Establishment and characterization of a new human esophageal cancer cell line (YES-1)

A new human esophageal cancer cell line (YES-1) was established from a subcutaneous tumor implanted into nude mice, which had been transplanted from a surgical specimen obtained 50-year-old Japanese male patient. This cell line has been maintained for 33 months through 94 passages with stable growth. YES-1 cells are mainly polygonal-to-spindle shaped, have eosinophilic cytoplasm and oval-to-round nuclei with some prominent nucleoli. There are also some cells having clear cytoplasm and round nuclei with prominent nucleoli. The cells proliferate in a pavement-like cell arrangement and show a lack of contact inhibition. The doubling time at the 33rd passage was 35.2 hours. YES-1 cells produce carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC antigen) and tissue polypeptide antigen (TPA) as tumor markers. Chromosome study have shown that the chromosome number ranges from 47 to 54 with a mode of 51. Tumorigenicity has been identified by the development of tumors after the subcutaneous injection of YES-1 cells into nude mice, which were found to be similar to the original tumor on histological examination. Thus, these findings indicate that the YES-1 cell line is available as a new human esophageal cancer cell line which should be useful for various studies.     

Primary culture of human bladder carcinomas and establishment of human bladder carcinoma cell line by serum-free culture

It was possible to cultivate cells from bladder carcinoma tissues in 4 cases out of 6 without the overgrowth of fibroblast. A new human transitional cell carcinoma cell line (HAMT-1) was established in longterm tissue culture by using serum-free medium (BEM-841) which had been developed by us. The tissue for culture was taken from a 61-year-old Japanese male with grade 3 transitional cell carcinoma of the bladder. The microscopic features of the cell in cultures and of the tumors developed in nude mice resembled closely that of the original tumor. Electron microscopy of the cultured cells and the tumors developed in nude mice revealed characteristics of the epithelial origin of these cells with microvilli, junctional complex and scarce filament formation. Blood TPA level of the nude mouse with the transplanted tumor was equally high as that of the patient from whom the original tumor had been taken. The cells were anchorage independent in the serum-free medium but anchorage dependent in the medium containing 5% FCS. Anchorage dependency could not be restored by the addition of collagen and fibronectin. The doubling time of the cells were 18-20 hours. The chromosome counts of the cell line ranged from 59-78 with a modal count of 74.     

Evaluation of CA19-9 as a tumor marker in urothelial malignancy

OBJECTIVE: Carbohydrate antigen 19-9 (CA19-9) is known to be a marker with a high positive rate in pancreatic cancer. There are limited data on the use of CA19-9 as a tumor marker in bladder carcinoma. We tested the expression of CA19-9 in transitional cell carcinoma (TCC) cell lines and bladder cancer patients to determine its usefulness in clinical applications. MATERIAL AND METHODS: The expression of CA19-9 was determined in six TCC cell lines and 42 bladder carcinoma tissues using two approaches: immunohistochemistry and enzyme immunoassay (EIA) analysis. EIA was used for testing CA19-9 levels in spent medium of cultured TCC cells and the urine of bladder tumor patients. RESULTS: The CA19-9 value was low in spent media of the MGH-U1, MGH-U1R and MGH-U3 cell lines, but high in that of reactivity in MGH-U4 cells, while negative reactivity was found in high-grade MGH-U1 and MGH-U1R cells, both of which were derived from a stage B, grade 3 TCC. High incidences of negative CA19-9 staining were found in high-grade and invasive tumor tissues: 69.6% (16/23) and 70.8% (17/24), respectively. The sensitivity and specificity of urinary CA19-9 for detecting tumor recurrence were 83.3% and 50.8%, respectively. However, urinary tract infection also resulted in a high false-positive rate. CONCLUSION: CA19-9 is promising for use as a biomarker for the detection and monitoring of low-grade and low-stage bladder cancer, with the proviso that patients to be tested should be free of infection.     

Development and characterization of a monoclonal antibody to human embryonal carcinoma

A monoclonal anti-testicular carcinoma antibody was obtained via the somatic cell fusion technique by immunization of BALB/c mice with freshly prepared single cell suspension from a patient with testicular embryonal carcinoma with choriocarcinoma components. The hybridoma supernates were screened against the testicular carcinoma cells used in the immunization as well as normal mononuclear white blood cells isolated from the same patient. An antibody (5F9) was selected which bound to fresh tumor cells from two patients with embryonal testicular carcinoma and failed to bind to fresh tumor cells from 24 patients (2 seminoma, 2 melanoma, 3 neck, 2 esophageal, 1 ovarian, 3 colon, 1 prostate, 2 breast, 1 liposarcoma, 3 endometrial, 1 kidney, 1 adrenal, 1 larynx and 1 bladder tumors) or cell suspensions prepared from normal liver, lung, spleen, ovary, testes, kidney, red blood cells or white blood cells. The antibody was tested for its binding to several well established cancer cell lines, and was found to bind to the BeWo human choriocarcinoma and two human embryonal carcinoma cell lines. The antibody did not react with 22 other cell lines or with hCG. The antibody was labeled with 131I and injected into nude mice bearing BeWo tumors and evaluated for tumor localization by performing whole body scans with a gamma camera 5 days later. Six mice injected with the antibody showed positive tumor localization without the need for background subtraction while six mice injected with MOPC-21, a murine myeloma immunoglobulin, demonstrated much less tumor localization. Tissue distribution studies performed after scanning showed specific tumor localization (8:1 tumor: muscle) for the monoclonal antibody and no specific localization for MOPC-21. This antibody thus has selective reactivity with the surface of tumor cells from embryonal carcinoma (testicle) and choriocarcinoma both in vitro and in vivo.     

Expression of gastrin-releasing peptide receptors in endometrial cancer

BACKGROUND: The Bombesin (BBS)-related peptide, gastrin-releasing peptide, and its cognate receptor are ectopically expressed by many cancers, in which they regulate tumor proliferation and metastasis. But, their role in endometrial cancers is unknown. The purpose of this study was to determine whether endometrial cancer cell lines express functional BBS receptors and to determine whether they were coupled to the regulation of vascular endothelial growth factor (VEGF-A) expression. STUDY DESIGN: Endometrial cancer cell lines (HEC-1A, KLE, and AN3CA) were cultured according to the recommendations of the American Tissue Culture Collection. Ishikawa cells were maintained in Dulbecco's modified Eagle's medium plus 10% fetal bovine serum. Before BBS treatment, all cell lines were placed in serum-free, phenol-free media for 24 hours. BBS-stimulated increases in intracellular Ca(2+) ([Ca(2+)]i) were used to assess functional BBS receptor status. VEGF-A mRNA expression was determined by Northern blotting. RESULTS: BBS (100 nM) stimulated an increase in [Ca(2+)]i in HEC-1A, Ishikawa, and KLE cells, indicating the presence of functional BBS receptors. This increase did not occur in AN3CA cells. BBS stimulated a time-dependent increase in VEGF-A mRNA expression in Ishikawa and KLE cells. Ishikawa cells exhibited a peak of VEGF-A mRNA expression between 8 and 12 hours with a partial decline by 24 hours. KLE cells showed a relatively small increase at 12 hours. In contrast, HEC-1A cells exhibited a high baseline level of VEGF-A mRNA expression and did not show a response to BBS. CONCLUSIONS: These data demonstrate that endometrial cancer cell lines express functional BBS receptors. In Ishikawa, KLE, and HEC-1A cells, BBS receptors are coupled to the regulation of VEGF-A mRNA expression.     

Establishment of a new human renal cancer cell line (TC-1) and its productivity of interleukin-6 (IL-6)]

We established a new cell line (TC-1) from primary site of a renal cell carcinoma (RCC) patient. Its doubling time in tissue culture was 20 hours at 45th passage and mycoplasma contamination test was negative. The karyotypic analysis demonstrated a human karyotype with a modal number of 70. A consistent chromosomal abnormality was noted such as No. 4 monosomy, No. 7 trisomy and a loss of Y chromosome. Electron microscopic examination showed a brush border, vacuoles and abundant glycogen granules in the cytoplasm, which was compatible with RCC cells. This cell line was transplantable to nude mice and the grown tumor closely resembled the original tumor, i.e. clear cell type and hypervascularity. High titer of interleukin-6 (IL-6) was detected in the supernatant of TC-1 cell culture (approximately 5 ng/ml) as well as in sera of nude mice bearing this tumor (260 pg/ml). Exogenous IL-6 did not enhance the TC-1 cell proliferation as determined by cell count. Flow cytometric analysis could not demonstrate the existence of IL-6 receptor on the cell surface. These results suggested the produced IL-6 did not act as an autocrine growth factor in the cell line. Additional IL-1 alpha to the culture medium induced 3-4 times higher concentration of IL-6 in the culture supernatant compared with that of non-stimulating cells, while exogenous TNF alpha did not stimulate IL-6 production.     

Characteristics of three human gastric cancer cell lines, NU-GC-2, NU-GC-3 and NU-GC-4

Three human gastric cancer cell lines, NU-GC-2, NU-GC-3 and NU-GC-4 were established in vitro from the cancer tissues obtained from 3 patients during surgery. The pathological findings of the gastric tumors of these cases revealed poorly differentiated adenocarcinoma (and partial signet-ring cell carcinoma in the case of NU-GC-4). NU-GC-2 and NU-GC-4 were originally obtained from metastatic paragastric lymph nodes and NU-GC-3 was obtained from a metastatic tumor in the brachial muscle. The cells of NU-GC-2 and NU-GC-3 are polygonal in shape and grow as a monolayer sheet. NU-GC-4 cells, however, are mainly spherical in shape with a few free floating cells. Electron microscopy revealed epithelial characteristics in all 3 cell lines. The average doubling time of NU-GC-2 was 36.1 hours, that of NU-GC-3 was 38.2 hours and that of NU-GC-4 was 29.9 hours. The modal chromosome number of NU-GC-2 was 62, that of NU-GC-3 was 58 and those of NU-GC-4 grown in in vitro and in vivo were 52-54 and 53, respectively. In vitro and in vivo lines of NU-GC-4 were established from the same tumor. These two cell lines are quite similar in morphology, but slightly different in karyotype. The in vitro sensitivity to anticancer agents was highest in NU-GC-4 and lowest in NU-GC-2. Of the anticancer agents, mitomycin C and adriamycin were most effective on the cells of all 3 cell lines.     

先天耐药人肾细胞癌细胞系RCC—925的建立及其生物学特性

自原发性人肾细胞癌组织取材，行体外培养，经４年连续传代培养，建立了人肾细胞癌细胞系ＲＣＣ９２５。细胞为贴壁生长的上皮样细胞，生长稳定，接触抑制消失，有重叠生长。超微结构具有恶性细胞特征。细胞群体倍增时间为３３．２６小时，细胞分裂第三天达高峰，分裂指数为３５‰，平均集落形成率为５０．５％。染色体众数为６６～７５条，具有明显的超二倍体特征。裸鼠移植瘤形态与原手术瘤组织相似。对ＣｏｎＡ有高凝集反应。ＬＤＨ同功酶显示特征酶谱。细胞对长春新碱高度耐药，对阿霉素低度耐药，具有先天多药耐药性，ｍｄｒ１基因的ｍＲＮＡ水平呈高表达。     

Cytotoxicity of lymphocytes sensitized by autologous tumor cells in vitro, against autologous lung cancer cell lines

In order to obtain killer lymphocytes that would have a strong cytotoxicity against autologous tumor cells, we conducted a cytotoxic assay of effector cells cultured from human peripheral blood lymphocytes (PBL) using 10 cultured lung cancer cell lines as target cells. We present this result. (1) Lymphocytes obtained by 17 days mixed lymphocyte tumor cell culture (MLTC: Fresh PBL from lung cancer patients was mixed with irradiated autologous tumor cells in a medium, and cultured for 3 days. Irradiated allogeneic lymphocytes were then added, and cultured for 14 days) were cytotoxic against 4 of the 10 autologous lung cancer cell lines. (2) Lymphocytes obtained by 14 days MLTC and 3 more days culture in a medium containing interleukin 2 were cytotoxic against all 10 of the autologous lung cancer cell lines. These lymphocytes proliferated to 4.13 times of the original cell number, and their surface marker was OKT3+. These killer lymphocytes are expected to be effective in adoptive immunotherapy as effector cells.     

Characteristics of three human gastric cancer cell lines,NU-GC-2, NU-GC-3 and NU-GC-4

Three human gastric cancer cell lines, NU-GC-2, NU-GC-3 and NU-GC-4 were establishedin vitro from the cancer tissues obtained from 3 patients during surgery. The pathological findings of the gastric tumors of these cases revealed poorly differentiated adenocarcinoma (and partial signet-ring cell carcinoma in the case of NU-GC-4). NU-GC-2 and NU-GC-4 were originally obtained from metastatic paragastric lymph nodes and NU-GC-3 was obtained from a metastatic tumor in the brachial muscle. The cells of NU-GC-2 and NU-GC-3 are polygonal in shape and grow as a monolayer sheet. NU-GC-4 cells, however, are mainly spherical in shape with a few free floating cells. Electron microscopy revealed epithelial characteristics in all 3 cell lines. The average doubling time of NU-GC-2 was 36.1 hours, that of NU-GC-3 was 38.2 hours and that of NU-GC-4 was 29.9 hours. The modal chromosome number of NU-GC-2 was 62, that of NU-GC-3 was 58 and those of NU-GC-4 grown inin vitro andin vivo were 52–54 and 53, respectively.In vitro andin vivo lines of NU-GC-4 were established from the same tumor. These two cell lines are quite similar in morphology, but slightly different in karyotype. Thein vitro sensitivity to anticancer agents was highest in NU-GC-4 and lowest in NU-GC-2. Of the anticancer agents, mitomycin C and adriamycin were most effective on the cells of all 3 cell lines.     

Interactions of human colorectal carcinoma cells with basement membranes. Analysis and correlation with differentiation

The abilities of colorectal carcinoma cell lines to adhere and invade through a basement membrane were examined. The four poorly differentiated cell lines studied were three to four times more adherent and spread to a greater extent following adherence to a basement membrane matrix than the three moderately well-differentiated (MWD) lines. One exception was the MWD cell line DLD-2, whose histologic features resembled a signet ring carcinoma. The ability of these cells to invade through a basement membrane model was measured. This assay showed that the poorly differentiated cell lines as well as DLD-2 were three times more invasive than the remaining MWD cell lines. These data indicate that tumor cell adherence can be correlated with invasion through basement membranes. In addition, the ability of colorectal carcinoma cells to interact with the basement membrane seems, in general, to be inversely related to the degree of cytodifferentiation.     

Establishment of three human renal cell carcinoma cell lines (SMKT-R-1, SMKT-R-2, and SMKT-R-3) and their characters

Summary We have established three new cell lines of human renal cell carcinoma (RCC), designated as SMKT-R-1, SMKT-R-2 and SMKT-R-3. These cell lines were derived from a primary lesion of the tumor or a tumor initially xenotransplanted in nude mice. These cell lines have maintained a stable growth in vitro for more than a year. They also exhibited characteristics showing a lack of contact inhibition of cells, colony formation in soft agarose and tumor formation in nude mice by a xenotranaplantation of cells, all of which suggested an epithelial origin. The tumors produced in nude mice by the innoculation of cell lines were demonstrated by light an electron microscopy tobe derived from RCC. The doubling time of these cell lines were 180.0 h, in SMKT-R-1, 56.4 h in SMKT-R-2 and 55.7 h in SMKT-R-3. The cell lines were aneuploid in their chromosomal analysis, and SMKT-R-2 and SMKT-R-3 also had three and two marker chromosomes, respectively. The different biological characters of these cell lines from the others so far established would be of benefit in the future study of human RCC.     

Differences in sensitivity to tumor-specific CTLs between primary and metastatic esophageal cancer cell lines derived from the same patient

Purpose

MHC antigens and adhesion molecules, such as the intracellular adhesion molecule (ICAM-I), play an important role in cellular immune response. We examined the expression patterns of these molecules in both primary and metastatic esophageal carcinoma cells from the same patient and evaluated the cellular immune responses against these cells.

Materials and methods

In the esophageal cancer patient (H122), tumor cell lines were established from primary and subcutaneous metastatic lesions. We compared the expression of cell surface molecules on the metastatic tumor cell line (H122SC) with that on the primary tumor cell line (H122ESO) using flow cytometry. Moreover, we analyzed the differences in cellular immune responses against these cell lines, which expressed similar levels of the Tara antigen, using the Tara antigen-specific CTL clone.

Results

H122SC ICAM-1 expression was significantly lower in H122ESO, and the Tara antigen-specific CTL clone produced lower levels of TNF in response to H122SC than H122ESO. ICAM-1 transfection into the H122SC rendered these cells as sensitive to the CTL clone as the H122ESO.

Conclusion

The metastatic tumor cells displayed lower regulated ICAM-1 expression levels and were less sensitive to specific CTLs. ICAM-1 downregulation may be one mechanism by which tumor cells escape immunologic surveillance.     

Establishment of a cell line (TSUS-1) derived from a human squamous cell carcinoma of the penis

We established a cell line ( TSUS -1) derived from a relatively well-differentiated squamous cell carcinoma of the penis. The cells had been subcultured 60 times during two years and five months. The average doubling time of this cell line was 38 hours. Chromosomal analysis indicated that the modal chromosome number was 75; seven marker chromosomes and a male karyotype were present. The cultured cells were easily transplantable into BALB/c nude mice and the histological picture of the inoculated tumor was similar to that of the original tumor.     

An autopsy case of thymic carcinoma producing various tumor markers and the examination of 222 autopsy cases of thymic malignant tumor in Japan

The autopsy of a 76-year-old Japanese female patient, which revealed thymic carcinoma with various tumor markers such as NSE, CYFRA, and CA-125, is presented. The patient died from hepatic failure because the liver was overtaken by the tumors. At autopsy, the thymic carcinoma was found to have metastased only in the liver. From microscopical analyses and electron microscopical findings, we diagnosed poorly differenciated squamous cell carcinoma of thymic origin. In the histochemical analyses, the tumor cells were positively stained in CA 125, CA 19-9, EMA, NSE, AE 1, AE 3, CEA, S-100, glimerius and Bcl-2. These date suggest that the tumor cells produced various tumor markers. In 222 autopsy cases of thymic malignant tumor observed in Japan over a period of 4 years, the dominant pathohistological image was squamous cell carcinoma. It is interesting that the greatest number of combined malignant tumors with thymic malignancies were thyroid papillary carcinomas.     

Activin A Causes Cancer Cell Aggressiveness in Esophageal Squamous Cell Carcinoma Cells

BACKGROUND: Expression of activin A is associated with lymph node metastasis and clinical stage in esophageal cancer. METHODS: To clarify the aggressive behavior of tumors with high activin A expression, we used the beta subunit of activin A to establish stable activin betaA (Act-betaA)-transfected carcinoma cells in two human esophageal carcinoma cell lines, KYSE110 and KYSE140. The biological behavior of these cells was compared with that in mock-transfected cells from the same cell lines. We focused our attention on cell growth and tumorigenesis, and proliferation and apoptosis. RESULTS: Both Act-betaA-transfected carcinoma cell lines showed a higher growth rate than the mock-transfected carcinoma cells. In an in vitro invasion assay and a xenograft analysis, the Act-betaA-transfected carcinoma cells showed far higher proliferation in vitro and a higher potency for tumorigenesis in vivo, respectively. Moreover, in an analysis of apoptosis via Fas stimulation, the Act-betaA-transfected carcinoma cells showed a higher tolerance to apoptosis compared with the mock-transfected carcinoma cells. Moreover, anti-activin-neutralizing antibody-treated squamous cell cancer cell lines inhibited their migration. CONCLUSIONS: Collectively, these data indicate that continuous high expression of activin A in esophageal carcinoma cells is not related to tumor suppression, but rather to tumor progression in vitro and in vivo. The inhibition of activin might be one of the methods to attenuate tumor aggressiveness.     

Establishment and characterization of cell lines derived from a human osteosarcoma

Continuously growing cell lines have been established in vitro from a human osteosarcoma after transplantation into athymic nude mice. These cell lines grew as an adherent monolayer and consisted of various types of cells. Twelve clones were colonially isolated from a cell line, HuO-3N1, and subdivided into three groups depending on the morphologic features. The cells of HuO-3N1 and its clones had alkaline phosphatase (ALP)-positive granules in the cytoplasm. Cytogenetic studies showed that these cell lines were human aneuploid lines. A tumor was produced by injection of HuO-3N1 cells into an athymic nude mouse. ALP activity increased in a clonal cell line, HuO-3N1 cl-2, when cells were treated with 1,25-dihydroxy-vitamin D3. The proliferation of cells was inhibited when the cells were cultured in a medium supplemented with L-homoarginine, which is an inhibitor of bone and liver-specific ALP. This cell line has an osteoblastic phenotype and provides a useful model for studies of human osteosarcoma and phenotypical expression of human osteoblastic cells.     

Ultrastructure of cultured normal bladder transitional epithelial cell and BBN induced rat bladder tumor cell lines

A normal rat bladder transitional epithelial cell line and seven tumor cell lines cultured from tumor induced by 0.05% N-Butyl-N-4-hydroxybutyl-Nitrosamine (BBN) were established and a comparative study on the doubling time and Ultrastructure of the cultured cell lines was performed. 1. Seven cultured cell lines (TU-B1 1-7) were obtained from bladder tumors induced by the administration of BBN for 32 weeks. 2. The doubling time of the cultured tumor cell lines were 43, 24, 28, 24, 25, 31, and 45 hours, suspectively. 3. These cultured tumor cell line were inoculated subcutaneously into nude mice and transitional epithelial carcinomas differing in degree of differentiation were observed. 4. Horizontal and tangential sections of these cultured tumor cell lines were examined by transmission electron microscopy. The development of intracellular organella and the presence of microvilli on the surface of the cells were noted. Intracellular adhesion was poor but the morphological characteristics of bladder transitional epithelium with compressed vesicles and other intracellular structures such as desmosome and free ribosomes were observed. 5. By applying the attached collagen gel method to our cell lines, it was possible to obtain cultured rat epithelial cells with the same morphology as in vivo, in addition to the tumor cell lines. 6. These cultured cell lines of normal rat transitional epithelium appear to be useful a experimental models with which one could clarify the function of the transitional epithelium and the mechanisms of bladder carcinogenesis.     

Expression of the SART3 tumor rejection antigen in renal cell carcinoma

PURPOSE: We recently reported that SART3 tumor rejection antigen is recognized by HLA class I restricted cytotoxic T lymphocytes from patients with esophageal cancer. We now investigate the expression of SART3 antigen in renal cell carcinoma to identify an appropriate molecule that may be used in specific immunotherapy of renal cell carcinoma. MATERIALS AND METHODS: Renal cell carcinoma and nontumorous kidney tissues were obtained at surgery. A section of each sample was minced with scissors and stored at -80C until use. SART3 antigen expression was examined in uncultured renal cell carcinoma and nontumorous kidney tissues. We also evaluated the ability of derived peptides to include cytotoxic T lymphocytes in peripheral blood mononuclear cells from patients with renal cell carcinoma. RESULTS: The SART3 antigen was detected in all renal cell carcinoma cell lines, primary cultures of renal cell carcinoma and nontumorous kidney tissues, and in the cytosol of 57% and 15% of renal cell carcinoma and nontumorous kidney tissues, respectively. HLA-A2402 restricted and tumor specific cytotoxic T lymphocytes (KE4) used in cloning of the SART3 gene were significantly cytotoxic to cells from renal cell carcinoma cell lines and primary cultures of renal cell carcinoma tissue but they did not lyse normal cells, including those from primary cultures of nontumorous kidney tissue. The SART3 peptides derived from positions 109-118 and 315-323 induced HLA-A24 restricted cytotoxic T lymphocytes to renal cell carcinoma cells from peripheral blood mononuclear cells of patients with renal cell carcinoma. CONCLUSIONS: The SART3 antigen and derived peptides may be applied to the specific immunotherapy of HLA-A24+ renal cell carcinoma.