Establishment and characterization of a tumorigenic trophoblast-like cell line from a human placenta

A trophoblast-like cell line, TL, was established from a normal-term human placenta. The TL cells were epithelial in morphology with relatively large vesicular nuclei, prominent nucleoli, and numerous microvilli on the cell surface. Cytoplasmic organelles were generally sparse but mitochondria and polysomes were abundant. The cells grew as compact sheets with close membrane approximation interconnected occasionally by desmosome-like junctions. TL cells contained placental alkaline phosphatase, a placenta-associated antigen, cytokeratin, and prekeratin, but not keratin. In parallel, they were negative for factor VIII, vimentin, and fibronectin. Population doubling time was estimated to be about 34 h. TL cells were tumorigenic in nude mice and an increase in tumorigenicity was observed after a certain number of passages in vitro. Chromosome analysis revealed that TL cells were highly heterogenous and had a female aneuploid karyotype with a hypotriploid mode. Unlike trophoblastic cell lines established from neoplastic tissues, TL cells did not synthesize human chorionic gonadotropin or other gonadal hormones, and only a small amount of ferritin (40.3 ng/10(6) cells) could be detected in the cell supernatant and cell extract. Based on various morphological and histochemical criteria, we suggest that the TL cells are derived from the Langhans cells (villous cytotrophoblast), and due to their special features, the cells may be valuable for the study of the differentiation and tumorigenesis of trophoblastic cells.     

Establishment and characterization of a tumorigenic cell line from areca quid and tobacco smoke-associated buccal carcinoma

A cell line, TW2.6, has been established from the surgically resected specimen of an untreated primary squamous cell carcinoma of the buccal mucosa from a 48-year-old man who was an areca quid chewer and tobacco smoker. TW2.6 cells exhibited morphological features of keratinocytes and replicated rapidly in culture with a doubling time of 24h. The karyotype showed human chromosomes with high hyperdiploidy and complex rearrangements. Western blotting showed pronounced expression of p53 and moderate expression of p21(CIP1). The baseline expressions of p27(KIP1) and p16(INK4a) were barely detectable. Low levels of Bax and Fas were found in TW2.6 cells but Bcl-2 expression was more readily observed. Mutational analysis of p53 gene revealed an A-->G transition at the second base of codon 220, resulting in amino acid substitution from tyrosine to cysteine in the protein. Functional analysis showed that TW2.6 was unable to activate the p53-specific PUMA promoter. Lipofectamine 2000 and calcium phosphate precipitation technique offer good transfection efficiencies for TW2.6 cells and may be used in future transfection experiments. A xenograft-SCID mouse tumor model was established for TW2.6. Histological examination demonstrated that the engrafted tumors maintained the morphological features of a squamous cell carcinoma. It is thought that the establishment of tumorigenic TW2.6 cell line provides a valuable model for AQ and tobacco smoke-associated buccal carcinoma.     

Establishment and characterization of a murine megakaryoblastic cell line growing in protein-free culture (L8057Y5)

A murine megakaryoblastic cell line growing in protein-free culture (L8057Y5) was established from an experimentally induced murine leukemia (MK8057). Most of the Y5 cells were small and blast-like, with 2-4N in DNA content. Also, large cells possessing a lobulated nucleus characteristic of megakaryocytes, which showed polyploidization to more than 4N up to 16N, were occasionally seen. Nearly 5% of the total number of Y5 cells were positive for acetylcholinesterase reaction. The survival time of C3H/He mice after injection with Y5 cells was longer than that of mice injected with the original MK8057 cells. The colony-forming ability of Y5 cells in the spleen of the lethally irradiated mouse was much lower, whereas the number of in vitro colonies derived from Y5 was greater than that of MK8057. The plating efficiency of colony formation in serum-free methylcellulose culture was higher at a low O2 tension. Conditioned medium of Y5 cells enhanced colony formation as well as 3H-TdR uptake by Y5 cells, which implies that Y5 cells may produce autocrine growth factor(s). mRNAs for IL-6, LIF, and INF-gamma were expressed in Y5 cells; these cytokines may have roles in the growth mechanisms of the cell line.     

Establishment and characterization of a human myeloma cell line (KMM-1)

A new cell line (KMM-1) was established from a subcutaneous plasmacytoma of a 62-year-old male with multiple myeloma. Immunological studies indicated that cultured cells were derived from the same clone of myeloma cells in vivo: smeared cells were stained with fluoresceinconjugated globulin of antisera monospecific to lambdachain, and lambda-chains in the cell extracts and in culture media were identical to the Bence-Jones protein found in the patient's urine. The cell line grew in suspension with a doubling time of 36-40 h and contained primitive plasmablastoid cells with prominent nucleoli and rough endoplasmic reticulum. Cells had the karyotype of 47, X, -Y. lq+, -2, +t(1:2) (cen:cen), +7, 12q+, 14q+, +mar and carried no Epstein-Barr virus-determined nuclear antigen. Surface markers were as follows; E rosette (-), IgG Fc receptor (-), C3 receptor (-), S-lg (+), TdT (-), asialo-GM, (-). The reasons for the successful establishment of the myeloma cell line are discussed.     

Establishment and characterization of a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung

We established a murine osteosarcoma cell line (LM8) with high metastatic potential to the lung from murine Dunn osteosarcoma using 8 repeated Fidler's procedures. We performed the biological characterization of the LM8 and the maternal Dunn cell lines in vitro and in vivo. Morphologically, LM8 possesses many fillopodial protrusions, lamellipodial structures surrounding the cell surface and membrane ruffles suggesting enhanced cell motility. The increased matrix metalloproteinase (MMP) 2 activity of this cell line might help cell invasion after penetration of the endothelial (mesothelial) cell layer. This cell line exhibited high in vitro invasive activity when seeded onto the mesothelial cell monolayer. Higher expression of VEGF mRNA in this cell line might facilitate neovascularization at the site of metastaisis, resulting in extremely high metastaic potency after i.v. injection. LM8 also showed a high metastic incidence (7/7) to the lung even after s.c. transplantation into the back space of mice. This cell line can provide an excellent tool for studying inhibitory agents against pulmonary metastasis as well as the various important factors involved in metastasis of osteosarcoma. Int. J. Cancer 76:418–422, 1998.© 1998 Wiley-Liss, Inc.     

Establishment of a highly tumorigenic LNCaP cell line having inflammatory cytokine resistance

Human androgen-dependent prostate cancer LNCaP cells are low tumorigenic even in immunodeficient mice and were killed by the synergistic effect of inflammatory cytokines, IL-beta and IL-6. To establish a highly tumorigenic LNCaP cell line, we isolated the cytokine-resistant LNCaP-CR cell line and examined the phenotypes. The parental LNCaP cells were induced to commit apoptosis by the addition of IL-1beta and IL-6, but LNCaP-CR cells showed strong resistance against the cytokine action. However, LNCaP-CR cells did not exhibit any resistance to various antitumor drugs investigated. While LNCaP cells formed only palpable tumors in SCID mice, LNCaP-CR cells readily made tumors and their growth was significantly higher than that of LNCaP cells. Moreover, LNCaP tumor-bearing mice gained the weight gradually, but LNCaP-CR tumor-bearing mice significantly lost their body weight. LNCaP-CR cells still responded to androgen action and expressed AR, erbB2, IL-1R, IL-6R, gp130, STAT3, p21, Bcl-2 and caspase-3 as well as LNCaP cells. These results indicate that LNCaP-CR cell line is a new type of tumorigenic LNCaP cell lines and should be useful for identifying responsible genes of tumorigenicity, cytokine resistance, and also cachexia.     

Establishment and characterization of a novel nasopharyngeal carcinoma cell line (SUNE2) from a Cantonese patient

The undifferentiated form of nasopharyngeal carcinoma (NPC) is the most common malignant head and neck cancer in South China, especially in Cantonese populations. However, few NPC cell lines have been established from the patients in this region. In this study, we established a new NPC cell line, termed SUNE2, from a Cantonese patient with undifferentiated NPC. This cell line had extremely low concentrations of Epstein-Barr virus (EBV) DNA in long-term culture and expressed low levels of latent membrane protein 1 (LMP1), latent membrane protein 2A (LMP2A), BamH1-A right frame 1 (BARF1), EBV-encoded RNA-1 (EBER1), and EBV-encoded RNA-2 (EBER2) in early passages. SUNE2 cells also showed much stronger transforming ability than 5-8F cells in colony formation assays and anchorage- independent growth assays in soft agar, and they only need 2 weeks to form tumors in nude mice. In summary, the SUNE2 cell line is a new in vitro model that can be used for further research on the mechanisms underlying the occurrence and development of NPC.     

Establishment and characterization of a human IgA-kappa-secreting plasma cell line (MT3)

We have established a new human plasma cell line from the peripheral blood of a patient with an IgA-kappa plasma-cell leukemia. Morphological, immunological, cytogenetic and molecular studies confirm that the cultured cells are derived from the same clone of leukemic plasma cell in vivo. The established cell line (MT3) grows in suspension, secretes high amounts of IgA kappa and exhibits morphological and ultrastructural characteristics of plasma cells. Surface marker analysis shows that both primary and cultured cells express the plasma-cell-associated antigens PCA-1 and T10, while specific B- and T-cell determinants and EBV nuclear antigen are undetectable. In the established cell line a few cells express Ia-like and CALLA antigens. Cytogenetic analysis of MT3 cells reveals a prevalent hypertriploid karyotype with constant chromosomal aberrations consisting of 14q+, 22q- and marker chromosomes.     

Establishment and characterization of a human EBV-negative B cell line (MN 60)

A permanent cell line, MN 60, was established from the peripheral blood of a patient with an acute lymphoblastic leukemia (ALL) classified morphologically as being of the L3 type. Cell growth started rapidly in vitro and no feeder cells were needed. Cells of the MN-60 line were identical to the original leukemic cells with respect to surface immunoglobulin (Ig) expression and karyotype, including the presence of four marker chromosomes [1q+, 6q-, t(8;14)]. Continuous proliferation was maintained in stationary suspension culture with a doubling time of 25 h. The cells were tumorigenic in athymic nude mice and had the capacity to form colonies in semi-solid medium in vitro. Monoclonal surface Ig (mu lambda) was demonstrated whereas no cytoplasmic immunoglobulin could be demonstrated. The MN-60 cells were Epstein-Barr virus (EBV) negative as evidenced by EBNA tests and by nucleic acid hybridization studies. The cells expressed HLA-A-C, HLA-DR. beta 2-Microglobulin and cALL, but not Fc gamma. C3, sheep and mouse red blood cell receptors. No reactivity was found with anti-glycophorin A or the anti-BL 38.13 monoclonal antibody. Cell growth was retarded in the G0/G1 phase of the cell cycle after incubation with leukocyte interferon, hydrocortisone, phorbol myristate acetate and dimethyl sulphoxide.     

Establishment and characterization of a new human Burkitt's lymphoma cell line (WSU-BL)

A new human cell line, WSU-BL, was established from a malignant ascitic fluid occurring in a patient with Burkitt's lymphoma. The established line grows in a single-cell suspension with a doubling time of 19 hours and expresses L3 morphologic features by the French-American-British classification. Immunologic study revealed that WSU-BL cells express IgM-lambda both in the cytoplasm and on the surface and react with monoclonal antibodies to B-cell antigens (B1, B4, BL3, BL4, HLA-DR, and common acute lymphoblastic leukemia antigen [CALLA]). These cells are negative for T-cell and myeloid/monocyte antigens as well as Epstein-Barr virus nuclear antigen (EBNA). These results suggest that WSU-BL corresponds to an intermediate stage of B-cell differentiation. Both fresh tumor and WSU-BL cells had a hyperdiploid karyotype carrying the 8;14 chromosome translocation. Molecular studies showed that WSU-BL has a rearrangement of c-myc proto-oncogene and expresses c-myc RNA. Phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) and interferon-gamma (IFN-gamma) were able to induce several phenotypic changes on WSU-BL cells. Two-dimensional gel electrophoresis of total cellular protein showed that either TPA or IFN-gamma induced both the synthesis or loss of several proteins. Analysis of the protein patterns indicated that some proteins were uniquely responsive to either TPA or IFN-gamma and others were common to both. This cell line should be valuable for future studies of cell proliferation, differentiation, and oncogenesis concerning this neoplasm.     

Establishment and characterization of a continuous lung squamous cell carcinoma cell line (U-1752)

A continuous cell line, U-1752, was established from a lung tumor originally diagnosed as a small cell carcinoma. The cell line has been in continuous in vitro passage for 29 months. The epithelial, rather than small cell nature of the U-1752 cells was demonstrated by the presence of desmosomes, prominent tonofilament bundles, by the reactivity with an anti-keratin antiserum and by the expression of cell surface receptors for epidermal growth factor (EGF). The U-1752 cells grow as monolayer cultures and have a population doubling time of around 36 hours at optimal growth in 20% calf serum. The most important neoplastic features of U-1752 were its aneuploidy, its capacity for colony formation in agarose and its tumorigenic potential subcutaneously in nude mice.     

Establishment and characterization of an oral melanoma cell line (ME)

A new cell line, ME, has been established from a melanoma of the palatal mucosa. The cultured monolayer of cells was fusiform and melanin-producing. The cells were highly tumorigenic and metastatic in nude mice. The xenographic tumors resembled the original tumor in morphology, melanin production, and the expression of S-100 and HMB-45 antigens. The metaphase karyotype of ME indicated multiple aberrations of chromosomes 2, 3, 5, 7-11, 13, 19, 21 and X. A homozygous loss of the p16/MTS1 gene during the establishment of ME correlated with karyotypic evidence of chromosome 9 abnormalities. Absence of nm23 protein expression and elevated expression of CD44 protein (indicative of metastatic phenotypes) were demonstrated in primary and xenographic tumors. ME cells could be valuable in developing novel therapeutic strategies for oral melanoma.     

Establishment and characterization of a woodchuck hepatocellular carcinoma cell line (WH44KA)

A continuous cell line was established from a hepatocellular carcinoma obtained from a woodchuck that was sero-positive for woodchuck hepatitis virus (WHV). The cell line, designated WH44KA, grows as an adhering monolayer with a doubling time of 36 hr in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. The cells not only showed epithelial origin on light and electron microscopic examination but also possess biosynthetic markers of the latter, such as albumin and alpha-fetoprotein, which were demonstrated in cultured cells. When they were transplanted into athymic nude mice, tumors developed at the site of inoculation. These tumors were shown to be hepatocellular carcinoma, similar in morphology to the original tumor from which the WH44KA cells were derived. Chromosome analysis revealed a chromosome number ranging from 31 to 126, with a modal number of 35. Integration of WHV DNA was shown by Southern blot analysis. However, WHV surface antigen was not demonstrated in the cultured cells or supernatant medium. The WH44KA cell line appears to be a useful in vitro model for the study of virus-induced hepatocellular carcinoma.     

Establishment and characterization of a human urinary bladder carcinoma cell line (TSGH-8301)

A cell line derived from a well-differentiated human transitional cell carcinoma of the urinary bladder, designated TSGH-8301, was established in vitro. The cultured epithelioid cells exhibited monolayer growth and loss of contact inhibition. The tumorigenicity of TSGH-8301 had been shown by growth in soft agar and tumor induction in athymic nude mice. A reverse ratio of lactate dehydrogenase (LHD) isoenzyme in the cell line and nude mouse-grown tumors was seen predominantly with LDH-V. Chromosomal analysis revealed a heterodiploid stem line with a modal number of 50. Sera of urinary bladder cancer patients reacted with membrane antigens of the TSGH-8301 cells, suggesting the existence of tumor-associated antigens in the cells. In vitro chemosensitivity tests of these cells may provide data valuable in the selection of proper anticancer drugs for the TSGH-8301 donor patient.     

Establishment and characterization of a human acute monocytic leukemia cell line (THP-1)

A human leukemic cell line (THP-1) cultured from the blood of a boy with acute monocytic leukemia is described. This cell line had Fc and C3b receptors, but no surface or cytoplasmic immunoglobulins. HLA haplotypes of THP-1 were HLA-A2, -A9, -B5, -DRW1 and -DRW2. The monocytic nature of the cell line was characterized by: (1) the presence of α-naphthyl butyrate esterase activities which could be inhibited by NaF; (2) lysozyme production; (3) the phagocytosis of latex particles and sensitized sheep erythrocytes; and (4) the ability to restore T-lymphocyte response to Con A. The cells did not possess Epstein-Barr virus-associated nuclear antigen. These results indicate that THP-1 is a leukemic cell line with distinct monocytic markers. During culture, THP-1 maintained these monocytic characteristics for over 14 months.     

Establishment and characterization of a human neuroblastoma cell line

A continuous human cell line RN-GA was established from a stage-III primary neuroblastoma prior to therapy. Light and electron microscopic analysis of the biopsy showed morphological features typical of neuroectodermal origin. Relative cellular DNA content and N-myc oncogene copy number were also analyzed in the biopsy tissue: the tumor cells presented a near-diploid genome with N-myc amplification. The derived tumor cell line expressed distinctive ultrastructural, cytogenetic and immunological markers of neuroblastoma. Moreover, cells from the culture could be serially transplanted into splenectomized-irradiated nude mice, where they formed a progressively growing solid tumor. Surprisingly, the cells in culture did not show any N-myc amplification, while they retained a near-diploid DNA content. We propose that several techniques (electron microscopy, oncogene analysis, flow cytometry, cytogenetics, tissue culture, cell antigen immunodetection) should be used to establish a firm diagnosis and a correct clinical grading of this tumor. The establishment of this continuous cell line should be valuable as an experimental in vitro system for further studies of neuroblastoma biology and morphology.     

Establishment and characterization of a leukemic murine cell line derived from MCF 247 MuLV-induced T-cell lymphoma

Understanding of the leukemic evolution of human non-Hodgkin's lymphomas is hindered by the lack of appropriate animal models. For this purpose, a highly leukemic cell line NQ22, derived from a MCF 247 murine leukemia virus (MuLV)-induced murine T-cell lymphoma, was established, and its preliminary characterization is described. The NQ22 cell line is easily transplantable subcutaneously (s.c.) into syngeneic AKR mice exhibiting early peripheral blood invasion and widespread dissemination with a leukemic pattern of infiltration. Such peculiar in vivo behavior is a stable phenotypic feature, probably determined genetically. Biological and differentiation characteristics of the NQ22 cell line were analyzed and compared to those of other non-leukemic T-lymphoma lines. In addition, no evidence of possible involvement of plasminogen activator (PA) enzymes and of their inhibitors (PAI) in the spreading ability of NQ22 cells was observed.     

Characterization of a tumorigenic murine T-lymphoid-cell line spontaneously derived from an IL-2-dependent T-cell line

The establishment of IL-2-independent T-cell lines spontaneously derived from long-term IL-2-dependent cytotoxic T-cell lines is described. Two lines (cloned and uncloned) studied in detail have shown the following characteristics: (1) Permanent loss of IL-2 dependence. (2) Partial or complete loss of both cytotoxic activity and the IL-2 receptor. (3) Increased expression of T-cell membrane markers (Thy1.2, Lyt1.2) compared with the parental line. (4) Lower level of DNA methylation than in freshly obtained lymphoid cells. (5) Different karyotypic pattern from the parental IL-2-dependent line, with a mean number of 39-40 chromosomes and a resemblance to T leukemic lines. (6) Leukemia caused in normal syngeneic C57BL/6 mice by the uncloned line, in contrast to the cloned IL-2-independent line or the parental dependent line. Unlike established leukemic lines, however, the independent line gave rise to tumors which regressed in some mice within a few days of their appearance. These findings suggest that T-cell lines maintained with IL-2 for prolonged periods of time (greater than 3 months) can undergo transformation and, therefore, should not be utilized for immunotherapeutic purposes.     

Establishment and characterization of a schwannoma cell line from a patient with neurofibromatosis 2

By using retroviral mediated gene transfer technique, a primary schwannoma culture from a 56-year-old Neurofibromatosis type 2 (NF2) patient was immortalized with HPV E6-E7 genes. This cell line, HEI193, has a unique splice site mutation of the NF2 gene. Both immunocytochemistry and molecular biology techniques were used to demonstrate that this cell line is of Schwann cell origin. Comparison of the primary tumor with HEI193 revealed the same NF2 mutation and an identical pattern of allele loss at multiple loci, indicating that the established cell line had maintained many of the properties of the original tumor. The immortalized cell line was non-tumorigenic in both severe combined immunodeficient (SCID) mice and nude mice, but has altered growth properties such as higher proliferation rate and independence of Schwann cell growth factors. To our knowledge, this is the first attempt to establish permanent cell lines from human NF2 patients. This Schwann tumor-derived cell line may provide a useful model system for the study of familial NF2 tumor pathogenesis, for elucidating NF2 functions and for testing new gene-based therapeutic approaches.     

Establishment and characterization of a cell line (T201) derived from a human larynx squamous cell carcinoma

The purpose of this report was the initiation and further maintenance of tumor cells from a primary larynx squamous cell carcinoma. A tumor fragment was mechanically dissociated, the cells were grown in RPMI medium, being the primary culture dependent on the presence of epidermal growth factor and insulin; during subsequent passages the adaptation to conventional growth conditions was obtained. Cells grew in monolayer with an epitheliod shape, showing a pavement-like arrangement; at confluence, cells piled up without contact inhibition maintaining the same morphology. Population doubling time was about 48 h with a colony-forming efficiency of 10%. Immunocytochemical characterization was performed with a panel of monoclonal antibodies reactive against tumor associated antigens, including mucin glycoproteins and related carbohydrate antigens, carcinoembryonic antigen (CEA), p53 as well as cytokeratins, vimentin and desmin. T201 expressed CEA, sialyl Lewis x, Lewis x, Lewis y, MUC1 mucin, Tn hapten, p53, vimentin and cytokeratins. On the other hand, a modal chromosome diploid number of 46 occurring in 74% of cells was detected. Present data confirmed that the methodology employed was adequate for the establishment and characterization of a new cell line which can provide a useful model to study biological and immunological aspects of larynx squamous cell carcinoma.