8-1A, a human monoclonal antibody that reacts with intact human chorionic gonadotropin.

The incidence of choriocarcinoma has decreased over time and therapeutic results have improved about 90% complete remission in patients without extensive metastasis. However, some choriocarcinomas metastasize to other organs and show resistance to chemotherapy, having a poor prognosis despite multidisciplinary treatment. Better methods of early diagnosis for recurrence or micrometastasis, and treatment against cases with intractable gestational trophoblastic neoplasia (GTN) are needed to improve the prognosis. Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits and a tumor marker to make a diagnosis and monitor therapeutic effect in GTN. Even when hCG levels in the serum become too low to measure with the hCG beta-CTP system which is the most sensitive assay, there are estimated to be approximately 10,000 trophoblastic cells in the body. Residual trophoblast cells may cause symptoms such as bleeding or undergo malignant transformation to choriocarcinoma. Since most monoclonal antibodies developed so far are murine, administration creates human anti-mouse antibodies, resulting in clinical failure. More recent mouse/human chimeric antibodies or humanized antibodies still possess substantial immunogenicity that makes repeated administration difficult. In the present study, KM mice that can produce completely human monoclonal antibodies were used to prepare hCG-specific human monoclonal antibody. This yielded 8-1A, a human monoclonal antibody capable of reacting with intact hCG. In the future, new diagnostic techniques and treatments for chorionic diseases may be developed using this kind of human monoclonal antibody.

Effects of ricin A chain conjugates of monoclonal antibodies to human alpha-fetoprotein and placental alkaline phosphatase on antigen-producing tumor cells in culture.

Two tumor-associated proteins, alpha-fetoprotein (AFP) and placental alkaline phosphatase (PLAP), were investigated as target proteins for antibody:cytotoxin conjugates. An AFP-producing hepatoma cell line (HepG2) and a PLAP-producing cervical carcinoma cell line (SKGIIIa) were used as target cells. Both cell lines were equally susceptible to the toxic effects of intact ricin. Immunofluorescent studies showed that AFP could be detected at the surface of the HepG2 cells in a speckled distribution, while PLAP was uniformly distributed over the surface of the SKGIIIa cells. The anti-AFP ricin A chain conjugate was not cytotoxic to either cell line at low concentrations and killed both types of cells at high concentrations. The anti-PLAP conjugate at low concentrations was 100-fold more toxic than the anti-AFP conjugate to the PLAP-producing SKGIIIa cells. At high concentrations, it also killed both types of cells. The enhanced toxicity of the anti-PLAP conjugate to the SKGIIIa cells was inhibited by an excess of unconjugated anti-PLAP but not anti-AFP, indicating that the uptake of the conjugate depends on specific cell surface binding to the antigen. The indiscriminate toxicity observed at high concentrations of either conjugate was not inhibited by unconjugated antibody, suggesting that this effect depends on conjugate uptake independent of the identity of the antigen. These results emphasize the importance of the properties of the target antigen to the cytotoxic effects of antibody conjugates as well as the need for caution in experiments using high concentrations of conjugates. They suggest that PLAP may be a suitable target for immunotoxin therapy of human cancer.

CA54/61 as a marker for epithelial ovarian cancer.

Using a new one-step, double-determinant enzyme immunoassay, we performed quantitative measurements of a mucin-type glycoprotein antigen (CA54/61) that we recently detected in sera of ovarian carcinoma patients. When the cutoff value was set at 12 units/ml, at which a high diagnostic efficiency was demonstrated [or at 20 units/ml (mean + 3 SD of healthy females)], the positive rates of ovarian serous, mucinous, clear cell, and endometrioid carcinomas were 76% (or 63%), 63% (or 55%), 57% (or 52%), and 50% (or 38%), respectively. Even in mucinous cystadenocarcinoma, more than one-half of the cases were positive, indicating the potential utility of the assay in the diagnosis of mucinous tumors. In sera from patients with benign ovarian tumors, only 9% (or 4%) of the cases were positive, indicating the quite high specificity of this test for ovarian carcinomas. To make a comparison between CA54/61 and CA125, we set the cutoff level of CA125 at 110 units/ml, at which value a high diagnostic efficiency was demonstrated [or at 35 units/ml (mean + 3 SD of healthy females)]. When both CA54/61 and CA125 were assessed in sera from 36 patients with mucinous cystadenocarcinoma, the positive rates of CA54/61 and CA125 were 64% (or 56%) and 36% (or 56%), respectively, suggesting that CA54/61 is of clinical value as a new tumor marker for ovarian cancers, including mucinous tumors.

Human uterine endometrial adenocarcinoma: characteristic acquirement of synthetic potentials for II3SO3-LacCer and ganglio series sulfoglycosphingolipids after transfer of the cancer cells to culture.

The acidic glycosphingolipid composition of human uterine endometrial adenocarcinoma was compared with those of normal uterine endometrium at the proliferative and the secretory phases. Upon chemical composition analysis, no significant transformation-associated change of these glycolipids was observed. However, when cancer cells from the patients with human uterine endometrial adenocarcinoma were transferred to culture, the composition of glycosphingolipids, particularly sulfoglycosphingolipids, was significantly altered after the 70th doubling time. I3SO3-GalCer, which was contained in the original tissues of uterine endometrial adenocarcinomas, disappeared completely from the cultured cells at the 70th doubling time, whereas II3SO3-LacCer and ganglio series sulfoglycosphingolipids, which were originally contained in a trace amount or not present at all in the cancer tissues, became the major components in the total acidic glycosphingolipids in the cultured cells. Also, among cell lines established from several gynecological cancers, which include uterine cervical squamous carcinoma, uterine endometrial adenocarcinoma, ovarian clear cell carcinoma, choriocarcinoma, uterine sarcoma, ovarian sarcoma, and vulvar melanoma, only those cells derived from uterine endometrial adenocarcinoma expressed II3SO3-LacCer and ganglio series sulfoglycosphingolipids and the synthetic activities of these sulfoglycolipids, indicating that uterine endometrial adenocarcinoma cells characteristically lose the sulfotransferase to GalCer and acquire the sulfotransferase to LacCer after being transferred to culture in vitro. Thus, the unique sulfoglycosphingolipids and sulfotransferase are useful markers for the characterization of uterine endometrial adenocarcinoma among human gynecological cancers.

Establishment of a quantitative assay of abnormal glycolipid expression in endometrial cells, and its diagnostic value for endometrial carcinoma.

We developed a new quantitative method for detecting abnormal glycolipid expression in endometrial cells using a monoclonal antibody (MSN-1) and analyzed the glycolipid antigen recognized by MSN-1 in 173 clinical endometrial cell samples (66 normal endometria, 39 endometrial hyperplasias, and 68 endometrial adenocarcinomas). The mean glycolipid antigen levels in normal endometrium, endometrial hyperplasia, and endometrial carcinoma were 0.42 +/- 1.37, 2.13 +/- 3.84, and 19.4 +/- 25.8 (mean +/- SD) units, respectively. If the cutoff rate of this assay was fixed at 1.8 units, the positivity rates for patients with normal endometrium, endometrial hyperplasia, and endometrial carcinoma were 6.1% (4/66), 28.2% (11/39), and 76.5% (52/68), respectively. In 35 endometrial carcinoma patients, endometrial smears were simultaneously performed, and there were 22 positive smears (62.9%). When the cytological diagnosis was combined with our assay, 94.3% (33/35) of the carcinomas were detected. Thus, this assay seems to be a supplementary diagnostic method for endometrial carcinoma.

Immunoglobulin variable region sequences of two human monoclonal antibodies directed to an onco-developmental carbohydrate antigen, lactotetraosylceramide (LcOse4Cer).

A human monoclonal antibody, 11-50, was generated and was shown to recognize an onco-developmental carbohydrate antigen, LcOse4Cer. The isotype of this antibody was IgM, lambda, similar to the previously known human anti-LcOse4 antibodies, such as IgMWOO and HMST-1. We raised a murine anti-idiotypic antibody G3 (IgG1, kappa) against 11-50, and tested its reactivity towards the affinity purified human polyclonal anti-LcOse4 antibodies prepared from pooled human sera using a Gal beta 1-->3GlcNAc beta-immobilized column. The results indicated that at least a part of the human polyclonal anti-LcOse4 antibodies shared the G3 idiotype with 11-50. We further analyzed the sequence of variable regions of the two anti-LcOse4 antibodies, 11-50 and HMST-1. Sequence analysis of the heavy chain variable regions indicated that the VH regions of these two antibodies were highly homologous to each other (93.5% at the nucleic acid level), and these antibodies utilized the germline genes VH1.9III and hv3005f3 as the VH segments, which are closely related germline genes of the VHIII family. It was noted that these germline VH genes are frequently utilized in fetal B cells. The JH region of both antibodies was encoded by the JH4 gene. For the light chain, the V lambda segments of the two antibodies were 96.3% homologous to each other at the nucleic acid level. The V lambda segments of both antibodies showed the highest homology to the rearranged V lambda gene called V lambda II.DS among reported V lambda genes, while the exact germline V lambda genes encoding the two antibodies were not yet registered in available sequence databanks. The amino acid sequences of the J lambda segments of both antibodies were identical. These results indicate that the two human antibodies recognizing the onco-developmental carbohydrate antigen Lc4 are encoded by the same or very homologous germline genes.

Phenotypic diversity and prognosis of adult T-cell leukemia.

We examined phenotypically 107 patients with adult T-cell leukemia (ATL), using a panel of monoclonal antibodies, in order to clarify the occurrence of aberrant phenotypes, and to determine the correlation between phenotypic diversity and prognosis. The incidence of the typical (CD4+.CD8-) phenotype, the double-negative (CD4-.CD8-), the double-positive (CD4+.CD8+), and the CD8-positive (CD4-.CD8+) phenotypes was 81%, 7%, 7%, and 4%, respectively. The median survival time (MST) for all patients was 10.0 months with 17% survival at 2 years. The patients with typical phenotypes had a 10.2 month MST with 20% survival at 2 years, significantly better than the patients with the unusual phenotypes whose MST were 4.9, 7.8, and 2.6 months, respectively, for the double-negative, double-positive, and CD8-positive phenotypes. Lack of antigens reactive with CD2, CD3, CD5, and WT31 monoclonal antibody panels was one factor in bad prognosis, but the presence of CD4 and CD8 antigen abnormalities was much more significant.

Expression mechanism of human uterine endometrial cancer-specific fucosylated carbohydrate chains - aberrant alpha-1-]4-fucosyl-transferases in uterine endometrial cancer-derived cell-lines with type-I carbohydrate chain.

Cells of uterine endometrial cancer cell line SNG-II were classified into two groups according to their reactivity with anti-uterine endometrial cancer monoclonal antibody (MSN-1), whose recognition antigen is mainly the Lewis(b) antigens; those that strongly reacted with MSN-1 (SNG-S group) and those that weakly reacted with it (SNG-W group). The SNG-S showed a higher activity of a 1-->4-fucosyltransferase activity than that of the SNG-W. The expression of Lewis(b) antigen was stronger in the SNG-S than that in the SNG-W. Therefore, the expression of uterine endometrial cancer-specific fucosylated carbohydrate could be mainly controlled by alpha-fucosyltransferase activities.

Expression of human beta 1,4-galactosyltransferase in gynecological cancer cell lines.

We examined the expression of beta 1,4-GT gene products in 11 gynecological cancer cell lines. A 4.7 kb mRNA and protein (54,000 Da and 57,000 Da) were detected by Northern blot and Western blot. Immunocytochemical staining revealed that beta 1,4-GT was localized in the Golgi or ER of tumor cells. An intense beta 1,4-GT mRNA signal was detected in ovarian and cervical cancer cells, whereas the level of beta 1,4-GT mRNA was very low in uterine endometrial cancer cells. We also confirmed that expression of beta 1,4-GT mRNA corresponded to expression of beta 1,4-GT protein. These results suggest that expression of the beta 1,4-GT gene products is higher in human cervical and ovarian cancer cells than in uterine endometrial cancer cells.

Production and characterization of a monoclonal antibody (MSN-3) for uterine endometrial and endocervical adenocarcinoma.

A monoclonal antibody (MSN-3) was raised using HEC-108 cells derived from poorly differentiated endometrial carcinoma as the immunogen. The immunoglobulin subclass of MSN-3 was IgGr1. The target antigen of MSN-3 was a protein with a molecular weight of 77 kDa, and it was shown to be localized in the cytoplasm. MSN-3 only reacted with 14% of normal proliferative endometrium cells, but it showed a high positivity rate of 66% for endometrial carcinoma. The target antigen of MSN-3 increased as endometrial cells became more malignant, and the possibility of changes in localization was also suggested. Moderately and poorly differentiated endometrial carcinoma showed a high positivity rate for MSN-3. MSN-3 reacted rarely or not at all with normal cervical glandular tissue, but the positivity rate for cervical adenocarcinoma (especially endocervical adenocarcinoma) was a high rate of 59%. The patterns of staining of endocervical adenocarcinoma by MSN-3 included diffuse staining of the whole cytoplasm and not only that near the glandular lumen, as well as staining of the basal cytoplasm. Changes in the localization of the target antigen were clearly associated with carcinogenesis of the cervical glandular cells. The MSN-3-positive rate was high in patients with lymph node metastasis and vascular invasion. Among the staining patterns, the basal and diffuse patterns tended to increase with malignacy. The basal pattern of staining was characteristic of MSN-3, suggesting that it might assist in the diagnosis of cervical adenocarcinoma.

Chronic administration of beta-adrenergic agonists can mimic the stimulative effect of cold exposure on protein synthesis in rat brown adipose tissue.

When mammals are exposed to a cold environment for a long time, the capacity of nonshivering thermogenesis by brown adipose tissue (BAT) increases in association with the stimulation of synthesis of some specific proteins and tissue hyperplasia, which are totally dependent on sympathetic innervation to this tissue. To clarify the roles of the adrenergic mechanisms for the cold-induced protein synthesis and hyperplasia in BAT, in this study, the effects of chronic treatment with adrenergic agonists using an osmotic mini-pump were examined in rats. Continuous administration of noradrenaline or isoproterenol (beta-agonist) for 10 days resulted in increased synthesis of the mitochondrial uncoupling protein and an isoform of glucose transporter (GLUT4), and tissue hyperplasia, in the same way as after cold exposure of the same duration. Phenylephrine (alpha-agonist) administration did not have any significant effect. Surgical sympathetic denervation completely abolished the effects of cold exposure, whereas it did not influence those of adrenergic agonists at all. These results indicate that the stimulative effects of cold exposure on protein synthesis and hyperplasia of BAT are attributable solely to the beta-adrenergic action of noradrenaline secreted from the sympathetic nerves in this tissue.

Selective cytotoxicity of adriamycin immunoconjugate of monoclonal antibody MSN-1 to endometrial adenocarcinoma in vitro and in vivo.

Missile therapy, which destroys cancer cells specifically, has been regarded as an effective treatment modality for carcinoma. The monoclonal antibody MSN-1 (IgM), which reacts strongly with endometrial adenocarcinomas, was combined with adriamycin (ADM) by a disulfide bond using N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) and 2-iminothiolane. Its selective cytotoxicity against SNG-II was examined in a colony formation in vitro, and on athymic mice in vivo. The results of our study suggest that the or IC50, of the MSN-1-ADM immunoconjugate against SNG-II to be 57 times that of ADM alone in vitro. The reductions in resected weights of target tumor cells, at the local site of the MSN-1-ADM immunoconjugate treatment, were 25% with caudal vein administration, and 38% with local administration, as compared with the untreated group, in vivo. There was no weight loss in treated mice. Our results suggest that this MSN-1-ADM immunoconjugate has potential clinical application in the treatment of endometrial adenocarcinomas.

Expression of GalCer sulfotransferase by human uterine endometrial carcinoma cell lines.

We investigated the expression of sulfoglycolipids in several gynecological cancer cell lines by metabolic labeling with S-35-sulfate as well as the activity of GalCer sulfotransferase (ST) and arylsulfatase A (ASA), enzymes which are responsible for the synthesis and degradation of sulfoglycolipids. The endometrial carcinoma cell lines expressed sulfoglycolipids and showed ST activity, indicating that increased synthesis of sulfoglycolipids due to elevated ST activity is a characteristic of endometrial carcinoma that distinguishes it from other carcinomas. These cell lines could provide a useful model for studying the functions of sulfoglycolipids as well as biological properties of ST in cancer cells.

Effects of gelonin immunoconjugate of monoclonal antibody MSN-1 to endometrial adenocarcinoma on antigen-producing tumor cells in vitro.

Missile therapy, which destroys cancer cells specifically, has been considered to be an effective modality for treatment of carcinoma. We have developed a monoclonal antibody MSN-1 (immunoglobulin class: IgM), of which the immunogen is the endometrial adenocarcinoma cell line SNG-II, which strongly reacts with endometrial adenocarcinomas. We describe an immunoconjugate consisting of the MSN-1 and a plant hemitoxin named gelonin which has revealed to assume selective cytotoxicity against the SNG-II in a colony formation assay in vitro. The results of our study suggest that the 'inhibitory concentration' or IC50, of the MSN-1-gelonin immunoconjugate against the SNG-II was 188 fold that of gelonin alone. These results indicated that the MSN-1-gelonin immunoconjugate exhibited highly selective cytotoxicity to endometrial adenocarcinoma, which expressed an epitope against the MSN-1, and it is suggested that the MSN-1-gelonin immunoconjugate has possibility of clinical application to treatment of endometrial adenocarcinomas.

Inhibition of attachment and chemotactic invasion of uterine endometrial cancer cells by a new vinca alkaloid, conophylline.

The effect of conophylline, a new vinca alkaloid that inhibits ras expression, on tumour cell adhesion and infiltration was evaluated using a human endometrial cancer cell line. When SNG-II, a highly differentiated human endometrial cancer cell line, was exposed to conophylline, the cells developed filamentous processes at concentrations which did not affect cell proliferation (0.03-0.3 microgram/ml). After exposure to conophylline (0.3 microgram/ml), cells adherent to matrigel- and type IV collagen-coated wells respectively decreased to 26.9% and 33.3% of the number in the untreated control culture (p < 0.01). In an in vitro invasion assay using a Boyden chamber, infiltration of cells exposed to conophylline decreased to 19.4% (0.3 microgram/ml) (p < 0.01) of the control. In a wound assay, conophylline inhibited the movement of cells at 24 hr after wounding. Flow cytometric analysis revealed that expression of integrin beta 1 was not altered by conophylline, but E-cadherin and CD44 were decreased. The expression of E-cadherin and CD44 could be changed by conophlline.

Establishment of a human ovarian clear cell carcinoma cell line (RMG-I) and its single cell cloning--with special reference to the stem cell of the tumor.

A cell line, designated RMG-I, has been established from ascites of a patient with ovarian clear cell carcinoma. RMG-I cells have grown well for more than 6 years with mirror ball formation. Chromosome analysis revealed aneuploidy with a modal number of 47 and 3 marker chromosomes. The histological characteristics of the transplanted tumor were similar to those observed in the original tumor which was composed of dark cells, clear cells, and hobnail cells. Thus, the RMG-I cell line was identified to be an ovarian clear cell carcinoma. Immunohistochemically, basic fetoprotein (BFP) and ferritin were demonstrated in the original tumor, the cultured cells, and the transplanted tumor. Furthermore, in order to clarify whether these 3 kinds of cells originate from one stem cell or not, monoclonal cell population were transplanted into nude mice, and its histological investigation revealed that 3 types of cells existed in the transplanted tumor, proving that they originate from one stem cell.

[Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer].

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.

Effect of diets supplemented with amino acids on intestinal sucrase and leucine aminopeptidase activities in rats.

In order to investigate the relationship between dietary amino acids and protein, and activities of intestinal sucrase [EC 3.2.1.26] and leucine aminopeptidase [EC 3.4.11.1, LAPase] in rats, the effect of supplementation of amino acids into a protein-free diet and a low casein diet containing sucrose as the carbohydrate source on these enzyme activities was studied. The segmental weights of the small intestine and its mucosa of rats fed the protein-free diet supplemented with L-methionine or with L-methionine and L-threonine at 0.1 or 0.2% levels were significantly higher than those of rats fed the protein-free diet or one supplemented with L-glutamic acid, but there was no difference in the segmental activities of the sucrase and LAPase among rats fed these diets. On the other hand, the supplementation of methionine or methionine plus threonine to the 5% or 10% casein diet produced remarkable increases in the segmental weights of the small intestine and its mucosa as well as in the segmental activities of the sucrase and LAPase. There was no difference between the segmental sucrase activity of rats fed the 10% casein diet supplemented with 0.2% methionine ad libitum and that of rats fed this diet under restricted feeding conditions, although the segmental LAPase activity was affected by the amount of food consumed.

Hyperplasia of brown adipose tissue after chronic stimulation of beta 3-adrenergic receptor in rats.

When mammals are exposed to a cold environment for a long time, the capacity of nonshivering thermogenesis by brown adipose tissue (BAT) increases in association with the increased expression of some specific proteins and tissue hyperplasia, which are totally dependent on sympathetic innervation to this tissue. To clarify roles of the beta-adrenergic mechanism in BAT hyperplasia, the effects of chronic administration of various beta-adrenergic agonists on BAT were examined in rats, especially focusing on some agonists to the beta 3-adrenoceptor which is present specifically in adipocytes. Chronic administration of noradrenaline or isoproterenol for 7-10 days produced a marked increase in the tissue contents of DNA, total protein, mitochondrial uncoupling protein, and insulin-regulatable glucose transporter protein. The trophic effects of noradrenaline and isoproterenol were mimicked by chronic administration of beta 3-adrenergic agonists, such as CL316,243, BRL 26830A, and ICI D7114. These results suggest that the beta 3-adrenoceptor plays important roles for hyperplasia of BAT, and thereby increasing in the capacity of thermogenesis.

[ATL (adult T-cell leukemia/lymphoma)].

128 cases of ATL (adult T-cell leukemia/lymphoma) were divided into 3 subgroups, 70 cases of acute type, 19 cases of chronic type and 39 cases of lymphoma type, and their prognosis were evaluated. Median survival time from the onset of acute type using Kaplan-Meier method was 8 months, that of lymphoma type was 14 months and that of chronic type was 50 months. Median survival time from the start of treatment of acute type was 5 months, that of lymphoma type was 11 months and that of chronic type was 22 months. Relatively short median survival time of chronic type may be due to they had some duration with observation only at first. No significant elongation of survival time could be obtained when acute type was divided into 2 stages, before and after 1982. Twelve patients with non-Hodgkins' lymphoma unresponsive to the first line combination chemotherapy were treated with combination therapy with cis-dichlorodiammineplatinum (CDDP). Ten patients were evaluable for response (4 cases of CR, 6 cases of PR). To stop the HTLV-1 carrier mothers milk for giving their children seems to be effective method to decrease the occurrence of ATL in future.