Establishment and characterization of a new cell line derived from human colorectal laterally spreading tumor

AIM： To study the molecular mechanism of laterally spreading tumor （LST）, a cell line [Laterally Spreading Tumor-Rectum 1 （LST-R1）] was derived and the characteristics of this cell line were investigated.
METHODS： A new cell line （LST-R1） originated from laterally spreading tumor was established, Properties of the cell line were characterized using scanning and transmission electron microscopy, immunohistochemistry method, cytogenetic analysis and nude mice xenograft experiments, In vitro invasion assay, cDNA microarray and Western blotting were used to compare the difference between the LST-R1 and other colorectal cancer cell lines derived from prudent colon cancer.
RESULTS： Our study demonstrated that both epithelial special antigen （ESA） and cytokeratin-20 （CK20） were expressed in LST-R1. The cells presented microvilli and tight junction with large nuclei. The karyotypic analysis showed hyperdiploid features with structural chromosome aberrations. The in vivo tumorigenicity was also demonstrated in nude mice xenograft experiments. The invasion assay suggested this cell line has a higher invasive ability, cDNA microarray and Western blotting show the loss of the expression of E-cadherin in LST-R1 cells.
CONCLUSION： We established and characterized a colorectal cancer cell line, LST-R1 and LST-R1 has an obvious malignant tendency, which maybe partially attributed to the changes of the expression of some adhesion molecules, such as E-cadherin. It is also a versatile tool for exploring the original and progressive mechanisms of laterally spreading tumor and the early colon cancer genesis.     

Kinetics of carcinoembryonic antigen and carbohydrate antigen 19-9 production in a human pancreatic cancer cell line (SUIT-2)

Kinetics of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) production in a human pancreatic cancer cell line (SUIT-2) were investigated. Production of CEA reached a maximum, 31.0 ng/1 X 10(6) cells, in the late stationary phase with transient decline during the early exponential phase and 15.5% of the produced CEA was released into the medium, while production of CA19-9 reached a maximum, 421 U/1 X 10(6) cells, in the early stationary phase and 68.8% of the produced CA19-9 was released into the medium. Accordingly, the kinetics of CEA and CA19-9 production of SUIT-2 in vitro might be independent. CEA was stained immunohistochemically in the cytoplasm of the cells forming small buds above the monolayer cell sheet. On the contrary, CA19-9 positive cells were observed scattered in the monolayer cell sheet and CA19-9 was stained in the cytoplasm, predominantly at the projections of the cell surface. CEA and CA19-9 were also detected in the sera of nude mice bearing SUIT-2 tumors and their concentrations correlated well with tumor volume. Correlation coefficients between tumor markers and tumor volume were 0.79 (p less than 0.01) for CEA and 0.90 (p less than 0.01) for CA19-9.     

Establishment and characterization of a spontaneously immortalized myofibroblast cell line derived from a human liver angiosarcoma

BACKGROUND/AIM: Fibrosis and/or cirrhosis are present in the precursor stages of most liver cancers. However, little is known about the reciprocal interactions of fibroblasts, mainly responsible for fibrosis, and the other liver cells. We report here the isolation of a new liver myofibroblast cell line from a human liver angiosarcoma and its characterization. METHODS: The cells were isolated by the explant technique and characterization was performed, on one hand, using immunohistochemical and ultrastructural analysis and, in the other hand, by determining their karyotype, ras and p53 status and their tumorigenic properties. RESULTS: To date, the cells have undergone approximately 170 population doublings and are still proliferating. Immunohistochemically, they were negative for desmin, smooth muscle myosin, cytokeratin 19 and von Willebrand factor, positive for vimentin and alpha-smooth muscle actin, with an important deposition of fibronectin around the cells. Ultrastructure showed particularly cytoplasmic microfilament bundles. Their chromosome number ranged from 38 to 168 with a bimodal population, near diploid and hypotetraploid. No mutations were found in codons 12, 13 or 61 of Ha-, Ki- and N-ras genes but a homozygous missense mutation in codon 179 (CAT-->CTT) was detected in the p53 gene. They were unable to form foci in soft agar or tumors in nude mice. CONCLUSIONS: Taken together, these results show that these cells, called BM 2.2.1, exhibited typical myofibroblast-like features. Although they contained a karyotype suggestive of tumoral cells and a homozygous mutated p53 gene, they were not tumorigenic. The nature of these cells and the abnormalities of the p53 gene and the karyotype, suggest that: i) they were a component of the tumor stroma, and ii) they could have been involved in angiosarcoma development. Thus, this cell line may be valuable for the study of cellular interactions in liver carcinogenesis.     

Establishment and characterization of a cultured cell line derived from nitrosamine-induced pancreatic ductal adenocarcinoma in Syrian golden hamsters

Seven kinds of pancreatic ductal adenocarcinomas induced by N-nitrosobis(2-hydroxypropil)amine in Syrian golden hamsters were established as transplantable tumor lines on syngeneic animals. These tumor lines were all well or moderately differentiated adenocarcinomas, showing various velocities of growth, unrelated to the grade of histological differentiation. A cell line, designated HaP-T1, was established in continuous tissue culture from one of these homografts. The cells grew in a monolayered sheet with an approximately 17-hour population doubling time. Chromosomal analysis revealed that the modal chromosomal number of the cell line was 44. HaP-T1 cells showed apparent tumorigenicity both on syngeneic hamsters and athymic nude mice, but they grew much faster when injected into the former animals. Morphological characteristics of HaP-T1 cells and tumors induced by HaP-T1 inoculation in both animals revealed apparent epithelial characteristics resembling ductal adenocarcinoma of the pancreas. These transplantable tumor models will contribute to the further investigation in the field of pancreatic cancer.     

Establishment and characterization of a new human leukemia cell line derived from M4E0

A new human leukemia cell line, designated as ME-1, was established from the peripheral blood leukemia cells of a patient with acute myelomonocytic leukemia with eosinophilia (M4E0). This cell line has the characteristic chromosome abnormality of M4E0, inv(16) (p13q22). When cultured in RPMI 1640 medium containing 10% fetal calf serum, ME-1 cells were monoblastoid, but with the addition of cytokines such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4, or medium conditioned by phytohemagglutinin-stimulated human peripheral leukocytes (PHA-LCM), the cells exhibited differentiation to macrophage-like cells. PHA-LCM also promoted eosinophilic-lineage differentiation of this cell line, although IL-5 did not do so. To elucidate the mechanism of proliferation and differentiation of ME-1 cells, we studied the effect of a potent inhibitor of protein kinase C, 1-(5-isoquinolinyl-sulfonyl)-2- methylpiperazine (H-7), on colony formation of ME-1 cells. H-7 inhibited colony formation of ME-1 cells by IL-3 or GM-CSF dose dependently, but had little inhibitory effect on colony formation by IL- 4. These results indicate that the proliferation and differentiation of ME-1 cells by IL-3 or GM-CSF were related to the activation of protein kinase C, while those by IL-4 involved other regulatory systems. ME-1 cells should be useful for studying the pathogenesis of M4E0 and the mechanisms of proliferation and differentiation of leukemic and normal progenitors by cytokines.     

Establishment of a human carcinoembryonic antigen (CEA)-producing cell line in a protein-free chemically defined medium

Summary To examine whether a human carcinoembryonic antigen (CEA)-producing cell can proliferate and sythesize CEA in vitro in culture without protein supplements, long-term cultivation of such cells was carried out in a protein-free chemically defined medium. Using stepwise decreases in fetal bovine serum concentration, continuous growth of the cells was established in a protein-free am's F-12 medium. The cells, designated as HLC-Yl, have been propagated in this medium for 3 years. The population doubling time of the cells is about 52 h. Addition of the serum stimulated the cell growth (population doubling time = 27 h). Saturation density was not increased by the addition of serum. The cells grown in a protein-free F-12 secrete large amounts of CEA (65.4±2.6 ng/10⁶ cells/24 h). Addition of serum did not stimulate the production of CEA. The cells produced tumours when inoculated into athymic nude mice. The mice bearing the tumour showed high serum CEA levels, and CEA was demonstrated in the tumour tissue by the immunoperoxidase method. The present study suggests that cells grown in a protein-free medium do not require serum components for their growth or CEA synthesis and provide an excellent model for better understanding the growth and production of CEA in human lung cancer cells.Abbreviations FBS fetal bovine serum - CEA carcinoembryonic antigen - PAP peroxidase anti-peroxidase - DAB Diaminobenzidine - PAS periodic acid-Schiff - PBS phosphate buffered saline - TEM transmission electron microscopy - SEM scanning electron microscopy     

Clonal preservation of human pancreatic cell line derived from primary pancreatic adenocarcinoma.

Adenocarcinoma of the pancreas generally remains an incurable disease by available treatment modalities, demanding the development of a suitable cell-culture/animal model and the discovery and evaluation of novel therapeutic agents. We report the clonal preservation of a human pancreatic cell line (KCI-MOH1) established from a 74-year-old African-American man diagnosed with pancreatic cancer. Initially the human primary tumor was grown as a xenograft in SCID mice and, subsequently, a cell line was established from tumors grown as a xenograft as reported in our earlier publication. The molecular characterization of the primary tumor, the tumors grown as xenograft, and the cell line all revealed similar genotypic properties. By using an automated DNA sequencer, a K-ras mutation (codon 12, GGT to CGT, Gly to Arg) was detected in the pancreatic tumor tissue taken from the patient, whereas no p53 mutation was detected. The same K-ras mutation and unaltered p53 was also found in the xenograft tumor and in the KCI-MOH1 cell line. Chromosome analysis of the cultured cells revealed: 42,XY,add(3)(p11.2),der(7)t(7;12) (p22;q12),-10,-12,add (14)(p11),-18,add (20)(q13),-22/84, idemx2, which is the same chromosome complement found in xenograft tumors. The KCI-MOH1 cell line grows well in tissue culture and forms tumors in the SCID mice when implanted subcutaneously, as well as in orthotopic sites. The KCI-MOH1 cell line-derived SCID mouse xenograft model was used for efficacy evaluation of bryostatin 1, auristatin-PE, spongistatin 1, and gemcitabine alone and in combination. Tumor growth inhibition (T/C expressed as percentage), tumor growth delay (T - C), and log 10 kill for these agents were 38%, 22 days, and 0.53; 15%, 30 days, and 0.80; 24%, 25 days, and 0.66; and 10%, 33 days, and 0.90, respectively. When given in combination, two of seven gemcitabine + auristatin-PE-treated animals were free of tumors for 150 days and were considered cured. Animals treated with a combination of bryostatin 1 and gemcitabine and a combination of spongistatin and gemcitabine produced remissions in only one of seven mice. From these results, we conclude that (a) this is the first study illustrating that clonal characteristics of primary pancreatic tumors remained unchanged when implanted in mice and as a permanent cell line grown in vitro; and (b) there is a synergistic effect between gemcitabine and selected marine products tested in this study, which is more apparent in the gemcitabine and auristatin-PE combination. The results of this preliminary study suggest that these agents should be explored clinically in the treatment of pancreatic cancer.     

Establishment and characterization of a new human eosinophilic leukemia cell line

A human eosinophilic leukemia cell line, designated as EoL, was established from the peripheral blood of a patient with Philadelphia chromosome-negative eosinophilic leukemia (EL). The EoL cell line grows in single cell suspension with a doubling time of 48 hours for about one year. The reactivity of these cells was tested with a panel of monoclonal antibodies; they were found to express surface IA antigen, myeloid antigen (IF10, MY9) and membrane receptors for interleukin 2 (IL-2, Tac antigen). Under standard culture conditions, a small percentage of cells having more typical eosinophilic characteristics was present. These cells had cytoplasmic granules and were positive for Luxol-fast-blue and eosinophil peroxidase. Under culture conditions to induce the maturation of myeloid cells, such as alkaline medium or addition of dimethyl sulfoxide (DMSO), the frequency of cells with typical eosinophilic features increased to about 40%. In addition, cytogenetic studies showed that cultured cells and original leukemic blasts presented similar chromosome abnormalities. EoL seems to be a unique leukemic line committed to the eosinophilic lineage and can provide a useful in vitro model for the study of malignant eosinophilic properties.     

Establishment and characterization of a new Epstein-Barr virus transformed cell line from a human B cell lymphoma

Summary We have established a new cell line from a patient with centrocytic B cell lymphoma. Highly purified peripheral blood B cells from patient DUL (WBC counts 158,000/µl) were infected in vitro with Epstein-Barr virus (EBV), and CD 20⁺ B cells were cloned into 96 well culture plates with the aid of a cell sorter autoclone device. As shown by GTG-banding and Southern blot analysis, outgrowing EBV-positive clones had the same chromosomal abnormalities and identical monoclonal IgH gene rearrangement as the original EBV-genome-negative leukemic B cell clone. Surface marker analysis with a panel of monoclonal antibodies revealed identical patterns on EBV-negative and -positive clones, with the exception of PCA 1 (reactive with plasma cells) which was negative on freshly explanted leukemic B cells but positive on EBV-converted clones.List of abbreviations E sheep erythrocytes - EBV Epstein-Barr virus - FCC-NHL follicular centrocytic non-Hodgkin's Lymphoma - FCS fetal calf serum - LCL lymphoblastoid cell line(s) - mab monoclonal antibody - MNC mononuclear cells - NHL non-Hodgkin's lymphoma This work was supported by the Deutsche Forschungsgemeinschaft (SFB 322/B 2)This work forms part of the Ph. D. thesis of O. Janssen     

Establishment and characterization of a new cell line (VHB-1) derived from a primary breast carcinoma

Summary A continous line of human breast carcinoma cells, VHB-1, was established in culture following collagenase treatment of an infiltrating duct cell carcinoma. The cells displayed an epithelial pattern and multiplied rapidly. Maintained in monolayer culture, the VHB-1 cells exhibited a 30-h doubling time and a plating efficiency of 20%. The cells possessed an abnormal karyotype with a mode of 70–74 chromosomes per cell. the karyotype was heavily rearranged and numerous marker chromosomes were found. Transplantation of the cells into nude mice produced tumors bearing histological resemblance to the original material. The VHB-1 cells contained significant levels of prolactin receptors, were steroid hormone (estrogen, progesterone, androgen, glucocorticoid) receptor positive, and were capable of functional differentiation in vitro. These characteristics make the VHB-1 cell line a suitable model for studying the biological properties of human breast tumors.     

Cloning and characterization of platelet factor 4 cDNA derived from a human erythroleukemic cell line

We report the isolation of a platelet factor 4 (PF4) cDNA clone from a lambda gt11 expression cDNA library which was derived from a human erythroleukemic (HEL) cell line. The sequence of the DNA insert includes the 3'-untranslated region, the entire amino acid coding region for the mature PF4 protein, and a 5' region containing coding information for an additional 18 amino acids. In addition, supplemental genomic DNA sequencing shows that the full-length leader sequence is 30 amino acids long plus an initial methionine and codes for a hydrophobic signal-like sequence which is probably involved in transmembrane transport. A single species mRNA of approximately 800 nucleotides was detected on blots of HEL cell poly(A) + RNA using a labeled PF4 cDNA probe. The human PF4 leader sequence shares some DNA, but no amino acid, homology with the 15 amino acids at the N-terminus of mature bovine PF4, suggesting rapid divergence in this region of PF4 between these two species. Sequence comparison of the coding regions of mature PF4 and gamma IP-10, a protein induced in a variety of cells following treatment with gamma-interferon, shows a corrected divergence of 76%. The divergence of a common ancestor protein into PF4 and gamma IP-10 may have accompanied the development of sophisticated immune and coagulation systems in vertebrates. The availability of cDNA and genomic DNA information for these genes in other species will be useful in studying the evolution of the coagulation and immune systems.     

Establishment,functional and genetic characterization of a colon derived large cell neuroendocrine carcinoma cell line

AIM To establish cell line and patient-derived xenograft(PDX) models for neuroendocrine carcinomas(NEC) which is highly desirable for gaining insight into tumor development as well as preclinical research includingbiomarker testing and drug response prediction.METHODS Cell line establishment was conducted from direct in vitro culturing of colonic NEC tissue(HROC57). A PDX could also successfully be established from vitally frozen tumor samples. Morphological features, invasive and migratory behavior of the HROC57 cells as well as expression of neuroendocrine markers were vastly analyzed. Phenotypic analysis was done by microscopy and multicolor flow cytometry. The extensive molecular-pathological profiling included mutation analysis, assessment of chromosomal and microsatellite instability; and in addition, fingerprinting(i.e., STR analysis) was performed from the cell line in direct comparison to primary patient-derived tissues and the PDX model established. Drug responsiveness was examined for a panel of chemotherapeutics in clinical use for the treatment of solid cancers.RESULTS The established cell line HROC57 showed distinct morphological and molecular features of a poorly differentiated large-cell NEC with KI-67 50%. Molecular-pathological analysis revealed a Cp G island promoter methylation positive cell line with microsatellite instability being absent. The following mutation profile was observed: KRAS(wt), BRAF(mut). A high sensitivity to etoposide, cisplatin and 5-FU could be demonstrated while it was more resistant towards rapamycin. CONCLUSION We successfully established and characterized a novel patient-derived NEC cell line in parallel to a PDX model as a useful tool for further analysis of the biological characteristics and for development of novel diagnostic and therapeutic options for NEC.