Establishment and characterization of human ovarian clear cell adenocarcinoma cell line (SMOV-2), and its cytotoxity by anticancer agents

A novel cell line derived from a surgically resected ovarian clear cell adenocarcinoma of 46 year-old Japanese woman was established and designated SMOV-2. Cells of this lineage were continuously propagated in vitro over 44 months and were grown in a mono-layered sheet with a doubling time of 48.2 hours. The histopathology of the transplanted tumor in nude mice showed two distinctive cell types, hobnail cells and clear cells, which demonstrated recognizable characteristics of clear cell adenocarcinoma, as compared to resected original tumors. At the molecular level, SMOV-2 cells had the wild type p53 genes that were free from missence mutations. Anticancer agents (cisplatin and paclitaxel) were examined for cytotoxity against these SMOV-2 cells in vitro. These examinations revealed that the chemotherapy-treated cells had decreased proliferation, cell cycle arrests, and induction of apoptosis by the anticancer agents. As can be gleaned from this research, SMOV-2 is a valuable model to study the mechanism of apoptotic responses of solid tumors to future anticancer agents.     

Establishment and characterization of a cisplatin-resistant cell line (IGSK-1) from a poorly differentiated gastric adenocarcinoma

We successfully established a spontaneously cisplatin-resistant tumor cell line (designated as IGSK-1) derived from original gastric carcinoma. The patient was a 75-year-old Japanese woman. The histopathological diagnosis was gastric poorly differentiated adenocarcinoma accompanied with metastatic foci in lymph nodes, pT3, N2 M0, stage IIIB. The IGSK-1 cells grew as adhesive and monolayered cultures on the bottom of dishes. The susceptibility of the IGSK-1 cells to anti-cancer drugs was examined using oxygen electrode apparatus (Daikin, Tsukuba, JPN), and the results suggested TXL was effective, and CDDP, CPT-11 and 5-FU were not effective. Gastrin and somatostatin secretions were confirmed by immunohistochemical staining and also radioimmunoassay. Immunohistochemistry and radioimmunoassay for serotonin suggested the IGSK-1 cells might incorporate serotonin from the growth media. Spontaneously cisplatin-resistant gastric carcinoma cell line secreted gastrin and somatostatin is very important material for chemotherapy.     

Histogenesis of carcinosarcoma and Establishment of leiomyosarcoma cell line (HTMMT) derived from human uterine carcinosarcoma

A cell line designated HTMMT was established from the human uterine carcinosarcoma (composed of leiomyosarcoma and adenocarcinoma) of a 66-year-old Japanese woman. The cell line grew well and 83 serial passages were successively done within 24 months. The cell line contained spindle- or fibrous-shaped cells that revealed neoplastic and pleomorphic features, and multipled rapidly without contact inhibition. These cells were characterized as possessing myofibrils. The karyotype exhibited hyperploidy and the chromosome number was ranged from 87 to 100. The cells were transplanted into an immune-depressed hamster cheek pouch or into nude mouse skin, but produced no tumors. We supported the combination theory for the histogenesis of the carcinosarcoma.     

Establishment and characterization of JHUCS-1 cell line derived from carcinosarcoma of the human uterus

The cell line designed JHUCS-1 was established from a carcinosarcoma (malignant mixed mesodermal tumor) of the uterus that was surgically removed from a 57-year-old Japanese woman. We carefully examined the histopathology of the original tumor after the cell line was established and noted differentiation into a neuroendocrine carcinoma within the tumor's epithelial components. Immunohistochemical staining of the tumorous tissue that had been heterotransplanted was positive for Leu7. Additionally, secretary granules were observed in the grafted cells as determined by electron microscopy. These results support the existence of neuroendocrine cells within the JHUCS-1 cell line.     

A new human hepatocellular carcinoma cell line (KYN-1) with a transformation to adenocarcinoma

Summary A human hepatocellular carcinoma (HCC) cell line (KYN-1) has been established from a resected HCC of a 58-yr-old Japanese, male patient with HCC. Original resected HCC was moderately differentiated and proliferated in a solid pattern with vague trabecular structure in part. This cell line has been maintained for 10 mo. through 50 passages. Morphological features of KYN-1 cells demonstrated one or more large, round-to-oval nuceli with prominent nucleoli and eosinophilic polygonal-to-spindle abundant cytoplasm. In addition, some of these cells contained mucicarmin-positive materials in the cytoplasm. The cells exhibited a typical epithelial feature with pavementlike cell arrangement, and lacked contact inhibition. The doubling times of the cells grown in a serum-containing and a serum-fre, medium were about 31 h and 10 to 11 d, respectively. Functonally, KYN-1 cells produced albumin, α-fetoprotein (AFP), carcinoembryonic antigen (CEA), ferritin, β₂-microglobulin (BMG), and α₁-anti-trypsin (AAT). Positive reactions for albumin, AFP, CEA, and ferritin were identified in the cells by immunohistochemical techniques. Chromosome study revealed the chromosome number in a range from 61 to 74 without mode. The tumorigenicity of KYN-1 cells was identified by the tumor formation after subcutaneous inoculation of the cells into nude mice. The developed tumor showed compact growth of the tumor cells with gland formations containing mucicarmin-positive materials. Features of adenocarcinoma were identified by electron microscopy. The tumor cells were also identified to contain albumin, AFP, CEA, ferritin, and AAT by immunohistochemical techniques. AFP, CEA, and BMG were detected in the sera of nude mice. Thus, KYN-1 cells represented the morphologic features of adenocarcinoma, retaining some characteristics of original HCC. These findings suggest that KYN-1 is a new human HCC cell line with transformation to adenocarcinoma, which will provide useful information to clarify the histogenesis of combined hepatocellular and cholangiocellular carcinoma. This study was supported in part by the Sarah Cousins Fund.     

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.     

Basement membrane material and tigroid background in a fine needle aspirate of clear cell adenocarcinoma of the cervix. A case report

BACKGROUND: Although cytologic findings of clear cell adenocarcinoma of the female genital tract have been reported sporadically, the background on the smear has received little attention. CASE: A 16-year-old female had a large, necrotic mass in the cervix. As cervical brushing cytology and a punch biopsy of the mass could not make a definitive diagnosis, fine needle aspiration cytology was performed from the mass. The smears revealed loose, three-dimensional clusters and sheet arrangements of atypical cells. Dispersed atypical cells were also seen. The atypical cells were large and had abundant, weakly stained cytoplasm and round or oval nuclei with large nucleoli. There were a few tumor cells with clear cytoplasm and distinct cell borders in Papanicolaou-stained smears. The background in Diff-Quik-stained smears revealed a tigroid background and basement membrane material. CONCLUSION: The malignant tumor, revealing both a tigroid background and basement membrane material, seems to have been clear cell adenocarcinoma. Both features are diagnostic clues to clear cell adenocarcinoma in the female genital tract. Our case indicates that aspiration cytology is also an effective method of diagnosing a cervical tumor when the tumor is polypoid and the surface is extensively necrotic.     

Establishment and characterization of a cell line derived from a human serous surface papillary carcinoma of the ovary

A novel serous surface papillary carcinoma of the ovary (SSPC) cell line, HYKSSPC, was established successfully. Carcinoma cells were obtained from ascitic fluid of a 60-year-old Japanese woman. The population doubling time was 51.4 h. A phase contrast micrograph showed a pavement stone-like arrangement without contact inhibition. The chromosome number showed a wide distribution of aneuploidy, and the mode was in 46-47. An immunocytochemical study showed that CA125, BerER4 and cytokeratin were positive and that CEA, calretinin and thrombomodulin were negative. This cell line preserved some characters of the adenocarcinoma while growing in vitro. A chemosensitivity test revealed that HYKSSPC cells were sensitive to CDDP (cis-platinum), 5-fluorouracil, mitomycin C, paclitaxel and irinotecan. To our knowledge, HYKSSPC is the first established cell line derived from SSPC, and it may offer some useful information for investigating this disease.     

Establishment and characterization of a lung cancer cell line,SMC-L001, from a lung adenocarcinoma

Lung cancer cell lines are a valuable tool for elucidating lung tumorigenesis and developing novel therapies. However, the majority of cell lines currently available were established from tumors in patients of Caucasian origin, limiting our ability to investigate how cancers in patients of different ethnicities differ from one another in terms of tumor biology and drug responses. In this study, we established a human non-small cell lung carcinoma cell line, SMC-L001, and characterized its genome and tumorigenic potential. SMC-L001 cells were isolated from a Korean lung adenocarcinoma patient (male, pStage IIb) and were propagated in culture. SMC-L001 cells were adherent. DNA fingerprinting analysis indicated that the SMC-L001 cell line originated from parental tumor tissue. Comparison of the genomic profile of the SMC-L001 cell line and the original tumor revealed an identical profile with 739 mutations in 46 cancer-related genes, including mutations in TP53 and KRAS. Furthermore, SMC-L001 cells were highly tumorigenic, as evidenced by the induction of solid tumors in immunodeficient mice. In summary, we established a new lung cancer cell line with point mutations in TP53 and KRAS from a Korean lung adenocarcinoma patient that will be useful for investigating ethnic differences in lung cancer biology and drug response.     

Establishment and characterization of a human malignant schwannoma cell line (HKMS)

The malignant schwannoma cell line (HKMS) was established from the subcutaneous tumor of Axilla region of a 48-year-old Japanese woman. The HKMS line has the following biological properties. 1. The HKMS cells were spindle in shape and showed neoplastic and pleomorphic features. The monolayer sheet of HKMS cells showed the resemble cell-arrangement with that of the original tumor tissue. 2. The cells showed a stable growth and the serial passages were successively carried out 150 times within 3 years. Their population doubling time is about 40 hours. 3. The chromosome number varied widely, and the modal number was stable at the 78-80. The marker chromosomes were present. 4. The cells were transplanted into the subcutis of nude mice and produced the malignant schwannoma.     

Establishment and characterization of an IGSK-2 cell line derived from ascitic fluid of recurrent hCG and somatostatin secreted adenocarcinoma of the stomach

We examined a 32-year-old Japanese man who was clinically diagnosed with gastric cancer, type 4, and histopathologically diagnosed with mucinous and poorly differentiated adenocarcinoma (mucinous > poorly) of the stomach. We successfully established and characterized a cell line (designated as IGSK-2) derived from the ascitic fluid of the patient with recurrent and cisplatin-resistant carcinoma. The IGSK-2 cells grew in multi-layered culture in culture dishes. The cells secreted 18 pg/mL somatostatin, 9.1 mIU/mL human chorionic gonadotrophin (hCG), 8000 U/mL carbohydrate antigen 19-9 and 410 ng/mL carcinoembryonic antigen over 4 days of culture. The population doubling time was approximately 83 h. The susceptibility test of anticancer drugs revealed that IGSK-2 cells were sensitive to Taxol, but were not sensitive to cisplatin, 5-fluorouracil and irinotecan. Immunohistochemical staining revealed that the IGSK-2 cells were positive against antihCG antibody and antiserotonin antibody, and negative against antisomatostatin antibody and antigastrin antibody.     

Establishment and characterization of a human malignant choroids plexus papilloma cell line (HIBCPP)

A cell line designated "HIBSPP" was established from a human malignant choroids plexus papilloma of 29-year-old Japanese woman. This line grew well without interruption for 3 years and was subcultivated over 70 times. The cells were spindle, oval, and polygonal in shape, and neoplastic and pleomorphic features, a jigsaw puzzle-like arrangement, multilayering and forming papillary structures without contact inhibition. The cells proliferated slowly, and the population doubling time was about 69 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the hypotetraploid range, and many marker chromosomes were observed. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.     

Basement membrane-like substance in cytologic diagnosis in clear cell adenocarcinoma of the minor salivary gland of the palate. A case report

BACKGROUND: Clear cell adenocarcinoma (CCA) of the minor salivary gland accounts for < 1% of all tumors of the salivary gland. CASE: A 32-year-old woman with a history of papillary carcinoma of the thyroid 1 year earlier complained of pain on the left side of the neck. After a detailed examination, the patient underwent the resection of a tumor located at the palate. Imprint cytology of the tumor revealed cohesive tumor cells of uniform size containing an abundant clear cytoplasm and round nuclei with extra but fine granular chromatin and conspicuous nucleoli. A basement membrane-like substance (BMS) was stained in light green with Papanicolaou staining and was positive for laminin with immunohistochemical staining. Histopathologic analysis confirmed the trabecular or nest-like arrangement of the cells with the clear cytoplasm and BMS substance surrounded by tumor cells, which were positive for laminin and AE1 immunohistochemically. CONCLUSION: Although CCA of the palate is extremely rare, an accurate cytologic diagnosis can be made if the characteristic findings of CCA, including BMS, are imaged.     

Advanced primary clear cell carcinoma of the vagina not associated with diethylstilbestrol

BACKGROUND: Primary vaginal clear cell carcinoma occurs in young women exposed to diethylstilbestrol (DES) in utero. Primary vaginal clear cell carcinoma not associated with DES is very rare. We report the clinicopathologic and cytopathologic features of a patient with advanced, sporadic primary vaginal clear cell carcinoma with metastases to liver, lung and paraaortic lymph nodes. CASE: A postmenopausal, 63-year-old woman presented to our department with genital bleeding. A hemorrhagic tumor found in the vagina was diagnosed as a clear cell carcinoma by cytopathologic examination of the tumor smear and by histopathologic examination of a biopsy specimen. A chest radiograph revealed multiple lung metastases, and metastases to the liver and paraaortic lymph nodes were noted on computed tomography and magnetic resonance imaging. The tumor was diagnosed as primary clear cell carcinoma of the vagina, stage IVb (FIGO) based on a normal cytopathologic examination of the cervix, endometrium and ascites; normal appearance of the uterus, ovaries and kidneys on magnetic resonance imaging; and absence of detectable tumor in the urinary tract. The patient died of respiratory failure 31 days after hospitalization. The tumor demonstrated overexpression of p53 protein and did not show microsatellite instability. CONCLUSION: This patient was the second reported Japanese woman with advanced primary vaginal clear cell carcinoma not associated with DES.     

Synthesis of type 1 and 2 lacto series glycolipid antigens in human colonic adenocarcinoma and derived cell lines is due to activation of a normally unexpressed beta 1----3N-acetylglucosaminyltransferase

Human colonic adenocarcinoma tissue and derived cell lines have been characterized by an abundance of different type 1 and 2 lacto series glycolipid antigens which are either low or not found in normal colonic mucosa. The enzymatic basis for the expression of contrasting glycolipid compositions between adenocarcinomas and normal colonic mucosa, as well as between derived cell lines, has been studied. The following results were of particular interest. (i) Abundant activities of beta 1----4galactosyltransferase associated with synthesis of both lactosylceramide and lactoneotetraosylceramide, beta 1----3galactosyltransferase for synthesis of lactotetraosylceramide, and an alpha 1----3/4fucosyltransferase responsible for synthesis of Lex and Lea antigens were found in normal colonic mucosa or in a normal mucosal epithelial cell line HCMC, or in both. Variable levels of these activities were found in adenocarcinoma tissues and in various established adenocarcinoma cell lines. In striking contrast, significant activity of a beta 1----3N-acetylglucosaminyltransferase responsible for synthesis of lactotriaosylceramide (Lc3) was found in various cases of colonic adenocarcinoma and cell lines, but was undetectable in normal colonic epithelial cells. (ii) In situ transfer of galactose to Lc3 was performed on histologic sections by preincubation of the tissue with acceptor glycolipid followed by incubation with UDP-galactose. The biosynthesized glycolipid was revealed by indirect immunofluorescence with the monoclonal antibody 1B2 which defines lactoneotetraosylceramide antigen. In these studies, histologic sections prepared from frozen normal proximal colon tissue were shown to lack native type 2 chain structures. However, transfer of galactose from UDP-galactose could be demonstrated in the epithelial cells of normal proximal colon after incorporation of Lc3 into the membranes, indicating the ability of normal colonic epithelial cells to synthesize type 2 chain core structures if the precursor Lc3 is available. In contrast, adenocarcinoma tissues showed significant native immunofluorescence with the antibody. These data suggest that an accumulation of both type 1 and 2 chain lacto series glycolipids with alpha 1----3- or alpha 1----4fucosyl substitution in human adenocarcinoma is due to enhanced beta 1----3N-acetylglucosaminyltransferase rather than enhancement of other enzymes. This enzyme may play a key role in regulating the level of various types of lacto series tumor-associated antigens with the lacto type 1 or 2 chain.     

Establishment and characterization of a human undifferentiated carcinoma cell line (HMG)]

The undifferentiated carcinoma cell line (HMG) was established from a nude mouse tumor which had been produced by transplantation of a intraperitoneal tumor of 27-year-old woman. The HMG cell line has the following biological properties. 1. The HMG cells are round to oval in shape and grow as floating cell aggregates like a rouleau or a cluster of grapes. 2. 100 passages have been carried out over a year, and the population doubling time is about 17 hrs. 3. In the original tumor, keratin and vimentin were expressed simultaneously, in HMG cells, however, only localization of vimentin was confirmed. 4. By chromosomal analysis, over 90% of the cells revealed 46, XX, with no karyological abnormalities, at passage 82. 5. When heterotransplanted into the subcutis of a nude mouse, HMG cells produced a undifferentiated carcinoma resembling the original tumor.     

Human renal cell carcinoma: Establishment and characterization of a new cell line (OS-RC-2)

Summary A cell line, designated as OS-RC-2, has been established from a renal cell carcinoma in a 52-yr-old Japanese male patient and maintained for 23 mo. through 60 in vitro passages. The OS-RC-2 formed monolayers of polygonal epithelial cells and lacked contact inhibition. Doubling time of cells was about 60 h at the 30th passage. Electron microscopic findings indicated numerous long microvilli on the cell surface and many glycogen granules in the cytoplasm of this cell line, which are characteristic structures of renal cell carcinoma. Chromosomal analysis revealed that a small portion of this cell line had a hypodiploid modal number of 40 and a large portion had a hypotetraploid modal number of 75. The characteristics of the karyotype were one detected marker chromosome and the translocation between the Chromosomes 2 and 13. Cell line OS-RC-2 was serially transplantable into nude mice, and histopathological findings of heterotransplanted tumor showed a close similarity to those of the original tumor. Histocompatibility antigens of OS-RC-2 were HLA-A9, Bw52, which were identical to those of the peripheral blood lymphocytes of the patient.     

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.     

Characterization of the state of differentiation of six newly established human non-small-cell lung cancer cell lines

Six new non-small-cell lung cancer (NSCLC) cell lines were established directly from human tissue or indirectly via nude mouse xenografts in serum-supplemented media with success rates of 8% and 13%, respectively. They comprised one adenocarcinoma (ADLC-5M2), two squamous cell carcinomas (EPLC-32M1, EPLC-65H), two large cell carcinomas (LCLC-97TM1, LCLC-103H), and one malignant biphasic mesothelioma (MSTO-211H). All cell lines grew adherent to culture vessels with population doubling times (PDT) of 16-40 h, formed colonies in soft agarose with efficiencies of 0.1%-5.1%, and all grew in athymic nude mice. Xenograft histologies appeared as follows: (a) undifferentiated carcinomas with feeble resemblance to the original tumors in the case of adenocarcinomas and squamous cell carcinomas; (b) large cell carcinoma with high resemblance to the original tumor; (c) an undifferentiated tumor with predominance of large epithelial cells and few fibrous cells in the case of mesothelioma. Human chorionic gonadotropin (HCG) was found by radioimmunoassay and high-affinity binding sites for epidermal growth factor (EGF) by radio-receptor assay in 4/4 cell lines. A very low activity of L-DOPA decarboxylase (DDC) was detectable only in the adenocarcinoma cell line. All cell lines overexpressed the c-myc protooncogene, and no gene rearrangement or amplification was observed. Chromosome analysis revealed modal chromosome numbers of 70-73 in ADLC-5M2, EPLC-32M1, EPLC-65H, and MSTO-211H. Cell lines derived from large cell carcinoma had modal values of 65 and 170 and a wider chromosome distribution than all other cell lines. A NSCLC specific chromosomal aberration has been undetectable until now. These cell lines may aid in elucidating the biology of NSCLC and its interrelationship to other lung tumors.