Tumor antigen and human chorionic gonadotropin in CaSki cells: a new epidermoid cervical cancer cell line.

Epidermoid cervical carcinoma cells (CaSki line) have been established in continuous culture. When leukocytes from cervical cancer patients were incubated with CaSki culture fluid concentrates, inhibition of leukocyte migration was observed in more than 70 percent of the patients tested. By contrast, significantly less inhibition was observed with normal donor leukocytes or leukocytes from patients with other types of cancer. These results were consistent with the expression of tumor-associated antigen by CaSki cells. Analysis of the serum from the donor of the cell line at the time of tumor biopsy, and of CaSki culture fluids, demonstrated the presence of the beta subunit of human chorionic gonadotropin.

Discordant synthesis and secretion of human chorionic gonadotropin and subunits by cervical carcinoma cells.

Epidermoid carcinoma of the cervix cell lines DoT and CaSki were examined for the production of human chorionic gonadotropin (HCG) and its subunits (HCG-alpha and HCG-beta). Additionally, the effect of butyrate and other regulatory agents on synthesis and secretion was investigated. Media and cell lysates were analyzed by radioimmunoassay for HCG and HCG-beta and by radioreceptor assay for intact HCG. Although DoT and CaSki cells secreted more HCG-beta than HCG, the cell lysates contained only one-half as much HCG-beta as HCG. This raises the possibility that HCG-alpha synthesis may be limiting to the formation of intact HCG in DoT and CaSki cell lines. Our results also indicate discordance in HCG and HCG-beta production in response to butyrate. Whereas the production of HCG-beta was increased by 5 mM sodium butyrate, the synthesis and secretion of intact hCG were decreased. These results support the concept of independent regulation of alpha- and beta-subunit synthesis and secretion.

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.

Use of glycosidase digested human chorionic gonadotropin beta-subunit to explain the partial binding of ectopic glycoprotein hormones to Con A.

Recent studies have demonstrated that ectopic glycoprotein hormones only partially bind Con A. To investigate the basis for these findings, the Con A binding of ectopic hCG beta from DoT cervical carcinoma cells was examined after digestion with various glycosidases. Ectopic hCG beta only partially bound Con A, as was the case after digestion with neuraminidase and beta-galactosidase. However, subsequent digestion with N-acetylhexosaminidase increased Con A binding to 96%. It is apparent that Con A binding of ectopic hCG can be inhibited by a residue removed by N-acetylhexosaminidase, probably extra beta-N-acetylglucosamine linked to beta-mannose on N-linked oligosaccharides. The method used involved the glycosidase digestion of glycoprotein alkylated under denaturing conditions and was first validated with milligram amounts of standard hCG beta.

Absence of the COOH-terminal peptide on ectopic human chorionic gonadotropin beta-subunit (hCG beta).

A comparison of the physical properties and immunoreactivity of ectopic hCG beta from cultured cervical cancer cells and standard hCG beta was performed. The heterogeneity of the preparations was examined by ion-exchange chromatography, and the molecular size by analytical gel filtration. Two distinct forms of ectopic hCG beta were identified. One form was indistinguishable from standard hCG beta, while the other, although larger, lacked the characteristic COOH-terminal peptide (CTP). This was shown by the failure of antisera specific for determinants on the cTP to recognize this molecule, and by the apparent absence of the 0-linked oligosaccharides and thermolysin cleavage site normally found in this region.

Human chorionic gonadotropin in BeWo trophoblastic cells after 12 years in continuous culture: retention of intact human chorionic gonadotropin secretion in mechanically versus enzyme-dispersed cells.

Gel filtration and radioimmunological studies were used to demonstrate that BeWo malignant trophoblastic cells, subcultured for more than 12 yr as roller tube colonies using dissection by scalpel, retained the ability to secrete large quantities of intact hCG. By contrast, BeWo cells that had been subcultured weekly for 12 hr using proteolytic enzyme dispersion lost their ability to secrete intact hCG. Rather, a large form of hCG-alpha was the major secretory product, along with lesser amounts of heterogeneous low molecular weight forms of hCG-beta. The gradual loss of hormone secretory ability and fidelity of hormone product by cells in continuous culture is a well known phenomenon. Our results suggest that the habitual use of trypsin-EDTA to subculture cells may accelerate this process.

Studies on human chorionic gonadotropin secretion: effects of EGTA, lanthanum, and the ionophore A23187.

Human malignant trophoblast cells that secrete human chorionic gonadotropin (hCG) in culture were employed to assess the calcium requirement for hormone production. Cellular and secreted hCG was measured by radioimmunoassay. Cells cultured for 7 h in Ca2+- and Mg2-free medium or in Ca2+-free medium, secreted less hCG than cells cultured in medium containing Ca2+. Addition of ethylene glycol bis (beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA), ethylenediamine tetraacetic acid (EDTA), or La2+ inhibited hCG release from the cells, but did not affect the amount of hCG in the cells. Inhibition of hCG release was dependent on time of incubation and on concentration of agent added. Inhibition of hCG secretion by EGTA was reversed upon removal of the EGTA from the culture fluid or by addition of equimolar Ca2+ to the fluid. These results demonstrated that divalent cations, probably Ca2+, are required for release and further synthesis of hCG. Addition of the divalent cation ionophore A23187 (0.1 to 10 muM) failed to increase hCG secretion by the malignant trophoblast, in contrast to the stimulatory effect of this agent in other secretory systems. Incubation of the cells for 15 to 60 min with 10 muM A23187 reduced hCG secretion, and this inhibition was reversed upon removal of the ionophore from the culture fluid. The studies with the ionophore supported other evidence indicating basic differences between hormone secretory mechanisms in the trophoblast compared to other endocrine tissues.

Ectopic secretion of complete human chorionic gonadotropin and beta-subunit by human cervical carcinoma cells lines.

The CaSki and DoT cervical cancer cell lines were reported to secrete the beta-subunit of hCG in the absence of complete hCG or alpha-subunit (Science 196: 1456, 1977; J Clin Endocrinol Metab 46: 69, 1978). In the present study, concentrates of fluids from large numbers of CaSki or DoT cultures were analyzed for the presence of hCG beta and complete hCG. Although some CaSki and DoT preparations contained no detectable complete hCG, others were found to contain as much as 18% hCG. The reason for the variation in the amount of hCG relative to hCG beta in CaSki and DoT cultures is unknown.

Regulation of human chorionic gonadotropin (hCG) secretion by serum and dibutyryl cyclic AMP in malignant trophoblast cells in vitro.

Human chorionic gonadotropin (hCG) secretion was studied in human malignant trophoblast cells (Jar line) in continuous culture. Radioimmunoassayable hCG in the cells and in the daily culture fluid was increased for at least 3 days by 10% newborn calf serum and by 1 mN N-6, 0-2'-dibutyryl cyclic adenosine 3', 5'-monophosphoric acid (dbcAMP) plus 1 mM theorphylline. Incubation of the cells in the presence of serum, dbcAMP and theorphylline resulted in additive stimulation of hCG secretion after 1 day. After 2 or 3 days, a supra-additive stimulation was observed, which was prevented by Actinomycin D. When cells were preincubated for 2 days in the absence of serum but with dbcAMP plus theorphylline, subsequent addition of serum resulted in supra-additive stimulation of hCG secretion within a single day; the continued presence of dbcAMP plus theophylline was not necessary for this stimulation by serum. These findings suggested that dbcAMP activates a slow process requiring RNA synthesis, resulting in increased hCG secretion. Serum appears to protect the hCG synthesizing system from degradation.

A procedure using immobilized antibody for the isolation of the beta-subunit of human chorionic gonadotropin from the culture fluid of a beta-subunit-secreting nontrophoblastic cell line.

Antibody produced against the beta-subunit of hCG was linked to Sepharose 4B and used to isolate ectopic hCG beta secreted by DoT cervical cancer cells in culture. beta-Subunit was eluted with 3 M guanidine-hydrochloride, pH 3.0 (three treatments, 15 min each, at 20 C). The eluted hCG beta was desalted with Sephadex G-25 and concentrated by lyophilization. A single batch of antibody-bound Sepharose was used repeatedly to adsorb hCG beta from liter quantities of spent culture fluid containing 10% fetal bovine serum. The material recovered from 15.75 liters of fluid was pooled and treated a second time with immobilized antibody. Using only immobilized antibody, purification of more than 200,000-fold was achieved, with an overall recovery of about 28%.

Differential occurrence of free beta and free alpha subunits of human chorionic gonadotropin (hCG) in pregnancy sera.

Levels of hCG alpha beta dimer, free alpha-subunit and free beta-subunit were measured in pregnancy sera. Dimer and free alpha were quantitated by radioimmunoassays (RIAs) using specific polyclonal antisera. Free beta was quantitated both by monoclonal anti-beta RIA and by polyclonal anti-beta RIA following the complete adsorption of cross-reacting hCG by immobilized alpha-antisera. Consistent with the findings of other laboratories, hCG levels in pregnancy sera peaked at around 10 weeks after the last menstrual period (post LMP), and declined thereafter. Free alpha levels rose as hCG levels declined and accounted for 30-40% of total serum alpha in the third trimester. Although free beta accounted for only a small proportion of the total beta-subunit at the time of the hCG peak and thereafter (2.4-3.6%), in early pregnancy serum samples, 4-6 weeks post LMP, when hCG was generally first detected, an average of 16% free beta was detected. At this time, the higher the hCG level (20-2000 ng/ml), the lower the percent free beta (54-3%). Thus, the free beta portion started high and declined prior to the hCG peak; the free alpha portion increased thereafter. To explain these findings, we propose a two phase regulation of hCG dimer formation. Up to the time of the hCG peak, supplies of alpha-subunit are limiting (hence the presence of free beta). Thereafter, beta-subunit levels drop, restricting dimer formation and leaving uncombined alpha.

A human parathyroid carcinoma that produces parathyroid hormone: long term maintenance in tissue culture.

Parathyroid carcinoma cells from a pulmonary metastasis of a patient with a serum Ca of 17 mg/dl and an immunoreactive parathyroid hormone (PTH) of 6.4 ng eq bovine (b) PTH/ml (normal 40--400 pg/ml) have been maintained in tissue culture for more than 2 1/2 years. The cells secrete PTH into the culture media that 1) during immunoassay dilutes in parallel to human hyperparathyroid serum, 2) has a molecular weight similar to intact highly purified bPTH, and 3) stimulates bone resorption in a manner that is equivalent and additive to synthetic bPTH-(1--34).

Clinical utility of human chorionic gonadotropin and alpha-subunit measurements.

Sensitive and quantitative radioimmunoassays of serum human chorionic gonadotropin (hCG) levels have expanded the utility of hCG measurements from a simple pregnancy test into a powerful tool applicable in an increasing number of clinical disorders. Proper management of gestational trophoblastic disease depends on reliable and sensitive hCG determinations. Information on the in vivo doubling time of hCG in early pregnancy can be used in more effective diagnosis of early pregnancy disorders such as ectopic pregnancy and spontaneous abortion. Serum hCG is an effective tumor marker in the staging and management of testicular cancer and potentially of nontrophoblastic cancers. Measurement of glycoprotein hormone alpha-subunit levels has clinical application in gestational trophoblastic disease, abnormal pregnancy, and some forms of nontrophoblastic malignancy.

Semiquantitative use of Biocept-G radioreceptorassay.

Serum samples (93 total) from 62 different patients were analyzed by quantitative radioimmunoassay (RIA) and by the Biocept-G radioreceptorassay (RRA) for detection of human chorionic gonadotropin (hCG). The ratio of sample counts to reference control counts (S/R) was determined for each RRA. For the 44 serum samples having S/R ratios between 0.295 and 1.20 (corresponding to hCG levels of 4600 and 89 mIU/ml, respectively), there was good correlation between the S/R ratio by RRA and the serum level of hCG by RIA (correlation coefficient = -0.92). When serum samples were analyzed in as many as 4 different Biocept-G kits, the same S/R ratios were obtained. The S/R ratio reliably predicted the trend in serum hCG titer in 7 patients during treatment for gestational trophoblastic disease. The usefulness of the S/R ratio in obtaining semiquantitative results with the Biocept-G RRA is discussed.

Discordant human chorionic gonadotropin results: causes and solutions.

This study was performed to demonstrate the phenomenon of discordant human chorionic gonadotropin (hCG) results, in which some serum specimens are positive in one hCG detection procedure but negative in another procedure. Nine different quantitative hCG procedures were used to document discordant hCG results in 22 cases. A two-site monoclonal antibody immunoradiometric assay had the least tendency to give aberrant low-positive hCG values in nonpregnant patients without neoplasms. Potential causes of discordant hCG results are discussed, and guidelines for dealing with them are suggested. Recommended approaches include analysis in an alternate hCG detection procedure that uses a different technology for collection of antigen-antibody complex, dilution analysis, and sequential hCG analysis.

Effect of Crude and Purified hCG on Lymphocyte Blastogenesis.

Crude APL hCG completely inhibited blastogenesis induced by phytohemagglutinin, pokeweed mitogen, or concanavalin A in normal lymphocytes and lymphocytes obtained from cancer patients. Phenol in quantities comparable to its concentration in commercial lyophilized APL hCG also completely inhibited all three mitogenic responses. By contrast, similar doses of highly purified hCG had no inhibitory effect; higher concentrations produced only slight inhibition. Beta-hCG had a partial (less than 50%) inhibitory effect that was dose-independent and nonreproducible.

Production of tumor-associated antigen, TA-4, by the CaSki cervical carcinoma cell line.

Previous Japanese studies described the purification of TA-4 from homogenates of tumor tissues excised from squamous cell carcinomas of the uterine cervix, and the development of a radioimmunoassay to detect TA-4 in sera of patients with this disease. The aim of the present investigation was to determine if TA-4 was produced by the CaSki cell line, established in culture ten years ago from epidermoid carcinoma of the uterine cervix. The radioimmunoassay detected the TA-4 antigen in the CaSki cells, but not in cell lines derived from either choriocarcinoma or breast carcinoma. The TA-4 concentration in the CaSki cell lysate exceeded that in the CaSki culture fluid by more than twentyfold, and exceeded the concentration of human chorionic gonadotropin beta-like immunoreactive material by nearly two orders of magnitude. Antiserum to TA-4 was used to immunoprecipitate biosynthetically labeled TA-4 from CaSki cultures that had been incubated with [3H]leucine. After electrophoresis and autoradiography, the immunoprecipitated material showed a major band corresponding in apparent molecular weight (48,000 daltons) to TA-4 originally isolated from squamous cell carcinoma tumors. It is concluded that the CaSki cell line constitutes an ideal model with which to investigate the biosynthesis and regulation of TA-4, and a source for large-scale production of TA-4 for characterization studies as well as development of clinical diagnostic reagents.

Alpha subunit and human chorionic gonadotropin in normal pregnancy and gestational trophoblastic disease.

The reported incidence of gestational trophoblastic disease is an order of magnitude higher in Nigeria than in the United States. Sera from a total of 283 pregnant black patients, 138 United States and 148 Nigerian pregnant patients, were analyzed for their serum levels of alpha subunit and human chorionic gonadotropin (hCG). The patterns of hCG secretion were similar in the two populations during normal pregnancy. However, the level of alpha subunit was persistently higher in Nigerian women than in comparable pregnant United States patients. A statistically significantly higher alpha subunit level in the Nigerian patients was found only in the ten- to 13-week gestational period (P less than .005). The higher level of alpha subunit in pregnancy in Nigerian women may signal a population of trophoblastic cells which may be at higher risk for malignancy development in the Nigerian woman.

Macromolecular synthesis is required for stimulation of estrogen synthetase activity by dibutyryl cyclic AMP plus theophylline in choriocarcinoma cell culture.

Estrogen secretion and estrogen synthetase (aromatase) activity are stimulated in human trophoblast cells (JAr line) after addition of 1 mM dibutyryl cyclic AMP plus 1 mM theophylline (dbT) to the growth medium. The data given here show that (a) the aromatase specific activity in homogenized cells increases linearly over a 96 hr incubation period after addition of dbT; (b) addition of inhibitors of macromolecular synthesis, cycloheximide or actinomycin D, to the culture medium at the time of addition of dbT abolishes the stimulation of aromatase activity; (c) mixing dbT-grown cells, containing increased aromatase activity, with control cells does not result in an aromatase specific activity higher than the expected average, suggesting that dbT-grown cells do not contain a factor present in excess which serves to stimulate aromatase in control cells; and (d) NADPH, included in vitro in the aromatase assay or incubated with the cells for 48 hr as well as being present in the aromatase assay, has no stimulatory effect on aromatase specific activity in homogenized cells.

Estrogen synthetase in choriocarcinoma cell culture. Stimulation by dibutyryl cyclic adenosine monophosphate and theophylline.

The stimulation of estrogen biosynthesis by N6,O2'--dibutyryl adenosine 3':5'-cyclic monophosphate and theophylline dbT) in cultures of the JAr line of choriocarcinoma cells was investigated by measuring the specific activity and kinetic constants of estrogen synthetase (aromatase) in the various subcellular fractions after differential centrifugation of homogenized cells in isotonic sucrose. The low speed (900xg) pellet, from cells grown with or without dbT and homogenized in isotonic sucrose, contains the majority of the aromatase activity and the highest aromatase specific activity. The aromatase specific activity in the homogenate of cells grown with dbT and in the various subcellular fractions is 4- to 10-fold higher than in cells grown without dbT. The Vmax of androstenedione (4-androstene-3,17-dione) aromatization in homogenates from dbT-stimulated cells (6.9 pmol estrogen/min per mg protein) is significantly increased over that measured in the absence of dbT (1.5 pmol estrogen/min per mg protein); the Km values, however, are not significantly different (average of 43.8nM in dbT-stimulated fractions; 53.2nM in control fractions). These results suggest that the increased aromatase specific activity in dbT-stimulated cells results from an increase in amount of active enzyme, rather than from an increase in affinity of the enzyme for its substrate.