Effects of progesterone on human endometrial carcinoma cells in vivo and in vitro.

The effects of progesterone on the growth and differentiation of human endometrial adenocarcinoma cells (cell lines SNG-P and SNG-M derived from primary and metastatic tumors, respectively) were assessed in vitro and in vivo. Progesterone suppressed their growth and induced cell differentiation in vitro. The suppressive effect of progesterone was stronger in the primary tumor cells than in the metastatic ones. Progesterone produced morphologic changes such as multinucleation, multinucleolation, vacuolation, extensive Golgi apparatus, and papillary arrangement of cells. The cells were transplanted sc into nude BALB/c mice where they produced undifferentiated adenocarcinomas in untreated mice and well-differentiated adenocarcinomas in progesterone-treated ones. Progesterone reduced tumor growth and decreased transplantability in nude mice. This hormone produced no change in the distribution of the chromosome numbers or in the karyology.

Establishment of HUOCA-II, a human ovarian clear cell adenocarcinoma cell line, and its angiogenic activity.

A cell line designated "HUOCA-II" was established from a human ovarian clear cell adenocarcinoma. The HUOCA-II cells, which were oval, spindle, or polygonal and had neoplastic and pleomorphic features, grew in multiple layers without contact inhibition. The cell line grew fast (population doubling time, 24 hr), and 55 serial passages were carried out within 11 months. The chromosomal number ranged around 46, and no karyological abnormality was found in G-band karyotyping. When heterotransplanted into the subcutis of BALB/c nude mice, HUOCA-II cells produced a poorly differentiated clear cell adenocarcinoma. The tumor angiogenesis factor (TAF) of a molecular weight of about 14,000 was purified from the conditioned medium of HUOCA-II cells, and neovascularization was detected by bioassay with the use of the chorioallantoic membrane of a chick embryo. This TAF also stimulated the growth of endothelial cells in an in vitro culture system.

Establishment and characterization of HUOT, a human ovarian malignant teratoma cell line producing alpha-fetoprotein.

A cell line designated HUOT was established from a recurrent tumor of human ovarian malignant teratoma. The cell line grew slowly and stably and serial passages were performed 50 times within 35 months. The cells, polygonal or spindle, with neoplastic and pleomorphic features grew in multiple layers and without contact inhibition. Population doubling time was 98 hours and the plating efficiency was less than 6%. The chromosome number varied from 43 to over 256, and the modal number was stable at the hyperdiploid range (52-56). The cultured cells produced anaplastic carcinomas by heterotransplantation into the subcutis of nude mice and were characterized as producing large amounts of alpha-fetoprotein, in vitro, at the stationary growth phase and as forming cystlike structures. Dibutyl cAMP suppressed the cellular proliferation and increased the production of alpha-fetoprotein. Therefore, this HUOT line is expected to have a wide application for various laboratory studies.

Establishment and characterization of an estrogen-producing human ovarian granulosa tumor cell line.

Cells designated HTOG and HTOT were established by long-term culture from a human ovarian granulosa cell and a theca cell tumor, respectively. The HTOG line grew well forming colonies and multilayered rapidly without contact inhibition; serial passages of HTOG were performed over 100 times successively within 25 months. HTOG were spindle cells, polygonal or spherical in shape, revealed neoplastic and pleomorphic features, and produced estrone (E1) and 17 beta-estradiol (E2). The chromosome number varied considerably and showed hyperploidy; the modal chromosome number was in the hypertriploid-tetraploid range. When HTOG cells were heterotransplanted into the subcutis of BALB/c nude mice, they produced a sarcomatous diffuse type of granulosa cell tumors. In contrast, HTOT cells grew slowly while forming monolayers and underwent five successive passages in about 100 days, but a theca cell tumor line could not be established. HTOT cells were fibroblastic in shape and also produced E1 and E2. The majority of the cells showed diploidy and karyologic normality.

Establishment and characterization of two human mixed mesodermal tumor cell lines from the same patient.

A cell line designated HIRS -BM was established from fluid aspirated from the sternal bone marrow of a 16-year-old female. Another cell line ( HIRS -PB) was derived from the peripheral blood of the same patient. Both lines grew well, multilayering rapidly without contact inhibition, and 62 serial passages were successively done within 28 months. Both cultures contained spindle- or fibrous-shaped cells that revealed neoplastic and pleomorphic features, and these cells were characterized as possessing cross-striations in the cytoplasm. The cross-striations were detected by phosphotungstic acid hematoxylin stain. Some elongated cells were stained positively with anti-myoglobin by use of periodic acid-Schiff methods. The primary tumor in the uterus was diagnosed as a mixed mesodermal tumor composed of adenocarcinoma and rhabdomyosarcoma cells. The karyotype exhibited hyperploidy and large submetacentric marker chromosomes, and the modal chromosome number was 84. No difference was found between the 2 cell lines except for growth behavior and heterotransplantability . HIRS -BM cells grew more rapidly and were highly transplantable. The HIRS -BM cells were transplanted into the subcutis of BALB/c nude mice and produced mixed mesodermal tumors resembling the uterine tumor, while the HIRS -PB cells could not be transplanted. Due to the histogenesis of the mixed mesodermal tumor being's obscure with histologic observations only, this study was performed to obtain data by tissue culture of the tumor and resulted in support of the combination theory reported in the literature in regard to tumor.

Effects of 17beta-estradiol and progesterone on growth and morphology of human endometrial carcinoma cells in vitro.

The effects of 17beta-estradiol and progesterone on the rate of growth and the morphological changes of human endometrial adenocarcinoma cells were studied in in vitro culture. 17beta-Estradiol enhanced their growth and produced no cellular morphological changes at low concentrations of less than 1 microgram/ml, whereas it suppressed their growth and produced such cellular changes as enlargement of nuclei, karyorrhexis, and karyolysis at high concentrations of more than 5 microgram/ml. On the other hand, progesterone did not affect the cells at less than 1 microgram/ml, but it suppressed their growth and induced differentiation at more than 5 microgram/ml. Specific morphological changes produced by progesterone were characterized by multinucleation, multinucleolation, prominent Golgi apparatus, occurrence of vacuoles, and papillary-like arrangement of cells. These features suggested that progesterone acted directly on the endometrial carcinoma cells and induced their histological differentiation. These changes could not be detected by the adminstration of 17beta-estradiol.

Development and characterization of established cell lines from primary and metastatic regions of human endometrial adenocarcinoma.

Cell lines designated SNG-P and SNG-M were established from operation specimens of primary and metastatic regions of human endometrial adenocarcinoma. The cell lines grew well without interruption for over 13 months and were subcultivated more than 65 times. They continue to exhibit stable growth. The cultured cells appeared epithelial in shape, showing a pavement arrangement and multilayering without contact inhibition. The cytology revealed anaplastic and pleomorphic features. Upon electron microscopic observation, most of the cultured cells were characterized by highly indented nuclei with multiple large nucleoli and by desmosomal cell contact. The chromosomal number varied widely and showed aneuploidy, but the modal chromosomal number was stable at the diploid range. No marker chromosome could be identified. Both of these cell lines, SNG-P and SNG-M, were transplanted to an immune-depressed hamster cheek pouch and produced a tumor resembling the original.

Histogenesis and culture of human uterine carcinosarcoma.

We set out to ascertain whether uterine carcinosarcoma represents: (a) a "collision tumor," i.e., a mixture of two histogenetically distinct malignant cell populations (endometrial carcinoma and sarcoma); (b) a "combination tumor" with both histological elements of common stem cell origin; or (c) a "composition tumor," i.e., an endometrial carcinoma with reactive, atypical stroma. In in vitro cultures of human uterine carcinosarcoma, we could separate two distinct, different cell types and succeeded in establishing adenocarcinoma cell lines (HWUA-1 and HWUA-2) and sarcoma cell lines (HWUS-1, HWUS-1a, and HWUS-2). These cell lines grew well for over 10 months. HWUS-1a was hypertetraploid, HWUA-1 and HWUA-2 were pseudodiploid, and HWUS-1 and HWUS-2 were hyperdiploid. These cell lines were transplanted into the subcutis of BALB/c nude mice and produced tumors. HWUA-1 and HWUA-2 cell produced poorly differentiated adenocarcinoma, HWUS-1 and HWUS-2 produced poorly differentiated sarcoma, and HWUS-1a produced well-differentiated leiomyosarcoma. These results support the combination tumor theory and reject the composition tumor theory as the cause of carcinosarcoma.

Establishment of a human leiomyosarcoma cell line.

A cell line designated SKN was established from the human uterine leiomyosarcoma of a 52-year-old female. The cell line has grown well and the serial passages were successively carried out 82 times within 12 months. The monolayer cultured cells revealed anaplastic and pleomorphic features, and they multipled rapidly without contact inhibition. Electron microscope studies revealed myoibrils but no virus-like particles, while chromosomal studies showed that all cultured cells were hyperploid, the modal number was 112, and the marker chromosome was present. The cells were transplanted into an immune-depressed hamster cheek pouch and produced a histological leiomyosarcoma resembling the original tumor.

Expression of CD44 in normal human versus tumor endometrial tissues: possible implication of reduced expression of CD44 in lymph-vascular space involvement of cancer cells.

Alternatively spliced variants of the CD44 molecule have been found to be associated with invasive and metastatic potential of cancer cells and poor prognosis in several types of carcinoma. We have examined expression of CD44 in normal and cancerous tissues of the endometrium as well as in cell lines established from patients with endometrial cancers by the combination of reverse transcription-polymerase chain reaction and Southern blot hybridization and by cell surface staining with antibodies to CD44. Of eight cancer cell lines tested, two lines, HOOUA and HEC50B, both of which are possibly potential candidates for metastasis, expressed a very small amount of mRNA for CD44. Variant forms of CD44 were expressed in 9 of 11 (81.8%) normal endometria, whereas 8 of 47 (17.0%) endometrial carcinomas showed expression of the variants. Hyperplasia samples displayed the variant expression in 42.9% of specimens (the value was between those of the normal and cancerous cells) and none of 3 in Müllerian mixed tumors. There was a significant difference in frequencies of CD44 variant expression between normal and cancerous tissues. Furthermore, lymph-vascular space involvement of cancer cells was observed to be statistically significant in the CD44-negative group as opposed to the positive group. Reverse transcription-polymerase chain reaction and Southern blot hybridization clearly demonstrated that normal endometrial tissues express the standard CD44 form as well as the variant form. Immunohistochemical examination of normal endometrium revealed that intense staining was seen on the gland cells at the basement membrane side, and less intense staining was seen between the gland cells. These results suggest that CD44 could play important roles in the function of normal endometrium and that reduced CD44 expression might be related to the metastasis of endometrial cancer cells through lymph-vascular space.

Coexpression of cholesterol sulfate and cytokeratin as tumor markers in well-differentiated squamous cell carcinoma of the human uterine cervix.

The expression of cholesterol sulfate (CS) is known to increase during squamous differentiation of keratinocytes and to activate the epsilon, eta, and zeta forms of protein kinase C as a signal transduction molecule for the subsequent expression of transglutaminase-1 (TG-1) and cytokeratins. To gain further insight into the regulation of cellular differentiation and tumorigenesis by CS, we examined the concentration and the potential for synthesis of CS in seven and four surgical specimens from human ovarian and uterine cervical cancer patients, respectively, and eight cell lines established from human uterine cervical cancer patients and compared them for the rate of expression of cytokeratin. CS was present in all of the uterine cervical cancer tissue specimens but only in the mucinous type of cystadenocarcinoma among ovarian cancer tissue specimens, and cytokeratin was highly expressed in the tissues with a high concentration of CS, which were classified as well-differentiated on the basis of morphological examination. Similarly, cells derived from a keratinizing type of well-differentiated cervical carcinoma demonstrated strong potential for synthesis of CS, stained positive with anti-cytokeratin antibody, and exhibited a higher specific activity of TG-1, whereas the cells without CS did not stain positive with anti-cytokeratin antibody and exhibited a lower specific activity of TG-1. These findings indicate that CS is coexpressed with TG-1 and cytokeratin in the well-differentiated types of squamous cell cancers as a tumor marker.

Characteristics of hepatocytes derived from early ES cells and treatment of surgically induced liver failure rats by transplantation.

BACKGROUND: Although liver transplantation has become a standard therapy for diseases such as fulminant hepatitis and cirrhosis, the lack of donor organs remains a major problem. One solution is the development of transplantable hepatocytes. The metabolic characteristics as well as function and adaptation of hepatocytes (R-EES-hep cell) derived from rat early embryonic stem cells were examined after transplantation into rats with surgically induced liver failure. METHODS: Rat hepatocyte cell lines were established from early embryonic stem cells cultured in the presence of embryotrophic factors by colony cloning methods. The cell lines were established from two cell embryos taken from spontaneous dwarf rats using the novel method of Ishiwata et al. Morphologic differentiation as well as albumin and bilirubin production were observed by immunostaining. R-EES-hep cells were transplanted into the spleens of 90% hepatectomized, surgically induced liver failure rats to analyze survival rates. RESULTS: When cultured in type I collagen gel the cells formed cordlike structures resembling the liver. Both albumin and bilirubin production were observed when transplanted; the spleen was converted into a liver-like structure with prolonged survival of the 90% hepatectomized rats for up to 3 months up to the time of killing. CONCLUSIONS: R-EES-hep cells showed many of the distinctive metabolic characteristics of the liver. These cells may be efficient for further research and application for hepatic cell transplantation to treat liver insufficiency patients and as biologic artificial organs.

Isolation of clones resistant to 6-thioguanine and G418 from HHUA endometrial carcinoma cells and their application to cell hybridization.

Two kinds of clones were isolated successfully from the HHUA 95 cells that were derived from a human well-differentiated adenocarcinoma of endometrium, with 6-thioguanine (6-TG) selection and transfection with plasmid containing the neo gene (pSV2 neo). One clone was resistant to the 6-TG (6-TGr 95) and the other to both the 6-TG and the G418 (6-TGr-neor 95). Karyotypes of these three kinds of cells were normal, even though random chromosome abnormalities were observed in some cells. Two types of cell fusion were performed: one consisted of the hybridization between 6-TGr 95 cells and normal human fibroblasts (HF), and the other, between 6-TGr-neor 95 and human choriocarcinoma cells (CC1). Tumorigenicity of both hybrid cell types was completely suppressed. Complementation for genetic lesions given by cell hybridization was assumed to be responsible for the suppression of tumorigenicity. These results suggest that genetic losses played an essential role in the evolution of the malignant phenotype of endometrial carcinoma cells. The data obtained from the endometrial carcinoma could not be used directly for the understanding of suppression mechanisms of choriocarcinoma.

[Tissue repair of uterine cervix--cell-biological properties of normal uterine cervical epithelia of transformation zone in vitro].

The objective of this study is to culture the epithelia of the transformation zone of the uterine cervix for long term and evaluate their biological characteristics, such as morphology, growth behavior, alkaline phosphatase activity and heterotransplantability. The epithelia of transformation zone of 15 cases of myoma uteri were cut into 1 x 1 x 1 mm fragments and placed directly on the cover glass. The explants were cultured at 37 degrees C in 5% CO2 and 95% air. In vitro outgrowth of squamous cells (squamous cell outgrowth pattern) was observed in 44, that of columnar cells (columnar cell outgrowth pattern) was observed 49, a mixture of squamous and columnar cell outgrowth patterns was 52 out of 198 explants of transformation zone. The squamous cells were polygonal in shape and showed a pavement-like cell arrangement. The glandular cells grew in whorled fashion. Along the margins of the outgrowth of glandular cells, two types of cells were seen after 2 weeks of culture. One type contained secretory vacuoles of glandular cell, and the other type contained a large number of tonofilaments of squamous metaplastic cells. These phenomena suggested that biological characteristics of the cells in vivo can well be retained in vitro for a relative long term (about 6 weeks).

Tumor angiogenesis factors produced by cancer cells.

Tumor angiogenic activity from tumor angiogenesis factors (TAFs) produced by 25 cell lines was assayed onto chorioallantoic membranes (CAMs). Neovascularization occurred prominently in such cell lines, as HTBOA (poorly differentiated ovarian carcinoma), HUOCA-II (poorly differentiated clear cell adenocarcinoma), HWUA (poorly differentiated endometrial adenocarcinoma), HKUS (uterine cervical small cell carcinoma), and in HOTHC (anaplastic thyroid carcinoma). The cell lines which secreted TAF showed high heterotransplantability in nude mice and produced rapidly growing tumors which were rich in blood vessels. The TAFs polypeptides of 14,000 and 78,000 molecular weight, were extracted and purified from the conditioned medium of HUOCA-II or W3UF (sub-line of HUOCA-II) lines, respectively. TAFs at concentrations of 10 ng/ml and 100 ng/ml promoted proliferation of the endothelial cells and induced tube formation. Microsequencing analysis revealed that TAF of 78,000 molecular weight has sequence identity with human hepatocyte growth factor (hHGF).

Effects of embryotrophic factors on the embryogenesis and organogenesis of mouse embryos in vitro.

In order to study embryogenesis and organogenesis in vitro, two cell mouse embryos were cultured with alpha-MEM supplemented 10% FCS and embryotrophic factors (ETFs). The ETFs were separated from the conditioned medium of a SKG-II-SF cell line derived from a human uterine cervical epidermoid carcinoma. IL-1 beta, IL-6, IL-8, EGF, GH, PDGF-AB, basic FGF, VEGF were also detected in the conditioned media of this cell line. Using ETFs and a 10% FCS supplemented culture medium, 23% of the mouse two cell stage embryos developed to the bilaminar disc stage, 13% to the trilaminar germ disc stage, 9% were observed with blood islets in the yolk sac, and the heart beat was noted in 7% (28 embryos) of the embryos. Furthermore, primordial organs, such as the liver, heart, kidney, notochord, retina-like structure, etc. were observed. Usually, structures associated with the primordial streak stage (bilaminar germ disc embryo) developed in vitro using ETFs from two cell stage embryos. These closely resemble structures found at the same stage in the normal embryo in vivo. After the primordial streak stage, the cultured embryos showed no resemblance to the same stage in normal embryos. None of the external appearances of these embryos appeared normal. On the other hand, trilaminar disc stage embryos never developed when using only a 10% FCS supplemented culture system.

Cloned transgenic mouse fetuses from embryonic stem cells.

Development of efficient efficient system for genetic modification and large-scale cloning of livestock is of importance for agriculture, biotechnology, or human medicine. The mouse, on the other hand, is an ideal model in the basic studies of genetic modification. In this study, we investigated about production of clone mice from established embryonic stem (ES) cell line by nuclear transfer. Further, we had try of production of cloned transgenic mouse fetuses/offspring using ES cells modified with a marker gene, EGFP. With the ES cell line TT2 which is at least 15 passages, reconstructed oocytes developed to 2-8 cell embryos, morulae, or blastocysts (44.8%), and 17.2% of them developed to term (19.5 days post-coitum, dpc). When 40 embryos with the marker gene transferred to 11 surrogate mothers (pseudopregnant females), 5 live fetuses were recognized in the uteli at 13.5 dpc and in these fetuses expression of GFP was observed, but none developed beyond 19.5 dpc. The present results suggest that ES cells can be used tg produce cloned mice.

[Establishment and characterization of a human malignant fibrous histiocytoma line serially transplanted in nude mice].

Serial heterotransplantation of human malignant fibrous histiocytoma (MFH) derived from tibia was attempted in BALB/c nu/nu mice, and HKMFH-nu transplantable tumor line was established. This line had the following biological properties. (1) Eighteen serial passages were carried out in 41 months. (2) Morphological changes of the grafts occurred in nude mice with serial passages: During the first 6 passages, histiological picture was consistent with the common type of MFH similar to that of the original tumor, then after the 7th passage, the myxoid type coexisted with the common type, and finally the myxoid type occupied the entire grafts to form large cysts. (3) The common type grafts grew more rapidly than the myxoid type grafts. (4) Granulocytosis (neutrophilia) was observed in mice bearing the common type tumor, but not in mice bearing the myxoid type tumor.

[Heterotransplantability of human anaplastic thyroid carcinoma line (HOTHC) in nude mice, and biological properties of the heterograft].

The heterotransplantability of HOTHC line (human anaplastic thyroid carcinoma) into the subcutis of BALB/c nude mice and the biological properties of grafts were discussed. (1) The HOTHC line showed high transplantability, and 1 x 10(4) cells produced anaplastic carcinoma (giant cell type) containing the colloid-like substance. (2) The grafted tumors grew rapidly and the mice were dead within 2 months after transplantation. (3) The number of leukocytes (neutrophils) of mouse peripheral blood increased as the tumor size increased, and the leukocyte count returned to a normal value after removal of the tumor. (4) The conditioned media of HOTHC line formed colonies of granulocytes (neutrophils) on soft agar. These phenomena revealed that HOTHC is a granulocyte-colony stimulating factor-producing line. (5) The conditioned media of HOTHC line showed promoting-effect on neovascularization on the chorioallantoic membrane.