The genetic differences between gallbladder and bile duct cancer cell lines

Biliary tract cancers carry dismal prognoses. It is commonly understood that chromosomal aberrations in cancer cells have prognostic and therapeutic implications. However, in biliary tract cancers the genetic changes have not yet been sufficiently studied. The aim of this study was to clarify the presence of mutations in specific chromosomal regions that are likely to harbor previously unknown genes with a significant role in the genesis of biliary tract cancer. The recently developed bacterial artificial chromosome (BAC) array comparative genomic hybridization (CGH) can facilitate detail analysis with high resolution and sensitivity. We applied this to 12 cancer cell lines of the gallbladder (GBC) and the bile duct (BDC) using a genome-wide scanning array. Cell line DNA was labeled with green colored Cy5 and reference DNA derived from normal human leucocytes was labeled with red colored Cy3. GBC, as well as BDC cell lines, have shown DNA copy number abnormalities (gain or loss). In each of the seven GBC cell lines, the DNA copy number was gained on 6p21.32 and was lost on 3p22.3, 3p14.2, 3p14.3, 4q13.1, 22q11.21, 22q11.23, respectively. In five BDC cell lines, there were DNA copy number gains on 7p21.1, 7p21.2, 17q23.2, 20q13.2 and losses were on 1p36.21, 4q25, 6q16.1, 18q21.31, 18q21.33, respectively. The largest region of gain was observed on 13q14.3-q21.32 ( approximately 11 Mb) and of loss on 18q12.2-q21.1 ( approximately 15 Mb), respectively. Both GBC and BDC cell lines have DNA copy number abnormalities of gains and/or losses on every chromosome. We were able to determine the genetic differences between gallbladder and bile duct cancer cell lines. BAC array CGH has a powerful potential application in the screening for DNA copy number abnormalities in cancer cell lines and tumors.     

Characterization of four newly established human colorectal adenocarcinoma cell lines from Chinese patients.

Four colon adenocarcinoma cell lines, CC-M2, CC-M3, CC-M4, and CC-M2NM, have been established from surgical specimens of 18 unselected patients without the use of "feeder" cells and additional growth factors (e.g., insulin, hydrocortisone, etc.) in the culture medium. The methods of primary cultivation of tissue explants are described. Studies of determination of morphology, growth curve, plating efficiency, chromosomal analysis, CEA and beta-HCG synthesis, and tumorigenicity, were done to characterize the cell lines. Significant variations have been found in one of the four cell lines, both in vitro and in vivo studies. There are distinct phenotypes in the established cell lines which may be useful in studying the cell differentiation and progression of colorectal cancer.     

c-myc amplification and expression in newly established human osteosarcoma cell lines

Three cell lines were isolated from a patient with osteosarcoma of the femur. These lines were obtained from the primary neoplasm before (HTLA145) and after (HTLA161) chemotherapy and from a metastasis of the lung (HTLA195) in the same patient. The three cell lines exhibited a similar morphology in culture and formed tumors in nude mice which demonstrated a histopathology similar to that which had been observed in the patient. High expression of the genes coding for the alpha-1 and alpha-2 chain of collagen Type I was found in vitro and in s.c. tumors growing in nude mice. The c-myc protooncogene was amplified in all three cell lines and extensive expression of c-myc was found in vitro and in vivo. No heterogeneity in regard to c-myc expression in vivo was detected by in situ localization in tumors growing in nude mice.     

Biologic properties of three newly established human esophageal carcinoma cell lines

Three epithelial cell lines, CE-48T/VGH, CE-69T/-VGH, and CE-81T/VGH, were established from human squamous cell carcinoma of the esophagus. The cells were polygonal with a high nucleus-to-cytoplasm ratio. Many cells were multinucleate. Electron microscopy revealed the presence of tonofilaments and desmosomes. Chromosome analysis showed that these 3 cell lines were heteroploids of human origin. When transplanted into BALB/c (nu/nu) mice, CE-69T/VGH and CE-81T/VGH produced tumors, the histology of which proved to be carcinomas. All 3 cell lines secreted carcinoembryonic antigen. However, the secretion patterns were different. These 3 cell lines may provide useful models for the study of human esophageal cancer.     

Differential characteristics of two newly established human breast carcinoma cell lines

Two human breast carcinoma cell lines, EP and MW, were established in culture from malignant pleural effusions. In addition to producing tumors in antithymocyte serum-immunosuppressed mice, both cell lines showed epithelial characteristics and anchorage-independent growth in soft agar. EP and MW differed in morphology (spindle-shaped versus round), chromosomal mode (hyperdiploid versus near triploid), estrogen receptor content (43.8 versus 5.1 fmol/mg protein), cloning efficiency (0.24 versus 15%), and activities (milliunits/10(6) cells) of creatine phosphokinase (25.7 versus 62.6) and lactate dehydrogenase (346.7 versus 778.5). Electron microscopy revealed that MW cells had more perinuclear filamentous material and more frequent intracytoplasmic vacuole formation than did EP cells. While having no effect on MW cells at the concentrations studied (10(-5) to 10(-11) M), beta-estradiol (10(-7) M) stimulated the growth of EP cells by 106% over the hormone-depleted control. In a variety of systems, EP was consistently the more drug sensitive of the two lines. In vitro, EP was significantly (p less than 0.001) more sensitive to methotrexate, vincristine, and 5-fluorouracil, respectively. In antithymocyte serum-mouse xenografts, EP displayed a greater response to three different dosages of a combination of cyclophosphamide, methotrexate, and 5-fluorouracil. One such dosage (cyclophosphamide, 32.0 mg/kg/day; methotrexate, 13.0 mg/kg/day; 5-fluorouracil, 190.0 mg/kg/day; for 1 day) reduced EP and MW tumor weights to 5.9 and 41% of controls, respectively. These results correlated well with the clinical responses.     

Characterization of six cell lines established from human pancreatic adenocarcinomas

BACKGROUND: Six human pancreatic carcinoma cell lines, designated as KMP-1 to KMP-6, were established and maintained in vitro for > 3 years. All were derived from pancreatic ductal adenocarcinomas. The six cell lines originated from either primary pancreatic tumors, metastatic liver tumors, or metastases to lymph nodes. METHODS: Each cell line was characterized by its morphology, doubling time, colony forming efficiency (CFE) on plastic dishes, tumorigenicity in nude mice, chromosomal analysis, and the amount of tumor markers secreted into the culture medium. Furthermore, mutations in the K-ras, p53, and p16/INK4a genes were analyzed. RESULTS: All cell lines grew as an adhering monolayer and were cultured in medium supplemented with 2% fetal bovine serum. The doubling time ranged from 16-70 hours, and the CFE ranged from 0.1-11%. Subcutaneous transplantation of these carcinoma cells into nude mice resulted in the formation of tumors. Chromosomal analysis showed that the modal numbers ranged from 43-124, and each karyotype was unique. Each cell line secreted detectable amounts of squamous cell carcinoma antigen, carcinoembryonic antigen, carbohydrate antigen 19-9, Dupan-II, and cytokeratin 19 fragment, respectively. Genetic alterations of the K-ras, p53, and p16 genes were detected in six, three, and five, respectively, of the six cell lines. CONCLUSIONS: The authors believe that these newly established pancreatic carcinoma cell lines will contribute to wide ranging studies regarding pancreatic carcinoma progression.     

Effect of radiation treatment on newly established human breast cancer cell lines MACL-1 and MGSO-3

The characterization of new cell lines is an important tool to understand the biological processes involved in cancer treatments. In the present study, we used two newly established epithelial human breast cancer cell lines from primary sites MACL-1 and MGSO-3 and compared their susceptibility to the treatment with ionizing radiation (IR) with the commercial cell line MDA-MB-231. In the doses used (10 or 20 Gy), IR induced a reduction in cell viability and cell death, measured as DNA fragmentation, at 48 and 72 h after treatment. In addition, 48 h after treatment with IR, we observed an enhancement in the percentage of apoptotic cells. The broad-range caspases inhibitor zVAD-FMK inhibited cytotoxicity induced by IR. After 24 h, treatment with IR activated caspase-9 in MACL-1 and MDA-MB-231 but not in MGSO-3 cells. Thirty hours after treatment with IR (20 Gy), we observed an activation of caspases 8 and 3. These results suggest the involvement of caspases in the cell death induced by IR in two newly established cell lines. These cells may be useful in studies of breast cancer in defining basic mechanisms in molecular and cellular radiobiology and may contribute to the rational design of future models of cancer therapies.     

Characterization of human neuroblastoma cell lines established before and after therapy

Six new cell lines have been established from human neuroblastomas. Cell line SMS-KAN, from primary tumor before therapy, and line SMS-KANR, from bone marrow after chemotherapy and radiotherapy, were established from the same patient. Cell lines SMS-KCN (from primary tumor before any therapy) and SMS-KCNR (from bone marrow after chemotherapy) were established from another patient. Two other lines (SMS-MSN and SMS-SAN) were established from different patients before any therapy was given. Cell lines established from recurrent disease after chemotherapy (SMS-KANR and SMS-KCNR) had significantly shorter doubling times and increased plating efficiencies compared to those of cell lines derived from the same patient before chemotherapy (SMS-KAN and SMS-KCN). All cell lines contained tyrosine hydroxylase, aromatic L-amino acid decarboxylase, and dopamine-beta-hydroxylase. Measurable amounts of choline acetyltransferase were also detected in SMS-KAN and SMS-KANR. Karyotype analysis showed all cell lines except SMS-MSN to be pseudodiploid with modal numbers of 46 and deletions of the short arm of chromosome 1; SMS-MSN had a modal number of 57-58 chromosomes. All cell lines had double-minute chromosomes, except SMS-KANR, which had abnormally banding regions. These new cell lines provide in vitro models of neuroblastoma suitable for the study of differences in neuroblastoma cell populations before chemotherapy as compared to the cell populations that proliferate after therapy.     

Serum-dependent growth patterns of two, newly established human mesothelioma cell lines

Two cell lines with different in vitro growth patterns were established from the pleural fluid of a patient with malignant epithelial pleural mesothelioma. The cell line established in RPMI 1640 supplemented with human AB serum had an epithelial morphology, while the cell line established in fetal calf serum-supplemented medium had a fibroblast-like morphology. Exposure of the fibroblast-like cell line to human AB serum-containing medium resulted in a nearly complete transformation of the morphology to the epithelial-like phenotype, and the epithelial-like cell line changed its phenotype to fibroblast-like upon exposure to fetal calf serum-supplemented medium. Both cell lines formed colonies in soft agarose and secreted hyaluronic acid into the culture medium. In both cell lines all the metaphases studied lacked chromosomes 5 and 9, demonstrating the same clonal origin. However, one marker and a missing chromosome 11 were found only in the fibroblast-like cell line. We conclude that human AB serum supplement can be used for the establishment of human tumor cell lines, and that the choice of serum can affect the in vitro morphology of the established mesothelioma cell lines. The mechanisms behind the different growth patterns seem to be a selective stimulation of different subpopulations of malignant cells as well as induction of changes in the morphology of individual cells.     

Antigenicity of newly established colorectal carcinoma cell lines

Cells from two adenocarcinomas, an adenoma and a metastatic node were isolated in soft agar. Expression of antigens, CEA, Y haptenic blood group and 791T-p72, defined by a range of candidate antibodies for tumour targeting was assessed. All of the cells expressed low levels of CEA but high levels of the Y haptenic blood group antigen although there was enormous inter and intraclonal variation. Of particular interest was the membrane expression of 791T-p72 antigen on all of the dividing tumour cells as previous studies had shown that 791T/36 antibody reacted with tumour stromal elements rather than malignant cell surfaces. The DNA content was abnormal in all of the cells whether they were derived from diploid or aneuploid primary tumours. They all grew readily in athymic mice and at least one monoclonal antibody, 791T/36, localised efficiently within these xenografts. Clonogenic cells therefore expressed the three tumour-associated antigens, several at higher levels than observed in the primary tumour. Monoclonal antibody 'cocktails' should therefore allow antibody mediated drug cytotoxicity to be effective at eradicating rapidly dividing tumour cells.     

Characterization of bile salt-induced apoptosis in colon cancer cell lines

Bile salts have been shown to be involved in the etiology of colorectal cancer. Although there is a large body of evidence for bile salts as a cocarcinogen in azoxymethane-induced colorectal cancer, bile salt-induced apoptosis of colorectal cancer cells has not yet been studied in detail. Therefore, we investigated the effects of different bile salts on apoptosis and apoptotic signaling in colon cancer cell lines. Incubation of colorectal cancer cell lines with physiological concentrations of deoxycholic acid led to a dramatic induction of apoptosis. Caspase cleavage and caspase activation occurred as early as 30 min after the addition of deoxycholate. Caspase-2 (Ich-1, Nedd2), caspase-3 (CPP-32, YAMA, Apopain), caspase-7 (Mch-3, ICE-LAP-3), and caspase-8 (FLICE, Mach-1, Mch5) are activated in HT-29, whereas caspase-1 (ICE) remained intact. Caspase activation and cellular apoptosis induced by bile salts were reversed by broad spectrum and selective caspase inhibitors. As opposed to hepatocyte death mediated by bile acids, CD95 was not involved in deoxycholate-induced apoptosis. The cytoprotective effect of ursodeoxycholic acid in hepatocytes or other tumor cell lines, which is mediated by inhibiting the mitochondrial permeability transition, was not observed in colon cancer cell lines as well. This points to distinct intracellular functions of ursodeoxycholate in different cancer cell types. Here we describe the specificity of bile salt-induced apoptosis in colon cancer cell lines. Differences from hepatocytes are shown. Bile acid-specific caspase activation is part of the apoptotic pathway induced by bile salts in colon cancer cell lines. Furthermore, a lack of cytoprotective function of ursodeoxycholate in these cells is demonstrated. Our data raise questions as to the role of bile salts in colorectal carcinogenesis.     

Characterization of two newly established EBV-containing lymphoblastoid cell lines from patients with myeloid leukemias

Two spontaneous outgrowing Epstein-Barr virus (EBV)-carrying lymphoblastoid cell lines (LCLs), CG2 and CG3, have been established from bone marrow cells of myeloid leukemia patients. CG2 was derived from a patient with chronic myelomonocytic leukemia (CMMoL) and who has a 45 XO karyotype. CG3 was derived from a patient with juvenile chronic myeloid leukemia (CML) and who carries a hypotetraploid karyotype, 91XXYY. Both CG2 and CG3 cells carry the same type of translocation; t(1;19)(q23;p13). Both cell lines are of an early B cell lineage as shown by their reactivities with monoclonal antibodies OKIa, B1, B2 and B4. The combination of horizontal discontinuous agarose slab gel and Southern hybridization results show CG2 and CG3 cells are of monoclonal origin and harbor episomal EBV genomes. Approximately 50 EBV genome equivalents were contained in CG2 and CG3 cells. Immunofluorescence studies demonstrate the expression of EBV-encoded antigen (EBNA) in almost all cells of these two lines. The expression of EA and VCA is only observed in a small percentage of cells and cannot be induced by treatment with TPA and SB. Therefore, CG2 and CG3 cells are probably nonproducer cell lines for EBV. The serum samples from both patients have been shown to contain elevated IgG antibody titers to EBV antigens. Both cells are found to be nontumorigenic in nude mice. These cells may provide an important tool in analyzing molecular epidemiological aspects of EBV infections in diseases such as CMMoL and juvenile CML.     

Growth inhibition of newly established human glioma cell lines by leukemia inhibitory factor

We have established three new cell lines deriving from malignant human gliomas. The cell lines were described in terms of both morphology and growth characteristics. Most cells in all three cell lines expressed the neuroepithelial marker protein GFAP. In terms of growth characteristics, the cells showed only slight differences. The cell lines showed no expression of the neural form of the c-src gene, pp60^{c-s rcN}, but did express the ubiquitous form, pp60^{c-s rc}. The established glioma cell lines were also examined for expression of members of the neuropoietic cytokine family, CNTF and LIF, and their respective receptor components CNTFR, LIFR and gp130. With the exception of CNTFR both the ligands and their receptor components were expressed in similar amounts in all three cell lines. The presence of ligand and receptor prompted us to study the effects of exogenously supplied factors on the growth of the glioma cell lines. Whereas LIF induced a high c-fos expression, only low c-fos induction was observed upon CNTF treatment. Accordingly, CNTF did not have any noticeable effects on glioma cell growth in culture, while LIF mediated an inhibiting effect on the growth of the three glioma cell lines in culture.     

Characterization of non-small-cell lung cancer cell lines established before and after chemotherapy

We established several in vitro drug-resistant cell lines after continuous, long-term exposure of each drug to elucidate mechanisms of drug resistance. Whether drug resistance in these in vitro resistant cell lines reflects clinical drug resistance still remains unanswered. In this study, a pair of lung cancer cell lines was established from one patient with squamous cell carcinoma of the lung, with one line being established before and one line after combination chemotherapy (cisplatin/ifosfamide/vindesine). Combination chemotherapy selected resistant EBC-2/R cells, which showed cross-resistance to 4-hydroxyifosfamide (3.2-fold), cisplatin (2.3-fold), and methotrexate (3.7-fold) and collateral sensitivity to vindesine (0.77-fold) compared with parent EBC-2 cells. EBC-2/R cells showed decrease in intracellular accumulation of cisplatin, increase in intracellular concentration of glutathione (GSH), and overexpression of multidrug resistance-associated protein (MRP) 3 when compared with EBC-2 cells. A single cycle of chemotherapy was not sufficient to select other mechanisms of drug resistance, such as multidrug resistance-1/P-glycoprotein, MRPs 1, 2, 4, and 5, lung resistance-related protein, metallothionein IIa, glutathione S-transferase pi, gamma-glutamylcysteine synthetase (light and heavy chain), and excision repair cross complementing 1. Sequentially we established two cell lines, which cell lines showed the differences of the cisplatin resistance, expression level of MRP3, intracellular GSH level and intracellular accumulation of cisplatin. A pair of cell lines will be useful to elucidate resistant mechanisms of cisplatin in heterogeneous lung cancer cells.     

Characterization of novel cell lines established from three human oral squamous cell carcinomas

Human oral squamous cell carcinoma cell lines (KOSCC-11, -25A, -25B, -25C, -25D, -25E, -33A, and -33B) were established by explantation culture from these oral squamous cell carcinomas. The histopathology of the primary tumors, in vitro growth characteristics, epithelial origin, in vitro anchorage-independency, in vivo tumorigenicity, the frequency of human papillomavirus (HPV) infections, and the status of proto-oncogenes, tumor suppressor genes, DNA mismatch repair genes, and microsatellite instability were investigated in the cell lines. KOSCC-11 is a well-differentiated oral squamous cell carcinoma (OSCC) derived from mandibular gingiva. KOSCC-25A, -25B, -25C, -25D, and -25E cell lines were derived from the same OSCC. KOSCC-33A and -33B were established from the same tumor that originated from the maxillary sinus. All tumor lines studied grew as monolayers and showed: i) epithelial origin by the presence of desmosome and keratin; ii) in vitro anchorage-independent growth ability; and iii) tumorigenic potential in nude mice. The cancer cell lines did not contain HPV DNA and did not express viral genes. Northern blot analysis revealed: i) overexpression of EGFR in four cell lines, ii) overexpression of c-H-ras in four cell lines, iii) overexpression of c-myc in three cell lines, iv) decreased expression of TGF-alpha in seven cell lines, and v) decreased expression of c-jun in five cancer cell lines compared with normal human oral keratinocytes. In all KOSCC cell lines and their corresponding tumor tissues, mutations were identified in highly-conserved functional regions of the p53 gene. The KOSCC-11 cell line contained a frameshift mutation and the other cell lines harbored an identical p53 mutation at codon 175 from CGC (Arg) to CTC (Leu). In five cell lines, a significant reduction of p21WAF1/Cip1 protein was evident. Cancer cell lines expressed higher level of Rb protein than normal human oral keratinocytes. DCC, a tumor suppressor gene, was not detected in KOSCC-25C. The KOSCC-33A cell line displayed microsatellite instability and showed a loss of hMSH2 expression. These well-characterized human OSCC cell lines should serve as useful tools for understanding the biological characteristics of oral cancer.     

Surface-glycoprotein patterns of established human lung cancer cell lines and primary cultures

The cell surface glycoprotein (GP) profiles of cell lines representing the four major histopathological lung cancer entities, squamous cell (SQC)-, small cell (SCC)-, adeno (ADC)-, and large cell carcinoma (LCC), and two primary cultures of SCC and LCC, respectively, have been examined by the galactose-oxidase tritiated sodium-borohydride cell surface labelling method, The SCC specimens (five cell lines and one biopsy) had a characteristic pattern of major surface GPs, with common GPs at apparent molecular weights (MWs) of 54 -kilo (k) Daltons (D) and 88 kD, which was discriminative from the group of non-SCC (SQC, ADC and LCC). The non-SCC group constantly expressed GPs at apparent MWs of 80 kD and 110 kD, both as established cell lines and in the primary LCC culture. The GP patterns of the SCC cell lines and the LCC cell line were retained in comparison to corresponding primary biopsy material. The propagation of an established SCC cell line without supplementation of serum did not alter the GP expression at the cell surface. Taken together, the surface GP patterns for SCC versus non-SCC appear to be reliable and reproducible markers for these tumor entities, both in biopsy material and in established cell lines.     

Responsiveness of three newly established human colorectal cancer cell lines to transforming growth factors beta 1 and beta 2.

We have established 3 new human colorectal cancer cell lines (LS411N, LS513, and LS1034) from clinical biopsy samples. These lines are tumorigenic and grow s.c. as adenocarcinomas in nude mouse xenografts. Specific marker chromosomes are observed in each line. Carcinoembryonic antigen is expressed at the surface of all 3 lines, but with marked quantitative differences. Indeed, less than 10% of the cells from the HT-29 line used as a reference express carcinoembryonic antigen while more than 90% of the LS1034 cells do so. LS513 and LS1034 consistently express HLA class I antigens and intercellular adhesion molecule 1 which are not detected at the surface of the LS411N cells. No expression of HLA class II antigens DR, DQ, and DP has been measured on any of the lines. All three lines grow well in 5% fetal calf serum medium without addition of exogenous growth factors. The LS1034 line has been adapted to growth in serum-free conditions and exhibits increased clonogenicity when cells are seeded in serum-free methylcellulose medium, as compared with medium containing 5% fetal calf serum. The LS513 and LS1034 lines have proved to be of particular interest since they respond to the growth-inhibitory action of TGF-beta 1 and TGF-beta 2 in both liquid and semisolid medium. Both factors were, at pM concentrations, equipotent inhibitors of LS1034 cell proliferation. In contrast, higher concentrations of TGF-beta 1 are inhibitory for proliferation of LS513 cells, whereas TGF-beta 2 has no effect on the growth of these cells in liquid assay. On this basis, using appropriate anti-TGF-beta 1 and anti-TGF-beta 1 IgY, we developed a bioassay for TGF-beta 1 and TGF-beta 2. Two of the three lines have indeed been shown to produce latent-TGF-beta 1 activity.