TSG101 expression in gynecological tumors: relationship to cyclin D1, cyclin E, p53 and p16 proteins.

Recent studies have shown that in vitro steady-state expression of the tumor susceptibility gene TSG101 is important for maintenance of genomic stability and cell cycle regulation. To determine the contribution of TSG101 expression in neoplastic formation, expression of TSG101 protein levels were evaluated in primary ovarian and endometrial adenocarcinoma tumors. Expression of TSG101 was also examined in various tumor cell lines (PA-1, AN3CA, HeLa, HS578T, HCT116). Full-length TSG101 protein was detected in these tumors and cell lines indicating that intragenic deletions were not characteristic of TSG101. In addition, TSG101 protein levels were compared with aberrations of prominent cell cycle regulatory molecules such as cyclin D1, cyclin E, p16 and p53. Reduced TSG101 protein was observed in 36% (8/22) of ovarian and 17% (1/6) of endometrial adenocarcinoma. Aberrant levels of p53, p16, cyclin D or E were comparable to published studies indicating that the clinicopathological distribution of these cases did not favor advanced stage tumors. Altogether, these findings suggest that a down-regulation of TSG101 is associated with tumorigenesis in a subgroup of gynecological tumors.

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.

Endometriosis with a pleural effusion and ascites. Report of a case treated with nafarelin acetate.

BACKGROUND: Although endometriosis is generally confined to the pelvis, it may occur at remote sites with unusual manifestations. Rare examples include pulmonary endometriosis and endometriosis associated with ascites. These complications represent diagnostic and therapeutic dilemmas, especially when both occur concurrently in the same patient. CASE: A 21-year-old nulligravida had a history of endometriosis and complained of severe dyspnea and weight loss. Pleural effusion and pelvic ascites associated with recurrent endometriosis were found. The patient was treated with thoracentesis and nafarelin acetate nasal spray. A remarkable improvement in her condition occurred, and the side effects of the spray were minimal during treatment. CONCLUSION: Gonadotropin-releasing hormone agonist therapy is recommended as the initial treatment for endometriotic ascites and/or pleural effusion.

Phosphorus-32-chromic phosphate for ovarian cancer: I. Fractionated low-dose intraperitoneal treatments in conjunction with platinum analog chemotherapy.

UNLABELLED: For many years, 32P-chromic phosphate (32P-CP) intraperitoneal instillations and platinum analogue chemotherapy have been used to treat disseminated ovarian cancer. To investigate possible enhancement of 32P-CP irradiation due to the concomitant administration of chemotherapy, in vitro studies were undertaken. Based on those laboratory investigations, a clinical regimen of combined 32P-CP and platinum analogue chemotherapy was developed. METHODS: In vitro enhancement of 32P-CP cytotoxicity by cisplatin was studied in cultured human ovarian adenocarcinoma (CHOA) cell lines and in a fibroblast cell strain. In addition, ovarian cancer cells obtained from the malignant abdominal ascites and pleural effusions of 10 individual patients were also studied ex vivo. As part of routine clinical care, 30 patients with disseminated ovarian adenocarcinoma underwent up to eight monthly cycles of platinum analogue chemotherapy with concomitant intraperitoneal instillation of 5 mCi of 32P-CP at each monthly chemotherapy cycle. RESULTS: There was an enhanced and possibly supra-additive effect of cisplatin on the cytotoxicity from 32P-CP irradiation. For the 30 patients, the survival rate at 3 yr was 63%. CONCLUSION: Phosphorus-32 CP low-dose intraperitoneal treatments in conjunction with platinum analogue chemotherapy is a promising approach for the treatment of disseminated intraperitoneal ovarian cancer.

Gestational and non-gestational trophoblastic neoplasms: new developments in DNA analysis, metabolic function, diagnosis and treatment.

Recent technological advances have made it possible to further define important molecular biological characteristics of gestational and non-gestational trophoblast neoplasm, as they relate to genetic origin and metabolic, endocrine, and diagnostic functions. Exciting new work with DNA analysis has defined origins of placental site trophoblastic tumors, a variant of choriocarcinoma not previously well understood. Coexistent hydatidiform mole and normal gestation have been diagnostically defined and carried out to viability. Finally, refinement of classification systems and expanded tumor markers offer promise of improved salvage in gestational neoplasms and an enlarging number of non-gestational neoplasms.

Radioimmunoassay of free beta-subunit of human chorionic gonadotropin in diagnosis of high-risk and low-risk gestational trophoblastic disease.

A radioimmunoassay was performed with monoclonal antibody 1E5, which distinguishes free beta-subunit of human chorionic gonadotropin in the presence of intact human chorionic gonadotrophin. Serum samples were obtained from 68 pregnant women, 9 with hydatidiform mole who underwent spontaneous remission, 12 with hydatidiform mole who developed gestational trophoblastic disease, 5 with metastatic gestational trophoblastic disease of high-risk category, and 1 with choriocarcinoma concomitant with pregnancy. The concentrations of free beta-subunit of human chorionic gonadotropin and total beta-subunit were determined on the sera. The assay data were expressed as a ratio of nanograms of free beta-subunit per 1000 mIU of total beta-subunit. The ratios, analyzed by the Wilcoxon two-sample test, indicated a highly significant correlation between high ratios and the eventual diagnosis of high-risk gestational trophoblastic disease (p = 0.0019). This study suggests that the excessive production of free beta-subunit of human chorionic gonadotrophin may identify patients with high-risk gestational trophoblastic disease much earlier and identify gestational trophoblastic disease in patients during pregnancy.

Trophoblastic neoplasms: occult disease.

Occult choriocarcinoma may be associated with low or undetectable levels of human chorionic gonadotropin. This article reviews the various methods of identifying the foci of choriocarcinoma that are currently available.

Bacillus Calmette-Guérin immunotherapy in ovarian cancer.

Forty-six advanced ovarian cancer patients treated with conventional modalities with the addition of bacillus Calmette-Guérin (BCG) immunotherapy showed prolonged survival when compared to controls not given BCG. Although the data suggest enhancement of survival with the addition of BCG to conventional treatment, the fact remains that disease recurrence ultimately claims the lives of most of these patients. Nonetheless, patients are surviving longer in the face of advanced disease.

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.

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.

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.

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.

Segregation patterns of polymorphic restriction sites of the gene encoding the alpha subunit of human chorionic gonadotropin in trophoblastic disease.

The gene encoding the alpha subunit of human chorionic gonadotropin contains at least two polymorphic sites in its 3' flanking region detected by restriction enzymes HindIII and EcoRI. We used these polymorphic sites as markers of tissue genotype in normal placenta, hydatidiform mole, choriocarcinoma, and peripheral leukocytes. As expected, inheritance patterns of most hydatidiform moles showed only a paternal genetic contribution. However, one uncommon DNA polymorphism pattern, homozygosity for the absence of the EcoRI site and the presence of the HindIII site, predominated in choriocarcinoma. Thus, our results suggest that moles which have this uncommon polymorphism pattern appear particularly likely to develop into choriocarcinoma.

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.

Cytogenetic analysis of choriocarcinoma cell lines.

Five permanent cell lines were established in vitro from patients with gestational choriocarcinoma. Chromosome study with Q-band technique showed a modal chromosome number per cell in the hypotetraploid range in all cell lines. There were unidentifiable, structurally altered chromosomes in each cell line. BeWo cell line from choriocarcinoma following delivery of a normal male baby lost identifiable Y chromosome, whereas Jar cell line following delivery of a normal male did have a Y chromosome. One of the two cell lines following complete hydatidiform mole had a Y chromosome. The significance of this finding is discussed.