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.     

Establishment and characterization of a novel human myoepithelial cell line and matrix-producing xenograft from a parotid basal cell adenocarcinoma

Myoepithelial cells exert important paracrine effects on epithelial morphogenesis and mitogenesis through direct cell-cell interactions and through synthesis of a basement membrane extracellular matrix. To study these effects further, this study established the first immortalized human myoepithelial cell line, HMS-1, and transplantable xenograft, HMS-X, from the rare parotid basal cell adenocarcinoma. The cell line exhibited a fully differentiated myoepithelial phenotype and the xenograft exhibited the rare property of accumulating an abundant extracellular matrix composed of both basement membrane and nonbasement membrane components with the latter predominating. With HMS-1 as a feeder layer, dramatic and specific induction of epithelial morphogenesis (spheroid formation) occurred with selected normal epithelial and primary carcinoma target cells. HMS-1 and HMS-X provide distinct advantages over the conventional murine matrices in existence. They will be invaluable in future studies of human tumor-myoepithelial and matrix interactions important for tumor cell growth, invasion, and metastasis.     

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.     

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.     

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 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 multipotential neural cell line that can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro

In the mammalian central nervous system (CNS), multipotential neural stem cells in the neuroepithelium generate the three major types of neural cells, namely, neurons, astrocytes, and oligodendrocytes. To explore the molecular mechanisms underlying proliferation and differentiation of these neural stem cells, we established a cell line named MNS-57 from the embryonic day 12 rat neuroepithelium by introducing the mycer fusion gene, in which c-myc can be conditionally activated by adding oestrogen to the culture medium. MNS-57 cells expressed nestin, vimentin, and the RC1 antigen, which are potential markers for neural stem cells. We show that under particular culture conditions, MNS-57 cells can conditionally generate neurons, astrocytes, and oligodendrocytes in vitro, indicating that they are likely to originate from multipotential neural stem cells. Incubating MNS-57 cells with either oestrogen, which activates mycer, or growth factors such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) stimulated their growth, and the combination of oestrogen and bFGF (or EGF) had a synergistically stronger mitogenic effect than the single factors. Furthermore, both c-myc activation and bFGF appeared to be necessary for the differentiation of MNS-57 cells, and only when stimulated by both signals simultaneously, the cells committed to generating multiple neural cell types. Thus, the property of the cell line is unique in that its differentiation into neurons and glia can be conditionally manipulated in vitro in an exogenous signal-dependent manner. We propose that the cell line described here will provide an useful in vitro model to understand genetic and environmental mechanisms that control the generation of neural cell diversity in the CNS.     

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.     

Establishment of ameloblastoma cell line, AM-1

For a number of years titanium osteointegrated implants have represented a major step forward in dentistry, plastic and maxillofacial surgery, providing a lively stimulus to research both in their application and in the experimental field. The study of osteointegration has led to a more detailed knowledge of bone remodelling regulation mechanisms and the biological mediators involved. In this review the authors examine the role played by cytokines in the homeostasis of bone tissue, paying special reference to the phase of osteoclast activation and its possible effects on peri-implant bone resorption. The authors focus in particular on the effects of interleukin-6 (IL-6) as an important osteotropic factor and a hypothetical target on which to act in order to modulate its effects for therapeutical purposes.     

Establishment of a highly differentiated thyroid cancer cell line of Hürthle cell origin

Hürthle cell carcinomas (HCC) of the thyroid are a variant of follicular thyroid tumors. In contrast to follicular thyroid carcinoma, HCC rarely take up radioiodine and frequently metastasize to the lymph nodes. Histologically they are indistinguishable from Hürthle cell adenomas except for evidence of invasion and metastasis. How these carcinomas develop and why they behave differently than other follicular tumors is not known. Although some differentiated thyroid cancer cell lines exist, none are from Hürthle cell tumors. We have established a well-differentiated thyroid cancer cell line from a metastasis of a HCC, designated XTC.UC1. In vitro, XTC cells display epitheloid morphology, grow with a population doubling time of 4.3 +/- 0.3 days, migrate, and invade through reconstituted basement membranes. The cells are immunoreactive for and release thyroglobulin, respond to thyrotropin (TSH) with increase of intracellular cyclic adenosine monophosphate (cAMP), proliferation, and invasion of reconstituted basement membrane, thus exhibiting characteristics of well-differentiated thyroid carcinoma. In vivo, xenografted XTC cells grow with a doubling time of 9.8 +/- 0.8 days. Tumors spontaneously metastasize to the lymph nodes and less frequently to the lungs and the liver. The cells retained their differentiated function in vivo as assessed by human thyroglobulin (hTG) secretion and immunohistochemistry. This is a first report of the establishment of a unique, highly differentiated thyroid carcinoma cell line derived from an HCC. Based on the ability to invade through reconstituted basement membrane in vitro and the potential to metastasize in vivo, this cell line may provide a unique model to study invasion and metastazation of well-differentiated thyroid 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 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 of an osteocyte-like cell line, MLO-Y4

Although osteocytes are the most abundant cells in bone, their functional role remains unclear. In part, this is due to lack of availability of osteocyte cell lines which can be studied in vitro. Since others have shown that cell lines can be readily developed from transgenic mice in which the SV40 large T-antigen oncogene is expressed under the control of a promoter which targets the cells of interest, we used this approach to develop an osteocyte cell line. We chose as a promoter osteocalcin, whose expression is essentially limited to bone cells and which is expressed more abundantly in osteocytes than in osteoblasts. From these transgenic mice, we isolated cells from the long bones using sequential collagenase digestion and maintained these cells on collagen-coated surfaces which are optimal for osteocyte maintenance and growth. We describe here the properties of a cell line cloned from these cultures, called MLO-Y4 (for murine long bone osteocyte Y4). The properties of MLO-Y4 cells are very similar to primary osteocytes. Like primary osteocytes and unlike primary osteoblasts, the cell line produces large amounts of osteocalcin but low amounts of alkaline phosphatase. The cells produce extensive, complex dendritic processes and are positive for T-antigen, for osteopontin, for the neural antigen CD44, and for connexin 43, a protein found in gap junctions. This cell line also produces very small amounts of type I collagen mRNA compared with primary osteoblasts. MLO-Y4 cells lack detectable mRNA for osteoblast-specific factor 2, which appears to be a positive marker for osteoblasts but may be a negative marker for osteocytes. This newly established cell line should prove useful for studying the effects of mechanical stress on osteocyte function and for determining the means whereby osteocytes communicate with other bone cells such as osteoblasts and osteoclasts.     

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.     

Isolation and characterization of apoptosis-resistant mutants from a radiosensitive mouse lymphoma cell line

This paper reports the findings of a research study funded by the English National Board for Nursing, Midwifery and Health Visiting (ENB), which explored the impact of community care reforms on mental health and learning disability nurses and their practice. In this study we were struck by the divergent views of our respondents about the nature of mental health and learning disability nursing as practice disciplines and the implications of these views for the future shape of preregistration educational preparation. We noted, in particular, the debate between those who advocate what is referred to as specialist educational preparation and others who favour generic preparation. The specialist-generic debate is relevant to nursing as a whole but was magnified in the context of our study because genericism was perceived by many of our respondents as a threat to the minority branches and especially to those (arguably mental health and learning disability) that are not rooted in the biomedical tradition of general nursing. This paper seeks to contribute to this debate as it impinges on the two nursing specialties by developing models of future nurse education grounded in the empirical data from our research and interrogating them to draw out their central features. Two models are clearly independent: the 'specialist' and the 'generic' models. Another three models are partial in that they draw upon the first two: the 'pragmatic' model, the 'unity-of-nursing' model, and the 'social care' model. A common feature of the pragmatic and unity-of-nursing models is that they support the existing DipHE programme, which is now the dominant form of preregistration nurse preparation in the UK. The social care model is applicable principally to learning disability nursing.     

Isolation, cloning and characterization of a putative type-1 astrocyte cell line

The high prevalence of cholesterol gallstone disease in hypertriglyceridemic patients may be associated with frequent metabolic defects in cholesterol and bile acid syntheses and in the concomitant formation of bile supersaturated with cholesterol. This study had the two aims: 1) to assess whether the defects as well as the degree of biliary cholesterol supersaturation in patients with hyperlipoproteinemia (HLP) can be estimated by the simultaneous determination of plasma mevalonate (MVL) and 7alpha-hydroxy-4-cholesten-3-one (C4); and 2) to assess the possible application of an estimated cholesterol saturation index ([CSI]E) as a means of evaluating the clinical effects of simvastatin on biliary lipid composition. Biliary cholesterol supersaturation was observed in patients with both IIa and IV HLP types. Consistent with the high activity and steady-state messenger RNA level of 3-hydroxy-3 methylglutaryl coenzyme A (HMG-CoA) reductase, plasma MVL was significantly higher in 86 patients with HLP (38 type IIa, 44.1 +/- 2.4 nmol/L and 48 type IV, 56.7 +/- 2.3; P < .01) than in 41 normolipidemic subjects (34.2 +/- 1.5), closely correlating with the molar percentage of cholesterol in bile (r = .61, P = .0001; n = 86). On the other hand, consistent with the high activity and messenger RNA level of cholesterol 7alpha-hydroxylase, plasma C4 was significantly higher in patients with HLP (type IIa, 28.8 +/- 2.3 nmol/L and type IV, 38.3 +/- 2.7; P < .01) than in normolipidemic subjects (17.4 +/- 1.5). Plasma C4 was closely correlated with plasma MVL (r = .40, P = .0001; n = 86), but was inversely correlated with the molar percentage of bile acids in bile (r = .49, P = .0001; n = 86). Assuming that cholesterol supersaturation in patients with HLP may be governed by both an enhanced cholesterol secretion (closely reflected by plasma MVL) and a decreased secretion of bile acids (closely reflected by plasma C4), the multivariate linear regression-analyses revealed that an index defined as estimated CSI ([CSI]E) (%) in patients with HLP was given by the following equation using plasma MVL and C4 (nmol/L): [CSI]E = 1[MVL] + 0.7[C4] + 44.4. Biliary cholesterol supersaturation in patients treated with simvastatin improved in a manner parallel to the time course of decreases in plasma MVL and C4. The [CSI]E before and at the end of treatment were correlated with biliary CSI. These results indicate that defects of hepatic cholesterogenesis, and bile acid synthesis, and the degree of biliary cholesterol supersaturation in patients with HLP can be estimated exactly by the simultaneous determination of plasma MVL and C4; furthermore [CSI]E may be adopted for clinical use as a convenient index of biliary CSI.     

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 and characterization of a novel in vitro angiogenesis model using a microvascular endothelial cell line, F-2C, cultured in chemically defined medium

The behavior of vascular endothelial cells (EC) is an important factor in the processes involved in angiogenesis, but the regulatory mechanisms of angiogenesis, especially underlying the tubulogenesis by EC are not yet clear. Although a number of in vitro experimental models of tubulogenesis have been developed by use of cultured EC, most of those models are too complex to be easily handled and further, the culture media are usually supplemented with serum, creating problems in interpretation of experimental results. To generate a simple in vitro angiogenesis study model under serum-free culture conditions, we adapted a murine microvascular endothelial cell line, F-2, to a chemically defined medium, Cos Medium 001, and successfully established a subline of F-2, designated F-2C, which revealed a unique growth pattern. In Cos Medium 001, F-2C proliferates in a cobblestone pattern at an early growth stage, but, at a late growth stage, spontaneously differentiates to form three-dimensional honeycomblike tubular structures without the supplementation of any specific factors. The cell aggregation activity of F-2C in the presence of Ca2+ was much greater than that of F-2. The amount of subendothelial matrix deposited by F-2C was significantly higher than that by F-2, and increased prominently after the F-2C cells reached the differentiating stage of tubulogenesis. These findings indicate that F-2C is a new EC line in which tubulogenesis is spontaneously induced by the marked deposition of basement membrane analog to the subendothelial matrix and by the enhancement of presumable cadherin activity. We suggest that this cell line, F-2C, represents a simple and useful in vitro angiogenesis model.