Phenotypic and cytogenetic characteristics of a new Epstein-Barr virus negative cell line (SKW 4) derived from a B-cell lymphoma

A new Epstein-Barr virus nuclear antigen (EBNA) negative cell line SKW 4 has been established in vitro from a patient with diffuse histiocytic lymphoma. The SKW 4 seems to be an authentic human tumour cell line as evidenced by its EBV negativity, monoclonality and aneuploidy tested during early in vitro passage. The cell line expresses surface mu and kappa-chains, HLA-DR antigen, C3 and Fc receptors and B-cell lineage antigens. The karyotypic analyses demonstrated many numerical and structural aberrations. No Burkitt lymphoma associated translocations (t8;14, t2;8, t;22) were detected, but most of the markers found are those commonly associated with various types of human cancer. The SKW 4 thus represents the most common type of 'histiocytic lymphoma', that with a B-lymphoid cell phenotype, but is unique among HL derived lymphoma lines in its strong expression of a Helix pomatia A agglutinin binding surface glycoprotein of an apparent molecular weight of 75 000 daltons.     

Establishment and characterization of a pancreatic carcinoma cell line derived from malignant pleural effusion

BACKGROUND/AIMS:A novel cell line, designated p34, was developed from the malignant pleural effusion of a patient with carcinoma of pancreas. The objective of this work was to characterize this cell line. METHOD: The in vitro studies included karyotype analysis, immunohistochemistry, XTT cell proliferation assay, analysis of the cell cycle by FACS and cell sensitivity to chemotherapeutic drugs and irradiation. Subcutaneous and intra-spleen inoculations into nude mice were carried out to study the tumorigenicity and the metastatic tendency of this cell line. RESULTS: The p34 cell line showed typical morphological characteristics of epithelial pancreatic tumor cells. The cells were hyperdiploid with a modal number of 48, and had two markers, deletion in the short arm of chromosome 2 and duplication of the short arm of chromosome 8. The doubling time was 16 h. Subcutaneous inoculation of the cells into nude mice yielded 100% tumorigenicity, and intra-spleen inoculation resulted in extensive intra-abdominal spread. The antiproliferative effect of chemotherapy (gemcitabine, cisplatin, taxol and vinorelbine), chemopreventive agents (celecoxib and curcumin) and radiotherapy showed dose-dependent cytotoxicity. CONCLUSIONS: This p34 cell line can be used as a new model for studying various aspects of the biology of human pancreatic cancer and potential treatment approaches for the disease.     

Establishment and characterization of a new cell line from primary human breast carcinoma

Summary A new cell line (BRC-230) was established from surgical material of primary ductal infiltrating breast carcinoma. The epithelial nature of this cell line was confirmed by ultrastructural analysis and demonstrated the retention of structural properties characteristic of the original tumor. The BRC-230 cell line induced tumor in athymic Cr1:nu/nu(CD-1)BR nude mice, it possessed an abnormal karyotype with a modal chromosome number between 60–61 with eight recurrent marker chromosomes, and it presented a doubling time of 30.5 hr. Scatchard analysis demonstrated that both primary tumor and BRC-230 cells were estrogen and progesterone receptor negative.Immunoenzymatic and radioimmunoassays showed a production of marker antigens (CEA, TPA, CA125, CA15-3, CA19-9) which was similar in the patient's serum and BRC-230 cells. Thein vitro drug sensitivity assay of the cell line and of the parental tumor tissue showed overlapping results to all tested antiblastic drugs. BRC-230 cells were resistant to 4-Idroperoxy-cyclophosphamide, Idarubicinol, Mitoxantrone, Etoposide, 4Epidoxorubicin, and Doxorubicin, showing a multiple drug resistance phenotype. Amplification or rearrangement of Her-2neu, Ha-ras, and C-myc genes was observed neither in the original tumor nor in BRC-230 cells; the mdr-1 gene was also present in a single copy.We conclude from these studies that the BRC-230 cell line maintains the same characteristics as the original tumor and may provide us with a good model to studyin vitro the biology of drug resistance of breast cancer.     

Cytokeratin characterization of human prostatic carcinoma and its derived cell lines

Two murine monoclonal anti-cytokeratin antibodies with defined specificity were shown to distinguish between basal cells and luminal cells in human prostate tissue. Forty-one biopsies or transurethral resection specimens were characterized using these two antibodies. In cases of benign prostatic hyperplasia, focal loss of the basal cell layer was noted in areas of glandular proliferation. Ten cases of adenocarcinoma of the prostate, varying in Gleason's histological grade from 2 to 4, were also studied. In each case the carcinoma was shown to represent the luminal cell phenotype with no evidence of involvement of the basal cell phenotype. An analysis of three established metastatic prostatic carcinoma cell lines (DU-145, PC-3, and LNCaP) using two-dimensional electrophoresis showed that the cytokeratin complement of each cell line was slightly different but retained the phenotype of the luminal cell. It was concluded that during both hyperplasia and neoplastic transformation of the prostate, the luminal cell phenotype is primarily involved and that the basal cell phenotype does not appear to contribute to either intraluminal proliferation or invasive cell populations.     

Phenotypic, cytogenetic and molecular characterization of a new B-chronic lymphocytic leukaemia (B-CLL) cell line

A lymphoid cell line was established from a patient with B-cell chronic lymphocytic leukaemia (B-CLL) by infecting blood lymphocytes with Epstein-Barr virus (EBV). Immunoglobulin gene rearrangement studies and the presence of a chromosome marker (isochromosome 17q) provided the formal proof that the line has originated from the neoplastic B cells. The morphology and phenotype indicate that the EBV-induced cell line has reached a plasma cell-like stage of differentiation.     

Establishment and characterization of continuous cell line MTC-SK derived from a human medullary thyroid carcinoma

Tumor cells of a human medullary thyroid carcinoma were isolated and propagated in tissue culture. Several cell lines with different morphology developed from the primary culture, among others a fibroblast-like growing cell line (MTC-F) and a cell line growing as a suspension of single cells and spherical cell clusters (MTC-SK). The MTC-SK cell line was serially propagated for 90 passages, over 3 years. When examined at different times throughout the in vitro period, MTC-SK exhibited properties characteristic of medullary thyroid carcinomas: the cells maintained their epithelioid morphology; endocrine granules were demonstrated in the cytoplasm by electron microscopy; in situ hybridization confirmed the production of calcitonin- and bombesin-mRNA (gastrin releasing peptide); the cells revealed positive immunoreactivity with antibodies to calcitonin, calcitonin gene-related peptide, and bombesin. The in vitro properties of the MTC-SK cells corresponded to the results obtained from the tissue of origin. Cytogenetic studies of the MTC-F cell line revealed a supernumerary metacentric chromosome (20?). In the MTC-SK cell line the predominant findings were terminal chromosomal rearrangements most frequently concerning chromosome 11p, i.e., the locus of the calcitonin and calcitonin gene-related peptide genes and the H-ras oncogene, and a characteristic instability of the centromeric region of chromosome 16 and somatic pairing of the homologous chromosomes 16.     

Establishment and characterization of a novel neuroendocrine carcinoma cell line derived from a human ascending colon tumor

The incidence of rare neuroendocrine tumors (NET) is rapidly increasing. Neuroendocrine carcinoma (NEC) is a NET with poorly differentiated histological features, high proliferative properties and associated poor prognoses. As these carcinomas are so rare and, thus, affect only a small number of patients allowing for few cell lines to be derived from patient biopsies, the histological, immunohistochemical, and clinical characteristics associated with colorectal NEC and NEC in other organs have yet to be clearly defined. Herein, we describe the establishment of a novel NEC cell line (SS‐2) derived from a tumor resection of the ascending colon from a 59‐year‐old Japanese woman. The histological, electron microscopic and immunohistochemical features of chromogranin A (CgA) as well as confirmation of synaptophysin positivity in this tumor were typical of those commonly observed in surgically resected colorectal NEC. Further, the Ki‐67 labeling index of the resected tumor was >20% and, thus, the tumor was diagnosed as an NEC of the ascending colon. The SS‐2 cell line maintained characteristic features to those of the resected tumor, which were further retained following implantation into subcutaneous tissues of nude mice. Additionally, when SS‐2 cells were seeded into ultra‐low attachment plates, they formed spheres that expressed higher levels of the cancer stem cell (CSC) marker CD133 compared to SS‐2 cells cultured under adherent conditions. SS‐2 cells may, therefore, contribute to the current knowledge on midgut NEC biological function while providing a novel platform for examining the effects of colorectal NEC drugs, including CSC.     

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.     

Establishment of a novel cell line derived from nasopharyngeal carcinoma

A novel epithelial cell line (designated as HNE-1), derived from nasopharyngeal carcinoma (NPC), was established and has passed more than 100 generations over one year. The NPC biopsy specimen was taken from a 27 year old man with poorly differentiated squamous cell carcinoma of the nasopharynx. The cultured cells showed polygonal shape and grew into multilayers under the inverted microscope. Electron microscopy showed that HNE-1 cells were characterized by bi-directional differentiation with some being poorly differentiated squamous carcinoma and the other poorly differentiated adenocarcinoma cells. A continuous positivity was showed by EBNA at subcultures 5-81. Tumorigenicity was demonstrated by heterotransplantation in BALB/c (nu/nu) mice, developing into well differentiated squamous carcinoma. Karyotype analysis showed aneuploidy with the modal chromosomal number 74-101 (5th-20th passages) and 15 marker chromosomes. The frequency of spontaneous sister chromatid exchange was very high in HNE-1 cells (87.6 +/- 0.4/cell).     

Establishment and characterization of a new cell line derived from a human chondrosarcoma

A new human cell line, CAL 78, derived from a dedifferentiated chondrosarcoma of the muscle of the thigh has been established in culture. Fibroblastoid morphology, vimentin expression and lack of epithelial antigens are in agreement with mesodermic origin of these cells. The xenograft of CAL 78 cells in nude mouse showed the characteristics of hyaline cartilaginous differentiation. Cytogenetic changes were numerous and complex, all the metaphases were tetraploid and no alterations described by other authors have been found. CAL 78 constitutes an appropriate model to evaluate efficiency of a new therapy for chondrosarcomas. Moreover, this cell line may be used to study some stage of chondrocytic differentiation.     

Biological and karyotypic characterization of a new cell line derived from human gliosarcoma

A cell line (NCE-G28) was established from the biopsy material of a human gliosarcoma of low histological differentiation. The initial cultures showed a mixed population of cells which in later stages became more uniform due to loss of slower growing constituents. The cells have been growing steadily for 20 months. A suspension of NCE-G28 cells injected s.c. as well as i.p. into nude mice produced solid tumors in all cases. Histologically these tumors closely resembled the original tumor. The original tumor, the nude mouse tumor, and NCE-G28 cells were immunochemically positive for glial fibrillary acidic protein as well as for neural plasma membrane antigen A2B5 expression. Two cell strains, 9B2C and 9B2E, were obtained by cloning of the initial cultures and another strain, NCE-G28T, was derived after explantation of a mouse heterotransplant. The two subclones were negative for glial fibrillary acidic protein expression but stained for cell surface fibronectin. NCE-G28T cells initially were positive for glial fibrillary acidic protein but lost this property within 8 months of cultivation. Karyotype analysis of NCE-G28 and the three strains revealed hyperdiploidy and six structurally altered marker chromosomes five of which were shared by nearly all cells. Receptors for epidermal growth factor were detected in all cell lines with the highest levels (about 300,000 receptors/cell) in the parental cell line. The epidermal growth factor receptors had an affinity of 2.5 nM (Kd) and by affinity cross-linking analysis a molecular weight of 170,000 was found. Initially, NCE-G28 cells responded to epidermal growth factor as well as fibroblast growth factor with increased rates of proliferation, while platelet derived growth factor had no effect. In higher passages the growth factor sensitivity was reduced. Using antibodies directed against synthetic protooncogene peptides the production of c-sis immunoreactive material was detected. NCE-G28 cells produce an autocrine factor which stimulated proliferation. This factor is present in conditioned medium and is active on cultured meningiomas and other glioma cell lines. NCE-G28 cells can be maintained in serum-free defined medium on plastic coated with fibronectin or an extracellular matrix from bovine corneal endothelial cells. The NCE-G28 cell line with its strains provide an in vitro model system in which the complexity of gliosarcoma cell populations and the interaction of the cloned cellular constituents can be studied.     

Isolation and partial characterization of surface components of cell line MDA-MB-231 derived from a human metastatic breast carcinoma

Neuraminidase (Vibrio cholerae) treatment of human metastatic mammary carcinoma MDA-MB-231 cells grown in culture released 0.60-0.63 mg of N-acetylneuraminic acid from 10(9) cells. Incubation of intact cells with a modified trypsin and fractionation by gel filtration gave mainly O-glycopeptides. The presence of O-glycosyl-linked chains having one or two carbohydrate residues was confirmed by treatment of the glycopeptide fractions with galactose oxidase, followed by reduction with alkaline sodium borotritide and fractionation. The major glycopeptide fraction, which consisted of 53% carbohydrate and 47% protein, and a minor glycopeptide fraction each inhibited hemagglutination by peanut lectin. These results suggest the presence of O-beta-D-galactopyranosyl-(1 leads to 3)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1 leads to 3)-L-serine (threonine) residues. The absorptive capacities for anti-HLA-A2 and anti-HLA-B8 antisera were slightly greater for intact than for lyophilized cells, which suggested that masking of these major histocompatibility antigens did not occur in intact cells.     

Establishment and characterization of a human prostatic carcinoma cell strain obtained from exfoliated tumor cells after prostatic massage

During a control campaign connected to our main program on early diagnosis of prostatic carcinoma through tissue culture of prostatic fluid samples obtained after prostatic massage (M. Bologna et al., Eur. Urol., 14, 474-476, 1988), we isolated and characterized a human prostatic carcinoma cell strain from a 58-year-old patient with a grade III prostatic carcinoma. The epithelial cell strain, named PMU-23, has been passaged in vitro for 31 subculture cycles during a period of approximately 8 months, after which cell proliferation slowed down irreversibly. The isolation of this cell strain constitutes a renewed confirmation of the validity of our method for the early diagnosis of prostatic carcinoma and demonstrates some intermediate features in the progression of prostatic tumors. In addition, the study of limited-lifespan tumor cell strains in culture may extend the knowledge on prostatic cell biology, particularly toward the identification of intermediate steps of tumor progression, for a better approach of tumor therapy and prevention of metastatic spread.     

Phenotypic analysis of an established cell line derived from a patient with Hodgkin's disease (HD)

This report describes the phenotypic analysis of a cell line obtained from a female patient with the nodular sclerosing subtype of Hodgkin's disease (HD). The cell line has a neoplastic karyotype and is stable in culture in the absence of feeder layers or growth factors. Phenotypic analysis of this cell line shows that it cannot easily be characterized as either a lymphocyte, macrophage or granulocyte but resembles in its characteristics certain HD lines already described in the literature. The cell line carries the antigen defined by the Ki-1 monoclonal antibody, shows myeloid markers on a proportion of cells and has cytoplasmic UCH-T1.     

Establishment and characterization of a cell line from smokeless tobacco associated oral squamous cell carcinoma

A cell line, AMOS-III has been established from the surgically resected specimen of an untreated primary human oral squamous cell carcinoma of the floor of mouth from a chronic smokeless tobacco consumer. Immunocytochemical analysis showed epithelial specific antigen, cytokeratins 5, 10, 13 and 16 and integrin ₆ markers in AMOS-III cells, confirming the epithelial lineage of the cell line. Analyses of morphology, ultrastructure, karyotype, anchorage independent growth and immunocytochemical properties of the cell line demonstrated the transformed phenotype of epithelial cells. AMOS-III cells have doubling time of 42–44 h. Giemsa-banding patterns of chromosomes confirmed the human origin of the AMOS-III cells. Molecular analysis of cancer-related gene products, p53 and p21^cip1/waf1 showed the presence of wild type p21^cip1/waf1 and truncated p53 proteins. The molecular mechanism underlying the action of retinoids in preventing the occurrence of second primary tumors in oral cancer patients remain to be clearly defined. Treatment of AMOS-III cells with all-trans retinoic acid (ATRA) at 10⁻⁴ μM resulted in 81% cell death. ATRA treatment resulted in enhanced expression of p21^cip1/waf1, nuclear translocation of retinoic acid receptors and apoptotic cell death. Thus, this cell line provides an in vitro model for elucidating the mechanism involving p53 inactivation and p21^cip1/waf1 overexpression in smokeless tobacco-induced oral cancer. Furthermore, the ATRA responsiveness of the cell line underscores its potential utility in identifying the retinoid responsive molecular targets in oral cancer cells.     

Establishment and characterization of a tumor cell line from human nasopharyngeal carcinoma tissue

An epithelial tumor cell line, CG1, was established from human nasopharyngeal carcinoma tissues. The CG1 cells are of an epithelial origin as shown by their reactivities with the epithelial-specific antikeratin antibodies and by the presence of the desmosome structure at cell-cell junctions. CG1 cells possess characteristics of tumor cells because these cells are tumorigenic in nude mice and also have reduced serum requirements for in vitro cultivation. The doubling time of CG1 cells is 20 h and these cells have been successfully cultured in vitro for more than 200 generations. The average chromosome number of these cells is 60. Slot and Southern blot hybridizations showed the presence of Epstein-Barr virus-DNA sequences in CG1 cells. This cell line provides us an in vitro system for the study of the role of Epstein-Barr virus in nasopharyngeal carcinoma.     

Cellular heterogeneity in a tissue culture cell line derived from a human bladder carcinoma

To study heterogeneity in a cell line derived from a human bladder carcinoma (EJ), 7 clones were isolated at low passage and examined for differences in culture behaviour, ability to grow in agar and tumorigenicity in nude mice. The parent EJ line had several distinct chromosome populations (both diploid and tetraploid), grew in agar and produced tumours in nude mice. Three of the clones had pseudodiploid modes and 4 had either hypo- or hypertetraploid modes. The 7 clones had 5 marker chromosomes in common but the combination of other marker chromosomes made each clone unique. No significant difference was found between the clones in the in vitro growth rate although analysis of in vitro culture behaviour showed heterogeneity in the pattern of cell movement on plastic substratum. Three clones were composed of static cells, one clone had very mobile cells; the other clones had rates of movement intermediate between the two. Differences were also found in the packing density of the cloned cells and in the cell size. All 7 clones grew in agar but heterogeneity was seen between the clones as shown by widely varying colony-forming efficiencies (0.5-13%). One clone had a high colony-forming ability in agar but failed to produce tumours in nude mice. The other clones were tumorigenic regardless of colony-forming efficiency in agar. Specific chromosome abnormalities were found to be associated with growth in agar and tumorigenicity but not with the growth pattern or the rate of movement of the cloned cells in culture.     

Phenotypic instability of drug sensitivity in a human colon carcinoma cell line

Colon cancer is one of the tumors most refractory to treatment by chemotherapy, and this may be due to inherent phenotypic instability of such tumor cells with respect to the biochemical determinants of drug sensitivity. To test this hypothesis, a clonal human colon carcinoma cell line, clone A, was passaged in culture in the absence of selection conditions or mutagens. During this time, sensitivity to several drugs was examined, and was found to decrease 4-fold during 30 weeks of culture. Five randomly selected subclones, having never been exposed to drug or mutagen, displayed a range of sensitivities to etoposide (50% inhibitory concentrations ranging from 1.5 to 4.9 microM) and to vincristine (9-fold range), but all had the same sensitivity to methotrexate. With time these sensitivities also changed, and subsequent subclones were chosen from the lines with highest and lowest drug sensitivity. Again a wide range of phenotypes was observed. Sensitivity to vincristine ranged 14-fold and to doxorubicin 3-fold. Several biochemical determinants of drug sensitivity had a broad range of expression between cell lines. Cellular accumulation of [3H]vincristine, as well as expression of multidrug resistance protein P170 and glutathione transferase activity all varied significantly between subclonal lines. This suggests that some human colon tumors may be phenotypically unstable with respect to drug sensitivity, and this could contribute to clinical resistance to chemotherapeutic compounds.