Establishment and characterization of a novel bone-marrow-derived macrophage-like accessory cell line

An accessory cell line, designated line A, was generated from bone marrow stem cells which differentiated in vitro in response to colony-stimulating factor (CSF) in substratum cultures. The cells were found to constitutively secrete large amounts of CSF, the activity of which was neutralized by anti-CSF-1 antibodies. Cells of line A and its supernatants potentiate the suboptimal response of thymocytes to PHA, manifesting an interleukin-1 (IL-1)-like activity. Culture fluids of this line also reconstitute the response to T cell mitogens of spleen cells depleted of adherent accessory cells. It was also found that cells of line A bear low levels of surface Ia, and they efficiently present soluble antigen to proliferating memory T cells. Constitutive prostaglandin secretion, which sometimes masks antigen-presenting capacity, was also demonstrated. Cells of line A are poorly phagocytic, do not secrete lysozyme, and lack Fc and complement receptors. However, they manifest strong cytoplasmic nonspecific esterase staining and an ectoenzyme profile resembling that of elicited inflammatory macrophages. In addition, the cell surface antigen Mac-2 was demonstrated, while stainings with anti-Mac-1 and anti-Mac-3 were negative. Thus, line A may represent a unique subpopulation of immunoregulatory accessory cells, the features of which are discussed.     

Establishment and characterization of a novel dedifferentiated liposarcoma cell line, NDDLS-1

We established a dedifferentiated liposarcoma cell line (NDDLS-1) that produces interleukin-6 (IL-6) and granulocyte-colony stimulating factor (G-CSF). The parental tumor showed high leukemoid reactions. The NDDLS-1 cell line was established from a pleural effusion associated with a lung metastasis. Pleomorphic tumor cells arranged in a haphazard growth pattern were seen in xenograft tumors. Numerous inflammatory cells including neutrophils or eosinophils were present throughout the tumor cells. This finding resembled the dedifferentiated area of the parental tumor. The mice bearing NDDLS-1 showed marked leukocytosis. In addition, the NDDLS-1 cells expressed IL-6 and G-CSF at both the mRNA and protein levels, while the NDDLS-1 cells produced near normal levels of tumor necrosis factor alpha (TNF-α). In the cytogenetic analysis, both the parental tumor and the NDDLS-1 cells showed a ring or giant marker chromosomes. The NDDLS-1 cell line demonstrated the amplification and expression of both MDM2 and CDK4 by fluorescence in situ hybridization and immunohistochemical analysis. The NDDLS-1 cell line is consistent with the parental dedifferentiated liposarcoma, and it should therefore be useful for further investigations of human dedifferentiated liposarcomas.     

小鼠髓母细胞瘤细胞系的建立与鉴定

目的 建立表达PARP-1和P53基因双缺失的小鼠髓母细胞瘤细胞系,为研究髓母细胞瘤（medulloblastoma,MB）发生机制提供细胞模型。方法 对小鼠髓母细胞瘤细胞进行原代培养,体外传代观察,传代30次以上对培养细胞进行形态学观察、细胞标志性蛋白的免疫荧光分析、用Western blot法检测PARP-1蛋白的表达、用含有PARP-1蛋白功能区域的重组质粒pEGFP-C1-Hparp-1和绿色荧光蛋白空质粒pEGFP-C1转染细胞进行检测。结果 新建立的小鼠髓母细胞瘤细胞系,呈多角形、短梭形,免疫荧光分析可见未成熟神经元标志性蛋白Vimentin、Dcx、 III-Tubulin等表达阳性,Western blot检测PARP-1蛋白阴性,导入外源PAPR-1基因后呈阳性。结论 成功的建立了DNA损伤修复蛋白功能缺陷的小脑神经源性髓母细胞瘤细胞系,有助于深入研究髓母细胞瘤的病因及发病机制。     

Establishment and characterization of a novel genetic hybrid cell line for propagation of four pathogens

Chromosomal DNAs were purified from human epidermoid carcinoma (HEP-2) cells and transfected into human embryonic lung (HEL) cells to establish a genetic hybrid cell line susceptible to infections by toxoplasma, rubella virus, cytomegalovirus, and herpes simplex virus. Karyotype analysis showed that the resultant hybrid cells, designated D3, had a chromosome number of 96, which was stable after passage for 100 generations. Direct microscopy and immunofluorescence showed that the D3 cells could be infected by the four pathogens with overt cytopathic effects. The toxoplasma and three viruses were purified from infected D3 cells by sucrose gradient centrifugation and used as the antigens for detection of specific IgG and/or IgM in serum samples from pregnant women with suspicious infections by the four pathogens, the results of which were consistent with those of commercial kits. These data indicate that a stable genetic hybrid cell line has been generated, which is a valuable tool for the isolation of the four intrauterine pathogens and for the preparation of antigens for serological tests.     

Establishment and characterization of a neuroendocrine skin carcinoma cell line

A neuroendocrine skin carcinoma cell line MKL-1 has been established from a nodal metastasis in a 26-year-old patient. The line grows as irregularly outlined, loosely packed floating aggregates lacking central necrosis. MKL-1 is hyperdiploid and has a mean doubling time of 120 hours. Xenografts of 2 X 10(7) MKL-1 cells produce tumors in nude mice at 4 to 6 weeks after subcutaneous inoculation. The xenografts were morphologically indistinguishable from the original skin primary and the nodal metastasis. Electron microscopy revealed sparse membrane-bound neurosecretory granules, and conspicuous, paranuclear aggregates of intermediate filaments. Immunohistochemical study showed diffuse and consistent staining with neuron-specific enolase, while bombesin, adrenocorticotrophic hormone, Leu-enkephalin, substance P, and vasoactive intestinal polypeptide displayed heterogeneous and variable expression. Uniform staining of all cells appearing as cytoplasmic fibrils and paranuclear aggregates was noted with antibodies to cytokeratin. Appreciable amounts of cytokeratin polypeptides 8, 18, and 19 and IT protein were seen on two-dimensional gel electrophoresis of cytoskeletal preparations from MKL-1 cells and from tumor-rich frozen sections. Immunostaining also showed coexpression of neurofilaments arranged in paranuclear aggregates; gel electrophoresis and immunoblotting demonstrated the presence in MKL-1 cells of prominent amounts of the small neurofilament polypeptide. Focal expression of desmoplakin was noted in the xenografts. The cells reacted with monoclonal antibodies anti-Leu-7 and anti-Leu-M1 but did not react with antibodies to human lymphocyte antigens (HLA)-A, HLA-B, and HLA-C. Cytogenetic analysis revealed the presence of 3 chromosomally abnormal cell lines with the majority of metaphase cells demonstrating a gain of an isochromosome of the short arm of chromosome 5. Thus, MKL-1 cell line shares several characteristics with small cell neuroendocrine bronchopulmonary carcinoma cell lines but shows distinct cytogenetic abnormalities.     

兔VX2肿瘤细胞系的建立及其生物学特性的观察

目的建立兔VX2肿瘤细胞系,观察其生物学特性。方法采用小块法对兔VX2肿瘤进行原代培养,体外传代观察,传代40次以上对培养细胞进行形态学观察、组织化学染色、细胞周期检查、核型分析、兔及裸鼠移植。结果新建立的兔VX2肿瘤细胞,呈多角形、短梭形,电镜下细胞内可见张力纤维、细胞间可见桥粒,细胞角蛋白阳性,体外连续培养10个月,传代70次以上,细胞倍增时间为34．5h,细胞周期测定G,期为69．3％,G：期为5．6％,S期为25．1％。染色体为亚三倍体核型,众数为58～62条。同种移植成瘤率100％,裸鼠移植成瘤率100％,无支原体污染。结论兔VX2肿瘤细胞系来源于兔鳞状细胞癌,可用于兔（较大动物）的肿瘤实验研究。     

Establishment and characterization of a new Ewing's sarcoma cell line.

A new human Ewing's sarcoma cell line (CADO-ES1) was established from the malignant pleural effusion of a 19-year-old woman. These cells grew both anchorage dependently and anchorage independently. When cultured in bacteriologic dishes, they grew as tightly packed multicellular tumor spheroids; they were also capable of proliferating in soft agar. Flow cytometric DNA analysis demonstrated a nearly diploid DNA content (DNA index = 0.902). Chromosomal studies of cultured cells showed an isodicentric chromosome 8 in all examined cells, but t(11;22)(q24;q12), a translocation reported previously in Ewing's sarcoma, was not detected. Under normal culture conditions, no morphologic evidence of neural differentiation was detected. In addition, immunocytochemical studies showed that vimentin was intensely positive, whereas neurofilament (NF) and neuron-specific enolase (NSE) were weakly positive. Treatment with cyclic AMP (cAMP) induced pronounced morphologic evidence of neural differentiation and strong expression of NF in cultured cells. S-100 protein, glial fibrillary acidic protein (GFAP), desmin, cytokeratin, and epithelial membrane antigen were not detected immunohistochemically in either untreated or cAMP-treated cells, however. These data suggest that this cell line is derived from a highly undifferentiated neural cell with high chromosomal clonality, differentiating into neural features under certain conditions.     

Establishment and characterization of a new highly metastatic human osteosarcoma cell line

Osteosarcoma is the most common primary malignancy of bone in children and young adults. There is a paucity of tumorigenic and highly metastatic human osteosarcoma cell lines that have not been further transformed by exogenous means. Here we establish and characterize a highly metastatic human osteosarcoma cell line that is derived from a poorly metastatic MG63 line through serial passage in nude mice via intratibial injections. The occasional pulmonary metastases developed from MG63 were harvested and repassaged in mice until a highly metastatic subline (MG63.2) was established. The parental MG63 and highly metastatic MG63.2 cells were further characterized in vitro and in vivo. MG63.2 cells demonstrated increased cell migration and invasion compared to the parental MG63 cells. Conversely, cell adhesion was significantly greater in MG63 cells when compared to the MG63.2 cells. MG63.2 cells grew at a slightly slower rate than that of the parental cells. When injected into nude mice, MG63.2 cells had a greater than 200-fold increase in developing pulmonary metastases compared to the parental MG63 cells. MG63.2 cells also formed larger primary tumors when compared to the parental MG63 cells. Further analysis revealed that ezrin expression was up-regulated in the metastatic MG63.2 cells. Interestingly, expressions of MMP-2 and MMP-9 were down-regulated, and expression of TIMP-2 was up-regulated in the MG63.2 cells. Taken together, we have established and characterized a highly metastatic human osteosarcoma cell line that should serve as a valuable tool for future investigations on the pathogenesis, metastasis, and potential treatments of human osteosarcoma.     

Establishment and characterization of a human gastric carcinoma cell line TMC-1

Established cancer cell lines are useful in the study of various cancers. We established a human gastric carcinoma cell line TMC-1 derived from the lymph node of a moderately differentiated adenocarcinoma of the stomach. TMC-1 cells grew in vitro as a mixture of attached and suspension cells, and exhibited spindle or ovoid morphology. They had a population doubling time of 15 h, a plating efficiency of 61%, formed colonies in semisolid agar, secreted the tumor marker CA 19-9, and were tumorigenic in athymic nude mice. The cells expressed E-cadherin and beta-catenin. The karyotypic analysis demonstrated hyperdiploid features with a modal chromosome of 53. The cell had the deletion at chromosome 18q and gains at chromosome 2p13-25, 5p15, 5q21-35, 7, 8q24, 9q, 11, 12p, 14q24-32 and 20. Analysis by fluorescence in situ hybridization showed the deletion at 7qtel and duplication at 7q11.2 at the rearranged chromosome 7. Growth of TMC-1 cells was inhibited by 27-32% by interferon-alpha (2,000 U/ml) and by interferon-gamma with an IC50 of 125 U/ml. The cell line is tumorigenic in vivo, and its growth is moderately inhibited by interferon-alpha and interferon-gamma. It can be used to develop new modalities of human gastric cancer treatment.     

Establishment and characterization of human metastatic hepatocellular carcinoma cell line

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with a poor prognosis. Recently, we established a HCC cell line from a metastatic HCC tumor. GTG banding analysis was performed and the karyotype showed that this metastatic HCC cell line is a hypertriploid (71-78 chromosomes) with a large marker chromosome containing a long homogeneously staining region (hsr). Comparative genomic hybridization was applied to characterize the chromosomal alterations in this metastatic HCC cell line. The results showed that the hsr was composed of amplified DNA sequences from 11q13. Further characterization of the hsr may lead to the isolation of the putative amplified oncogene at 11q13.     

Establishment and characterization of a cytotrophoblast cell line from normal placenta of human origin

A cell line has been established from human placentae at thefirst trimester of normal pregnancy. The cell line was obtainedby culture of purified cytotrophoblast cells in serum-free mediumsupplemented with epidermal growth factor, insulin, dexamethasoneand 0.1% bovine serum albumin. The cells can be subculturedfor >30 passages in one to three splits. All the cells weremononuclear epithelial-like cells positive to cytokeratin 18,gonadotrophin-releasing . hormone (GnRH), neuropeptide Y, neurotensin,leucine-enkephalin, dopamine and 5-hydroxytryptamine inununo-cytochemicalstaining. The cells secreted GnRH, progesterone and oestradiol(in the presence of testosterone) but little human chorionicgonadotrophin and no -endorphin. The cell line showed humankaryotypes and had a population doubling time of 48 h in serum-freemedium. However, the cells would stop growing in the mediumcontaining fetal bovine serum. A normal cytotrophoblast cellline established in serum-free medium will be particularly usefulin the study of cytotrophoblast cell proliferation and differentiation.     

Establishment and characterization of a new human synovial sarcoma cell line, HS-SY-II.

BACKGROUND: The line of differentiation in synovial sarcoma still remains controversial. Thus far, only a few human synovial sarcoma cell lines have been described. However, their morphologic characteristics have not been fully established. EXPERIMENTAL DESIGN: We established a new synovial sarcoma cell line (HS-SY-II) from pleural effusion with lung metastasis in a typical example of the monophasic spindle cell type. The HS-SY-II cells, in vitro and in vivo, were examined by light microscopy, immunohistochemistry, electron microscopy, and cytogenetics. RESULTS: The HS-SY-II cells showed a hypertriploid karyotype with complex chromosome abnormalities including pathognomonic t(X;18)(p11;q11), and have been stably maintained for more than 40 months in vitro, showing rather small spindle or polygonal shape without conspicuous pleomorphism. Histologic features of initially and serially transplanted tumors in nude mice were essentially the same as those of the original sarcoma, corresponding to the monophasic spindle cell variant with a prominent palisading pattern and calcified foci in parts. The HS-SY-II cells in vitro and in vivo similarly expressed vimentin and cytokeratin by immunohistochemistry, and also exhibit the same ultrastructural features such as irregularly shaped nuclei with prominent nucleoli, many paranuclearly running intermediate filaments, and filopodia-like processes. CONCLUSIONS: This HS-SY-II cell line retaining the distinct morphological characteristics as the monophasic spindle cell type of synovial sarcoma therefore will be extremely useful for various pathomorphologic investigations on synovial sarcoma.     

Establishment and characterization of a human glioblastoma cell line with a stable karyotype and nullisomy 13

A permanent cell line (HeRo) with a stable karyotype (80-84,XXYY) and with defined numerical and structural chromosome aberrations was established from a human glioblastoma, a highly malignant brain tumor. Transformation of these cells with SV40 led to a second permanent cell line (HeRo-SV) with a reduced, but also stable, karyotype (72-74,XXYY). The morphological appearance of the glioblastoma line was similar to the main component of the original tumor tissue. The transformed cells differed from their counterparts in accelerated growth, enhanced growth in soft agar, reduced growth conditions, expression of SV40 T antigen, and altered epitheloid morphology. Both cell lines have been grown in continuous culture for more than 2 years. The stability of both the biologic properties and the karyotypic changes induced by SV40 is quite remarkable. Both lines show a nullisomy 13.