Characteristics of antigens from an HCG alpha secreting cell line (ElCo) derived from human breast carcinoma in a Native American patient.

OBJECTIVE: Our purpose is to report a cell line derived from a Native American patients that could serve as a model for ethnic diversity in cell culture research. STUDY DESIGN: Tumor biopsy specimens from reproductive tract malignancies were explanted in the cell culture laboratory. Characterization of a successfully established line included a parameter commonly observed in minority populations in rapid moving Type A glucose-6-phosphate dehydrogenase enzyme (G6-PD). Multiple other standard characterization studies were carried out. RESULTS: A breast cancer cell line with a G6-PD-A enzyme was derived from an American Indian patient; this pattern is commonly observed in African-American populations. Results of lymphocyte-mediated cytotoxicity tests indicated that lymphocytes from patients with breast cancer were cytotoxic to ElCo cells. On the other hand, lymphocytes from patients with other types of cancer were not significantly different from healthy donor lymphocytes in their cytotoxicity. These studies indicate that ElCo cells express tumor-associated antigen(s). Many antigens of tumor cell origin were detected with use of antisera raised in rabbits in concentrates of ElCo cell culture fluid. One of these antigens was the alpha subunit of human chorionic gonadotropin. CONCLUSIONS: Chemotherapy testing, vaccine production, and various new treatment schemes are most commonly developed in cell culture systems. These systems should reflect characteristics of the target population so that desired outcomes best fit the population being served. Thus the cell line being reported, which was derived from a Native American woman, represents a tool that can be used in cell culture research as a model for diversity.

Gestational trophoblastic disease and human chorionic gonadotropin measurement.

Improved radioimmunometric assays for the glycoprotein human chorionic gonadotropin, its whole molecule and free beta and alpha subunits have improved the capability for trophoblast tumor detection and monitoring. New heights in survival rates have been reached with these improvements, particularly in high-risk disease.

Characterization of human chorionic gonadotropin in urine of patients with trophoblastic diseases by Western blotting using specific antibodies.

Human chorionic gonadotropin (hCG) from urine of patients with trophoblastic diseases was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by Western blotting using specific antibodies. Western blotting using anti-hCG beta carboxy-terminal peptide (CTP) revealed that the molecular weights of the beta subunits of the three molar hCG samples were identical to that of standard hCG beta, but those of choriocarcinoma hCG samples were individually different. In the five choriocarcinoma hCG samples, the beta subunits of three samples were apparently larger than standard hCG beta, while those of two samples were smaller than standard hCG beta but larger than desialylated standard hCG beta. These results suggest that structural changes of carbohydrates of hCG, which may be induced by malignant transformation of the trophoblast, can be observed as differences in molecular weight. In SDS-PAGE under nonreducing conditions. Western blotting using anti-hCG beta-CTP revealed a single band corresponding to hCG beta. Under reducing conditions with dithiothreitol (DTT), however, a second low-molecular-weight material (called here CTP') could be observed with anti-hCG beta-CTP. All five choriocarcinoma hCG samples were much more susceptible than standard hCG to cleavage of CTP' by DTT. We conclude that estimations of molecular weight and susceptibility to DTT reduction of urinary hCG by Western blotting using specific antibodies are useful in the diagnosis of choriocarcinoma.

Discordant induction of tumor-associated antigen, TA-4 and chorionic gonadotropin beta in cultured cervical carcinoma cells.

The CaSki cell line derived from an epidermoid carcinoma of the uterine cervix produces and releases two types of tumor-associated antigen. One is a eutopic antigen, TA-4 and the other is an ectopic antigen, hCG beta-like material. The aim of the present investigation was to elucidate a possible difference in the induction mechanism of production of TA-4 and hCG beta-like material in the CaSki cells in relation to cellular differentiation and gene modulation. The exposure to epidermal growth factor (EGF, 100ng/ml) for two days greatly increased TA-4 production by the cultured CaSki cells without affecting cell growth and immunoreactive hCG beta production. The EGF-stimulated increase in TA-4 production required a lag period of approximately 24 hours. The exposure to sodium butyrate (2.5mM) stimulated immunoreactive hCG beta production by the cells, but decreased TA-4 production. The stimulatory effect of sodium butyrate on immunoreactive hCG beta production occurred only during the exposure to the agent. These discrepant effects of EGF and sodium butyrate on the production of TA-4 and hCG beta-like material by the CaSki cells suggest that a fundamental difference exists in the induction mechanism for the expression of these two types of tumor-associated antigen in cervical squamous carcinoma cells.

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.

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.

Optimization of the production of hCG beta-like material by CaSki human cervical carcinoma cells in roller bottle culture.

This study was aimed at optimizing large-scale roller bottle culture conditions for CaSki human cervical carcinoma cells, to produce ectopic hCG beta-like material in quantities sufficient for subsequent characterization studies. Several cell culture techniques contributed to the achievement of this goal: (1) use of serum-free culture medium; (2) use of intermittent recovery periods in presence of serum; and (3) ultrafiltration of the serum-free medium pool for initial concentration of 100-fold.

Estrogen synthetase stimulation by hemin in human choriocarcinoma cell culture.

The ability of hemin to stimulate estrogen synthetase (aromatase) in cultured human trophoblast cells and in cellular homogenates was investigated and compared with aromatase stimulation by dibutyryl cAMP [(Bu)2 cAMP]. Cells grown with hemin for 24 h, or homogenates incubated for 45 min with hemin, showed maximal aromatase stimulation (150 to 200% of activities in the absence of hemin) at 25 microM and 0.1 microM, respectively. Aromatase stimulation in culture by 25 microM hemin was observed within 4 h after hemin addition, while (Bu)2 cAMP required more than 6 h. Intracellular heme and porphyrin levels were higher (160 to 185%) in 96 h (Bu)2 cAMP-grown cells than control cells.

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.

Phototoxic effects of hematoporphyrin derivative and its chromatographic fractions on hormone-producing human malignant trophoblast cells in vitro.

The in vitro phototoxicity of HPD on malignant cells relative to normal cells has been examined. Two human malignant cell lines were studied: the BeWo line of choriocarcinoma cells, which secrete the tumor marker human chorionic gonadotropin (hCG) and its alpha-subunit; and the CaSki line of human cervical carcinoma cells, which secrete hCG and its beta-subunit. Trophoblast-derived, hCG-secreting cells from human amniotic fluid were used as normal controls. In all experiments with HPD plus light, a close correlation was found 24 h after light between cell number and RIA-detectable marker concentration in the medium. Phototoxicity was greater when HPD was introduced in serum-free rather than serum-containing medium. No toxicity was observed in light and dark controls. Cells in Leighton tubes were incubated 1 h with HPD (25 micrograms/ml) in serum-free medium, then rinsed and incubated with medium containing 10% serum. At 2 and 3 days after contact with HPD, flasks were exposed to cool white fluorescent light (1 mW/cm2) for 5 min. Viable cell counts taken 1 day after the light dose indicated that HPD is significantly more phototoxic to BeWo than to CaSki cells; and that both malignant cell types are more photosensitive than amniotic fluid cells, presumably because the latter retain HPD less effectively. In another aspect of this work BeWo cells were used as a model system for comparing the phototoxic effects of the fast (F) and slow (S) eluting fractions of HPD obtained by Bio-Gel P-10 chromatography. Cells in light-shielded tubes were sensitized by incubating with porphyrins for 20 h in media containing 10% calf serum. At 2, 3, or 4 days after removal of porphyrin, with daily replacement of serum-containing medium, flasks were irradiated (see above), and then incubated in the dark for 2 to 4 additional days. Daily culture fluids were analyzed for hormone levels (hCG alpha), and cell counts were performed 2 or 3 days after the light dose. HPD-S (25 micrograms/ml) had no effect on either hormone secretion or cell viability in any of the flasks, whether exposed to light or not. HPD-F at low concentrations (0.25 or 2.5 micrograms/ml) had no detectable effect on cell count or hormone secretion in irradiated flasks. At 10 micrograms/ml, HPD-F was innocuous in dark controls, but caused a large decrease in cell count and hormone output in irradiated flasks.(ABSTRACT TRUNCATED AT 400 WORDS)

The hCG assay in the treatment of trophoblastic disease.

The most important criterion in monitoring the treatment of patients with trophoblastic disease is the quantitative measurement of hCG levels. It is particularly important to have a sensitive hCG-detection procedure that reliably distinguishes low positive hCG levels from negative ones. Reliable monitoring of serum hCG levels minimizes unnecessary chemotherapy in patients entering remission and provides an early indication for additional chemotherapy if and when the hCG levels again become detectable. We evaluated a two-site monoclonal-antibody immunoradiometric assay (IRMA) for its reliability in the management of trophoblastic disease. The assay utilizes a solid-phase, monoclonal antibody that recognizes the alpha subunit of complete hCG and a 125I monoclonal antibody that recognizes the beta subunit. The minimal effective concentration of hCG detectable with the IRMA is 3-4 mIU/ml. The study population consisted of 6 choriocarcinoma patients, 49 hydatidiform mole patients, 37 patients being monitored after spontaneous abortion or blighted ovum and 80 patients in whom the possibility of trophoblastic disease was being ruled out. Each serum specimen was analyzed with the IRMA and in one or more of a number of different radioimmunoassays. The results were correlated with the patient's clinical course. Of the procedures evaluated, the IRMA was the most reliable in identifying trophoblastic-disease patients who required additional chemotherapy. Furthermore, the IRMA yielded no persistent low positive hCG values in patients without clinical evidence of trophoblastic disease. Therefore, the two-site IRMA is recommended for accurately distinguishing a clinically relevant low positive hCG level from an undetectable one.

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.

Choriocarcinoma: blocking factor and monoclonal antibody iodine 131 imaging.

Postoperative iodine 131 monoclonal antibody localization in metastatic choriocarcinoma was accomplished in this study. The monoclonal antibody was prepared to male choriocarcinoma which cross reacted with gestational choriocarcinoma. The antibody was raised against whole choriocarcinoma cells and human chorionic gonadotropin (hCG) cross reactivity was excluded. The purified antibody was iodinated with 131I and successfully imaged BeWo choriocarcinoma transplanted in nude mice; however, imaging of choriocarcinoma in a patient was verified only after resection. It is our belief that failure to sufficiently concentrate the antibody in the tumor before operation was due to blocking factor in the serum of the patient. Blocking factor and hCG dropped postoperatively. Blocking factor activity in 15 patients with metastatic trophoblastic disease was monitored and, like hCG, was found to be a sensitive indicator of the presence of disease. Its efficacy may be in the small number of patients without hCG but with persistent disease.

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.

The carbohydrate on human chorionic gonadotropin produced by cancer cells.

Using the methods described, it is not possible to determine the number of N- and O-linked oligosaccharides on ectopic hCG beta. On standard hCG beta there are two NeuAc residues on each N- and O-linked oligosaccharide, so that the number of NeuAc residues is proportional to the number of oligosaccharides. Ectopic hCG beta and desialylated ectopic hCG beta are of similar molecular size to the standard preparations (gel filtration and RIA with anti-CTP antisera, data not presented). This suggests that ectopic hCG beta is sialylated to a similar extent as standard hCG beta, so the number of oligosaccharides on ectopic hCG beta could be similar to the number on standard hCG beta. There is a Fuc attached to the N-linked oligosaccharides of standard hCG beta (Fig. 3). Using the methods described, it was not possible to determine if this residue is also found on the N-linked oligosaccharides of ectopic hCG beta. Recently, a second form of ectopic hCG beta was identified (22). This form lacks the characteristic hCG beta carboxyterminal peptide, and as such is unrecognized by the RIA used in this study. Like the ectopic hCG beta described herein, and that produced by other cancers, this molecule only partially binds to Con A, and binds to Ricinus communis-120 following neuraminidase digestion. Intact hCG and free hCG subunits, which only partially bind to Con A, are found in cancer tissues, cancer sera, and the medium of cultured trophoblastic and nontrophoblastic cancer cells (Table 1). Our studies with DoT cancer of the cervix cells clearly indicate that the partial binding could be the consequence of the linkage of extra beta G1cNAc residues.

Stability of human chorionic gonadotropin and its alpha subunit in human blood.

The stability of human chorionic gonadotropin (hCG) and its alpha-subunit in whole blood, plasma, and serum under a variety of sample handling conditions commonly encountered in clinics, hospital wards, physician's offices, and clinical service laboratories was investigated with the use of radioreceptor assay, radioimmunoassays, as well as hormone integrity determinations. The results clearly demonstrate that hCG and its alpha-subunit are stable in unfrozen whole blood, plasma, and serum for at least 6 days and in frozen plasma and serum samples for at least 6 months. Repeated freezing and thawing of the samples during this period had no effect. Separation of plasma or serum from erythrocytes is not needed for at least 12 hours. Hemolysis in samples resulted in a 20% to 30% decrease in hCG and its alpha-subunit levels, which may be attributable to sample dilution.