Establishment and characterization of six new human endometrial adenocarcinoma cell lines

The endometrial carcinoma cell lines EC-MZ-1, 2, 3, 5, 9, and 11 were established between 1986 and 1990. Four cell cultures were started from endometrial tissue, one from ascites, and one from a lymph node metastasis. Lines have to date been subcultured up to 180 times and the doubling time varies between 26 hr and 3 weeks. Immunocytochemically the coexpression of cytokeratin (predominantly simple-epithelial cytokeratin polypeptides) and vimentin intermediate filaments was detectable in all cell lines, but three lines (EC-MZ-5, 9, 11) expressed vimentin only at low level. By transmission electron microscopy the tumor cells exhibited features of epithelial differentiation. After subcutaneous transplantation into nude mice three lines (EC-MZ-1, 2, 5) produced slow-growing tumors. The histological classification of these tumors ranged from moderately differentiated adenocarcinoma to undifferentiated carcinoma and closely corresponded to the original tumor. Even after long-term in vitro culture, without any addition of estrogens to the culture medium, the moderately differentiated receptor-positive cell line (EC-MZ-2) retained its morphological differentiation. The cells were propagated without estrogens in the culture medium. The estrogen and progesterone receptor levels of cultured cells were determined. Three lines (EC-MZ-1, 2, 3) were positive for the progesterone receptor in low passage number only, the other cell lines were negative for both receptors. The transplantable lines were investigated for hormonal receptor expression in ovariectomized nude mice. In the moderately differentiated cell line (EC-MZ-2) we observed an enhanced expression of the estrogen receptor under optimal stimulation of the nude mouse with estradiol benzoate. There was no effect on the expression of the progesterone receptor.     

Establishment and characterization of high- and low-lung-metastatic cell lines derived from murine colon adenocarcinoma 26 tumor line

We established and characterized high- (LuM1) and low-lung-metastatic (NM11) cell lines derived from murine colon adenocarcinoma 26 tumor line. LuM1 cell line was established as a clonal cell line from a cultured cell mixture derived from a lung-metastatic nodule after 7 sequential subcutaneous transplantations of lung-metastatic tumors in the abdominal wall of BALB/c mice. NM11 cell line was established from a cultured cell mixture derived from a subcutaneous transplant of murine colon adenocarcinoma 26 tumor cells. LuM1 cells showed marked spontaneous lung metastases, but NM11 cells rarely did. High invasive potential of LuM1 cells was revealed by in vitro invasion assay using Matrigel reconstituted membranes. Rapid retraction was observed in monolayers of human umbilical vein endothelial cells and bovine aortic endothelial cells when LuM1 cells were added on the monolayers. Gelatin zymography and immunochemical examinations with monoclonal antibodies against gelatinase B (Mr 95,000 type IV collagenase) showed secretion of large amounts of the gelatinase by LuM1 cells.     

Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma

A canine lymphoma cell line (CL-1) was established in culture from tumor cells found in the pleural fluid of a 7-year old female Japanese terrier with thymic form lymphoma. The CL-1 cells were positive for CD45 and MHC class II and negative for CD4, CD5, CD8, Thy-1 and B-cell specific antigen and surface immunoglobulin. The CL-1 cells had a rearranged T-cell receptor beta-chain gene and a germ-line form immunoglobulin gene, indicating that the CL-1 cells represented a monoclonally expanded population of canine alpha beta T-cell lineage.     

Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: a novel mechanism of tumor angiogenesis

Intussusceptive microvascular growth refers to vascular network formation by insertion of interstitial tissue columns, called tissue pillars or posts, into the vascular lumen and subsequent growth of these columns, resulting in partitioning of the vessel lumen. While intussusception has been reported in normal developing organs, its existence in solid tumors has not been previously documented. By observing the growth of the human colon adenocarcinoma (LS174T) in vivo for a period of 6 weeks, we demonstrate that intussusception is an important mechanism of tumor angiogenesis. At the leading edge of the tumor, vascular growth was found to occur by both intussusception and endothelial sprouting. In the stabilized regions, intussusception led to network remodeling and occlusion of vascular segments. The formation of some tissue pillars appears to depend on intravascular blood-flow patterns or changes in intravascular shear stress. The rapid vascular remodeling by intussusception could possibly contribute to intermittent blood flow in tumors.     

Establishment and characterization of a megakaryoblast cell line with amplification of MLL

A new cell line with megakaryoblastic features, designated UoC-M1, was established from the malignant cells of a 68-year-old patient with acute myeloid leukemia. The patient's leukemic cells reacted with alpha-naphthyl acetate esterase and acid phosphatase and expressed CD7, CD24, CD34, CD38, CD45, HLA-DR and CD61. Cytogenetic analysis of the patient's malignant cells (and of the UoC-M1 cells) showed a human, male hypodiploid karyotype with many chromosome rearrangements and marker chromosomes. Spectral karyotyping (SKY) analysis complemented the G-banded karyotyping and clarified several chromosomal translocations and identified the marker chromosomes. Fluorescence in situ hybridization (FISH) and SKY analysis demonstrated that one marker chromosome contained three segments of chromosome 9 interspersed with three segments of chromosome 11, as well as a portion of chromosome 19. FISH analysis with a probe for MLL revealed that the UoC-M1 cells contained four copies of the MLL gene. Southern blot analysis determined that the MLL gene had a germline profile while Northern and Western analyses showed that the MLL mRNAs and protein were of the appropriate sizes. This is the first report of amplification of the MLL gene which may be an additional mechanism of leukemogenesis or disease progression.     

Homologous complement activation on drug-induced apoptotic cells from a human lung adenocarcinoma cell line

Activation of the alternative pathway of homologous complement (C) was observed in a human lung adenocarcinoma cell line, CADO 43, after the cells had become apoptotic following treatment in vitro with vincristine and predonisolone. Deposition of C3b and C3bi on the serum-treated apoptotic cells was revealed by flow cytometry with anti-C3b and -C3bi-specific antibodies and immunoblotting with anti-C3 antibody immunoprecipitates extracted from solubilized fractions of serum-treated apoptotic cells. Two molecular mechanisms were found to be responsible for this post-apoptotic C-activation. Firstly, all C regulators, decay accelerating factor (DAF), membrane cofactor protein (MCP) and C3b/C4b receptor (CR1), were diminished on the cell surface concomitantly with the apoptotic process. Secondly, unidentified molecules which potentially activate homologous C and accept C3b/C3bi fragments became expressed on the cell surface during the apoptotic process. These findings may explain the mechanism whereby tumor cells are efficiently eliminated through chemotherapy.     

Purification and characterization of a cell growth factor from a human leukemia cell line: immunological identity with ferritin

We have succeeded in long-term cultivation of a human erythroleukemia cell line, K-562-T1 (T. Okabe, M. Fujisawa, and F. Takaku, Proc. Natl. Acad. Sci. USA, 81: 453-455, 1984). The cells grown in a protein-free chemically defined medium have been shown to produce cell growth factors (A. Mihara et al., In Vitro Cell. Dev. Biol., 23: 317-322, 1987). In this study, we have purified a cell growth factor from the conditioned medium that stimulates the proliferation of human leukemia cells, HL-60. In the purified factor, two major protein bands of 24 kDa and 22 kDa were identified on a sodium dodecyl sulfate-polyacrylamide gel. The 22 kDa protein was stained with a monoclonal antibody to the light chain of ferritin. The growth-promoting activity of the purified factor was coprecipitated with a monoclonal antibody to the light chain or heavy chain of human ferritin. These results suggest that K-562-T1 cells produce a cell growth factor that is related to ferritin.     

The human myoepithelial cell is a natural tumor suppressor

Myoepithelial cells, which surround ducts and acini of glandular organs, form a natural border separating proliferating epithelial cells from basement membrane and underlying stroma. Myoepithelial cells in situ and in vitro constitutively express high amounts of proteinase inhibitors that include tissue inhibitor of metalloproteinase 1, protease nexin-II, alpha-1 antitrypsin, and maspin. Human myoepithelial xenografts (HMS-X, HMS-3X, and HMS-4X), which our laboratory has established, accumulate an abundant extracellular matrix containing sequestered proteinase inhibitors. Humatrix, a gel that we have derived from HMS-X, inhibits tumor cell invasion (down to 25% +/- 10% of Matrigel control; P < 0.01), and our recently established human myoepithelial cell lines, HMS-1, HMS-3, and HMS-4, inhibit tumor cell invasion in cellular invasion (down to 42% +/- 7% of control; P < 0.05) and in conditioned media assays (down to 30% +/- 8% of control; P < 0.01). The anti-invasive effects of HMS-1, HMS-3, and HMS-4 can be enhanced by phorbol 12-myristate 13-acetate (down to 2% +/- 1% of control) by a maspin-dependent mechanism and abolished by dexamethasone (up to 95% +/- 5% of control) by a maspin-independent mechanism (P < 0.01). HMS-X, HMS-3X, HMS-4X, and Humatrix inhibit tumor invasion and metastasis in severe combined immunodeficient mice (P < 0.001). The cumulative data suggest that myoepithelial cells are natural paracrine suppressors of invasion and metastasis and may specifically inhibit the progression of precancerous disease states to invasive cancer in vivo.     

Establishment of human parotid pleomorphic adenoma cells in culture: morphological and biochemical characterization

This study reports the establishment of alpha-amylase-producing human parotid pleomorphic adenoma cell lines (2HP and 2HP1) which have been maintained in culture for over 1 yr. The procedures required preparation of cellular clumps from tumor tissue and plating them on plasma clot or precoated dishes. During the initial phase of growth they required modified MCDB-153 medium without serum. When cells showed signs of degeneration they were changed to MCDB-153 medium containing first 2% and then 10% heat inactivated fetal bovine serum. Although cells grew well in MCDB-153 containing 10% serum, the epithelial cell morphology was not distinct. Therefore, the growth and morphology of cells grown in MCDB-10% serum were compared with those in RPMI growth medium containing 10% fetal bovine serum and F12 containing 10% agammaglobulin newborn bovine serum. Although the growth of cells was a little slower in F12 medium than those in MCDB and RPMI, the epithelial cell morphology was maintained better than in other growth media. The cells of 2HP and 2HP1 produce low levels of alpha-amylase and relatively high levels of alpha-amylase mRNAs of 1176 and 702 bp and contain neurofilament-160, a neuronal-specific marker. The cells of 2HP1 are tumorigenic when tested in athymic mice, but the cells of 2HP are not. The establishment of amylase-producing human parotid adenoma cell lines of different characteristics in culture provides a new opportunity to study the mechanisms of differentiation and transformation, and regulation of alpha-amylase in these cells.     

Establishment and characterization of human gastric carcinoma cell lines

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.     

Establishment and characterization of human ocular melanoma cell lines

Five continuous cell lines have been established from 29 ocular melanomas and maintained for periods ranging from 3 to 9 years in medium identical to that in which 3 concomitantly studied lines of cutaneous melanoma cells were cultured as controls. The long-term problems to be overcome in establishing uveal cell lines are related to cell-doubling times which ranged from 72 to 432 hr, and plating efficiency, which ranged from 0.5%-6.5%. Tumors and cell lines were found to contain melanosomes. The morphology of uveal cells during the early subcultures exhibited multiple changes. Two different established cell lines were obtained from one ciliary-body tumor. Biochemical studies revealed markers of melanogenesis and neuroendocrine compounds. Cytogenetic studies revealed chromosomal abnormalities that differed between uveal and conjunctival melanomas.     

Establishment and characterization of a new erythropoietin-dependent acute myeloid leukemia cell line, AS-E2

We have established an erythropoietin-dependent human leukemia cell line, AS-E2, from a patient with acute myeloid leukemia. These cells have many characteristics of late erythroid progenitor cells, they are positive for CD36, Glycophorin A, and CD71 but negative for CD41, and positive for benzidine and PAS staining. These cells express GATA-1 and have low affinity erythropoietin (EPO) receptor on their surface. Interestingly, AS-E2 cells are strictly dependent on EPO for their growth and survival; other cytokines including GM-CSF, stem cell factor, or IL-3 cannot support the growth of this cell line. These features are similar to late erythroid lineage cells, like normal BFU-E or CFU-E, and we have demonstrated that EPO stimulation induces the tyrosine phosphorylation of several proteins in AS-E2 cells including the EPO receptor and JAK2 kinase. This new cell line is a useful reagent to study biological and molecular events during the late stages of erythropoiesis, and to understand transforming events in human erythroid cells.     

Identification and characterization of a 14 kDa immunosuppressive protein derived from IIB-MEL-J, a human melanoma cell line

The causes of decreased immune competence in melanoma patients as well as in other cancer patients are incompletely understood. The identification of the factor(s) responsible for this behaviour remains elusive. The present report demonstrates that an immunosuppressive activity (ISA), manifested in vitro as an inhibition of proliferation of human peripheral blood lymphocytes (PBL) induced both by phytohemagglutinin (PHA) or the cytokine IL-2, was exhibited by serum-free conditioned medium (SFCM) from the human melanoma cell line IIB-MEL-J, as well as by two other melanoma cell lines, IIB-MEL-LES and IIB-MEL-IAN, established in our laboratory. The ISA was found to be exerted by a protein, which co-eluted with serum albumin in anionic exchange Mono-Q, gel filtration chromatography and Blue Sepharose columns. It showed a molecular weight (Mw) of 14 kDa when separated from albumin traces by means of a Sephacryl S 200 column. It is not recognized by a pan-specific anti-transforming growth factor-beta (TGF-beta) antibody as determined by Western blots assays performed on the SFCM. The immunosuppressive factor (ISF) is secreted by IIB-MEL-J cell line in soluble from and in very scarce amounts, non-detectable by polyacrylamide gel electrophoresis (PAGE). This characteristic difficults its obtention in adequate quantities to sequentiation. Since this inhibitory factor may have a role in protecting melanoma tumors from attack by the host immune system, preparative isolation will be attempted. But we consider these results only as preliminary one's.     

Establishment and preliminary characterization of human malignant mesothelioma cell lines

In this retrospective study, 47 patients with clinical diagnosis of central nervous system metastases of breast cancer were evaluated by computerized tomography (CT), magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) examination. The patients were divided in 2 groups: 1, without leptomeningeal neoplasm and 2, with leptomeningeal neoplasm. In the group 2, the time interval between the primary disease and the central nervous system metastasis as well as the survival time were shorter than in group 1 (40 and 4.3 months in group 2 versus 57 and 10 months respectively, in group 1). In both groups the most common neurological symptoms and signs were intracranial hypertension and motor deficits. The most sensitive diagnostic methods were CT and MRI in group 1, and the CSF examination in group 2. The use of the tumor markers CEA and CA-15.3 in the routine examination of CSF showed promising results, mainly in leptomeningeal forms.     

Establishment of a diploid cell line of human lung (PHuE-1)

The obtention of a diploid cell line from human lung of great concern for the virological diagnosis and research is reported. Certain aspects about its application and characterization are discussed.     

Establishment and characterization of a human T-lymphocyte cell line immortalized by SV40 and with abnormal expression of TCR/CD3

OBJECTIVE: Elderly women with proximal femur fracture show a prolonged increase in plasma cortisol, which could have undesirable catabolic effects. Suppression of cortisol by dexamethasone is impaired, suggesting resistance to glucocorticoid effects at feedback inhibitory sites. We therefore wished to find out whether peripheral glucocorticoid sensitivity is normal. DESIGN: Peripheral blood mononuclear leucocytes were used as a model tissue. Blood samples were taken from elderly women about 2 weeks after hip fracture and from elderly control women. Each patient was then given 1 mg dexamethasone at 2300 h followed by further sampling at 0800 and 1600 h the next day. METHODS: Glucocorticoid-receptor binding parameters were measured by incubating whole cells with [3H]dexamethasone for 2 h at 37 degrees C. Inhibition of cell proliferation by dexamethasone was assessed by addition of [3H]thymidine to cells cultured for 65 h with concanavalin A. Cortisol and dexamethasone concentrations were measured in the dexamethasone suppression test. RESULTS: As expected, the hip-fracture patients had raised morning cortisol concentrations and impaired suppression by dexamethasone. The cells of the patients had similar numbers of glucocorticoid receptors to those of the control subjects but higher values for Kd (i.e. a lower binding affinity). The cells of the patients incorporated less [3H]thymidine than the control cells in the absence of dexamethasone. The percentage inhibition by a saturating concentration of dexamethasone was unchanged but the concentration giving half-maximal inhibition was decreased (sensitivity was increased) at the higher of the two concanavalin A concentrations used. CONCLUSIONS: These experiments in mononuclear leucocytes give no evidence of peripheral resistance to glucocorticoids in hip-fracture patients with impaired suppression of cortisol by dexamethasone.     

Establishment and characterization of bone marrow stroma-dependent leukemia cell line, HB-1

The suppressive effect of hydrocortisone and dexamethasone on salivary cortisol was investigated in a 2-year study of pituitary-adrenal function in a variety of child psychiatric patients and healthy controls. Symptomatology was assessed using the Child Behavioral Checklist (CBCL). Cortisol day profiles were assessed at 2-h intervals from 0800 to 2000 h on three occasions. Dexamethasone and hydrocortisone were administered orally twice at 2000 h, the doses being adjusted for bodyweight according to the standard dexamethasone suppression test. Fifty-one patients, including patients with dysthymia, oppositional defiant disorder, pervasive developmental disorder, and attention deficit hyperactivity disorder, and ten age and sex matched controls participated. Basal cortisol levels in patients were generally lower than in controls. Both dexamethasone and hydrocortisone were effective in suppressing salivary cortisol, although dexamethasone was somewhat more potent and its effect lasted longer. Hyporesponsiveness to hydrocortisone, but not to dexamethasone, distinguished patients with dysthymia and oppositional defiant disorder from controls. Responsiveness to hydrocortisone was correlated with the symptom clusters social problems and anxious/depressed. The data support the idea that there exist syndrome aspecific disturbances in feedback activity beyond the level of the pituitary, i.e. at the hypothalamic level, at an early age. From this perspective, hydrocortisone suppression is a useful tool for studying pituitary-adrenal function in children. Behavioral correlates of these disturbances of pituitary-adrenal function should be determined.