthesis. These data suggest that the differential effects of dibutyryl cyclic AMP and butyrate on trophoblastic and nontrophoblastic cells reflect differences in the transcription or translation of HCG subunit genes induced by these agents in the two cell types.

Production of human chorionic gonadotropin and its subunits by human tumors growing in nude mice.

The production and secretion of human chorionic gonadotropin (HCG) and its subunits by human tumors growing in nude mice have been examined. JAR choriocarcinoma cells growing in nude mice produce both free alpha subunit and complete HCG, but there is a decrease in the amount of free alpha subunit relative to complete HCG produced in vivo compared to HCG subunit production by these cells growing in culture. Cell lines that produce only free alpha subunit in culture (HeLa cervical carcinoma, ChaGo bronchogenic carcinoma, and BT-20 breast carcinoma) continue to produce primarily free alpha subunit in vivo, but a small amount of HCG-beta/HCG is detectable in the 24-hr urine collected from mice bearing HeLa or ChaGo tumors. CBT cells derived from a glioblastoma multiforme produce both alpha and HCG-beta/HCG in vivo. This represents a distinct shift from the pattern of HCG subunit production by CBT cells in culture because cultured CBT cells produce only free beta subunit and do not synthesize either free alpha or complete HCG. Thus, for human tumors growing in nude mice, there appears to be a shift toward more complete HCG production and a decrease in free subunit production as compared to the pattern observed for cultured cells.

Carcinoembryonic antigen for monitoring patients with small cell carcinoma of the lung during treatment.

Carcinoembryonic antigen (CEA) was measured at specific intervals in the plasma of 56 patients with small cell carcinoma of the lung. Of these patients, 47 had serial analyses for varying periods during their illness, 42 had pretreatment CEA levels, and 17 of the latter patients had determinations throughout the entire course of their disease. Pretreatment CEA levels were elevated above 2.5 ng/ml for 74% of the 42 patients and above 5.0 ng/ml for 48%. Although exceptions were noted, in general, a direct relationship was found between pretreatment CEA levels and extent of disease or tumor burden. Initial stage of disease, however, was more predictive of survival than was the pretreatment CEA level. With response to therapy, a corresponding decrease in CEA levels occurred for patients with an elevated pretreatment level. For those patients with a pretreatment CEA level below 5.0 ng/ml, an immediate slight increase in level was often seen associated with response and followed by a subsequent fall after one month. A rising CEA level was usually found with recurrence or progression of disease after initial response and occurred frequently prior to clinical evidence of progression. In combination with careful clinical evaluation, serial CEA measurements can aid in assessing tumor changes associated with treatment in patients with small cell carcinoma of the lung particularly at the times of recurrence or disease progression following a partial or complete response.

Content of gonadotropins in cultured human malignant cells and effects of sodium butyrate treatment on gonadotropin secretion by HeLa cells.

Twenty-one of 82 human cell lines examined for production of human chorionic gonadotropin and its subunits (HCG-alpha and HCG-beta) produced either one or both subunits at some phase in their growth. Of these, 14 produced an excess amount of free alpha subunit, and seven produced HCG-beta or complete HCG without evidence for free alpha subunit synthesis. Five of the HCG-producing cell lines also contained or secreted the beta subunit of human luteinizing hormone. CBT cells derived from a glioblastoma multiforme and JAR choriocarcinoma cells secreted significant amounts of the beta subunit of human luteinizing hormone, while three other cell lines (breast carcinoma MCF-7, HeLa S3, and melanoma A375) produced small amounts of the beta subunit of human luteinizing hormone but did not appear to secrete it. Two cell lines (the melanoma line A375 and the SV40-transformed line SV80) appeared to contain small amounts of human follicle-stimulating hormone. Sodium butyrate caused a 40-fold induction in the secretion of both HCG-alpha and HCG-beta by HeLa S3 cells, but the total amount of HCG-alpha secretion induced was 800-fold greater than that of HCG-beta. Induction was blocked by actinomycin D (1 microgram/ml) and cycloheximide (5 microgram/ml) but was not affected by 1-beta-D-arabinofuranosylcytosine at a concentration (5 microgram/ml) that blocked DNA synthesis 99%. These results indicate that a number of malignant human cell lines produce the subunits of both placental and pituitary gonadotropins and that there is frequently an excess secretion of the free alpha subunit common to these hormones.

A distinctive form of human chorionic gonadotropin beta-subunit-like material produced by cervical carcinoma cells.

The DoT and CaSki human cervical carcinoma cell lines ectopically produce material immunologically similar to the beta-subunit of human chorionic gonadotropin (hCG beta). Culture fluids were analyzed by gel filtration chromatography and radioimmunoassay (RIA) using (a) antiserum directed to conformation-specific (core-directed) determinants not involving the carboxyl-terminal peptide (CTP) in hCG beta purified from urinary hCG (i.e., standard hCG beta) or (b) antiserum directed to the CTP in standard hCG beta. CTP-directed RIA recognized a peak of hCG beta-like immunoreactive material that eluted in the same position as standard hCG beta. However, core-directed RIA recognized additional hCG beta-like material (i.e., ectopic beta-II), most of which eluted before standard hCG beta. CaSki cells were incubated with [3H]mannose, [3H]proline, and [3H] leucine, and the spent medium was immunoprecipitated and analyzed by gel electrophoresis. Several labeled peaks were detected in the lane from the anti-hCG beta X Sepharose immunoprecipitate, one of which corresponded in mobility to standard hCG beta, with two more intense components migrating at higher apparent molecular weights. Carboxypeptidase Y digestion released only 0.2 mol equivalents each of [3H]proline and [3H]leucine from the labeled CaSki material immunoprecipitated with anti-hCG beta X Sepharose, compared to 1 mol equivalent each in similar analysis of standard hCG beta. These findings were consistent with the absence of the 4-carboxy-terminal amino acids from 80% of the hCG beta-like immunoreactive material secreted by CaSki cells. The affinity purified ectopic beta-II failed to combine with standard hCG alpha under conditions in which combination of standard hCG beta with standard hCG alpha was essentially complete. Neither aggregation nor proteolytic degradation was the cause of failure of ectopic beta-II to combine with hCG alpha. We conclude that both the DoT and CaSki cervical carcinoma cell lines secrete a distinctive form of hCG beta-like material, ectopic beta-II. Lack of recognition by CTP-directed antisera and amino acid analysis suggest that ectopic beta-II may lack the CTP, despite its apparent larger size relative to standard hCG beta.

Biosynthesis and secretion of laminin and laminin-associated glycoproteins by nonmalignant and malignant human keratinocytes: comparison of cell lines from primary and secondary tumors in the same patient.

Laminin biosynthesis was compared in four pairs of human squamous cell carcinoma cultures derived from primary and recurrent or metastatic tumors in four patients with cancer of the larynx and hypopharynx to determine if changes in laminin production accompany tumor progression. Laminin profiles of the malignant cells were compared with laminin biosynthesized by nonmalignant human keratinocytes. Pulse-chase biosynthetic labeling of the cultures with [35S]methionine established that all of the squamous carcinoma cell lines synthesize immunoreactive A (Mr 400,000), B1 (Mr 205,000), and B2 (Mr 200,000) laminin subunits; assemble them to form the intact laminin molecule (Mr 950,000); and secrete a portion of the laminin they produce into the culture media. One aspect of laminin expression unique to keratinocytes, both malignant and nonmalignant, was the occurrence of three additional glycoprotein forms (Mr 195,000, 170,000, and 160,000) in the laminin immunoprecipitates. In contrast to the laminin subunits, these glycoproteins were not immunoreactive with the anti-laminin antiserum on Western blots. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis without and with reduction of disulfide bonds revealed that the laminin immunoprecipitates contained a family of oligomeric molecules. These ranged in apparent molecular weight from 370,000 to 950,000 and were composed of laminin subunits and the glycoprotein forms linked by interchain disulfide bonds. The malignant keratinocyte cell lines from different patients were distinguishable in terms of the array of laminin and glycoprotein forms displayed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the rate of [35S]methionine incorporation into laminin during the pulse-labeling, the fraction of [35S]methionine-labeled laminin secreted into the medium during the chase incubation, and the absolute amount of laminin secreted into the culture medium as determined by enzyme-linked immunosorbent assay. However, cell lines established from primary and metastatic or recurrent cancer in the same patient were indistinguishable in their profile of laminin biosynthesis and secretion. In comparison to primary cultures of nonmalignant foreskin basal keratinocytes, the malignant cells secreted into the culture medium a larger fraction of the laminin that they produce. This is an indication that the malignant keratinocytes in culture deposited a less stable basal lamina-like extracellular matrix than their malignant counterparts. The diminished integrity of the basal lamina matrix may be an important factor in the invasive growth of human epithelial cancer.

Partial purification and properties of a chromatin-associated phosphoprotein kinase from rat liver nuclei.

A phosphoprotein kinase (EC 2.7.1.37) KIVb, from rat liver nuclei, was purified 75-fold by phosphocellulose chromatography and gel filtration on Sephadex G-200. The enzyme, which has an apparent molecular weight of 55 000, phosphorylates casein and chromatin-bound nonhistone proteins more readily than histones or ribosomal proteins. It exhibits an absolute requirement for divalent cation with optimum activity at 15--20 mM Mg2+. Maximal kinase activity is achieved at 100 mM NaCl. The pH vs. activity curve is biphasic with optima at pH 6.5 and pH 8.0. The Km value for casein is 280 mug/ml and the Km for ATP is 6-10(-6) M. Kinase KIVb phosphorylates numerous nonhistone nuclear proteins as shown by electrophoretic analysis. The addition of kinase KIVb to reaction mixtures containing nonhistone proteins results in the phosphorylation of a spectrum of polypeptides similar to those that are phosphorylated by endogenous nuclear kinases. Nonhistone proteins bound to chromatin appear to be better substrates for KIVb than nonhistones dissociated from chromatin. A comparison of nuclear phosphoproteins phosphorylated either in the intact animal or in vitro (by the addition of kinase KIVb) indicates some differences and some similarities in the patterns of phosphorylation.

Dissociation of early folding events from assembly of the human lutropin beta-subunit.

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common alpha-subunit and beta-subunits that confer biological specificity. LHbeta and CGbeta share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHbeta assembles with the alpha-subunit more slowly than does hCGbeta, and only a fraction of the LHbeta synthesized is secreted, whereas CGbeta is secreted efficiently. To understand why the assembly and secretion of these related beta-subunits differ, we studied the folding of LHbeta in CHO cells transfected with either the LHbeta gene alone, or in cells cotransfected with the gene expressing the common alpha-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pbeta1, to the second major folding form, pbeta2, did not differ significantly from the pbeta1-to-pbeta2 conversion of CGbeta, suggesting that variations between the intracellular fates of the two beta-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGbeta, where the folding to pbeta2 results in an assembly-competent product, apparently greater than 90% of the LH pbeta2 recovered from LHbeta-transfected CHO cells was assembly incompetent, accounting for inefficient LHbeta assembly with the alpha-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGbeta, all 12 LHbeta cysteine residues formed S-S linkages as soon as pbeta2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pbeta2 and excess urinary alpha-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGbeta, LHbeta homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective beta-subunits.

Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.

There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects.

Patterns of human chorionic gonadotropin expression in untreated and 8-bromoadenosine-treated JAR choriocarcinoma cells.

Although the biosynthesis and secretion of hCG by both normal and neoplastic trophoblasts have been documented, the regulation of these events is not well understood. We have used the JAR choriocarcinoma cell line to study the biosynthesis and secretion of this hormone. Using immunofluorescence, we have determined that less than 5% of cells expressed detectable hCG at a given time, and about 30% of hCG-producing cells were morphologically differentiated. Treatment of the cells with 8-bromoadenosine produced a 2- to 5-fold increase in hCG synthesis and secretion and increased the number of cells expressing hCG by 4- to 6-fold, without altering the percentage of morphologically differentiated cells expressing hCG. The effect on hCG biosynthesis was dose dependent and was induced maximally with a 24-h exposure to 8-bromoadenosine. However, exposure of JAR cells to 8-bromoadenosine for 2 to 6 h was sufficient to initiate a response. Treatment of JAR cells with the adenosine A2-receptor agonist N-ethylcarboxamidoadenosine did not induce hCG biosynthesis. The effect of 8-bromoadenosine on hCG synthesis did, however, parallel the dose-effect curve for inhibition of thymidine incorporation and for decreased cell proliferation. We conclude that induction of hCG biosynthesis by 8-bromoadenosine occurs by inhibiting trophoblast cell proliferation, rather than by an adenosine receptor-mediated event. The observed increase in hCG production may be due to induction of an intermediate differentiated cell type or an increase in the number of cells in an hCG-producing cell cycle phase.

The effect of carbonyl cyanide trifluoromethoxyphenylhydrazone and methylamine on the processing and secretion of the glycoprotein hormone chorionic gonadotropin by human choriocarcinoma cells.

Carbonyl cyanide trifluoromethoxyphenylhydrazone (FCCP), a protonophore, and methylamine, a weak base, agents that dissipate hydrogen gradients across cellular membranes, were used to probe the coupling of hydrogen gradients to the processing and secretion of the glycoprotein hormone hCG by human choriocarcinoma cells (JAR) in culture. Both drugs disrupted the processing of asparagine-linked oligosaccharides such that the secreted hCG forms contained mostly high mannose rather than complex oligosaccharide chains. As the concentrations of FCCP were increased above 1 microgram/ml and those of methylamine above 12.5 mg/ml, the secretion of the labeled hCG dimer and free alpha-subunit was progressively inhibited. Both FCCP and methylamine also inhibited the incorporation of [35S] methionine and [3H]mannose into hCG subunits. Nevertheless, the inhibition of secretion was clearly apparent as an intracellular accumulation of the hCG subunit precursors in spite of the diminished incorporation of radioactive substrates. The intracellular hCG precursors that accumulated in the drug-treated cells contained predominantly Man8-9GlcNAc2 units, structures characteristic of glycoproteins localized in the endoplasmic reticulum. Both FCCP and methylamine inhibited hCG secretion at concentrations that did not lower the cellular content of ATP. We postulate on the basis of these results that a hydrogen gradient across the membrane either of the rough endoplasmic reticulum or the transitional vesicle is coupled to the rough endoplasmic reticulum to Golgi translocation step such that dissipation of the proton gradient blocks the secretion of hCG.

Asparagine-linked oligosaccharides facilitate human chorionic gonadotropin beta-subunit folding but not assembly of prefolded beta with alpha.

To determine the role of asparagine (N)-linked oligosaccharide chains in protein folding and assembly, the well established hCG-beta in vitro folding and assembly assays were used to analyze how the human CG (hCG) beta-subunit devoid of one or two N-linked glycans folds and assembles under different conditions. Two approaches were used: 1) site-specific mutagenesis of hCG-beta synthesized in Chinese hamster ovary cells transfected with beta-mutants lacking the asparagine glycosylation sites; and 2) enzymatic deglycosylation of hCG-beta synthesized in JAR cells with peptide N-glycosidase F or endoglycosidase H. In both cases, [35S]cysteine-labeled beta-subunits were used as substrates to measure the conversion of the hCG-beta folding intermediate p beta 1 into p beta 2 and assembly of p beta 2 with urinary alpha. Using the mutated substrates from Chinese hamster ovary cells, it was found that 60% of wild-type p beta 1 (two N-linked glycans), 60% of p beta 1 missing the Asn13-linked glycan, 40% of p beta 1 missing the Asn30-linked glycan, and 10% of p beta 1 missing two N-linked glycans were converted to the corresponding p beta 2, respectively. With the enzymatically deglycosylated substrate from JAR cells, 90% of p beta 1 (two N-linked glycans), 70% of p beta 1(1) (one N-linked glycan), and 10% of p beta 1(0) (without N-linked glycan) folded into p beta 2 under cysteamine and cystamine redox conditions with or without protein disulfide isomerase. These data demonstrate that at least one N-linked glycan is required for efficient folding of hCG-beta and that the Asn30-linked glycan is more important than Asn13-linked glycan for hCG-beta folding. It also was shown that the composition of N-linked glycans of hCG-p beta 1 did not change protein folding, since hCG-beta substrates with high mannose oligosacharides folded as efficiently as beta-substrates containing sialylated complex oligosaccharides. Moreover, assembly of the already folded, assembly-component folding intermediate, p beta 2, was not affected by removal of one or both of the N-linked glycans of the beta-subunit. These data thus show that N-linked glycans play their most important role in the folding component of the folding and assembly pathway for hCG-beta.

Combination of the chorionic gonadotropin free beta-subunit with alpha.

The rate-limiting event for combination of hCG alpha- and beta-subunits in JAR choriocarcinoma cells is the rate of disulfide bond formation in the beta-subunit. This is accompanied by a conformational change that produces a combination-competent form of the beta-subunit. The combination reaction, however, is incomplete, and 50% of the synthesized beta molecules remain uncombined (free). In addition, 70% of biosynthetically labeled free beta is degraded in the cell. Possible explanations for incomplete dimer formation include 1) biochemical differences between the free and combined beta-subunits that limit combination of free beta, and 2) an inefficient combination reaction due to low intersubunit affinities or limiting concentrations of combination-competent subunits within the cell. To examine whether the biochemical differences between free and combined beta-subunits that we have previously observed affect the combined beta-subunits that we have previously observed affect the combination competence of free beta, free and dimer beta-subunits were purified from the culture medium and lysates of JAR cells and examined for their ability to combine with alpha purified from pregnancy urine in an in vitro combination assay. Secreted free and dimer beta obtained from culture medium combined to the same extent with urinary alpha. Although the combination efficiencies were lower for the intracellular forms, the free and dimer beta-subunits purified from cell lysates also combined to the same extent with urinary alpha. Thus, biochemical differences that exist between the beta forms do not prevent combination of free beta with alpha in an in vitro combination assay. To examine the second possibility, we speculated that if high concentrations of hCG subunits remained in the rough endoplasmic reticulum (ER) for extended periods of time, the extent of dimer formation would increase in the cell. To increase the residence time of hCG subunits in the ER, JAR cells were treated with carbonyl cyanide trifluoromethoxyphenylhydrazone, an agent that inhibits the translocation of hCG subunits from the ER to the Golgi. Treatment of cells with trifluoromethoxyphenylhydrazone in long and short term pulse-chase labeling studies did not result in an increase in the extent of dimer formation. Thus, the subunit combination reaction in JAR cells may be incomplete due to subtle conformational differences in the free beta-subunit; however, these differences do not inhibit the combination of the free beta-subunit in vitro.

O-glycosylation of the alpha-subunit does not limit the assembly of chorionic gonadotropin alpha beta dimer in human malignant and nonmalignant trophoblast cells.

Biosynthetic experiments were carried out in cultures of human malignant trophoblast cells (the JAR cell line) and in explants of normal first trimester human placental tissue to test the hypothesis that the O-glycosylation of the glycoprotein hormone alpha-subunit at Thr-39 regulates the assembly of the CG alpha beta dimer. This modification of alpha has been shown to ablate its ability to combine in vitro with the beta-subunit of bovine LH and might explain why JAR cells and placental explants secrete uncombined alpha- and beta-subunits in addition to the hCG alpha beta dimer. We have previously detected an O-linked carbohydrate chain at Thr-39 in preparations of secreted free alpha-subunit, but not dimer CG alpha from JAR culture medium. We report here evidence that the O-glycosylation of alpha does not regulate the biosynthetic assembly of the hCG dimer in cultures of JAR choriocarcinoma cells or first trimester placental explants. The intracellular precursor forms of alpha and beta that accumulate in the endoplasmic reticulum and combine in that compartment are not yet modified with O-linked carbohydrate, as determined by measurements of their [3H]galactosamine content after biosynthetic labeling of amino sugars with [3H]glucosamine. Furthermore, only half of the free alpha-subunit secreted by JAR cells and less than 10% of free alpha secreted by 10-week-old placental explants received the O-linked chain. This was shown by determining the ratio of the unglycosylated and glycosylated forms of the tryptic peptide from free alpha that contains the O-glycosylation site (residues 36-42). Based on these findings, we make the following conclusions. 1) O-Glycosylation of alpha-subunit is a late event in the secretory pathway of trophoblasts compared to the rapid combination in the rough endoplasmic reticulum of hCG subunit precursors to form alpha beta dimer. 2) Association of alpha with beta precludes the subsequent addition of the glycan to alpha at Thr-39. 3) The alpha molecules that fail to combine with beta in the endoplasmic reticulum are substrates for the later addition of O-linked carbohydrate, presumably in the Golgi complex, but only a fraction of the free alpha molecules are modified with O-linked carbohydrate.

Detection of the free beta subunit of human chorionic gonadotropin (HCG) in cultures of normal and malignant trophoblast cells, pregnancy sera, and sera of patients with choriocarcinoma.

Immunoaffinity adsorption techniques, utilizing specific antisera for hCG and its subunits bound to Sepharose 4B, have been employed to separate hCG alpha beta dimer and free subunits of hCG. As previously reported by this and a number of other laboratories, trophoblast cells (in vivo and in vitro) produce free alpha subunit in addition to hCG dimer. We have now shown that cultured JAr choriocarcinoma cells also secrete free beta subunit: 37% of the total beta subunit (combined and free) secreted by JAr cells is in the free form. Moreover, in pooled sera from choriocarcinoma patients 15% of total beta subunit is free, and in media from placental explant cultures and in pooled first trimester pregnancy sera 11% and 6.5%, respectively, of total beta subunit are in the free form. The free beta s are all of similar mol wt to the combined forms, and associate with urinary hCG alpha to form hCG. Free alpha s, which are larger than the combined forms, are unable to associate with urinary hCG beta to form hCG. We propose that the supply of combinable alpha subunit, rather than beta, limits dimer formation.

Bacterial expression and in vitro folding of the beta-subunit of human chorionic gonadotropin (hCG beta) and functional assembly of recombinant hCG beta with hCG alpha.

A bacterial expression system for the beta-subunit of hCG (hCG beta) has been developed to produce suitable amounts of this protein for structural and biological studies. To produce hCG beta in Escherichia coli, the nucleotide sequence that encodes the amino acid leader sequence was removed from the hCG beta complementary DNA, and the gene was cloned into a pET expression vector. After induction of protein synthesis in host bacteria, recombinant hCG beta (rhCG beta) accumulated in inclusion bodies in an unfolded state. The inclusion bodies were purified from induced cultures of E. coli, solubilized in urea, and fractionated by reverse phase HPLC. In this way, 6-7 mg unfolded hCG beta were recovered from 1 liter culture, rhCG beta was folded in the presence of 6.4 mM cysteamine and 3.6 mM cystamine at pH 8.7 at a final concentration of 0.02 mg/ml protein. The folded protein assembled with urinary hCG alpha and the purified rhCG beta/urinary alpha dimer bound to and activated the human LH/CG receptor permanently expressed in a cell line, indicating that it was a functional hormone. The rhCG beta/urinary alpha dimer also stimulated in vivo ovulation in rats, thus confirming the biological activity of bacterially expressed hCG beta. Because E. coli lacks the ability to glycosylate proteins, these activity results indicate that the N-linked and O-linked oligosaccharides of hCG beta are not required for protein folding, subunit assembly, or full biological activity. The success of producing hCG beta in bacteria and of folding it in vitro implies that the beta-subunits of the other members of the glycoprotein hormone family, LH, FSH, and TSH, can also be produced in this manner. This may facilitate structural studies of these hormones as well as lead to the production of recombinant hormones for biological studies and clinical use.

A naturally occurring genetic variant in the human chorionic gonadotropin-beta gene 5 is assembly inefficient.

The hCGbeta gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGbeta gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGbeta gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGbeta gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in beta gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyrosine). To determine whether betaV79M hCG had biological properties that differed from those of wild-type hCG, a beta-subunit containing the V79M substitution was created by site-directed mutagenesis and was coexpressed with the glycoprotein hormone alpha-subunit in Chinese hamster ovary cells and 293T cells. When we examined betaV79M hCG biosynthesis, we detected atypical betaV79M hCG folding intermediates, including a betaV79M conformational variant that resulted in a beta-subunit with impaired ability to assemble with the alpha-subunit. The inefficient assembly of betaV79M hCG appeared to be independent of beta-subunit glycosylation or of the cell type studied, but, rather, was due to the inability of the betaV79M subunit to fold correctly. The majority of the V79M beta-subunit synthesized was secreted as unassembled free beta. Although the amount of alphabeta hCG heterodimer formed and secreted by betaV79M-producing cells was less than that by wild-type beta-producing cells, the hCG that was secreted as alphabeta V79M heterodimer exhibited biological activity indistinguishable from that of wild-type hCG.

Conformational intermediates in the production of the combinable form of the beta-subunit of chorionic gonadotropin.

Human trophoblastic cells synthesize and secrete hCG as well as uncombined forms of the alpha- and beta-subunits of hCG. We have previously reported that the rate-limiting step in alpha beta-dimer assembly in cultured JAR choriocarcinoma cells is a conformational change in beta-subunit accompanied by the formation of intramolecular disulfide bonds. We now report on the intermediate steps in the acquisition of this combinable conformation by the beta-subunit. The earliest biosynthetically labeled form of beta detected in JAR cells is a precursor termed p beta 1 that lacks at least one of the intramolecular disulfide bonds found in mature beta-subunit, that does not combine with alpha-subunit, and that does not react with a monoclonal antibody specific for free beta. The p beta 1 precursor rapidly assumes (within 5 min) a new conformation termed p beta 2 that, in contrast to p beta 1, migrates more slowly on nonreduced sodium dodecyl sulfate-polyacrylamide gels, combines with alpha to form the hCG dimer, and reacts with the monoclonal anti-free beta antibody. Pulse-chase kinetic experiments support the following sequence of events: p beta 1----uncombined p beta 2----combined p beta 2. The transition of p beta 1 to uncombined p beta 2 involves the formation of at least one intramolecular disulfide bond coincident with the conformational shift of the p beta molecule. Furthermore, treatment of the nonreduced subunits with trypsin releases a [35S]cysteine-labeled peptide from p beta 1, but not from either form of p beta 2. This peptide presumably contains one of the two crucial cysteine residues that participate in forming the disulfide bond that distinguishes p beta 1 from the p beta 2 forms. Dimer p beta 2 differs from both p beta 1 and uncombined p beta 2 in that it contains an O-linked N-acetylgalactosamine, which represents the first step in the formation of the O-linked glycans of beta-subunit. Dimer p beta 2 is, therefore, the most fully processed and kinetically the latest of the three p beta forms that appear in JAR cell lysates. We conclude that formation of an appropriate array of intramolecular S-S bonds accompanies the acquisition of a combinable conformation of beta-subunit, and we have identified intermediate steps in the pathway leading to this conformational change. The data suggest that it is the achievement of this conformation by beta-subunit that limits the alpha beta combination reaction rather than the amount or conformation of alpha-subunit.

Human chorionic gonadotropin-beta gene expression in first trimester placenta.

The hCGbeta gene family contains six genes linked in tandem on chromosome 19 and labeled beta genes 7, 8, 5, 1, 2, and 3. Previous studies on a small number of placentas have indicated that beta gene 5 was the most highly expressed gene during the first trimester of pregnancy, followed by genes 3 and 8. Beta genes 7, 1, and 2 were expressed at very low levels. The purpose of this study was to determine 1) whether this pattern of expression was typical during normal pregnancy by sampling a large number of first trimester placentas, and 2) whether there was a correlation between gestational age and the pattern of hCGbeta gene expression. Total RNA from 27 first trimester placentas varying in age from 6-16 weeks was reverse transcribed into complementary DNA. The complementary DNA was amplified by PCR, and the amount of DNA representative of each hCGbeta gene was quantified by Genescan analysis. In 14 of the 27 placentas, hCGbeta gene 5 accounted for 50% or more of the total beta messenger RNA expressed. Beta gene 3 was expressed at levels ranging from 1-42% of the total, and beta gene 8 expression ranged from 12-32% of the total. Gene 7 expression was less than 3% of the total beta expression in all 27 placentas. Although there appeared to be a trend toward lower expression of beta gene 3 in placentas beyond 10 weeks gestational age, there was no correlation of the pattern of beta expression with placental age. Beta gene expression was also examined in two blighted ova, a spontaneous abortion sample, and a hydatidiform mole as well as in cultured JAR choriocarcinoma cells. With the exception of JAR cells, these abnormal tissues had low levels of gene 3 expression, but these levels were within the range of the patterns observed in normal placentas. These data suggest that it is the total amount of hCGbeta gene expression rather than the expression of individual beta genes that is important for the maintenance of normal pregnancy.